WO2025221925A1 - Rna ligase mediated oligonucleotide synthesis - Google Patents

Rna ligase mediated oligonucleotide synthesis

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Publication number
WO2025221925A1
WO2025221925A1 PCT/US2025/025017 US2025025017W WO2025221925A1 WO 2025221925 A1 WO2025221925 A1 WO 2025221925A1 US 2025025017 W US2025025017 W US 2025025017W WO 2025221925 A1 WO2025221925 A1 WO 2025221925A1
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Prior art keywords
amino acid
seq
stranded rna
rna ligase
single stranded
Prior art date
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Application number
PCT/US2025/025017
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French (fr)
Inventor
Stephanie Marie FORGET
Anders Matthew KNIGHT
Nikki D. KRUSE
Nicholas Porter
Amani SHOUBBER
Ljubica Vojcic
Jonathan VROOM
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Codexis Inc
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Codexis Inc
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Publication of WO2025221925A1 publication Critical patent/WO2025221925A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01003—RNA ligase (ATP) (6.5.1.3)

Definitions

  • the oligonucleotides are designed primarily to target pre-mRNA, mRNA, or non-coding RNA (e.g., miRNA) to promote RNA degradation, modulate splicing, interrupt translation, or in some instances activate expression.
  • Small interfering RNAs are a class of oligonucleotide therapeutics that are typically double stranded oligonucleotides that act via RISC (RNA-induced silencing complex) pathway, where the strand complementary to an RNA target, also referred to as the guide strand, targets the RNA for degradation or translation inhibition.
  • RISC RNA-induced silencing complex
  • Antisense oligonucleotides are single-stranded and designed to bind sequence specifically to a target RNA and modulate protein expression via several different mechanisms.
  • Targets of ASOs include pre-mRNA, mRNA, and non-coding RNA.
  • siRNA and ASO oligonucleotides are designed to include various modifications to improve in vivo stability, cellular delivery, specificity, and efficacy.
  • Sugar modifications include 2’-O-methyl, 2’-O-ethyl, 2’- O-methoxyethyl, 2’-fluoro, and locked nucleic acid (LNA); modification of internucleoside linkages include phosphorothioate and phosphoramidate morpholino linkages; and nucleobase modifications include 5’- methylcytosine and G-clamp.
  • conjugating targeting moieties to the oligonucleotide such as GalNac and lipid groups, can enhance delivery to cells and tissues.
  • Modified oligonucleotides are generally synthesized chemically by solid-phase synthesis using phosphoramidite chemistry.
  • RNA ligase polypeptides and compositions thereof and methods of using the single-stranded RNA ligases for synthesis of oligonucleotides, including modified oligonucleotides with conjugate moieties.
  • a method of extending an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor.
  • the nucleotide donor, the oligonucleotide(A), or both the nucleotide donor and the oligonucleotide(A) comprise a modified nucleotide.
  • the oligonucleotide (A) comprises at least one modified nucleoside, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker.
  • the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.
  • the reactive group comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.
  • the modified nucleoside on the oligonucleotide (A) is at the 5’-terminal nucleotide, an internal nucleotide, or the 3’-terminal nucleotide.
  • the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide (A) .
  • the conjugate moiety or reactive group is attached to the nucleoside via a linker.
  • the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A).
  • the linker comprises a cleavable linker.
  • the oligonucleotide(A) comprises one or more terminal groups.
  • the terminal group is at the 5’-terminal nucleotide of the oligonucleotide(A).
  • the terminal group is a 5’-phosphonate (E- or Z-vinylphosphonate), 4’-amino, 4’-aminoalkyl, abasic nucleotide, or inverted abasic nucleotide.
  • the oligonucleotide(A) comprises a 5’- OH or a 5’-blocking group that inhibits ligation by the single-stranded RNA ligase.
  • the oligonucleotide (A) comprises the formula (I): A 1[•A2]m•A3-OH (I) wherein each of A 1 , A 2 and A 3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage.
  • each of A2 is the same or different nucleoside.
  • the oligonucleotide(A) comprises a modified nucleoside of at least one or more of A1, A2, or A3.
  • At least one of A1, A2, or A3 is modified with a conjugate moiety or conjugate reactive group.
  • Docket Number CX10-269WO4 [0018]
  • one or more of the internucleoside linkage is a modified internucleoside linkage.
  • the modified internucleoside linkage is a phosphorothioate or a phosphorodithioate.
  • the modified nucleoside on the oligonucleotide(A) comprises the formula (II): A-[L] g -[M] h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0.
  • the oligonucleotide (A) is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.
  • L is attached to the nucleobase or the sugar moiety of the nucleoside.
  • the nucleotide donor comprises a nucleotide(D) or an oligonucleotide(D).
  • the nucleotide(D) or oligonucleotide(D) comprises a modified nucleoside.
  • the modified nucleoside of nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or linker.
  • the conjugate moiety on the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.
  • reactive group on the nucleotide donor comprises an amino, -CN (cyano), N 3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.
  • the modified nucleoside on the oligonucleotide(D) is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.
  • the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the modified nucleoside.
  • the conjugate moiety or the conjugate reactive group is attached to the nucleoside via a linker L.
  • the linker comprises a cleavable linker.
  • the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) Docket Number CX10-269WO4 wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120.
  • the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.
  • each of D 2 is the same or different nucleoside.
  • the nucleotide donor comprises a modified nucleoside
  • at least one or more of D 1 , D 2 , or D 3 is modified.
  • at least one of D 1 , D 2 , or D 3 is modified with a conjugate moiety, reactive group, or linker.
  • D comprises a modified nucleoside.
  • D is modified with a conjugate moiety, reactive group, or linker.
  • the modified nucleoside on nucleotide(D) or oligonucleotide(D) has the formula (IV): D-[L] q -[M] r (IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0. [0033] In some embodiments, wherein when q is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside. In some embodiments, wherein when q is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside.
  • the nucleotide (D) or pD includes a 3’-phosphate (e.g., pDp).
  • the nucleotide donor i.e., nucleotide(D) or oligonucleotide(D)
  • the reaction with the single-stranded RNA ligase and a 3’-blocked nucleotide(D) or 3’-blocked oligonucleotide(D) results in a 3’-blocked extended oligonucleotide product.
  • the 3’- blocking group comprises a reversible blocking group.
  • the product is a 3’-blocked extended oligonucleotide
  • the 3’-blocked extended oligonucleotide is separated or removed from the single-stranded RNA ligase.
  • the single-stranded RNA ligase is inactivated.
  • the method further comprises removing or cleaving the 3’-blocking group on 3’-blocked extended oligonucleotide with a deblocking agent to form an unblocked extended oligonucleotide.
  • the method further comprises inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent.
  • the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase.
  • the method further comprises one or more cycles of extension with a nucleotide donor; separation of 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivation of the single-stranded RNA ligase; removing or cleaving the reversible 3’-blocking group with a deblocking agent; and separating the unblocked extended oligonucleotide, wherein each cycle uses a new nucleotide donor.
  • the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors, such as a mixture of different pD or pDp.
  • the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor (e.g., nucleotide(D) or oligonucleotide(D)) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence.
  • the nucleotide(D) comprises a selected or predetermined nucleotide(D) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence.
  • the single-stranded RNA ligase comprises RNA ligase 1.
  • the single-stranded RNA ligase comprises a recombinant single-stranded RNA ligase described herein.
  • the single-stranded RNA ligase is immobilized on a support medium.
  • the oligonucleotide acceptor (oligonucleotide(A)) and nucleotide donor are provided in solution or aqueous phase.
  • the oligonucleotide acceptor (oligonucleotide(A)) is immobilized is attached to a support medium.
  • the single-stranded RNA ligase and the nucleotide donor are provided in solution or aqueous phase.
  • the reaction further comprises a pyrophosphatase for cleaving of pyrophosphate product.
  • the reaction further comprises an ATP recycling system.
  • the suitable reaction conditions comprises one or more of an NTP, a divalent metal ion, and buffer. [0049] In some embodiments of the method, the suitable reaction conditions comprises a reaction temperature of 5-60 °C. Docket Number CX10-269WO4 [0050] In some embodiments of the method, the suitable reaction conditions comprise a reaction pH of about 5-8.
  • the present disclosure provides a recombinant single-stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the amino acid sequence of the recombinant single-stranded RNA ligase comprises the sequence comprising amino acid residue 12 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the single-stranded RNA ligase is the single-stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to S
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 254, 240, 118, 347, 167, 281, 303, 205, or 50, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/276, 246/276/302, 246/279/280,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/254/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference Docket Number CX10-269WO4 sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the Docket Number CX10-269WO4 reference sequence corresponding to SEQ ID NO: 32, 44,
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246,
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant single stranded RNA ligase comprising an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244- 912, and 934-1526.
  • the recombinant single stranded RNA ligase exhibits single stranded RNA ligase activity and at least an improved property as compared to a reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase exhibits an improved property selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), as compared to a reference single stranded RNA ligase.
  • the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768.
  • the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or an amino acid sequence corresponding to SEQ ID NO: 14.
  • the recombinant single-stranded RNA ligase further comprises a fusion protein.
  • the recombinant single stranded RNA ligase is provided as a purified preparation.
  • the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding any of the recombinant single stranded RNA ligase disclosed herein.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO.
  • polynucleotide sequence of the recombinant polynucleotide is codon- optimized for expression of the encoded recombinant single stranded RNA ligase.
  • polynucleotide sequence encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO.
  • the polynucleotide sequence of the recombinant polynucleotide encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or a polynucleotide sequence comprising SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499.
  • the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding any of the single stranded RNA ligases described herein.
  • the expression vector comprises a control sequence operably linked to the recombinant polynucleotide.
  • the control sequence comprises a promoter, particularly a heterologous promoter.
  • the present disclosure provides a host cell comprising an expression vector for expression or production of the recombinant single stranded RNA ligase.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell.
  • the present disclosure provides a method of producing a recombinant single stranded RNA ligase, the method comprising culturing a host cell described herein under suitable culture conditions such that the encoded recombinant single stranded RNA ligase is produced.
  • the method further comprises recovering the recombinant single stranded RNA ligase polypeptide from the culture and/or host cell.
  • the method further comprises purifying the recombinant single stranded RNA ligase polypeptide.
  • the present disclosure provides a composition comprising a recombinant single stranded RNA ligase.
  • the composition further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and/or one or more polynucleotide substrates for the single stranded RNA ligase.
  • the composition further comprises at least an oligonucleotide acceptor and/or a nucleotide donor (e.g., nucleotide (D) or oligonucleotide (D) ).
  • the oligonucleotide donor comprises a modified oligonucleotide donor.
  • the nucleotide donor nucleotide (D) comprises a modified nucleotide (D) .
  • the nucleotide donor oligonucleotide (D) comprises a modified oligonucleotide (D).
  • the present disclosure further provides a kit comprising a recombinant single stranded RNA ligase described herein.
  • the kit further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and/or one or more polynucleotide substrates for the single stranded RNA ligase.
  • a buffer nucleotide cofactor, divalent metal, ligation enhancer, and/or one or more polynucleotide substrates for the single stranded RNA ligase.
  • the single-stranded RNA ligases are used to produce modified oligonucleotide, particularly modified oligonucleotides having a conjugate moiety or a reactive group.
  • modified oligonucleotide particularly modified oligonucleotides having a conjugate moiety or a reactive group.
  • EC Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).
  • NC-IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.
  • ATCC refers to the American Type Culture Collection whose biorepository collection includes genes and strains.
  • NCBI refers to National Center for Biological Information and the sequence databases provided therein.
  • Protein “Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
  • amino acids and “amino acid” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission.
  • alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), Docket Number CX10-269WO4 threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).
  • the amino acid may be in either the L- or D-configuration about ⁇ -carbon (C ⁇ ).
  • “Ala” designates alanine without specifying the configuration about the ⁇ -carbon
  • “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively.
  • upper case letters designate amino acids in the L-configuration about the ⁇ -carbon
  • lower case letters designate amino acids in the D-configuration about the ⁇ -carbon.
  • Fusion protein refers to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins.
  • fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).
  • RNA ligase refers to enzymes that covalently joins the 5’-phosphoryl termini of RNA or DNA to the 3’-hydroxyl termini of RNA or DNA. Families of known RNA ligases include RNA ligase 1, also referred to as single-stranded RNA ligase or ssRNA ligase, which catalyzes the covalent joining of single-stranded 5’- phosphoryl termini of RNA or DNA to single-stranded 3--hydroxyl termini of RNA or DNA.
  • RNA ligase 2 also referred to as double stranded RNA ligase or dsRNA ligase, also catalyzes the covalent joining of a 3’- hydroxyl terminus of RNA to a 5’-phosphorylated RNA or DNA but shows preference for double stranded substrates.
  • RNA ligases include those enzymes classified in EC 6.5.1.3. It is to be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates also includes nucleotide substrates that contain modified nucleotides and/or nucleotide analogs.
  • Polynucleotide “nucleic acid,” or “oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides.
  • the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U).
  • nucleosides may be either ribonucleosides or 2’-deoxyribonucleosides.
  • the nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis.
  • a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated.
  • DNA refers to deoxyribonucleic acid.
  • RNA refers to ribonucleic acid.
  • polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions.
  • the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amide Docket Number CX10-269WO4 linkages, etc.; nucleosides with modified and/or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-al
  • Nucleobase refers to means an unmodified nucleobase or a modified nucleobase.
  • an “unmodified nucleobase” is adenine (A), thymine (T). cytosine (C). uracil (U). or guanine (G).
  • a “modified nucleobase” refers to a group of atoms other than unmodified A, T, C, U. or G capable of pairing with at least one unmodified nucleobase.
  • Nucleoside refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified.
  • internucleoside linkage refers to as a linkage that covalently couples two nucleosides together.
  • internucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides.
  • Non-limiting examples of internucleoside linkages include phosphodiester -O-P(O) 2 -O- linkages and modified internucleoside linkages, such as phosphorothioate -O-P-(O, S)-O- and phosphorodithioate -O-P(S) 2 -O-.
  • Modified oligonucleotide refers to an oligonucleotide which contains at least one modified internucleoside linkage and/or a modified nucleoside, or a modified terminal group.
  • Modified nucleotide refers to a nucleotide (e.g., NMP, NDP, NTP) in which at least one of the phosphate is a modified phosphate group and/or a modified nucleoside.
  • “Modified nucleoside” or “nucleoside modification” refers to a nucleoside modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase.
  • the modified nucleoside comprises a modified nucleobase and/or a modified sugar residue.
  • modified nucleoside may also be used herein interchangeably with the term “nucleoside analogue.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the nucleobase of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing.
  • “Modified internucleoside linkage” refers to as a linkage other than a phosphodiester (PO) linkage that covalently connects two nucleosides together.
  • PO phosphodiester
  • exemplary modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
  • Other modified phosphorus-containing internucleoside linkages include phosphotriesters, methylphosphonates, and phosphoramidates (P-NH 2 ). See, e.g., Clave et al., RSC Chem Biol., 20212(1): 94–150).
  • internucleoside linkages having a chiral atom can be prepared as a mixture of the stereoisomers, or as separate stereoisomers.
  • “Phosphorothioate internucleoside linkage” refers to an internucleoside linkage in which one of the oxygen atom in a phosphodiester linkage is replaced with a sulfur atom.
  • a phosphorothioate linkage may be represented as -O-P(O,S)-O-, wherein one of the non-bridging oxygen atoms is replaced with a sulfur atom.
  • Phosphorothioate internucleoside linkages are chiral (see, for example, Jahns et al.2022, Nucleic Acids Research Vol.50, No 3, 1221-1240), with right-handed (Rp) and left-handed (Sp) isomers.
  • the Rp diastereomer may be referred to as an R-PS internucleoside linkage or an srP internucleoside linkage.
  • the Sp diastereomer may be referred to as an S-PS internucleoside linkage or ssP internucleoside linkage.
  • the oligonucleotide comprises one or more srP internucleoside linkages.
  • the oligonucleotide comprises one or more ssP internucleoside linkages.
  • that phosphorothioate internucleoside linkage may be either an srP linkage or an ssP linkage.
  • “Non-bridging phosphorothioate internucleoside linkage” refers to a phosphorothioate internucleoside linkage in which the sulfur atom attached to the phosphorous atom is in place of a non-bridging oxygen atom.
  • Non-bridging phosphorodithioate internucleoside linkage refers to a modified internucleoside linkage which is a non-bridging phosphorodithioate internucleoside linkage.
  • a non-bridging phosphorodithioate internucleoside linkage has two identical sulfur atoms attached to the phosphorous atom, achieved by replacing the non-bridging oxygen atom in the phosphorothioate linkage with a sulfur atom.
  • “Abasic sugar moiety” refers to a sugar moiety of a nucleoside that is not attached to a nucleobase.
  • abasic sugar moieties are referred to as “abasic nucleoside.”
  • “Inverted nucleoside” refers to a nucleotide having a 3’ to 3’ and/or 5’ to 5’ internucleoside linkage.
  • inverted sugar moiety refers to the sugar moiety of an inverted nucleoside or an abasic sugar moiety having a 3’ to 3’ and/or 5’ to 5’ internucleoside linkage.
  • LNA nucleoside or “locked nucleoside” refers to 2'-modified nucleoside which comprises a biradical linking the C2' and C4' of the ribose sugar ring of said nucleoside (also referred to as a "2'- 4' bridge"), which restricts or locks the conformation of the ribose ring.
  • These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • BNA bicyclic nucleic acid
  • Non-limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med. Chem. Lett.2002, 12, 73-76, Seth et al. J.
  • Terminal group refers to a group located at the first or last nucleoside in a polynucleotide or oligonucleotide.
  • a 5’-terminal group refers to the terminal group bonded to 5′-or 4’-carbon atom of the first nucleoside within a polynucleotide.
  • a 3’-terminal group is a terminal group bonded to 3′- carbon atom of the last nucleoside within a polynucleotide or oligonucleotide.
  • “5’-blocking group” as used herein refers to a moiety or chemical group that prevents or inhibits attachment of another nucleoside, nucleotide or oligonucleotide to the 5’-terminal nucleoside.
  • a 5’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide or oligonucleotide to the to the 5’-terminal nucleoside, particularly the 5’-OH of the 5’-terminal nucleoside.
  • “3’-blocking group” refers to moiety or chemical group that prevents or inhibits attachment off another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside.
  • a 3’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside, particularly the 3’-OH of the 3’-terminal nucleoside.
  • “Reversible blocking group” refers to a blocking group that can be removed or cleaved off to provide a free 3’-OH.
  • the blocking group is removable with a deblocking agent, which can be a chemical or enzymatic deblocking agent.
  • Enzymatically reversible blocking group refers to a blocking group that is susceptible to removal or cleaving by an enzyme.
  • Duplex and “ds” refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]).
  • “Complementary” is used herein to describe the structural relationship between nucleotide bases that are capable of forming base pairs with one another.
  • a purine nucleotide base present on a polynucleotide that is complementary to a pyrimidine nucleotide base on a polynucleotide may base pair by forming hydrogen bonds with one another.
  • Complementary nucleotide bases can base pair via Watson/Crick base pairing or in any other manner than forms stable duplexes or other nucleic acid structures.
  • “Watson/Crick Base-Pairing” refers to a pattern of specific pairs of nucleobases and analogs that bind together through sequence-specific hydrogen-bonds, e.g., A pairs with T or U, and G pairs with C.
  • “Annealing” or “Hybridization” refers to the base-pairing interactions of one nucleobase polymer (e.g., poly- and oligonucleotides) with another that results in the formation of a double-stranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick base- pairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing.
  • the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers.
  • Docket Number CX10-269WO4 “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
  • Wild-type and “naturally-occurring” refer to the form found in nature.
  • a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
  • Coding sequence and synonymously “encoding” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
  • Percent (%) sequence identity refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as Docket Number CX10-269WO4 the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915).
  • Reference sequence refers to a defined sequence used as a basis for a sequence comparison.
  • a reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.
  • a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide.
  • two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences
  • sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.
  • Comparison window refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence.
  • the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.
  • “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as that of a recombinant primase, Docket Number CX10-269WO4 can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences.
  • “Mutation” refers to the alteration of a nucleic acid sequence.
  • mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation).
  • the mutation comprises a substitution, such that a different amino acid is produced.
  • the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence.
  • the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence.
  • “Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence.
  • the amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based.
  • the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide).
  • the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.
  • the amino acid difference e.g., a substitution
  • an amino acid residue difference or substitution may be a deletion and may be denoted by a “-“ where appropriate.
  • the phrase “an amino acid residue nB” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence.
  • a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence.
  • amino acid substitution set and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions.
  • a substitution set refers to the set of Docket Number CX10-269WO4 amino acid substitutions that is present in any of the variant primase polypeptides listed in any of the Tables in the Examples.
  • the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set.
  • the individual substitutions are separated by a semicolon “;” or slash “/”.
  • “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.
  • another aliphatic amino acid e.g.,
  • Non-conservative substitution refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain.
  • exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
  • “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide.
  • Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and/or retaining the improved properties of an recombinant primase.
  • Deletions can be directed to the internal portions and/or terminal portions of the polypeptide.
  • the deletion can comprise a continuous segment or can be discontinuous.
  • Insertions refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.
  • “Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which Docket Number CX10-269WO4 it is being compared (e.g., a full length recombinant primase of the present invention) and that retains substantially all of the activity of the full-length polypeptide.
  • “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides).
  • the term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).
  • the recombinant primase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations.
  • the recombinant primase polypeptides provided herein are isolated polypeptides.
  • substantially pure polypeptide refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.
  • a substantially pure primase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition.
  • the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the isolated recombinant primase polypeptides are substantially pure polypeptide compositions.
  • “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism.
  • the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome.
  • the polynucleotides encoding the primase are codon optimized for optimal production from the host organism selected for expression.
  • Control sequence refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure.
  • Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide.
  • control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such Docket Number CX10-269WO4 that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest.
  • “Promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence.
  • the promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Suitable reaction conditions or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co- solvents, co-factors, etc.) under which an RNA ligase is capable of attaching a nucleotide donor to a nucleotide acceptor. Exemplary “suitable reaction conditions” are provided herein (see, the Examples).
  • “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the primase polypeptide on the substrate.
  • “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).
  • “Vector” is a recombinant construct for introducing a polynucleotide of interest into a cell.
  • the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide or a polypeptide encoded in the polynucleotide.
  • an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.
  • “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • “Produces” refers to the production of proteins and/or other compounds by cells.
  • heterologous refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.
  • “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., a polynucleotide sequences encoding a recombinant primase).
  • the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art.
  • Alkyl refers to straight or branched chain hydrocarbon groups having the number of carbon atoms designated, for example 1 to 20 carbon atoms (C1-C20), particularly 1 to 12 carbon atoms (C1-C12 or C1-12), and more particularly (C1-C8 or C1-8) carbon atoms.
  • Exemplary “alkyl” includes, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl.
  • Alkenyl refers to straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 20 carbon atoms (C2-C20), particularly 2 to 12 carbon atoms (C2-C12 or C2-12), and most particularly 2 to 8 (C2-C8 or C2-8)carbon atoms, having at least one double bond.
  • alkenyl includes, but are not limited to, vinyl ethenyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl.
  • Alkynyl refers to a straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 12 carbon atoms (C 2 -C 12 or C 2-12 ), particularly 2 to 8 carbon atoms (C 2 -C 8 or C 2- 8 ), containing at least one triple bond.
  • alkynyl includes ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, 4-hexynyl and 5-hexynyl.
  • Alkylene alkenylene
  • alkynylene refers to a straight or branched chain divalent hydrocarbon radical of the corresponding alkyl, alkenyl, and alkynyl, respectively.
  • alkylene alkenylene and “alkynylene” may be optionally substituted, for example with alkyl, alkyloxy, hydroxyl, carbonyl, carboxyl, halo, nitro, and the like.
  • “Lower” in reference to substituents refers to a group having between one and six carbon atoms.
  • Heteroalkyl heteroalkenyl
  • heteroalkynyl refers to the corresponding alkyl, alkenyl, and akynyl in which one or more of the carbon atoms is replaced with a heteroatom, such as O, S and N.
  • Cycloalkyl refers to any stable monocyclic or polycyclic system which consists of carbon atoms, any ring of which being saturated.
  • Cycloalkenyl refers to any stable monocyclic or polycyclic system which consists of carbon atoms, with at least one ring thereof being partially unsaturated. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicycloalkyls and tricycloalkyls (e.g., adamantyl).
  • Heterocycloalkyl or “heterocyclyl” refers to a substituted or unsubstituted 3 to 14 membered, mono- or bicyclic, non-aromatic hydrocarbon, wherein 1 to 3 carbon atoms a (e replaced by a heteroatom.
  • Heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR’-, -PH-, -S(O)-, -S(O) 2 -, -S(O) NR’-, -S(O) 2 NR’-, and the like, including combinations thereof, where each R’ is independently hydrogen or lower alkyl.
  • Examples include oxiranyl, oxetanyl, azetidynyl, oxazolyl, thiazolidinyl, thiazolyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyranyl, tetrahydrofuranyl, tetrahydropyranyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, azapanyl, and the like.
  • Aryl refers to a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Examples of “aryl” groups include phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, and the like.
  • Heteroaryl refers to an aromatic heterocyclic ring, including both monocyclic and bicyclic ring systems, where at least one carbon atom of one or both of the rings is replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur, or at least two carbon atoms of one or both of the rings are replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur.
  • the heteroaryl can be a 5 to 6 membered monocyclic, or 7 to 11 membered bicyclic ring systems.
  • heteroaryl groups include pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, quinolyl, and the like.
  • Bridged bicyclic refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge.
  • a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen).
  • a bridged bicyclic group has 5 to 12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • bridged bicyclic groups include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include: , [0201] In some embodiments, a locked nucleoside is a bridged bicyclic compound. [0202] “Fused ring” refers a ring system with two or more rings having at least one bond and two atoms in common.
  • a “fused aryl” and a “fused heteroaryl” refer to ring systems having at least one aryl and heteroaryl, respectively, that share at least one bond and two atoms in common with another ring. Docket Number CX10-269WO4 [0203] “Carbonyl” refers to -C(O)-. The carbonyl group may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones. For example, an -C(O)R’, wherein R’ is an alkyl is referred to as an alkylcarbonyl.
  • R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • Halogen or “halo” refers to fluorine, chlorine, bromine and iodine.
  • Haloalkyl refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms.
  • “Hydroxy” refers to –OH.
  • Oxy refers to group -O-, which may have various substituents to form different oxy groups, including ethers and esters.
  • the oxy group is an –OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • acyl refers to -C(O)R’, where R is hydrogen, or an optionally substituted alkyl, heteroalkyl, cylcoalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl as defined herein.
  • exemplary acyl groups include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.
  • Alkyloxy or “alkoxy” refers to —OR’, wherein R’ is an optionally substituted alkyl.
  • Aryloxy refers to –OR’, wherein R’ is an optionally substituted aryl.
  • Carboxy refers to –COO- or COOM, wherein H or a M + counterion.
  • Carbamoyl refers to -C(O)NR’R’, wherein each R’ is independently selected from H or an optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocylcoalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl.
  • Cyano refers to –CN.
  • R is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocyclolalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • SiR SiR’R’R’, where R’ is as defined in the specification.
  • each R’ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • any heterocyloalkyl or heteroaryl group present in a silyl group has from 1 to 3 heteroatoms selected independently from O, N, and S.
  • “Thiol” or “sulfhydryl” refers to –SH.
  • “Disulfied” refers to -S-S- groups.
  • Sulfanyl refers to –SR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • R is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • -SR wherein R is an alkyl is an alkylsulfanyl.
  • “Sulfonyl” refers to -S(O)2-, which may have various substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones.
  • -S(O)2R’, wherein R’ is an alkyl refers to an alkylsulfonyl.
  • R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl.
  • Amino or “amine” refers to the group –NR’R’ or –NR’R’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, heterocycloalkyl, alkyloxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkyloxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like.
  • amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like.
  • “Optional” or “optionally” refers to a described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not.
  • “optionally substituted alkyl” refers to an alkyl group that may or may not be substituted and that the description encompasses both substituted alkyl group and unsubstituted alkyl group.
  • “Substituted” as used herein means one or more hydrogen atoms of the group is replaced with a substituent atom or group commonly used in pharmaceutical chemistry. Each substituent can be the same or different.
  • substituents include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, OR ’ (e.g., hydroxyl, alkyloxy (e.g., methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, arylalkyloxy, ether, ester, carbamate, etc.), hydroxyalkyl, alkyloxycarbonyl, alkyloxyalkyloxy, perhaloalkyl, alkyloxyalkyl, SR ’ (e.g., thiol, alkylthio, arylthio, heteroarylthio, arylalkylthio, etc.), S + R ’ 2, S(O)R ’ , SO2R ’ , NR ’ R ” (e.g., primary amine (i.e., NH2)
  • substitutions will typically number less than about 10 substitutions, more preferably about 1 to 5, with about 1 or 2 substitutions being preferred.
  • “Stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable.
  • “stereoisomer thereof” with respect to a compound includes any stereoisomer of the compound and mixtures of stereoisomers, and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposable mirror Docket Number CX10-269WO4 images of one another.
  • a compound may have more than one chiral center such that the compound may exist as either an individual diastereomer or as a mixture of diastereomers.
  • Ligation of Oligonucleotides with single-stranded RNA Ligases [0225]
  • the present disclosure provides a method of ligating an acceptor to a donor substrate of single-stranded RNA ligase to form a ligated oligonucleotide product.
  • the acceptor or donor, or both the acceptor and donor of the single-stranded RNA ligase substrates are modified to generate a ligated modified oligonucleotide product.
  • a method of synthesizing an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor to form an extended oligonucleotide.
  • the oligonucleotide(A), the nucleotide donor, or both the oligonucleotide(A) and the nucleotide donor comprise a modified nucleotide.
  • the single stranded RNA ligase can be used to synthesize, among others, antisense oligonucleotides (ASOs), polynucleotide strands for use in synthesis of double stranded polynucleotides (e.g., siRNA compounds), and guide RNA used in CRISPR or related genome editing technology, and the like.
  • ASOs antisense oligonucleotides
  • polynucleotide strands for use in synthesis of double stranded polynucleotides e.g., siRNA compounds
  • guide RNA used in CRISPR or related genome editing technology
  • the single stranded RNA ligase is used to add a nucleotide comprising a conjugate moiety, such as cell targeting moieties (e.g., GalNAc, lipids, steroids, etc.), to an oligonucleotide.
  • cell targeting moieties e.g., GalNAc,
  • the oligonucleotide acceptor (oligonucleotide(A)) comprises a free 3’-OH group, or a functional form thereof, at the 3’-terminal nucleotide suitable as an acceptor for or reaction with the nucleotide donor in the single-stranded RNA ligase reaction.
  • the oligonucleotide acceptor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the oligonucleotide acceptor is up to 122 nucleotides in length.
  • the oligonucleotides is up to 150 or more nucleotides in length.
  • the oligonucleotides is up to 200 or more nucleotides in length.
  • the oligonucleotide acceptor is 2 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 3 nucleotides in length. In some embodiments, the oligonucleotide acceptors is 4 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 5 nucleotides in length. In some embodiments, the oligonucleotide is 6 nucleotides in length. In some embodiments, the oligonucleotide is 7 nucleotides in length. In some embodiments, the oligonucleotide is 8 nucleotides in length.
  • the oligonucleotide is 9 nucleotides in length. In some embodiments, the oligonucleotide is 10 nucleotides in length. Docket Number CX10-269WO4 [0232] In some embodiments, the oligonucleotide acceptor comprises a 5’-OH group. In some embodiments, the presence of a 5’-OH group prevents the oligonucleotide acceptor from acting as a nucleotide donor for another RNA ligase reaction.
  • the oligonucleotide acceptor comprises a 5’-blocking group that prevents or inhibits the oligonucleotide acceptor to act as a nucleotide donor in another RNA ligase reaction, as further described herein.
  • use of an oligonucleotide acceptor with a 5’-OH or a 5’-blocking group in combination with a nucleotide donor having a 3’-blocking group can be used to direct or guide the ligation reaction to generate specific ligated products.
  • the oligonucleotide acceptor comprises at least a modified nucleotide.
  • the modified nucleotide on the oligonucleotide acceptor comprises a modified internucleoside linkage, a modified nucleoside, a 5’-terminal group, or any combinations thereof.
  • a modified nucleoside on the oligonucleotide acceptor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.
  • the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide acceptor, as limited by the length of the oligonucleotide acceptor.
  • all nucleosides of the oligonucleotide acceptor comprises a modified nucleoside.
  • the modified 3’-terminal nucleoside of the oligonucleotide acceptor comprises a modified sugar moiety.
  • the sugar moiety is modified at the 2’-position.
  • the modified 3’-terminal nucleoside is a 2’-fluoro adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro thymidine, 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine.
  • the oligonucleotide acceptor comprises at least one modified nucleoside or modified terminal group, wherein the modified nucleoside or terminal group comprises a conjugate moiety, a reactive group, or a linker.
  • the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.
  • the reactive group comprises an amino, -CN (cyano), N 3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.
  • the reactive group includes those used in click chemistry, such as copper-free click chemistry, and include, by way example and not limitation, azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups.
  • the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the nucleotide acceptor. In some embodiments, the conjugate moiety or reactive group is attached to the nucleoside via a linker. In some embodiments, the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide (A) . Docket Number CX10-269WO4 [0242] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 2’-position of the sugar moiety.
  • the conjugate moiety, reactive group, or linker is attached to the nucleobase and/or 2’-position of the sugar moiety.
  • the conjugate moiety, reactive group, or linker is attached to the 5’-OH group, a 5’-phosphate group, 4’-carbon of the sugar moiety, or through an abasic nucleoside, or an inverted abasic nucleoside on the 5’-terminal nucleotide.
  • the oligonucleotide acceptor comprises one or more terminal groups.
  • the terminal group is present at the 5’-terminal nucleotide of the nucleotide acceptor.
  • the terminal group present on the oligonucleotide acceptor is a 5’-phosphonate, (e.g., E or Z vinylphosphonate), 5’-phosphoalkyl (e.g., 5’-phosphomethyl or 5’-phosphoethyl, etc.), 4’-amino, 4’- aminoalkyl, abasic nucleoside, or inverted abasic nucleoside.
  • the oligonucleotide acceptor comprises one or more modified internucleoside linkages.
  • the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or a mixture of Sp and Rp stereoisomer. [0247] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 5’ terminal region with a modified internucleoside linkage.
  • the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages. [0248] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages.
  • the oligonucleotide acceptor comprises the formula (I): A1[•A2]m•A3-OH (I) wherein each of A1, A2 and A3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage.
  • m is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • each of A2 is same or different.
  • Docket Number CX10-269WO4 [0251]
  • at least one or more of A1, A2, or A3 comprises a modified nucleoside.
  • A1 is modified.
  • at least one of A2 is modified.
  • at least 2, 3, 4, 5, 6, or all of A2 is modified.
  • A3 is modified. In some embodiments, at least one of A1, A2, or A3 is modified with a conjugate moiety, reactive group, or linker. [0252] In some embodiments, A3 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position.
  • A3 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.
  • at least one internucleoside linkage “•“ comprises a modified internucleoside linkage.
  • the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
  • the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp or Rp stereoisomer
  • the linker on the modified oligonucleotide acceptor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like.
  • the linker comprises an C 2 -C 20 alkylene or polyethylene linker.
  • the modified nucleoside on the oligonucleotide acceptor comprises the formula (II): A-[L]g-[M]h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0.
  • the oligonucleotide acceptor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length.
  • nucleotide Donor or Donor Substrate Docket Number CX10-269WO4 Nucleotide Donor or Donor Substrate Docket Number CX10-269WO4
  • the nucleotide donor or donor substrate for the RNA ligase comprises a 5’- phosphate group, or functional form thereof, at the 5’-terminal nucleotide suitable for attachment or ligation to any of the oligonucleotide acceptors described herein.
  • the nucleotide donor comprises a single nucleotide donor, also referred to herein as nucleotide(D) or D, or an oligonucleotide donor, also referred to as oligonucleotide(D).
  • the oligonucleotide donor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the oligonucleotide donor is up to 122 nucleotides in length. In some embodiments, the oligonucleotides donor is up to 150 or more nucleotides in length. In some embodiments, the oligonucleotide donor is up to 200 or more nucleotides in length. [0261] In some embodiments, the oligonucleotide donor is 2 nucleotides in length. In some embodiments, the oligonucleotide donor is 3 nucleotides in length. In some embodiments, the oligonucleotide donor is 4 nucleotides in length. In some embodiments, the oligonucleotide donor is 5 nucleotides in length.
  • the oligonucleotide donor is 6 nucleotides in length. In some embodiments, the oligonucleotide donor is 7 nucleotides in length. In some embodiments, the oligonucleotide donor is 8 nucleotides in length. In some embodiments, the oligonucleotide donor is 9 nucleotides in length. In some embodiments, the oligonucleotide donor is 10 nucleotides in length. [0262] In some embodiments, the nucleotide donor, either as a nucleotide(D) or oligonucleotide(D), comprises a modified nucleotide.
  • the oligonucleotide(D) comprises at least one modified nucleotide.
  • the modified nucleotide on nucleotide(D) comprises a modified nucleoside, a 3’-terminal group, or any combinations thereof.
  • the modified nucleotide on an oligonucleotide (D) comprises a modified internucleoside linkage, a modified nucleoside, a 3’-terminal group, or any combinations thereof.
  • a modified nucleoside on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.
  • the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide donor, as limited by the length of the oligonucleotide donor.
  • all nucleosides of an oligonucleotide donor comprises a modified nucleoside.
  • a modified 5’-terminal nucleoside of the oligonucleotide donor comprises a modified sugar moiety.
  • the sugar moiety is modified at the 2’-position.
  • the modified 5’-terminal nucleoside is a 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O- methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.
  • the modified nucleoside of nucleotide donor nucleotide (D) or oligonucleotide (D) comprises a conjugate moiety, a reactive group, or a linker. Docket Number CX10-269WO4 [0268]
  • the conjugate moiety on the modified nucleoside of the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof.
  • the reactive group on the modified nucleoside comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group.
  • the reactive group used in click chemistry including copper-free click chemistry, such as azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups.
  • the linker on the modified nucleotide donor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like.
  • the linker comprises a C 2 -C 20 alkylene or polyethylene linker.
  • linkers suitable for the modified nucleoside of the nucleotide donor are described herein and also known in the art.
  • the nucleoside modified with a conjugate moiety, reactive group, or linker on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside.
  • the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide (D) , as limited by the length of the oligonucleotide donor.
  • the conjugate moiety, reactive group, or linker is attached to the nucleobase, the sugar moiety of a nucleoside of a nucleotide donor, or a 3’-terminal group.
  • the nucleotide donor comprises nucleotide(D)
  • the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or 3’-position of the sugar moiety, a 3’- terminal group, or any combinations thereof.
  • the conjugate moiety, reactive group, or linker can be attached to the nucleobase or the 2’-position of the sugar moiety of the 5’-terminal nucleoside and/or the internal nucleoside of oligonucleotide (D) .
  • the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or the 3’-position of the sugar moiety, or a 3’-terminal group, or any combinations thereof, of the 3’-terminal nucleotide.
  • the nucleotide donor comprises one or more terminal groups.
  • the terminal group is present on nucleotide (D) , or where the nucleotide donor comprises oligonucleotide (D) , at the 3’-terminal nucleotide of nucleotide (D) or oligonucleotide (D) .
  • the terminal group present on the nucleotide donor is a 3’-phosphate, 3’-phosphorothioate, 3’- phosphorodithioate, 3’-phosphonate (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 3-phosphomethyl or 3’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside.
  • the 3’-terminal group comprises a 3’-phosphate group for enhancing the efficiency of ligation of the nucleotide donor to the oligonucleotide acceptor, e.g., pDp, where the prefix p is a 5’-phosphate group and suffix p is a 3’-phosphate Docket Number CX10-269WO4 group.
  • a 3’-phosphate group can also act as a 3’-blocking group, either on the single nucleotide donor or oligonucleotide donor.
  • the oligonucleotide(D) comprises one or more modified internucleoside linkages.
  • the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage.
  • the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
  • the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or mixtures thereof.
  • the oligonucleotide (D) has at least 1 nucleotide at the 5’-terminal region with a modified internucleoside linkage.
  • the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages.
  • the oligonucleotide (D) has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage.
  • the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages.
  • the nucleotide donor comprises a 3’-blocking group.
  • a 3’-blocking group prevents or inhibits reiterative extensions or attachments of the nucleotide donor to another nucleotide donor.
  • the 3’-blocking group is present on the 3’-OH group or the nucleobase of the 3’-terminal nucleoside of the nucleotide donor.
  • the 3’-blocking group is a reversible blocking group, which is removable or capable of being cleaved with a deblocking agent to produce a free 3’-OH.
  • a deblocking agent to produce a free 3’-OH.
  • Various reversible 3’-blocking agents are described herein, and known in the art.
  • the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120.
  • n is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • each of D2 is same or different nucleoside.
  • D comprises a modified nucleoside.
  • D is modified with a conjugate moiety, reactive group, or a linker.
  • At least one or more of D 1 , D 2 , or D 3 comprises a modified nucleoside.
  • D 1 is modified.
  • at least one of D 2 is modified.
  • at least 2, 3, 4, 5, 6 or all of D 2 is modified.
  • D 3 is modified.
  • at least one of D1, D2, or D3 is modified with a conjugate moiety, reactive group, or linker.
  • D1 comprises a modified sugar moiety.
  • the sugar moiety is modified at the 2’-position.
  • D1 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine.
  • at least one internucleoside linkage “•“ comprises a modified internucleoside linkage.
  • the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp and Rp stereoisomers.
  • the modified nucleoside on the nucleotide donor comprises the formula (IV): D -[L]q-[M]r (IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0.
  • q is 1, L is attached to the nucleobase or the sugar moiety, or when q is 0, M is attached to the nucleobase or the sugar moiety.
  • L when q is 1, L can be attached to the 2’-position or 3’-position of the sugar moiety, as appropriate for D, either for nucleotide(D) or for oligonucleotide(D).
  • L can be attached to 2’-position or 3’-position of the sugar moiety.
  • D or D 3 where D is D1 or D2, L can be attached to the 2’-position of the sugar moiety. Where D is D3, the L can be attached to the 2’- or 3’- position of the sugar moiety.
  • D or D 3 further comprises one or more 3’-terminal groups.
  • the terminal group is a 3’-phosphate, 3’-phosphorothioate, 3’-phosphorodithioate, 3’- phosphonate, (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 5-phosphomethyl or 5’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside.
  • D of the nucleotide donor comprises a 3’-phosphate group, where the nucleotide donor comprises pDp representing the 5’-phosphate and the 3’-phosphate groups on the nucleoside D.
  • the pDp is pUp, pTp, pCp, pAp, or pGp.
  • the Docket Number CX10-269WO4 pDp comprises a 2’-modification.
  • pDp is pmUp, pmTp, pmCp, pmAp, or pmGp, with “m” representing 2’-O-methyl.
  • pDp is pfUp, pfTp, pfCp, pfAp, or pfGp, with “f” representing 2’-F.
  • the nucleobase of nucleoside comprises a nucleobase selected from the list described below.
  • the nucleotide donor comprises the formula pD1[-D2]n-D3p, wherein the pD1 represents the 5’-phosphate on the 5-terminal nucleoside D1, and D3p represents the 3’- phosphate on the 3’-terminal nucleoside D3.
  • D further comprises a 3’-blocking group.
  • D 3 further comprises a 3’-blocking group.
  • the 3’- blocking group prevents or inhibits attachment of the nucleotide donor to another nucleotide donor in the RNA ligase reaction, or reiterative extensions or attachments of a nucleotide substrate using a terminal nucleotidyl transferase.
  • the 3’-blocking group is present on the 3’-OH group or the nucleobase of D or D 3 .
  • the 3’-blocking group is a reversible blocking group.
  • the 3’-blocking is removable or capable of being cleaved with a deblocking agent, as further described herein.
  • the method of synthesizing an oligonucleotide comprises reacting an oligonucleotide acceptor and a nucleotide donor in presence of a single-stranded RNA ligase under reaction conditions suitable for ligation of the nucleotide donor to the oligonucleotide acceptor, wherein the nucleotide donor comprises a 3’-blocking group to form a 3’-blocked extended oligonucleotide.
  • the presence of the 3’-blocking groups provides for attachment of a single nucleotide donor to the oligonucleotide acceptor. In some embodiments, attachment of 3’-blocked nucleotide donor allows for attaching one nucleotide(D) or one oligonucleotide(D) to the oligonucleotide acceptor. In some embodiments, where the nucleotide nucleotide(D) comprises a conjugate moiety, reactive moiety, and/or a linker, the presence of a 3’-blocking group provides for attachment of one nucleotide(D) comprising a conjugate moiety, reactive moiety, and/or a linker.
  • the 3’-blocking group present on the nucleotide donor is a reversible 3’- blocking group, thereby resulting in formation of a reversible 3’-blocked extended oligonucleotide by the single-stranded RNA ligase reaction.
  • the reversible 3’-blocking agent on the blocked extended oligonucleotide is removable or cleavable with a deblocking agent appropriate for the particular 3’- blocking group.
  • an exemplary 3’-blocking groups comprises a 3’-phosphate, where the deblocking agent is a phosphatase.
  • the method further comprises removing or separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase.
  • the method further comprises degrading or inactivating the by- products (e.g., unreacted nucleotide donor, pyrophosphate, etc.) of the single-stranded RNA ligase reaction.
  • the method further comprises removing or cleaving the 3’-blocking group from the 3’-blocked extended oligonucleotide to form an unblocked extended oligonucleotide, i.e., an extended oligonucleotide with a free 3’-OH group.
  • the removing or cleaving of the 3’-blocking group is carried out with a deblocking agent.
  • the deblocking agent is inactivated or the unblocked extended oligonucleotide is separated from the deblocking agent.
  • the removing or cleaving the 3’-blocking group with the deblocking agent and the degrading or inactivating of the by-products of the single-stranded RNA ligase reaction are done concurrently.
  • the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase under suitable reaction conditions for attachment or ligation of the second nucleotide donor to the unblocked extended oligonucleotide.
  • the method further comprises one or more cycles of: extension with a nucleotide donor comprising a reversible 3’-blocking agent; separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase; removing or cleaving the 3’-blocking group with a deblocking agent; separating the unblocked extended oligonucleotide from the deblocking agent or inactivating the deblocking agent, wherein each cycle is with a new nucleotide donor. Repeating the cycle allows for step-wise extension of the extended oligonucleotide.
  • nucleotide donor is nucleotide (D)
  • repeating each cycle with new nucleotide (D) allows for step-wise extensions of a single nucleotide with each cycle.
  • nucleotide donor is oligonucleotide (D)
  • repeating each cycle with new oligonucleotide (D) allows for step-wise extensions with oligonucleotide (D) .
  • the 3’-blocked nucleotide donor is used in combination with a 5’-OH containing or 5’-blocked oligonucleotide acceptor.
  • each nucleotide donor is a selected or predetermined nucleotide donor for producing an extended oligonucleotide with a defined nucleotide sequence.
  • the use of a 3’-blocked nucleotide donor comprising a conjugate moiety, reactive moiety, or linker, where the 3’-blocking group is a reversible blocking group allows for sequential or stepwise addition of nucleotides with the conjugate moiety, reactive group, or linker.
  • the method comprises sequential or stepwise attachment or addition of at least two nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; at least three nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; or at least four nucleotide donors comprising a conjugate moiety, reactive moiety, or linker.
  • the nucleotide donor in the sequential or stepwise addition of nucleotides is nucleotide(D) comprising a conjugate moiety, reactive moiety, or linker. Docket Number CX10-269WO4 [0305]
  • the unblocked extended oligonucleotide can serve as a substrate for a terminal nucleotidyl transferase.
  • the method further comprises reacting the unblocked extended oligonucleotide with a nucleotide substrate in presence of a terminal nucleotidyl transferase under reaction conditions suitable for the extension by or addition of at least one nucleotide to form at least a single nucleotide extended oligonucleotide. In some embodiments, more than one nucleotide is attached or added to the extended oligonucleotide by the terminal nucleotidyl transferase.
  • the nucleotide substrate for the terminal nucleotidyl transferase comprises a 3’-blocked nucleotide substrate (i.e., a nucleotide substrate comprising a 3’-blocking group), providing for a single nucleotide addition or attachment to the oligonucleotide or the extended oligonucleotide to form a 3’- blocked single nucleotide extended oligonucleotide.
  • the method further comprises removing or cleaving the 3’-blocking group to form an unblocked single nucleotide extended oligonucleotide.
  • the removing or cleaving of the 3’-blocking group is done with a deblocking agent appropriate for the specific 3’-blocking group.
  • the method further comprises degrading the by-products (e.g., unreacted NTP, unreacted 3’-blocked NTP, NDP, NMP, 3’-blocked NMP, etc.) of the terminal nucleotidyl transferase reaction.
  • the 3’-blocking group is a 3’-phosphate
  • a phosphatase can be used as the deblocking agent.
  • the deblocking agent is inactivated or the deblocking agent separated from the unblocked single nucleotide extended oligonucleotide. In some embodiments, the inactivation and degrading of the by-products is done concurrently.
  • the method further comprises repeating one or more cycles of reacting the unblocked extended oligonucleotide with a nucleotide substrate comprising a 3’-blocking group in presence of the terminal nucleotidyl transferase, removing or cleaving the 3’-blocking moiety (i.e., deblocking the 3’- blocked nucleoside) on the extended oligonucleotide with a deblocking agent, and inactivating the deblocking agent or separating the deblocking agent from the unblock extended oligonucleotide, where each cycle is with a new nucleotide substrate.
  • each cycle of extension, deblocking, and inactivation/separation is with a new nucleotide substrate for step-wise, sequential attachment or addition of nucleotides to the extended oligonucleotide by the terminal nucleotidyl transferase.
  • each nucleotide substrate for the terminal nucleotidyl transferase reaction is a selected or predetermined nucleotide substrate for producing an extended oligonucleotide with a defined nucleotide sequence.
  • the use of a modified nucleotide substrate allows for stepwise, sequential addition of modified nucleotides to the extended oligonucleotide.
  • Reaction Conditions and Process Docket Number CX10-269WO4 [0313]
  • the single-stranded RNA ligase reactions are carried out under suitable conditions for the ligase mediated attachment of the oligonucleotide acceptor to the nucleotide donor.
  • the suitable reaction conditions include a nucleotide co-factor used by the single-stranded RNA ligase to catalyze the joining reaction.
  • the nucleotide cofactor is ATP. In some embodiments, the nucleotide cofactor is about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, or more as appropriate. In some embodiments, the nucleotide cofactor or substrate (e.g., ATP) is present at a concentration of about 0.05-25 mM, 1-20 mM, 2-18 mM, or 5-15 mM.
  • the nucleotide cofactor or substrate e.g., ATP
  • the nucleotide cofactor or substrate is present at a concentration of about 0.5 mM, 1 mM, 2 mM, 3 mM 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM.
  • the reaction conditions for the ligation include additional components, such as a divalent metal (e.g., Mg +2 ), buffer, and/or salts. Exemplary reaction components are provided in the Examples.
  • the salt in the reaction conditions include, among others, NaCl, KCl, ammonium salts (e.g., NH 4 Cl), and acetate salts (e.g., sodium acetate).
  • the salt is present at 0.5 mM-300 mM, 1 mM-250 mM, 2 mM-200 mM, 5 mM-150 mM, 10 mM-100 mM, 20 mM-80 mM, or 40 mM-60 mM.
  • the salt is present at about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM. In some embodiments, the salt is present at sufficient concentration to reduce degradation or other undesired products.
  • the reaction conditions also include a ligation enhancing reagent, including, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000).
  • a ligation enhancing reagent including, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000).
  • the single-stranded RNA ligase reaction is carried out at a suitable temperature and reaction time period for the ligation of the oligonucleotide acceptor and the nucleotide donor.
  • the RNA ligation reaction temperature is from about 2° C to about 60° C.
  • the RNA ligation reaction temperature is from 4 °C to 55 °C, 4 °C to 50 °C, 4 °C to 45 °C, or 10 °C to 40 °C. In some embodiments, the RNA ligation reaction temperature is 2 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 37 °C, 40 °C, 45 °C, 50 °C, 55 °C, or 60 °C. In some embodiments, the reaction temperature is chosen based on the thermostability of the single-stranded RNA ligase and/or the efficiency of the ligation at defined temperatures and reaction conditions.
  • the single-stranded RNA ligase reaction time can be a sufficient time for ligation of the oligonucleotide acceptor to the nucleotide donor.
  • the ligation reaction time is from 0.5-72 hr or longer.
  • the ligation reaction time is 1-72 hr, 2-48 hr, or 2-24 hr.
  • the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48, or 72 hr or longer.
  • the reaction conditions comprise a suitable pH.
  • the desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition.
  • the pH of the reaction mixture can be controlled before and/or during the course of the reaction.
  • the suitable reaction conditions comprise a solution pH from about 4 to about Docket Number CX10-269WO4 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8.
  • the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.
  • the pH of the reaction mixture may change during the reaction.
  • the pH of the reaction solution is maintained at a desired pH or within a desired pH range, such as by the addition of an acid or a base, before and/or during the course of the reaction.
  • the pH is controlled by using an appropriate buffer.
  • Suitable buffers to maintain desired pH ranges include, by way of example and not limitation, borate, phosphate, 2-(N- morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like.
  • the buffer concentration is from 1 to 500 mM, 1 to 400 mM, 1 to 300 mM, 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM.
  • the buffer concentration is about 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM.
  • the concentration of oligonucleotide acceptor and nucleotide donor can be varied based on, among others, the reaction conditions, the activity of the single-stranded ligase, factors affecting efficiency of the oligonucleotide acceptor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the acceptor, presence of secondary structure, 3’-terminal properties, etc.), and the efficiency of the nucleotide donor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the nucleotide donor, presence of secondary structure, 5’-terminal properties, etc.).
  • factors affecting efficiency of the oligonucleotide acceptor as substrate e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the nucleotide donor,
  • additional considerations include, among others, density of the oligonucleotide acceptor on the support medium, effect of support medium on the single-stranded RNA ligase reaction, and access of the single-stranded RNA ligase and other enzymes (e.g., pyrophosphatase, terminal nucleotidyl transferase, etc.) to the oligonucleotide acceptor bound to the support medium.
  • enzymes e.g., pyrophosphatase, terminal nucleotidyl transferase, etc.
  • the oligonucleotide acceptor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, oligonucleotide acceptor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM.
  • the oligonucleotide acceptor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the nucleotide donor.
  • the nucleotide donor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, nucleotide donor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM.
  • the nucleotide donor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, Docket Number CX10-269WO4 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the oligonucleotide acceptor.
  • the ratio of nucleotide donor to oligonucleotide acceptor is 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or higher.
  • the single stranded RNA ligase is provided at concentrations from about 0.01 g/L to about 50 g/L; about 0.01 to about 0.1 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/L to about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40 g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1 g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about 10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; or about 0.1 g/L to about 2 g/L.
  • the oligonucleotide acceptor is bound to a support medium (e.g., a solid support), and the ligation reaction carried out with the single-stranded RNA ligase and the nucleotide donor in solution to ligate the nucleotide donor to the support bound oligonucleotide acceptor.
  • the oligonucleotide is conjugated to the support medium via the 5’-terminal nucleotide to provide for a ligase accessible free 3’-OH.
  • the single-stranded RNA ligase, any unreacted nucleotide donor, and by- products of the single-stranded RNA ligase reaction are removed or separated from the immobilized extended oligonucleotide.
  • the extended oligonucleotide is released or cleaved from the substrate medium to yield the extended oligonucleotide in solution.
  • the releasing agent is a chemical cleavage agent appropriate for the covalent attachment to the substrate medium.
  • a biological releasing or cleaving agent is used, for example a nuclease that can cleave at a specific site in the oligonucleotide bound to the substrate medium.
  • a nuclease that can cleave at a specific site in the oligonucleotide bound to the substrate medium.
  • the ligation reaction is carried out with the single-stranded RNA ligase and a 3’-blocked nucleotide donor in solution to form a 3’-blocked extended oligonucleotide bound to the support medium.
  • the single-stranded RNA ligase, any unreacted nucleotide donor, and by-products of the single-stranded RNA ligase reaction are removed or separated from the immobilized 3’-blocked extended oligonucleotide.
  • the 3’-blocking group is desired in the extended oligonucleotide, for example, a conjugate moiety, reactive group, or linker attached to the 3’-OH group of the sugar moiety, no further extensions are carried out and the extended oligonucleotide is released or cleaved from the support medium.
  • a reversible 3’-blocking group is used and the support bound 3’- blocked extended oligonucleotide reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide.
  • the unblocked extended oligonucleotide is then released or cleaved from the support medium.
  • the 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution.
  • the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide bound to the support medium.
  • the deblocking agent is inactivated or the unblocked extended oligonucleotide separated from the deblocking agent.
  • the by-products of the single-stranded RNA ligase reaction are degraded with a degrading agent.
  • the cycle of reaction with the extended oligonucleotide with RNA ligase and deblocking and associated inactivation/separation of deblocking agent and degrading of by-products is repeated for each nucleotide donor.
  • the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide.
  • the unblocked extended oligonucleotide is then released or cleaved from the support medium.
  • the desired 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution to yield the final extended oligonucleotide product.
  • the final extended oligonucleotide product in solution can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.).
  • the ligation reaction is carried out with a single-stranded RNA ligase bound to a support medium, e.g., immobilized single-stranded RNA ligase.
  • a support medium e.g., immobilized single-stranded RNA ligase.
  • the oligonucleotide acceptor and nucleotide donor in solution can be contacted with the support-bound single-stranded RNA ligase to ligate the oligonucleotide acceptor to the nucleotide donor.
  • the substrates in solution are passed through a bed (e.g., a column) of support-bound single-stranded RNA ligase until a desired level of extended product has formed.
  • the extended oligonucleotide product is removed or separated from the immobilized single-stranded RNA ligase.
  • the nucleotide donor comprises a 3’-blocked nucleotide donor to form a 3’-blocked extended oligonucleotide in solution.
  • the method further comprises removing or cleaving the 3’-blocking group with a deblocking agent to form an unblocked extended oligonucleotide in solution.
  • the method further comprises inactivating and/or separating the deblocking agent from the unblocked extended oligonucleotide.
  • the deblocking agent comprises an enzyme, e.g., phosphatase
  • the enzyme can be on a support substrate (e.g., immobilized Docket Number CX10-269WO4 deblocking enzyme) to facilitate reaction with the blocked extended oligonucleotide in solution and its separation from the deblocking agent following the deblocking reaction.
  • the method further comprises degrading the by-products in the reaction containing the extended oligonucleotide in solution. In some embodiments, the degrading of by-products is achieved concurrently or subsequent to the deblocking of the extended oligonucleotide.
  • the degrading agent e.g., an enzyme
  • the degrading agent is immobilized to a support medium to facilitate degradation of by-products in solution and separation of the extended oligonucleotide from the degrading agent.
  • the 3’-blocked extended oligonucleotide in solution is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide, and following the deblocking reaction, the deblocking agent is inactivated or the deblocking agent separated from the unblocked extended oligonucleotide.
  • the cycle of reaction with the extended oligonucleotide with immobilized single-stranded RNA ligase and deblocking and associated inactivation/separation of deblocking agent and degrading of by-products is repeated for each new nucleotide donor.
  • the final 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to from the unblocked extended oligonucleotide.
  • use of an immobilized single-stranded RNA ligase and an immobilized deblocking agent e.g., a phosphatase
  • an immobilized deblocking agent e.g., a phosphatase
  • uses an immobilized single-stranded RNA ligase and an immobilized deblocking agent e.g., a phosphatase
  • the final unblocked extended oligonucleotide can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.).
  • chromatography e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography
  • HPLP and UPLC HPLP and UPLC
  • affinity techniques e.g., capture tags, hybridization, etc.
  • the oligonucleotide acceptor, nucleotide donor, or the combination of Docket Number CX10-269WO4 oligonucleotide acceptor and nucleotide donor can have various modifications. In some embodiments, the modifications occur on the nucleobase, the sugar moiety, terminal groups, or any combination thereof. The various modifications that can be used are described below.
  • the oligonucleotide acceptor and/or the nucleotide donor comprises a modified nucleoside, wherein the modification is on the sugar moiety of the nucleoside.
  • the modified sugar moiety is a modified furanosyl sugar moiety, for example ribose or deoxyribose.
  • the furanosyl sugar moiety is modified or substituted at the 2’, 3’, or a combination of 2’ and 3’ positions.
  • Modifications at the 4’, and/or 5’-positions are described below as “terminal group.”
  • the modification is at the 2’-position of the sugar moiety.
  • substitutions at the 2’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 2’-alkoxy (e.g., O-methyl, O-ethyl, etc.).
  • other modifications at the 2’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF 3 , OCF 3 , SCH 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , and NH 2 .
  • substituent groups at the 2’-position include, among others, O-(C 1 - C 10 )alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N- alkynyl, O-alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C 1 -C 10 alkyl or C 1 -C 10 alkenyl and alkynyl.
  • substituent groups at the 2’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl.
  • the substitution at the 2’-position is a phosphate (see, e.g., Current Protocols in Nucleic Acid Chemistry, 13.1.1- 13.1.31, John Wiley & Sons (2003).
  • the modified 2’-position of the sugar moiety is halo, 2’-O-R’, or 2’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl.
  • R’ is a C1-C4alkyl.
  • the modified 2’-position is a 2’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl.
  • the 2’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH 3 or -CH 2 CH 3 or -CH 2 CH 2 OCH 3 .
  • the modified 2’-position is 2’- O-(2-methoxyethyl), 2’-O-allyl, 2’-O-propargyl, 2’-O-ethylamine, 2’-O-cyanoethyl, -2’-O-amine, or 2’-O- acetate ester.
  • a modification at the 2’-position comprises a locked nucleoside.
  • locked nucleosides comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735–8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138–1142).
  • the ribose moiety of the locked nucleotide is in the C3’-endo (beta-D) or C2’-endo (alpha-L) conformation.
  • the bridge is a methylene bridge.
  • the bridge is an ethylene bridge, also referred to as ENA (see, e.g., Morita et al., Bioorg Med Chem Lett., 2002, 12(1):73-6).
  • ENA ethylene bridge
  • Other locked nucleoside are described in International patent publication WO 2121249993, incorporated by reference herein. Docket Number CX10-269WO4 [0349]
  • other locked nucleosides include, among others, 5’-methyl-LNA, 2’-amino- LNA, alpha-L-LNA, and thio-LNA.
  • a modification at the 2’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker or a linker, as described herein.
  • the modification is at the 3’-position of the sugar moiety.
  • the 3’-modification is on the 3’-terminal nucleoside of the nucleotide donor.
  • the modification at the 3’-position are similar to those at the 2’-position.
  • substitutions at the 3’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 3’-alkoxy (e.g., O-methyl, O-ethyl, etc.).
  • other modifications at the 3’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, and NH2.
  • substituent groups at the 3’-position include, among others, O-(C1-C10)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O- alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl.
  • substituent groups at the 3’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl.
  • the substitution at the 3’-position is a phosphate.
  • the modified 3’-position of the sugar moiety is halo, 3’-O-R’, or 3’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl.
  • R’ is a C 1 -C 4 alkyl.
  • the modified 3’-position is a 3’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl.
  • the 3’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3.
  • the modified 3’-position is 3’- O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O-ethylamine, 3’-O-cyanoethyl, -3’-O-amine, or 3’-O- acetate ester. Docket Number CX10-269WO4 [0353] In some embodiments, the modifications at the 3’-position is a reversible or cleavable 3’-blocking group.
  • removal or cleaving of the reversible or cleavable 3’-blocking group results in a free 3’-OH group, which in some embodiments can serve as an acceptor for single-stranded RNA ligase or a terminal nucleotidyl transferase.
  • exemplary reversible or cleavable 3’-blocking groups include, among others, 3’-O-azidomethyl, 3’-O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O- ethylamine, 3’-O-cyanoethyl, -3’-O-amine, 3’-O-acetate ester, 3’-phosphate, 3’-diphosphate, or 3’- triphosphate.
  • the 3’-blocking group is paired with the corresponding deblocking agent used in the deblocking or cleavage of the 3’-blocking group.
  • a modification at the 3’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker’ or a linker, as described herein.
  • the modified sugar moiety comprises an unlocked nucleoside. In some embodiments, in the unlocked nucleoside, the furanosyl ring is opened to result in the structure below: where B represents the nucleobase.
  • the oligonucleotide acceptor and/or nucleotide donor comprises one or more nucleosides comprising a modified nucleobase.
  • modified nucleobase that is capable of hydrogen bonding to form Watson and Crick type base pairing is selected.
  • nucleobase comprise an inosine nucleoside (i.e., nucleosides comprising a hypoxantine nucleobase).
  • the modified nucleobase is 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2. N-6 and O-6 substituted purines.
  • the modified nucleobase is 2-aminopropyladenine.5- hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6- N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine.5-propynyl uracil, 5- propynylcytosine.6-azouracil, 6-azocytosine, 6-azothymine.5-ribosyluracil (pseudouracil), 4-thiouracil.8- halo purine, 8-amino purine, 8-thio purine, 8-thioalkyl purine, 8-hydroxy purine, 8-aza purine, 5- bromocytosine.5-trifluoromethylcytosine, 5-halouracil, 5-halocytosine, 7-methylguanine, 7-methyla
  • modified nucleobases include tricyclic pyrimidines, e.g., 1,3-diazaphenoxazine-2-one.1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1.3- diazaphenoxazine-2-one (G-clamp). Docket Number CX10-269WO4 [0358]
  • the modified nucleobase includes, among others, nucleobases based on 2,4- dihalotolene and benzimidazole groups.
  • the modified nucleobase is 4- methylbenzimidazole, 2,4-difluorotoluene, 9-methylimidazo[(4,5)-b]pyridine, 2,4-dibromotoluene, benzimidazole, 5-nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole.
  • the modified nucleobase is 7-azaindole, and isocarbostyril (see, e.g., Berdis et al., Front. Chem.10:1051525).
  • Other modified nucleobases are described in, among others, patent publication WO2021249993.
  • a nucleobase that does not have a nucleobase also referred to as an abasic nucleoside.
  • the abasic nucleoside is present in the internal portion of an oligonucleotide acceptor.
  • an abasic nucleoside is attached to the 3’- or 5’-terminal end, which is in certain embodiments grouped as a terminal group.
  • the modified nucleobase is present on the 5’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, 3’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, and/or present on the internal nucleosides of the oligonucleotide acceptor or oligonucleotide donor.
  • the blocks or contiguous stretches of nucleosides in the oligonucleotide acceptor or oligonucleotide donor have modified nucleobases.
  • the oligonucleotide acceptor and/or nucleotide donor comprises a terminal group.
  • the oligonucleotide acceptor comprises a terminal group at the 5’-terminal nucleoside.
  • the terminal group is attached to the 5’-OH or 4’-carbon atom of the terminal nucleoside.
  • the terminal group comprises a C-4’ modification of the 5’-terminal nucleoside, including among others, 4’-thio-C2’ modifications, 4’-aminoalkyl, C4’-guanidino-C2’- modifications, and C4’-O-methyl (see, e.g., Gangopadhyay et al., RNA Biology, 2022, 19:1, 452-467)
  • the 5’-terminal group is a 5’-phosphate modification.
  • the 5'-phosphate modification includes, among others, 5’-C-methyl, particularly S isomer; 5’-(E or Z)- vinylphosphonate, or 5’-methylenephosphonate.
  • the 5’-terminal group comprises an abasic nucleotide attached to the 5’-OH.
  • the 5’-terminal groups comprises an inverted abasic nucleotide (5’-5’) attached to the 5’-OH of the 5’-end nucleoside.
  • the nucleotide donor comprises a 3’-terminal group.
  • the 3’-terminal group comprises a 3’-phosphate, which can also function as a reversible blocking group.
  • the 3’-phosphate is modified, such as with 3’-(E or Z)-vinylphosphonate, or 3’- methylenephosphonate.
  • the 3’-terminal group on the nucleotide donor comprises an abasic nucleoside.
  • the 3’-terminal group comprises an inverted abasic nucleotide (3’- 3’).
  • the modified oligonucleotide e.g., oligonucleotide acceptor and/or oligonucleotide donor, comprises at least one modified, non-naturally occurring internucleoside linkage.
  • the modified oligonucleotide has 1%, 2%, 5%, 10% 20%, 30%, 40%, 50%, or 60% or more modified internucleoside linkages.
  • all of the internucleoside linkages are modified internucleoside linkages.
  • the modified internucleoside linkage is a phosphorous containing modified internucleoside linkage.
  • exemplary phosphorous-containing internucleoside linkages include, among others, phosphotriesters, alkylphosphonates (e.g., methyl phosphonate, ethyl phosphonate, etc.), phosphoramidates, phosphorothioate, and phosphorodithioate.
  • the modified internucleoside linkage is a non-phosphorous containing internucleoside linkage.
  • the modified internucleoside linkage is amide linkage, such as those of glycine nucleosides or nucleoside ⁇ -amino acids (see, e.g., Banerjee et al., Bioconjugate Chem., 2015, 26, 8, 1737–1742).
  • the modified internucleoside linkages provides for a chiral center.
  • a phosphorothioate or alkylphosphonate internucleoside linkage can be in the Rp or Sp stereomeric configuration.
  • the oligonucleotide acceptor and/or oligonucleotide donor have a mixture of stereoisomers in the internucleoside linkage. In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor have greater than 50% of the internucleoside linkages as Rp or Sp configuration. In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration.
  • the modified internucleoside linkages are present in the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments at least 1 or 2 phosphorothioate internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor.
  • the phosphorothioate linkage is a non-bridging phosphorothioate internucleoside linkage.
  • the modified internucleoside linkages are present in the 3’-terminal region of the oligonucleotide acceptor or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 3’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor.
  • At least 1 or 2 phosphorothioate internucleoside linkages are present at the 3’-terminal region of an oligonucleotide acceptor or oligonucleotide donor.
  • the modified internucleoside linkages are present in the internal portions of the oligonucleotide acceptor or oligonucleotide donor.
  • the oligonucleotide acceptor and/or oligonucleotide donor comprises at least a phosphorothioate internucleoside linkage, where the phosphorothioate linkage is in the Sp configuration, the Rp configuration, or a mixture of Sp and Rp configurations in the population of the oligonucleotide acceptor and/or oligonucleotide donor.
  • Conjugate moiety In some embodiments, the oligonucleotide acceptor, or nucleotide donor comprises a conjugate moiety.
  • the nucleotide donor comprises a conjugate moiety that is compatible with single-stranded RNA ligase activity and that does not interfere with the nucleotide donor acting as a substrate for the single-stranded RNA ligase.
  • the conjugate moiety i.e., non-nucleotide moiety
  • the conjugate moiety includes, among others, carbohydrates (e.g. GalNAc), lipids, sterols, drug substances, hormones, polymers (e.g., polyethylene glycol, etc.), proteins, peptides, toxins (e.g.
  • the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and/or oligonucleotide cell targeting.
  • the conjugate moiety can be attached to the 5’-terminal nucleotide, the 3’- terminal nucleotide, or in an oligonucleotide an internal nucleotide.
  • the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, for example, to the 2’-OH.
  • the conjugate moiety is attached to the 3’-position of the sugar moiety of the nucleoside, for example 3’-OH.
  • the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res.2019 Feb 20; 47(3): 1082–1096).
  • the conjugate moiety is attached directly or attached using a linker.
  • the conjugate moiety comprises a C6-C22 alkyl, C6-22 alkenyl, or C6-C22 alkynyl.
  • the conjugate moiety comprises a C 6 -alkyl, C 7 -alkyl, C 8 -alkyl, C 9 -alkyl, C 10 - alkyl, C 11 -alkyl, C 12 -alkyl, C 13 -alkyl, C 14 -alkyl, C 15 -alkyl, C 16 -alkyl, C 17 -alkyl, C 18 -alkyl, C 19 -alkyl, C 20 -alkyl, C21-alkyl, or C22-alkyl.
  • the conjugate moiety comprises a C6 alkenyl, C7 alkenyl, C8 alkenyl C 9 alkenyl, C 10 alkenyl, C 11 -alkenyl, C 12 -alkenyl, C 13 -alkenyl, C 14 -alkenyl, C 15 -alkenyl, C 16 -alkenyl, C 17 -alkenyl, C 18 -alkenyl, C 19 -alkenyl, C 20 -alkenyl, C 21 -alkenyl, or C 22 -alkenyl.
  • the conjugate moiety comprises a C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11-alkynyl, C12- alkynyl, C13-alkynyl, C14-alkynyl, C15-alkynyl, C16-alkynyl, C17-alkynyl, C18-alkynyl, C19-alkynyl, C20- alkynyl, C21-alkynyl, or C22-alkynyl.
  • the conjugate moiety comprises a heteroalkyl, heteroalkenyl, or heteroalkynyl.
  • the heteroalkyl, heteroalkenyl or heteroalkynyl has one or more carbon atoms replaced with a heteroatom, such as O, S, or N.
  • the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group.
  • the cycloalkyl includes, among others, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl.
  • the heterocycloalkyl includes, among others, 1-(1,2,5,6-tetrahydropyridyfh l-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- Docket Number CX10-269WO4 morpholinyl, tctrahydrofuran-2-yl, tctrahydrofuran-3-yl, tetrahydrothicn-2-yl, tetrahydrothien-3-yl, l- piperazinyl, and 2-piperazinyl.
  • the conjugate moiety comprises an aryl or heteroaryl moiety.
  • the aryl group includes, among others, phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, anthracenyl, fluorenyl, indenyl, and azulenyl.
  • a heteroaryl group includes, among others, pyridyl, furanyl, thienyl, pynolyl, oxazolyl, oxadiazolyl, imidazolyl ihiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isoihiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, and indazolyl.
  • the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl-heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenyl-heterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl-.
  • the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid.
  • the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid.
  • the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans- vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, eicosapentaenoic acid, decanoic acid, docosahexaenoic acid (DHA), and docosanoic acid (DCA) conjugate moieties (see, e.g., Kubo et al., ACS Chem.
  • the conjugate moiety comprises a sterol.
  • the sterol comprises cholesterol, alpha-cholesterol, cholesterol ester (e.g., cholesteryl palmitate, etc.), cholesterol sulfate, phytosterol, cholic acid, or lithocholic acid.
  • the conjugate moiety comprises a phospholipid.
  • the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid.
  • the conjugate moiety comprises a carbohydrate, particularly a carbohydrate moiety acting as a ligand for a cellular receptor for cellular targeting of the oligonucleotide.
  • the carbohydrate moiety comprises galactose or galactose derivatives.
  • the carbohydrate moiety is attached to the nucleoside via a linker.
  • exemplary carbohydrates that can be used include the following.
  • the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety.
  • the oligonucleotide acceptor and/or nucleotide donor may be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety.
  • the conjugate moiety is a monovalent, divalent, trivalent or tetravalent, GalNAc.
  • the GalNAc moiety has the following structure, where L is a linker, and W is a heteroatom (e.g., O or N).
  • the W is the 2’-OH of the sugar moiety of a nucleoside.
  • the L includes a 3’-phosphate at the 3’-terminal nucleoside.
  • An exemplary monovalent GalNAc moiety is wherein the monovalent GalNAc is attached via the linker to the 2’-position of a nucleoside, such as adenine or guanine.
  • conjugate moieties can be present in contiguous nucleotides in an oligonucleotide (see, e.g., WO2024/040041). Docket Number CX10-269WO4 [0388]
  • the conjugate moiety is a trivalent GalNAc.
  • Tri-valent N-acetylgalactosamine conjugate moieties are described in, for example, WO 2014/076196, WO 2014/207232 and WO 2014/179620.
  • the term “trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. Exemplary trivalent GalNAc conjugate moiety is depicted below:
  • the conjugate is GalNAc targeting moiety L96.
  • the conjugate moiety comprises a reporter molecule.
  • reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6- carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X- rhodamine (ROX), liss
  • fluorescein and fluorescein dyes e.g., fluorescein isothi
  • the reporter moiety is a chemiluminescent moiety, for example acridinium esters, ruthenium derivatives (e.g., tris(2,2′-bipyridyl) ruthenium), and dioxetanes.
  • the conjugate moiety comprises an affinity or capture tag.
  • Exemplary affinity or capture tag includes, among others, biotin, desthiobiotin, digoxigenin, 3-amino-3-deoxydigoxigenin, and a hapten (e.g., dinitrophenol, Alexa Fluor 40, Alexa Fluor 488, dansyl, Lucifer yellow, Oregon Green 488, fluorescein). Docket Number CX10-269WO4 [0393]
  • the conjugate moiety comprises a peptide.
  • the peptide comprises a cellular targeting peptide and/or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide.
  • CCPP cell penetration peptide
  • the cell penetrating peptide is attached via a linker, including a cleavable linker.
  • Cell penetrating peptides include among others, TAT, penetratin, MAP, transportan/TP10, VP22, polyarginine, MPG, Pep-1, pVEC, YTA2, YTA4, M918, and CADY.
  • the conjugate moiety comprises an RGD (Arg-Gly-Asp) peptide.
  • the peptide can be attached using a thiol group on the 5’-phosphate of a polynucleotide or oligonucleotide.
  • a thiol group on the 5’-phosphate of a polynucleotide or oligonucleotide is shown below:
  • the modification comprises a reactive group that is conjugated to a nucleoside.
  • the reactive group is attached to the nucleoside via a linker.
  • the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans- Docket Number CX10-269WO4 cyclooctene, or tetrazine.
  • the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below: amino [0397] Other reactive groups used in click chemistry, particularly for nucleic acids, is described in Fantoni et al., Chem. Rev.2021, 121, 7122 ⁇ 7154, incorporated by reference herein.
  • RNA ligase substrates with a reactive moiety is provided below: Docket Number CX10-269WO4 wherein R 1 is H or phosphate; R 2 is a blocking group or H;; and R 3 is H, -OR, or halo, e.g., F, Br, or Cl.
  • Linker [0399] In some embodiments, as described above, the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker. Various linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups. [0400] In some embodiments, mixtures of linkers are used.
  • different linker types are connected to form a longer linker or linkers with branched or dendritic structure.
  • an alkylene linker is connected to a polyethylene linker through a functional group, e.g., an amide; an arylene linker is attached to an alkylene linker.
  • a functional group e.g., an amide
  • an arylene linker is attached to an alkylene linker.
  • different combinations of linker types can be connected to provide for longer linkers and/or branched linkers, for example for attaching multiple conjugate moieties.
  • linkers include, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene, Docket Number CX10-269WO4 and heteroarylalkenylene.
  • the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol.
  • the linkers have functional groups for conjugation.
  • the L has the structure below: , Docket Number CX10-269WO4
  • the linker comprises a substituted or unsubstituted polyethylene glycol linker.
  • the polyethylene glycol linker has the formula: [0406] In some embodiments, n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. [0407] In some embodiments, the polyethylene linker has the structure below: , , [0409] In some embodiments, the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety.
  • Example of a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g., Docket Number CX10-269WO4 Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2 nd Ed., S.S. Mark ed., 2011, Humana Press).
  • enzymatically cleavable linkers e.g., peptide linkers
  • photocleavable linkers see, e.g., Docket Number CX10-269WO4 Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2 nd Ed
  • bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra).
  • an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate.
  • the activating group can be a group that is derivable from a known coupling reagent.
  • coupling reagents include, but are not limited to, N,N′- dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N′- ethylcarbonate (EDC), (denzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-1-yl- N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU).
  • DCC N,N′- dicyclohexylcarbodimide
  • HOBt hydroxybenzotriazole
  • EDC
  • the single-stranded RNA ligase reaction further includes a pyrophosphatase for degrading pyrophosphate generated in the single-stranded RNA ligase reaction.
  • the presence of a pyrophosphatase in the ligase reaction can enhance the forward reaction for attachment of the nucleotide donor to the oligonucleotide acceptor by degrading the pyrophosphate generated in the ligase reaction.
  • the pyrophosphatase is present concurrently with the single-stranded RNA ligase.
  • the pyrophosphatase can be free in solution, co-immobilized with the single-stranded RNA ligase on the substrate medium, or immobilized on a substrate medium separately from the single-stranded RNA ligase.
  • the pyrophosphatase and the single-stranded ligase can be prepared as a fusion protein, and in some embodiments, immobilized on a support medium.
  • a pyrophosphatase can also be used in conjunction with the attachment of a nucleotide substrate to the oligonucleotide using a terminal nucleotidyl transferase.
  • the pyrophosphatase can be free in solution in the terminal nucleotidyl transferase reaction, co-immobilized with the terminal nucleotidyl transferase on the substrate medium, or immobilized on a substrate medium separately from the terminal nucleotidyl transferase.
  • a wide variety of pyrophosphatases can be adapted for the coupled reactions.
  • the pyrophosphatase is a Type 1 group of pyrophosphatases. In some embodiments, the pyrophosphatase is a Type II group of pyrophosphatases. In some embodiments, pyrophosphatases useful in coupled reactions with single-stranded RNA ligase or terminal nucleotidyl transferase reaction are disclosed in U.S. provisional application titled “Uses of Type II Inorganic Pyrophosphatases, filed April 16, 2024, incorporated by reference herein in its entirety.
  • the RNA ligase reaction further includes a nucleotide substrate regeneration system to regenerate ATP used as a co-factor in the RNA ligase reaction.
  • the ATP regeneration system is used to convert product AMP to ATP.
  • the regeneration of ATP is used to increase ligated product formation (e.g., product yield) in the single stranded RNA ligase reaction.
  • the ATP recycling or regeneration system includes a nucleoside monophosphate kinase for converting AMP to ADP in presence of a phosphate donor.
  • nucleoside monophosphate kinases can be used for the conversion of AMP to ADP, including various homologs of nucleoside monophosphate kinases. In some embodiments, more than one nucleoside monophosphate kinase can be used in the regeneration system. In some embodiments, the nucleoside monophosphate kinase is an adenosine monophosphate kinase (e.g., adenylate kinase), cytidine monophosphate (CMP) kinase, uridine monophosphate (UMP) kinase, and/or guanylate-monophosphate (GMP) kinase.
  • adenosine monophosphate kinase e.g., adenylate kinase
  • CMP cytidine monophosphate
  • UMP uridine monophosphate
  • GMP guanylate-monophosphate
  • a nucleoside monophosphate kinase useful in the ATP regeneration reaction is a cytidine monophosphate kinase.
  • cytidine monophosphate kinases are known in the art. These include homologs of cytidine monophosphate kinases.
  • a cytidine monophosphate kinase useful in the regeneration reactions include, among others, the cytidine monophosphate kinase of Thermus thermophilus (Q5SL35), Pyrococcus furiosus (Q8U2L4), Pseudomonas putida (AFO48857.1), Escherichia coli K-12 MG1655 (P0A6I0), Clostridium acetobutylicum (Q97I08), Halobacterium salinarum (Q9HPA5) Bacillus acidicola (WP_066270173), Acetobacter aceti (WP_010667744), Acidithiobacillus thiooxidans (WP_024892761.1), Acidithiobacillus ferrooxidans (WP_064220349.1), Metallosphaera sedula (WP_011921264.1), Amphibacillus xylanus (WP_0150099
  • a nucleoside monophosphate kinase useful in the ATP regeneration reactions is a uridine monophosphate kinase.
  • uridine monophosphate kinase Various suitable uridine monophosphate kinases are known in the art. These include homologs of uridine monophosphate kinases.
  • a uridine monophosphate kinase useful in the regeneration reactions includes, among others, the uridine monophosphate kinase of Pyrococcus furiosus (Q8U122), Thermus thermophilus (P43891), Pseudomonas putida (I7BW46), Escherichia coli K-12 MG1655 (P0A7E9), Aspergillus niger (A2R195), Saccharomyces cerevisiae (P15700), Clostridium acetobutylicum (Q97I64) ATCC 824 PyrH Halobacterium salinarum (Q9HNN8), Picrophilus torridus (WP_048059653), Metallosphaera sedula (WP_012021705), Thermoplasma acidophilum (WP_010900913), Sulfolobus solfataricus (WP_009992427), Acetobacter acet
  • HK1 WP_081759172.1
  • Amphibacillus xylanus WP_015010200.1
  • Vibrio psychroerythus Q485G8
  • Pseudoalteromonas haloplanktis Q3IIX6
  • Psychrobacter arcticus Q4FRH5
  • Psychromonas ingrahamii ABSM04676.1
  • Pseudomonas syringae Q4ZWS6
  • Halobacterium salinarum Q9HNN8
  • the nucleoside monophosphate kinase useful in the ATP regeneration reactions is a guanosine monophosphate kinase (guanylate kinase).
  • guanylate kinase guanosine monophosphate kinase
  • suitable guanylate kinases are known in the Docket Number CX10-269WO4 art. These include homologs of guanylate kinases.
  • a guanylate kinase useful in the regeneration reactions includes, among others, the guanylate kinase of Thermotoga maritima (Q9X215), Thermus thermophilus (Q5SI18), Pseudomonas putida (I7C087), Escherichia coli K-12 (P60546), Aspergillus niger (A2QPV2), Saccharomyces cerevisiae (P15454), Clostridium acetobutylicum (Q97ID0), Acidithiobacillus ferrooxidans (WP_064219869.1), Acidithiobacillus thiooxidans (WP_010637919.1), Bacillus acidicola (WP_066264774.1), Acetobacter aceti (WP_018308252.1), Amphibacillus xylanus (WP_015010280.1), Thioalkalivibrio s
  • the nucleoside monophosphate kinase useful in the ATP regeneration reactions is an adenosine monophosphate kinase (adenylate kinase).
  • adenylate kinase adenosine monophosphate kinase
  • suitable adenylate kinases are known in the art. These include homologs of adenylate kinases.
  • the adenylate kinase is a bacterial, fungal, plant, or animal adenylate kinase.
  • an adenylate kinase useful in the regeneration reactions includes, among others, adenylate kinases of Thermus thermophilus (Q72125), Pyrococcus furiosus (Q8U207), Pseudomonas putida (17CAA9), Escherichia coli K - 12 W3110 (P69441), Aspergillus niger CBS 513.88 (A2QPN9), Saccharomyces cerevisiae (P07170), Clostridium acetobutylicum (Q97E39), Halobacterium salinarum (Q9HPAT), Acidithiobacillus thiooxidans (WP_024894015.1), Acidithiobacillus ferrooxidans (WP_064218420.1), Bacillus acidicola (WP_066267988.1), Sulfolobus solfataricus (WP_009991241.1), Saccharomyces cerevisiae (
  • the adenylate kinase is an engineered adenylate kinase described in International patent application No. PCT/US2024/051084, filed October 11, 2024, incorporated herein by reference.
  • the ATP regeneration system includes at least an enzyme and a phosphate donor for the conversion of ADP to ATP.
  • the ATP regeneration system includes, among others, an acetate kinase, adenylate kinase, pyruvate kinase, creatine kinase, or polyphosphate kinase (see, e.g., Endo et al., Adv. Synth.
  • the phosphate donor for the conversion of ADP to ATP is selected based on the ATP regenerating enzyme employed.
  • the phosphate donor is acetyl-phosphate.
  • the phosphate donor is phosphoenolpyruvate.
  • the phosphate donor is creatine phosphate. If polyphosphate kinase is used for the conversion of ADP to ATP, the phosphate donor is inorganic polyphosphate. [0423]
  • the ATP regenerating system includes pyruvate kinase and phosphoenolpyruvate. In some embodiments, the ATP regenerating system includes creatine kinase and creatine phosphate. In some embodiments, the ATP regenerating system includes polyphosphate kinase and inorganic polyphosphate. In some embodiments, the ATP regenerating system includes acetate kinase and acetyl phosphate.
  • the ATP regenerating system includes acetate kinase and acetyl phosphate, where the acetate kinase is an acetate kinase of Escherichia coli str. K-12 substr. MG1655 (NP_416799.1), Corynebacterium jeikeium K411 (WP_011272972.1), Lactococcus cremoris subsp.
  • cremoris KW2 (WP_011835968.1), Lactococcus lactis (WP_004254593.1), Marinitoga sp.38H-ov (WP_165147355.1), Thermotoga sp. KOL6 (WP_101510533.1), Thermosipho melaniensis (WP_012057479.1), Thermotoga sp. RQ7 (WP_041844042.1), and Thermosipho africanus (WP_004102380.1).
  • the acetate kinase is an engineered acetate kinase described in, among others, International patent application No.
  • Single-stranded RNA (ssRNA) Ligases Single-stranded RNA (ssRNA) Ligases, Polynucleotides Encoding the Ligases, and Host Cells Single-stranded RNA (ssRNA) ligase polypeptides [0425]
  • the ligation reactions are mediated by a single-stranded RNA ligase.
  • the single-stranded RNA ligase comprises a viral single-stranded RNA ligase, a bacterial single-stranded RNA ligase, a fungal single-stranded RNA ligase, or a mammalian single-stranded RNA ligase.
  • the single-stranded ligase is a bacteriophage single-stranded RNA ligase.
  • the bacteriophage single-stranded RNA ligase is single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Phage TS2126, or Rhodothermus phage RM378.
  • the single-stranded RNA ligase is a bacterial single-stranded RNA ligase.
  • the single-stranded RNA ligase is an archael single-stranded RNA ligase (see, e.g., Nucleic Acids Res.2008 Nov; 36(19): 6218–6227). In some embodiments, the single-stranded RNA ligase is a Methanobacterium or Thermococcus single-stranded RNA ligase.
  • exemplary bacterial single-stranded RNA ligase is a single-stranded RNA ligase of Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, or Thermovibrio ammonificans HB-1.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 4.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 6, or to a reference sequence corresponding to SEQ ID NO: 6.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 10.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 16, or to a reference sequence corresponding to SEQ ID NO: 16.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Docket Number CX10-269WO4 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 18, or to a reference sequence corresponding to SEQ ID NO: 18.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 20, or to a reference sequence corresponding to SEQ ID NO: 20.
  • the single-stranded RNA ligase comprises one or more amino acid differences relative to the reference sequence corresponding to a sequence from amino acid residues 12 to the carboxyl terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20.
  • the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring single-stranded RNA ligases, and changing the amino acid residue of one sequence to the different amino acid residue present in the amino acid sequence of another naturally occurring single-stranded RNA ligase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence.
  • the recombinant single-stranded RNA ligase comprises an amino acid sequence comprising amino acid residues 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the single-stranded RNA ligase comprise variants of single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.
  • the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the sequence corresponding to amino acid residues 12 to the carboxy terminal residue is amino acid residues 12 to 387 of the referenced SEQ ID NO.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: S
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104,
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V31I, L32M, E33R, R34S/Y, N36D, V38M/T/V, V40L, S41V, Y42A, R43G/T, Y44R, N45Y, A46R, F48W, V49I, F50W, L54T, R56L, V84L, R91D, L92I, R93H/N, G94D, P109G/S, N113G, K115E, L118F/M, Q121K, K122C/I/L/M/S/T/V/Y, G123S, S125Q/R/T/V, P127L/Q/V, E130I, L135I, K138A, N141D, T146I, N151E, K152D, P156A/S, L160R, I161V, S16
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 90E/S, 93
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N/S/Y, 35L, 35S, 38T/V, 45Y, 48W, 49I, 54T, 66T, 90E/S, 93P, 115S, 118L/M, 121K, 122L/S, 127I/L/V, 162W, 165K, 167V, 170R, 177V, 197E/T, 205M, 213V, 220A/R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A/F/Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A/G, 337E, 351Y, 354E, 357
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue R34Y, V38T/V, N45Y, F48W, V49I, L54T, L118M, Q121K, G123S, P127L, S162W, F165K, L167V, I205M, L213V, I220A/R, N222G, R223G, S225G, K236H, K237G, H255F?Q, Y256R, F258L, T259S, F269L, I272G, G276H, I281L, G320A/G, K337E, K354E, R358G, T362E, or K365N, or any combinations thereof, wherein the amino acid positions are relative to the Docket Number CX10-269WO4 reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 121K, 45Y, 41V, 34Y, 269L, or 380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Q121K, N45Y, S41V, R34Y, F269L, or T380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34/45/269, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34Y/45Y/269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set or amino acid residues R34Y/N45Y/F269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 162, 236, 237, 320, 337, 358, or 362, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 162W, 236H, 237G, 320A, 337E, 358G, or 362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S162W, K236H, K237G, G320A, K337E, R358G, or T362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • Docket Number CX10-269WO4 the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 162/236/237/320/337/358/362, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 162W/236H/237G/320A/337E/358G/362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues S162W/K236H/K237G/G320A/K337E/R358G/T362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 123, 256, or 320, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 123S, 256R, or 320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue G123S, Y256R, or A320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 123/256/320, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 123S/256R/320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues G123S/Y256R/A320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 48, 49, 118, or 220, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: Docket Number CX10-269WO4 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38V, 48W, 49I, 118M, or 220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue T38V, F48W, V49I, L118M, or R220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38/48/49/118/220, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38V/48W/49I/118M/220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues T38V/F48W/V49I/L118M/R220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 54, 205, or 258, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38T, 54T, 205M, or 258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V38T, L54T, I205M, or F258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38/54/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38T/54T/205M/258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence Docket Number CX10-269WO4 corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues V38T/L54T/I205M/F258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 222, 223, 225, 255, 272, or 276, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127L, 222G, 223G, 225G, 255Q, 272G, or 276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue P127L, N222G, R223G, S225G, F255Q, I272G, or G276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127/222/223/225/255/272/276, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127L/222G/223G/225G/255Q/272G/276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues P127L/N222G/R223G/S225G/F255Q/I272G/G276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 165, 259, or 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 165K, 259S, or 281L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue F165K, T259S, or I281L, or any combinations thereof, wherein the Docket Number CX10-269WO4 amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 165/259/281, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 165K/259S/281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues F165K/T259S/I281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 238, 255, 359, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127V, 238G, 255A, 359D, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, E238G, Q255A, I359D, or K381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127/238/255/359/381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127V/238G/255A/359D/381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues L127V/E238G/Q255A/I359D/K381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 35, 170, 271, or 357, or any combinations thereof, Docket Number CX10-269WO4 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 35L, 170R, 271G, or 357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue E35L, P170R, N271G, or V357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 35/170/271/357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 35L/170R/271G/357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues E35L/P170R/N271G/V357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 118, 127, 275, 351, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 34S, 35S, 118L, 127I, 275W, 351Y, 374L, or 376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, N34S, L35S, M118L, V127I, Q275W, N351Y, F374L, or T376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34/35/118/127/275/351/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of Docket Number CX10-269WO4 the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34S/35S/118L/127I/275W/351Y/374L/376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues N34S/L35S/M118L/V127I/Q275W/N351Y/F374L/T376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 66, 90, 93, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 66T, 90S, 93P, or 197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue A19V, K66T, E90S, R93P, or E197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 19/66/90/93/197, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 19V/66T/90S/93P/197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues A19V/K66T/E90S/R93P/E197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 90, 197, 289, or 316, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 90E, 197E, 289Q, or 316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy Docket Number CX10-269WO4 terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S90E, T197E, N289Q, or S316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 90/197 and/or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 90E/197E and/or 289Q/316R wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues S90E/T197E and/or N289Q/S316R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, Docket Number CX10-269WO4 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence having a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO:
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 90E/S, 93
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N/S/Y, 35L, 35S, 38T/V, 45Y, 48W, 49I, 54T, 66T, 90E/S, 93P, 115S, 118L/M, 121K, 122L/S, 127I/L/V, 162W, 165K, 167V, 170R, 177V, 197E/T, 205M, 213V, 220A/R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A/F/Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A/G, 337E, 351Y, 354E, 357
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 44- 64, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 34Y/45Y/269L, 34Y/45Y/173E/297C, 34Y/173E/269L/380S, 173E/269L, 156S/269L/380S, 173E/269L/380S, 34Y/173E, 269L, 34Y/380S, 34Y/269L/380S, or 173E/380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R34Y/N45Y/F269L, R34Y/N45Y/K173E/R297C, R34Y/K173E/F269L/T380S, K173E/F269L, P156S/F269L/T380S, K173E/F269L/T380S, R34Y/K173E, F269L, R34Y/T380S, R34Y/F269L/T380S, or K173E/T380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or to the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 66- 118, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, Docket Number CX10-269WO4 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 207E, 237G, 94D/263D, 220V, 236H, 92I, 91D, 94D, 204E, 185E, 213V, 199L, 152D, 196D, 203V, 141D, 138A, 156A, 93N, or 181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R207E, K237G, G94D/N263D, I220V, K236H, L92I, R91D, G94D, K204E, K185E, L213V, K199L, K152D, N196D, I203V, N141D, K138A, P156A, R93N, or I181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or to the reference sequence corresponding to SEQ ID NO: 100, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 120- 216, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 283L/337E, 347E, 323D, 354V, Docket Number CX10-269WO4 343D, 347P, 118M, 347G, 345G, 347M, 354E, 314G, 345T, 268R/269F, 363S, 356M, 358P, 343P, 358G, 348S, 348L, 358T, 314S, 162W, 324T/330H, 354S, 347L, 346P, 323T, 348T, 160R, 362E, 361I, 341S/349S, 354H, 358D, 347S, 353T, 314P, 369L, 248H, 146I/346G, 332L, 346G, 314L, 170R
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) S283L/K337E, K347E, K323D, K354V, F343D, K347P, L118M, K347G, K345G, K347M, K354E, K314G, K345T, K268R/L269F, G363S, A356M, R358P, F343P, R358G, I348S, I348L, R358T, K314S, S162W, F324T/R330H, K354S, K347L, L346P, K323T, I348T, L160R, T362E, K361I, R341S/Q349S, K354H, R358D, K347S, L353T, K
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or to the reference sequence corresponding to SEQ ID NO: 140, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 244- 398, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 162W/337E/358C/362E, 162W/236H/237G/320A/337E/358G/362E, 151E/231I/237G/337E, 199L/231I/237G/337E, 231I/237G/314L/337E, 310G/314L/337E, 115E/162W/310G/314S, 199L/237G/337E, 151E/199L/205V/310G/314L, 199L/204E/205V/231I/236H/310G/314L, 320A/337E/358T/362E, 135I/320A/337E/358T/362E, 151E/212S/214E/345T/347L/358D, 135I/337E/358P/362E, 16
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) S162W/K337E/R358C/T362E, S162W/K236H/K237G/G320A/K337E/R358G/T362E, N151E/L231I/K237G/K337E, K199L/L231I/K237G/K337E, L231I/K237G/K314L/K337E, S310G/K314L/K337E, K115E/S162W/S310G/K314S, K199L/K237G/K337E, N151E/K199L/I205V/S310G/K314L, K199L/K204E/I205V/L231I/K236H/S310G/K3
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or to the reference sequence corresponding to SEQ ID NO: 246, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 400- 490, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 255F, 255M, 109G, 256R, 260I, Docket Number CX10-269WO4 260R, 273Y, 260V, 46R, 252R, 161V/162S, 311I/320G, 123S, 320G/326G, 174P, 256M, 162S/167V, 32M, 125V, 311C/320G, 162S/166H, 320G, 320G/326M, 260L, 256V, 283Q, 330I, 278L, 311F/320G, 320G/326T, 303A, 125T, 283T, 320G/326W, 281A, 333E/337K, 281W, 283K, 277T, 260T, 254R, 2
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) H255F, H255M, P109G, Y256R, Y260I, Y260R, F273Y, Y260V, A46R, V252R, I161V/W162S, L311I/A320G, G123S, A320G/E326G, D174P, Y256M, W162S/L167V, L32M, S125V, L311C/A320G, W162S/Q166H, A320G, A320G/E326M, Y260L, Y256V, S283Q, R330I, V278L, L311F/A320G, A320G/E326T, N303A, S125T, S283T, A320G/E326W, I281A, K333E
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 492- 510, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 123S/256R/320G, 260I, 256R/260I, 256R, 260I/281V, 320G, 123S/260I, 123S/320G, 256R/281V, or 260I/273Y, wherein the amino Docket Number CX10-269WO4 acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G123S/Y256R/A320G, Y260I, Y256R/Y260I, Y256R, Y260I/I281V, A320G, G123S/Y260I, G123S/A320G, Y256R/I281V, or Y260I/F273Y, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or to the reference sequence corresponding to SEQ ID NO: 492, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 512- 584, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 217P, 220A, 221P, 220S, 220R, 220P, 229L, 246F, 171S, 285R, 217G, 285G, 286Q, 165P, 285P, 226A, 168S, 177L, 226P, 285A, 223N, 224P, 118M/123G, 285S, 248M, 268A, 286P, 229A, 225P, 171P, 165T, 171R, 226Q, 84L, 226T, 284L, or 225T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L217P, I220A, E221P, I220S, Docket Number CX10-269WO4 I220R, I220P, F229L, V246F, Y171S, V285R, L217G, V285G, T286Q, F165P, V285P, I226A, V168S, F177L, I226P, V285A, R223N, E224P, L118M/S123G, V285S, S248M, K268A, T286P, F229A, S225P, Y171P, F165T, Y171R, I226Q, V84L, I226T, R284L, or S225T, wherein the amino acid positions are relative to the reference sequence
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 586- 602, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 254R, 240V, 118M, 347P, 167V, 281V, 303G, 205T, or 50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue H254R, F240V, L118M, K347P, L167V, I281V, N303G, I205T, or F50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or to the reference sequence corresponding to SEQ ID NO: 594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 604- 700, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 118M/220A, 118M/220A/303G/K347L, 347L, 118M/220A/254S/347L, 220A, 38V/220A, 220A/254S/303G/347L, 220A/254S, 220A/303G/347L, 48W/220A/347L, 49I/220A, 217G/220A/347L, 38V/220A/254S, 49I/118M/220A/254S/347L, 49I/220A/254S, 38V/48W/49I/118M/220A, 49I/217G/220A/254S, 49I/220A/347L, 49I/347L, 225G, 280S, 118F, 279R, 165K, 273A, 272S,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L118M/R220A, L118M/R220A/N303G/K347L, K347L, L118M/R220A/H254S/K347L, R220A, T38V/R220A, R220A/H254S/N303G/K347L, R220A/H254S, R220A/N303G/K347L, F48W/R220A/K347L, V49I/R220A, L217G/R220A/K347L, T38V/R220A/H254S, V49I/L118M/R220A/H254S/K347L, V49I/R220A/H254S, T38V/F48W/V49
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or to the reference sequence corresponding to SEQ ID NO: 634, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 702- 736, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 38M/54T/127V/205M/254A, 165M/255A/258L/259A, 205M/254A/258L, 33R/38T/127V/165M/254A, 38T/54T/205M/258L, 127V, 127V/165M/258L, 127V/205M/254A/255A/258L/259A, 205M/258L, 33R/38T/254A/255A, 127V/254A/258L/259A, 127V/165M, 38T/127V/165M/259A, 38M/127V/258L/259A, 38M/254D/255V, 127V/205M/254A/258L/259A, 127V/165M/258L/259A, or 165M/205M/2
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) V38M/L54T/P127V/I205M/H254A, F165M/F255A/F258L/T259A, I205M/H254A/F258L, E33R/V38T/P127V/F165M/H254A, V38T/L54T/I205M/F258L, P127V, P127V/F165M/F258L, Docket Number CX10-269WO4 P127V/I205M/H254A/F255A/F258L/T259A, I205M/F258L, E33R/V38T/H254A/F255A, P127V/H254A/F258L/T259A, P127V/F165M, V38T/P127V/
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or to the reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 738- 754, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 127L/222G/223G/225G/255Q/272G/276H, 127L/255Q/259S, 276H, 255Q/259S, 127L/255Q, 127L/162S/223G/255Q, 127L/162S/255Q/259S/272G/276H, 127L/255M, or 259S/272L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) P127L/N222G/R223G/S225G/F255Q/I272G/G276H, P127L/F255Q/T259S, G276H, F255Q/T259S, P127L/F255Q, P127L/W162S/R223G/F255Q, P127L/W162S/F255Q/T259S/I272G/G276H, P127L/F255M, or T259S/I272L, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or to the reference sequence corresponding to SEQ ID NO: 738, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 756- 760, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 165K/259S/281L, 127P, or 259S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) F165K/T259S/I281L, L127P, or T259S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or to the reference sequence corresponding to SEQ ID NO: 756, wherein the amino acid sequence comprises one or more substitutions Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 762- 810, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 177I, 362R, 44R, 365N, 46R, 279Q/281I, 303A, 165F/166S, 281I/282L, 302I, 362A, 360L, 295R, 246I, 127Q, 299R, 125Q, 238G, 295T, 56L, 109S, 125R, 38V, 295A, or 31I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) F177I, E362R, Y44R, K365N, A46R, D279Q/L281I, N303A, K165F/Q166S, L281I/F282L, T302I, E362A, A360L, E295R, V246I, L127Q, K299R, S125Q, E238G, E295T, R56L, P109S, S125R, T38V, E295A, or V31I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or to the reference sequence corresponding to SEQ ID NO: 768, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 812- 880, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/276, 246/276/302, 246/279/280,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 44R/118F, 44R/118F/246I/272L/276G, 44R/118F/246I/280S, 44R/118F/246I/280S/302I, 44R/118F/272L/276G, 44R/246I, 44R/246I/272L/276G/302I, 44R/246I/276G/279R/280S, 44R/246I/276G/280S, 44R/272L/276G/280S, 44R/272L/279R, 44R/272L/280S, 44R/276G, 44R/276G/279R, 44R/276G/280S/295R/302I, 44R/280S, 44R/302I, 118F/246I/276G/280S/302I, 118F/246I/280S/295R/302I, 44R/
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) Y44R/M118F, Y44R/M118F/V246I/G272L/H276G, Y44R/M118F/V246I/D280S, Y44R/M118F/V246I/D280S/T302I, Y44R/M118F/G272L/H276G, Y44R/V246I, Y44R/V246I/G272L/H276G/T302I, Y44R/V246I/H276G/D279R/D280S, Y44R/V246I/H276G/D280S, Y44R/G272L/H276G/D280S, Y44R/G272L/H276G/D280S, Y44R/G
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or to the reference sequence corresponding to SEQ ID NO: 844, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 882- 912, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 42A, 43G/T, 122C/I/L/M/S/T/V/Y, 271G/M, 272G, 278P, or 279Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Y42A, R43G/T, K122C/I/L/M/S/T/V/Y, N271G/M, L272G, V278P, or D279Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or to the reference sequence corresponding to SEQ ID NO: 882, wherein the amino acid sequence comprises one or more substitutions Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 934- 990, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 38V/127V/238G/284F, 38V/127V/255A, 38V/127V/255A/359D, 38V/127V/284F, 38V/238G/255A, 38V/238G/255A/359D, 38V/238G/255A/359D/381R, 38V/238G/284F/359D, 127V, 127V/212H/238G/284F/359D, 127V/238G/255A, 127V/238G/255A/359D/381R, 127V/238G/284F/359D, 127V/238G/284F/359D, 127V/238G/284F/359D, 127V/238G/284F/359D/381R, 127V/238G/359D, 127V/255A/359D/381R, 238
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) T38V/L127V/E238G/R284F, T38V/L127V/Q255A, T38V/L127V/Q255A/I359D, T38V/L127V/R284F, T38V/E238G/Q255A, T38V/E238G/Q255A/I359D, T38V/E238G/Q255A/I359D/K381R, T38V/E238G/R284F/I359D, L127V, L127V/R212H/E238G/R284F/I359D, L127V/E238G/Q255A, L127V/E238G/Q255A/I359D/K381R, L127V/E238G/R284F, T38V/L
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or to the reference sequence corresponding to SEQ ID NO: 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 992- 1094, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 34K/35L/38V, 34K/38V/170R, 34K/135M/170R/271G/357L, 34K/170R/271G, 34K/271G, 35L/38V/170R/357L, 35L/38V/271G/357L, 35L/170R/271G/357L, 38V/170R, 38V/170R/271G, 38V/170R/357L, 56M/135M/170R/357L, 56M/135M/271G/357L, 135M/170R/271G, 170R, 170R, 170R/271G/272R, 170R/271G/272R/357L, 170R/271G/357L, 170R/357L, 254R/260R, 254R/281M, 271G, or 357L
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) Y34K/E35L/T38V, Y34K/T38V/P170R, Y34K/L135M/P170R/N271G/V357L, Y34K/P170R/N271G, Y34K/N271G, E35L/T38V/P170R/V357L, E35L/T38V/N271G/V357L, E35L/P170R/N271G/V357L, T38V/P170R, T38V/P170R/N271G, T38V/P170R/V357L, R56M/L135M/P170R/V357L, R56M/L135M/N271G/V357L, R56M/L135M
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 9D/369V, 34K/38V/357L, 34K/135M/170R/271G/357L, 34K/271G, 56M/135M/170R/357L, 56M/135M/271G/357L, 113D, 115S, 141G, 144T, 145Y, 177C, 177V, 214A, 214C, 214S, 254R/260R, 254R/281M, 260R, 274I, 340C, 341A, 350A, 350C, 350R, 354Q/359I, 359I/360V, 359I/362Q, 368G, 369C, 369L, 371R, 377L/381K, 381K/384C, or 381K/384S, wherein the amino acid positions are relative to the amino acid positions are relative to the amino
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G9D/Y369V, Y34K/T38V/V357L, Y34K/L135M/P170R/N271G/V357L, Y34K/N271G, R56M/L135M/P170R/V357L, R56M/L135M/N271G/V357L, N113D, K115S, N141G, N144T, F145Y, F177C, F177V, K214A, K214C, K214S, H254R/Y260R, H254R/L281M, Y260R, L274I, G340C, R341A, E350A, E350C, E350R, E354Q/D359I, D359I/A360V, D3
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO: 992, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1096-1206, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/254/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 113D, 113D/115S, 113D/115S/177V/254R/281M, 113D/115S/177V/254R/281M/350A/359I, 113D/115S/177V/254R/350R, 113D/115S/177V/254R/359I, 113D/115S/177V/281M/359I, 113D/115S/254R, 113D/115S/254R/281M, 113D/115S/254R/350R, 113D/115S/254R/359I, 113D/115S/350R, 113D/115S/254R/359I, 113D/115S/350R, 113D/115S/359I, 113D/177V/254R/350R, 113D/177V/281M/359I,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) N113D, N113D/K115S, N113D/K115S/F177V/H254R/L281M, N113D/K115S/F177V/H254R/L281M/E350A/D359I, N113D/K115S/F177V/H254R/E350R, N113D/K115S/F177V/H254R/D359I, N113D/K115S/F177V/L281M/D359I, N113D/K115S/H254R, N113D/K115S/H254R/L281M, N113D/K115S/H254R, N113D/K115S/H254R/L281M, N113D/K115S/H254R/E350R, N113D/K115
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or to the reference sequence corresponding to SEQ ID NO: 1104, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1208-1254, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 27F, 27W, 27Y, 34G, 34N, 34R, 34S, 35A, 35S, 122A, 127I, 255S, 259N, 275W, 349V, 351F, 351Y, 354S, 356V, 363V, 374I, 374L, 374V, or 376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K27F, K27W, K27Y, Y34G, Y34N, Y34R, Y34S, L35A, L35S, L122A, V127I, A255S, S259N, Q275W, Q349V, N351F, N351Y, E354S, A356V, G363V, F374I, F374L, F374V, or T376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or to the reference sequence corresponding to SEQ ID NO: 1222, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 27W/127I/374I/376E, 27W/127I/374L, 27W/275W/356V, 27W/351Y, 34S/35S/118L/127I/275W/351Y/374L/376E, 122A, 127I/275W/374V, or 275W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K27W/V127I/F374I/T376E, K27W/V127I/F374L, K27W/Q275W/A356V, K27W/N351Y, N34S/L35S/M118L/V127I/Q275W/N351Y/F374L/T376E, L122A, V127I/Q275W/F374V, or Q275W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 21A, 66A, 69A, 151L, or 199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K21A, K66A, S69A, N151L, or K199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 25G, 54I, 54L, 54V, 65Q, 65S, 66A, 66P, 66Q, 66R, 66T, 90S, 93P, or 151L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue A19V, N25G, T54I, T54L, T54V, R65Q, R65S, K66A, K66P, K66Q, K66R, K66T, E90S, R93P, or N151L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 21A, 25W, 65P, 65S, 66T, 69C, 88W, 91L, 93A, 93H, 93P, 97L, 97P, 97V, 157L, 190L, 195T, 197T, or 198A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K21A, N25W, R65P, R65S, K66T, S69C, K88W, R91L, R93A, R93H, R93P, I97L, I97P, I97V, I157L, Y190L, E195T, E197T, or I198A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1308-1364 and 1396-1436, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 19V, 19V/21A/65Q/66T/90S/93P, 19V/21A/65S, 19V/21A/65S/66A, 19V/21A/65S/66Q/93P, 19V/21A/65S/66T, 19V/21A/65S/93P, 19V/21A/65S/190L, 19V/21A/65S/197T, 19V/21A/66Q, 19V/21A/190L, 19V/65Q/66A/197T, 19V/65S, 19V/65S/66A, 19V/65S/66A/93A, 19V/65S/66Q, 19V/65S/66Q/90S/93P/190L, 19V/65S/66Q/190L, Docket Number CX10
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) A19V, A19V/K21A/R65Q/K66T/E90S/R93P, A19V/K21A/R65S, A19V/K21A/R65S/K66A, A19V/K21A/R65S/K66Q/R93P, A19V/K21A/R65S/K66T, A19V/K21A/R65S/R93P, A19V/K21A/R65S/Y190L, A19V/K21A/R65S/E197T, A19V/K21A/K66Q, A19V/K21A/Y190L, A19V/R65Q/K66A/E197T, A19V/R65S, A19V/R65S/K66A, A19V/R65S/E197T, A19V
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237A, 240W, 246I, 289Q, 306F, 306M, 310A, 310D, 323G, 323N, 323Y, 330I, 334I, 334N, 334S, 336E, 336N, 351N/352S, 351N/352V, 351N/353V, or 351N/358V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237A, F240W, V246I, N289Q, T306F, T306M, S310A, S310D, K323G, K323N, K323Y, R330I, K334I, K334N, K334S, R336E, R336N, Y351N/Y352S, Y351N/Y352V, Y351N/L353V, or Y351N/G358V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237R, 240W, 288T, 291L, 299I, 306M, 314E, 316R, 325M, 325P, 332G, 336E, 351N/352G, 351N/352L, 351N/352M, 351N/352N, 351N/352R, 351N/352V, 351N/353V, 351N/355K, 372A/374F/376T, 372L/374F/376T, or 372P/374F/376T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237R, F240W, E288T, V291L, K299I, T306M, K314E, S316R, D325M, D325P, V332G, R336E, Y351N/Y352G, Y351N/Y352L, Y351N/Y352M, Y351N/Y352N, Y351N/Y352R, Y351N/Y352V, Y351N/L353V, Y351N/E355K, T372A/L374F/E376T, T372L/L374F/E376T, or T372P/L374F/E376T, wherein the amino acid positions are relative to the reference sequence corresponding to
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or to the reference sequence corresponding to SEQ ID NO: 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 21A/54I, 21A/65Q/66K/151L, 21A/90E/93R/122A, 21A/93R, 54I/58M, 65Q/93R, 65Q/151L, 66K/90E/151L/190L/197E, 90E/93R/122A, Docket Number CX10-269WO4 90E/190L/197E, 90E/197E, 93R, or 93R/151L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K21A/T54I, K21A/R65Q/T66K/N151L, K21A/S90E/P93R/L122A, K21A/P93R, T54I/L58M, R65Q/P93R, R65Q/N151L, T66K/S90E/N151L/Y190L/T197E, S90E/P93R/L122A, S90E/Y190L/T197E, S90E/T197E, P93R, or P93R/N151L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or to the reference sequence corresponding to SEQ ID NO: 1474, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237A, 240W, or 289Q/316R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237A, F240W, or N289Q/S316R, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or to the reference sequence corresponding to SEQ ID NO: 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 27A, 30R, 36R, 40I, 139R, 141A, 141P, 141R, 172P, or 223T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K27A, K30R, N36R, V40I, N139R, N141A, N141P, N141R, D172P, or G223T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, Docket Number CX10-269WO4 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140,
  • the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492,
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246,
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant single stranded RNA ligase comprises an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244- 912, and 934-1526.
  • the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions.
  • the recombinant single stranded RNA ligase has single stranded RNA ligase activity. In some embodiments, the recombinant single stranded RNA ligase has single stranded RNA ligase activity and exhibits at least an improved property as compared to a reference single stranded RNA ligase. [0626] In some embodiments, the recombinant single stranded RNA ligase exhibits increased ligase activity as compared to a reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity as compared to the reference single stranded RNA ligase. Exemplary increases in ligase activity is provided in the Examples. [0627] In some embodiments, the recombinant single stranded RNA ligase exhibits increased activity on modified oligonucleotide substrates.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with polynucleotide or oligonucleotide acceptor having a 3’-terminal modified nucleoside as compared to the reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase has increased activity with an oligonucleotide acceptor having a 3’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro- Docket Number CX10-269WO4 guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl-thymidine.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a nucleotide donor having a 5’-terminal modified nucleoside compared to the reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase has increased activity with an nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O- methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl- thymidine.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a polynucleotide or oligonucleotide donor having a 5’-terminal modified nucleoside compared to the reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase has increased activity with an nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro- uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O- methyl-uridine, 2’-O-methyl-thymidine.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a nucleotide donor pNp having a modified nucleoside compared to the reference single stranded RNA ligase.
  • the recombinant single stranded RNA ligase has increased activity with an nucleotide donor pNp having a modified nucleoside of 2’- fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O- methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl- thymidine.
  • the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity as compared to the reference single stranded RNA ligase with oligonucleotide acceptor having a 3’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’- fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O- methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine, and a nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine
  • the recombinant single stranded RNA ligase has increased activity with nucleotide donors of a single nucleotide, or short oligonucleotide donor of 2, 3 or 4 nucleotides in length as compared to the reference single stranded RNA ligase.
  • the single nucleotide donor, or the short oligonucleotide donor comprises at least a modified 5’-terminal nucleoside. Exemplary modified single stranded RNA ligase acceptors and donors are provided in the Examples.
  • the single stranded RNA ligase acceptor and donor for comparative activity are provided in Tables 4.1, 5.1, 6.1, 7.1, 8.1, 10.1, 11.1, 12.1, 13.1, 14.1, 14.2, 15.1, 15.2, 16.1, 16.2, 17.1, 18.1, 19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, 26.1, 27.1, 28.1, 29.1, 32.1, 37.1, 38.1, 39.1, 40.1, 42.1, and 43.1. List of donors and acceptors for the single stranded RNA ligase reaction is listed in the Examples.
  • the single stranded RNA ligase exhibits increased thermostability. In some embodiments, the single stranded RNA ligase exhibits increased thermostability at 58 °C as compared to a reference single stranded RNA ligase. In some embodiments, the comparison is made at a defined temperature for 30 min, 1 hr, 2 hr, 3 hr, or 4 hr or more.
  • the recombinant single stranded RNA ligase exhibits an improved property selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), as compared to a reference single stranded RNA ligase.
  • the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or an amino acid sequence corresponding to SEQ ID NO: 14.
  • the single-stranded RNA ligase is provided as a fusion protein.
  • the single-stranded RNA ligase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, immobilization on a support medium, or fusion to another protein.
  • the fusion protein of the single-stranded RNA ligase comprises a glycine-histidine or histidine-tag (His-tag).
  • the single stranded RNA ligase comprises one or more lysine residues inserted or fused to RNA ligase polypeptide, e.g., 1-25 lysine residues.
  • the single-stranded RNA ligase is fused to a polylysine (e.g., 2-25 or 2-10 lysine residues), for example, for conjugation to a support medium via the amino group of the polylysine.
  • the fusion protein of the single-stranded RNA ligase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA).
  • the fusion protein of the single-stranded RNA ligase comprises a GST, SUMO, Strep, MBP, or GFP tag.
  • the fusion is to cell localization signals (e.g., secretion signals).
  • the fusion is to the amino (N-) terminus of single-stranded RNA ligase polypeptide.
  • the fusion is to the carboxy (C-) terminus of the single-stranded RNA ligase polypeptide.
  • the fusion is selected or designed to preserve the activity of the single-stranded RNA ligase.
  • the single-stranded RNA ligase described herein is an isolated composition.
  • the single-stranded RNA ligase polypeptide is purified.
  • the Docket Number CX10-269WO4 recombinant phosphatase is provided in solution, as a lyophilizate, or immobilized on a substrate or support medium, as further discussed herein.
  • the present disclosure further provides functional fragments or biologically active fragments of single-stranded RNA ligase described herein.
  • a functional fragment or biologically active fragment of the single-stranded RNA ligase is provided herewith.
  • a functional fragment or biologically active fragments of a single-stranded RNA ligase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the single-stranded RNA ligase polypeptide from which it was derived (i.e., the parent single-stranded RNA ligase).
  • functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the phosphatase.
  • the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids.
  • a functional fragment of a single-stranded RNA ligase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the single-stranded RNA ligase.
  • the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.
  • the functional fragments or biologically active fragments of the recombinant single stranded RNA ligase described herein include at least a mutation or mutation set in the amino acid sequence of the recombinant single stranded RNA ligase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the recombinant single stranded RNA ligase displays the enhanced or improved property associated with the mutation or mutation set in the parent single stranded RNA ligase or parent recombinant single stranded RNA ligase.
  • Polynucleotides encoding single-stranded RNA ligases, expression vectors, and host cells [0642]
  • the present disclosure provides recombinant polynucleotides encoding the single- stranded RNA ligases described herein.
  • the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the single-stranded RNA ligases.
  • the present disclosure provides methods and compositions for the production of each and every possible variation of Docket Number CX10-269WO4 polynucleotides that could be made that encode the single-stranded RNA ligases described herein by selecting combinations based on the possible codon choices, and all such variations of polynucleotides are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples and in the accompanying Sequence Listing.
  • the codons are preferably optimized for utilization by the chosen host cell for protein production.
  • preferred codons in bacterial cells are used for expression in bacterial cells.
  • preferred codons in fungal cells are used for expression in fungal cells.
  • preferred codons in insect cells are used for expression in insect cells.
  • preferred codons in mammalian cells are used for expression in mammalian cells.
  • codon optimized polynucleotides encoding a recombinant single stranded RNA ligase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region.
  • a recombinant polynucleotide of the present disclosure encodes a single-stranded RNA ligase described herein.
  • the polynucleotide sequence of the recombinant polynucleotide is codon optimized.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising the single stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to a sequence from nucleotide residue 34 to the 3’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19, or to a reference sequence corresponding to an odd numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence comprising the sequence from nucleotide residue 34 to the 3’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19, or comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, Docket Number CX10-269WO4 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 162, 236, 237, 320, 337, 358, or 362, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 123, 256, or 320, or any combinations thereof, wherein the amino acid Docket Number CX10-269WO4 positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 38, 48, 49, 118, or 220, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 38, 54, 205, or 258, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 127, 222, 223, 225, 255, 272, or 276, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 165, 259, or 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 127, 238, 255, 359, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 35, 170, 271, or 357, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 115, 177, 254, or 359, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 34, 35, 118, 127, 275, 351, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 66, 90, 93, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 90, 197, 289, or 316, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, Docket Number CX10-269WO4 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, or 1500, or to the reference sequence
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference Docket Number CX10-269WO4 sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or to the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 66-118, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, Docket Number CX10-269WO4 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or to the reference sequence corresponding to SEQ ID NO: 100, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 120-216, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or to the reference sequence corresponding to SEQ ID NO: 140, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 244-398, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or to the reference sequence corresponding to SEQ ID NO: 246, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 400-490, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, Docket Number CX10-269WO4 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 492-510, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or to the reference sequence corresponding to SEQ ID NO: 492, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 586-602, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or to the reference sequence corresponding to SEQ ID NO: 594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 604-700, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 2
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or to the reference sequence corresponding to SEQ ID NO: 634, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 702-736, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or to the reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 738-754, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or to the reference sequence corresponding to SEQ ID NO: 738, Docket Number CX10-269WO4 wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 756-760, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or to the reference sequence corresponding to SEQ ID NO: 756, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 762-810, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or to the reference sequence corresponding to SEQ ID NO: 768, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 812-880, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/276/279/280/302, 246
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or to the reference sequence corresponding to SEQ ID NO: 844, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or to the reference sequence corresponding to SEQ ID NO: 882, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 934-990, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/3
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or to the reference sequence corresponding to SEQ ID NO: 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 992-1094, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO: 992, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1096-1206, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or to the reference sequence corresponding to SEQ ID NO: 1104, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or to the reference sequence corresponding to SEQ ID NO: 1222, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino Docket Number CX10-269WO4 acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or to the reference sequence corresponding to SEQ ID NO: 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or to the reference sequence corresponding to SEQ ID NO: 1474, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or to the reference sequence corresponding to SEQ ID NO: 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a Docket Number CX10-269WO4 substitution at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474,
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 13
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 8
  • the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934- 1526, or comprising an even-numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO.
  • the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523.
  • the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or a polynucleotide sequence comprising SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499.
  • the recombinant polynucleotide is codon-optimized for expression of the encoded recombinant single stranded RNA ligase.
  • the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, insect cell, or mammalian cell.
  • a recombinant polynucleotide encoding any of the single-stranded RNA ligase herein is manipulated in a variety of ways to facilitate expression of the single-stranded RNA ligase polypeptide.
  • the recombinant polynucleotide encoding the single-stranded RNA ligase comprises expression vectors where one or more control sequences is present to regulate the expression of the phosphatase encoding polynucleotides and/or encoded polypeptides.
  • the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.
  • suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E.
  • Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci.
  • promoters for filamentous fungal host cells include, but are not limited to promoters obtained Docket Number CX10-269WO4 from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acet
  • Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos
  • Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, p10, ELT, OpIE2, and hr5/ie1 promoters.
  • Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken ⁇ -actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, and from Gallus ⁇ -actin.
  • CMV cytomegalovirus
  • SV40 Simian vacuolating virus 40
  • the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription).
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the single-stranded RNA ligase polypeptide.
  • Any suitable terminator which is functional in the host cell of choice finds use in the present invention.
  • the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813.
  • Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
  • Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3- phosphate dehydrogenase.
  • terminators for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8(6):423-88).
  • Exemplary terminators for insect cells and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.
  • the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is used for translation by the host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the single-stranded RNA ligase polypeptide.
  • Any suitable leader sequence that is functional in the host cell of choice find use in the present invention.
  • Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- Docket Number CX10-269WO4 phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
  • Suitable leaders for mammalian host cells include but are not limited to the 5 ⁇ -UTR element present in orthopoxvirus mRNA.
  • control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA).
  • a polyadenylation sequence i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA.
  • Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
  • Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990).
  • Useful polyadenylation and 3’ UTR sequences for insect and mammalian host cells include, but are not limited to, OpIE2 polyA sequence, D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), SV40 polyA signal sequence, and the 3 ⁇ -UTRs of ⁇ - and ⁇ -globin mRNAs harboring sequence elements that increase the stability and translation of mRNA.
  • control sequence comprises a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway).
  • a signal peptide i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway.
  • the 5’ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide.
  • the 5’ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence.
  • Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA.
  • effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
  • Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
  • Useful signal peptides for insect and mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.
  • the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is referred to as a Docket Number CX10-269WO4 “proenzyme,” “propolypeptide,” or “zymogen.”
  • a propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha- factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • aprE Bacillus subtilis alkaline protease
  • nprT Bacillus subtilis neutral protease
  • Saccharomyces cerevisiae alpha- factor e.g., Rhizomucor mie
  • regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems.
  • suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system.
  • suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.
  • the present disclosure provides a recombinant expression vector comprising a recombinant polynucleotide encoding a single-stranded RNA ligase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced.
  • the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce recombinant expression vectors capable of expressing the encoded single-stranded RNA ligase.
  • the recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the single-stranded RNA ligase encoding polynucleotide.
  • the choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome).
  • the vector may contain any means for assuring self-replication.
  • the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.
  • the recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid.
  • the non-replicating expression vector or plasmid can be Docket Number CX10-269WO4 based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol.7, Article 75).
  • the expression vector contains one or more selectable markers, which permit selection of transformed cells.
  • a “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A.
  • the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a single-stranded RNA ligase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the recombinant phosphatase enzyme(s) in the host cell.
  • Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention include but are not limited to, bacterial cells, such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells.
  • bacterial cells such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells
  • fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2
  • Exemplary host cells also include various Escherichia coli strains (e.g., W3110 ( ⁇ fhuA) and BL21).
  • the present disclosure provides a method of producing the single-stranded RNA ligase, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the single-stranded RNA ligase under conditions suitable for expression of the polypeptide such that the single- stranded RNA ligase is produced.
  • the method further comprises isolating the single- stranded RNA ligase polypeptides from the culture and/or host cells.
  • the method further comprises purifying the expressed single-stranded RNA ligase polypeptide, as described herein.
  • the single-stranded RNA ligase expressed in a host cell is recovered from the cells and/or the culture medium using any one or more of the known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra- centrifugation, and chromatography, such as described herein.
  • Chromatographic techniques for isolation/purification of the single-stranded RNA ligase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion- exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the single-stranded RNA ligase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular Docket Number CX10-269WO4 shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the phosphatase.
  • compositions of single-stranded RNA (ssRNA) ligases [0787]
  • the present disclosure provides compositions of the RNA ligases disclosed herein.
  • the composition comprises a recombinant single stranded RNA ligase polypeptide described herein.
  • the recombinant single stranded RNA ligase polypeptide in the compositions is isolated or purified.
  • the recombinant single stranded RNA ligase is combined with other components and compounds to provide compositions and formulations comprising the engineered RNA ligase polypeptide as appropriate for different applications and uses.
  • the composition further comprises one or more of a buffer, a nucleotide cofactor (e.g., ATP), salt, and/or at least one or more substrates, for example an oligonucleotide or nucleotide substrate with modified nucleotides.
  • the components in the composition can comprise components described in the reaction conditions for the single stranded RNA ligase.
  • the composition comprises a buffer.
  • the buffer comprises, among others, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl- propane-1,3-diol (Tris), and the like.
  • the buffer concentration is from 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM.
  • composition comprises a salt, including, among others, NaCl, KCl, ammonium salts (e.g., NH 4 Cl), and acetate salts (e.g., sodium acetate).
  • the salt is present at 0.5 mM-300 mM, 1 mM-250 mM, 2 mM-200 mM, 5 mM-150 mM, 10 mM-100 mM, 20 mM-80 mM, or 40 mM-60 mM.
  • the salt is present at about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM.
  • the composition comprises nucleotide cofactor concentration of about 0.1-10 mM.
  • the nucleotide cofactor concentration is about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, or more as appropriate.
  • the cofactor is present in the form of a salt, such MgCl 2 , MnCl 2 , or CoCl 2 .
  • the cofactor is a nonnatural or non- preferred cofactor for the recombinant single stranded RNA ligase and/or the second enzyme.
  • the cofactor is a natural or preferred cofactor for the recombinant single stranded RNA ligase and/or the second enzyme. In some embodiments, the cofactor is a preferred or natural cofactor for a second enzyme and a nonpreferred or nonnatural cofactor for the recombinant single stranded RNA ligase. In some embodiments, the cofactor is present at a higher concentration compared to an in vivo concentration. Docket Number CX10-269WO4 [0792] In some embodiments, the composition further comprises a divalent metal ion. In some embodiments, the divalent metal ion, such as Ni 2+ , Mg 2+ , Mn 2+ , or Co 2+ .
  • the cofactor is present in the form of a salt, such MgCl2, MnCl2, or CoCl2.
  • the divalent metal is present at a concentration of 0.1 mM to 5 mM.
  • the composition comprises one or more of EDTA, DTT, and glycerol, such as for storage of the engineered single stranded RNA ligase.
  • the composition comprises an additive, such as a preservative, a buffer, or cryoprotectant.
  • the additive comprises a preservative/cryoprotectant, for example, dextran, polyethylene glycol, or glycerol.
  • the additive comprises a surfactant, for example, polysorbate (e.g., polysorbate 20 or 80).
  • the composition comprises a single-stranded RNA ligase and a nucleotide donor.
  • the nucleotide donor is a nucleotide (D) , or an oligonucleotide donor (oligonucleotide (D) ).
  • nucleotide (D) is a modified nucleotide (D) , including a modified nucleotide (D) comprising a conjugate moiety, reactive group, or a linker.
  • the oligonucleotide donor is a modified oligonucleotide donor, including a modified oligonucleotide donor comprising at least a modified internucleoside linkage, a modified nucleobase, a modified sugar moiety, or comprising a conjugate moiety, reactive group, or linker, as described herein.
  • the nucleotide donor e.g., nucleotide(D) or oligonucleotide(D) comprises a 3’-blocking group).
  • the composition comprises a single stranded RNA ligase and an oligonucleotide acceptor (oligonucleotide (A) ).
  • the oligonucleotide donor is a modified oligonucleotide acceptor, including a modified oligonucleotide acceptor comprising at least a modified internucleoside linkage, a modified nucleobase, a modified sugar moiety, or comprising a conjugate moiety, reactive group, or linker, as described herein.
  • the oligonucleotide acceptor comprises a 5'-OH or a 5'-blocking group.
  • the composition further comprises an additive or ligation enhancing agent, including, among others, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000).
  • the composition comprises 1% to 40% v/v of DMSO.
  • the composition comprises 0.1 M to 3 M betaine.
  • the composition comprises 0.5% to 20% w/v of PEG (e.g., PEG6000 or PEG8000).
  • the composition further comprises an RNase inhibitor, including, among others, porcine RNase inhibitor, human placental RNase inhibitor, human liver RNase inhibitor, mouse RNase inhibitor, rat RNase inhibitor, and recombinantly expressed RNase inhibitors thereof.
  • RNase inhibitor including, among others, porcine RNase inhibitor, human placental RNase inhibitor, human liver RNase inhibitor, mouse RNase inhibitor, rat RNase inhibitor, and recombinantly expressed RNase inhibitors thereof.
  • a recombinant single stranded RNA ligase described herein is provided in solution, as a lyophilizate, or is immobilized on a substrate.
  • the substrate is a solid substrate, porous substrate, membrane, or particles. The enzyme can be entrapped in matrixes or membranes.
  • matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, agarose or derivatives thereof (e.g., cross-linked agarose), and collagen, or solid matrices, such as activated carbon, porous ceramic, and diatomaceous earth.
  • the matrix is a Docket Number CX10-269WO4 particle, a membrane, or a fiber.
  • Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.
  • the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support material.
  • the enzyme is immobilized on the support material by covalent attachment.
  • Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.
  • the composition comprises a recombinant single stranded RNA ligase and another enzyme, such as a pyrophosphatase, such as a Type I or Type II pyrophosphatase, or an enzyme used in ATP recycling system.
  • another enzyme such as a pyrophosphatase, such as a Type I or Type II pyrophosphatase, or an enzyme used in ATP recycling system.
  • the composition further comprises a buffer.
  • Suitable reaction buffers are well known in the art and include but are not limited to, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2- hydroxymethyl-propane-1,3-diol (Tris), and the like. Any suitable buffer may be used. In some embodiments, the buffer is present at a concentration of 10-100 mM, 50-500 mM, or 300 mM- 1 M.
  • Terminal Nucleotidyl Transferase (TnT)
  • terminal nucleotidyl transferases are used to attach or incorporate one or more nucleotides to an oligonucleotide having an accessible 3’-OH group, for example, following attachment of a nucleotide donor to a nucleotide acceptor using the single-stranded RNA ligase
  • various terminal nucleotidyl transferases can be used in the methods.
  • the terminal nucleotidyl transferase is a terminal deoxynucleotidyl transferase (TdT).
  • the terminal nucleotidyl transferase comprises a poly(N) polymerase, including variants thereof.
  • the poly(N) polymerase comprises a poly(U) polymerase (see, e.g., Kwak et al., RNA.2007 Jun; 13(6): 860–867), a poly(A) polymerase, and poly(G) polymerase.
  • the terminal nucleotidyl transferase comprises polymerase theta ( ⁇ ).
  • Various forms of polymerase ⁇ are described in U.S. Patent No.11390856; International patent publication WO2018175436; U.S.
  • arrays and Kits [0806]
  • arrays comprising the recombinant single-stranded ligases, for example, for screening of nucleotide acceptors and nucleotide donors.
  • a kit comprising at least one recombinant single-stranded RNA ligase.
  • the kit further comprises one or more of a buffer, a nucleotide cofactor (e.g., ATP), and/or one or more nucleotide donors and/or oligonucleotide acceptors.
  • a nucleotide cofactor e.g., ATP
  • the kit further comprises an additive or ligation enhancing agent, including but not limited to, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000).
  • coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FPLC (fast protein liquid chromatography); ddH 2 O (double distilled water); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl ⁇ -D-1- thiogalactopyranoside); FIOPC or FIOP (fold improvements over positive control or parent); LB (Luria- Bertani); TB (Terrific-Broth).
  • CGSC Coli Genetic Stock Center
  • HTP high throughput
  • HPLC high pressure liquid chromatography
  • FPLC fast protein liquid chromatography
  • ddH 2 O double distilled water
  • PBS phosphate buffered saline
  • a single colony from each culture was Docket Number CX10-269WO4 transferred to 5 mL of LB broth with 1% glucose and 30 ⁇ g/mL chloramphenicol.
  • the cultures were grown for 20 h at 30 °C, 250 rpm, and subcultured at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 ⁇ g/mL of chloramphenicol, to a final OD600 of about 0.05.
  • the cultures were incubated for approximately 195 min at 30 °C, 250 rpm, to an OD600 of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM.
  • the induced cultures were incubated for 20 h at 30 °C, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M ⁇ 110L). The crude lysate was pelleted by centrifugation (11,000 rpm for 60 min at 4 °C), and the supernatant was then filtered through a 0.2 ⁇ m PES membrane to further clarify the lysate.
  • a Microfluidizer cell disruptor Microfluidizer cell disruptor
  • the cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-110L).
  • the crude lysate was pelleted by centrifugation (10,000 rpm for 90 min at 4 °C) in an Avanti J-series centrifuge and JLA-16.250 fixed rotor (Beckman Coulter).
  • the supernatant was then filtered through a 0.2 ⁇ m PES membrane to further clarify the lysate.
  • the clarified lysates were then supplemented with 20 mM imidazole and 500 mM NaCl.
  • ssRNA ligase lysates were supplemented with 1/10th volume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v/v Triton X-100 reagent) per well. Lysates were then purified using an AKTA Pure purification system and a 5 mL HisTrap FF column (GE Healthcare) using run parameters provided below.
  • the SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v/v Triton X-100 reagent.
  • Example 2 Capillary Electrophoresis (CE) Analysis of Oligonucleotides Sample preparation for reaction analysis using CE [0814] For analysis of the reaction samples, capillary electrophoresis was performed using an ABI 3500xl Genetic Analyzer (ThermoFisher). Reactions (1 ⁇ L) were quenched by the addition of 99 ⁇ L of 1 mM aqueous EDTA.
  • Quenched reactions were diluted in water to 1.25 nM FAM-labeled oligonucleotide, and a 2- ⁇ L aliquot of this solution was transferred to a new 96-well MicroAmp Optical PCR plate or 384-well MicroAmp Optical PCR plate containing 18 ⁇ L Hi-DiTM Formamide (ThermoFisher) containing an appropriate size standard (LIZ or Alexa633).
  • the ABI3500xl was configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre-run settings were 18KV for 50 sec. Injection was 10KV for 2 sec, and the run settings were 19KV for 620 sec.
  • ssRNA ligases [0815] Synthetic genes encoding an N-terminal 6-histidine tagged version of multiple wild-type (WT) ssRNA ligase enzymes were cloned into the pCK110900 vector system (See e.g., US Pat. No.9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in an E. coli strain derived from W3110.
  • Cells transformed with the ssRNA ligase expression constructs were grown at shake-flask scale as described in Example 1. Cells were then lysed, purified, and dialyzed into storage buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol). After overnight dialysis, protein samples were removed, and enzyme concentrations were measured by absorption at 280 nm using a NanoDropTM 1000 spectrophotometer.
  • Soluble protein concentrations are summarized in Table 3.1 below, showing a fold improvement in soluble protein production following shake-flask purification relative to the enzyme isolated from Escherichia phage vB_EcoM_VR25 (SEQ ID NO: 2).
  • SEQ ID NO: 2 Source organism of Relative Soluble Enzyme Production (nt/ ) RNA li n n (R l tiv t SEQ ID NO: 2) xamp e Activity of SEQ ID NO: 2-8 in a Ligation with a Methoxy-modified RNA donor Activity of shake-flask purified ssRNA ligases [0817] ssRNA ligase variants SEQ ID NO: 2, 4, 6, and 8 were produced in shake flask and purified as described in Example 1.
  • Reactions were performed in 96-well format 200 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 100 ⁇ M oligonucleotide donor, 1 mM ATP, 10 mM DTT, 25 vol % ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 50 mM TRIS-HCl (pH 7.5), and 10 mM MgCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Reactions included 100 ⁇ M oligonucleotide acceptor, 200 ⁇ M oligonucleotide donor, 1 mM ATP, 25 vol % ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Table 5.1 Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 5.2.
  • Reactions included 50 ⁇ M oligonucleotide acceptor, 100 ⁇ M oligonucleotide donor, 200 ⁇ M ATP, 25 vol % ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 1 mM CoCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • the structure of /5Phos/rC/3p-alkyne-1/ is below: Docket Number CX10-269WO4 [0826] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product(s) divided by the sum of the total peak area.
  • Table 6.2 SEQ ID NO: ssRNA ligase % conversion with % conversion with % conversion with A 0, xamp e Activity of SEQ ID NO: 6 with ATP recycling system
  • ssRNA ligase variant SEQ ID NO: 6 was produced in shake flask and purified as described in Example 1. A reaction with sub-stoichiometric ATP and an enzymatic ATP-recycle using adenylate kinase and acetate kinase enzymes to convert ligase reaction product AMP to ATP was compared to a reaction with excess ATP. The effect of each reaction condition on the generation of reaction by-products was compared.
  • Reactions were performed in 96-well format 200 ⁇ L BioRad PCR plates.
  • Reaction 1 with excess ATP Reactions included 100 ⁇ M oligonucleotide acceptor, 200 ⁇ M oligonucleotide donor, 250 ⁇ M ATP, 6.2 ⁇ M ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 10 mM MgCl2.
  • Reaction 2 with recycle enzymes Reactions included 100 ⁇ M oligonucleotide acceptor, 200 ⁇ M oligonucleotide donor, 0.005 ⁇ M ATP, 10 mM lithium acetyl phosphate, 6.2 ⁇ M ssRNA ligase solution, 1 uM acetate kinase (SEQ ID NO: 24), 1 uM adenylate kinase (SEQ ID NO: 26), 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 10 mM MgCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for LCMS analysis as described in Example 2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) heat-treated ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 6.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Table 8.1 Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product(s) divided by the sum of the total peak area. The results are shown in Table 8.2.
  • coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 ⁇ g/mL chloramphenicol. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom NUNCTM (Thermo-Scientific) plates filled with 180 ⁇ l/well LB medium supplemented with 1% glucose and 30 ⁇ g/mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner).
  • NUNCTM Thermo-Scientific
  • Example 10 Improvements over SEQ ID NO: 32 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0835] The ssRNA ligase of SEQ ID NO: 32 was selected as the parent Ligase enzyme.
  • Reactions included 50 ⁇ M oligonucleotide acceptor, 200 ⁇ M oligonucleotide donor, 0.5 mM ATP, 25 vol % ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 10.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 32 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 32 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.2.
  • SEQ ID NO: 44 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1. [0839] Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 50 ⁇ M oligonucleotide acceptor, 100 ⁇ M oligonucleotide donor, 0.5 mM ATP, 25 vol % ssRNA ligase solution, 1 ⁇ M IPP (SEQ ID NO: 22), 200 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 11.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 44 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 44 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 12.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 150 ⁇ M oligonucleotide donor, 0.5 mM ATP, 25 ⁇ M ssRNA ligase, 0.2 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl 2 and 1 mM CoCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Reactions included 100 ⁇ M oligonucleotide acceptor, 150 ⁇ M oligonucleotide donor, 0.5 mM ATP, 6.25 ⁇ M ssRNA ligase, 0.2 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl 2 and 1 mM CoCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Table 14.1 Reaction temperature - 50 °C; Reaction Time (min)- 180 min; Reaction volume ( ⁇ L)- 1; ssRNA ligase s [0849] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 14.2.
  • Table 14.2- ssRNA ligase activity improvements a) Docket Number CX10-269WO4 Table 14.2- ssRNA ligase activity improvements Acceptor Donor product 13/14 31/32 43/44 SEQ ID NO: 217 /5Phos/mAmUmA SEQ ID NO: 226 ++ ++ +++ p Activity of SEQ ID NOs: 14, 32, and 44 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule Activity of shake-flask purified ssRNA ligases [0850] The ssRNA ligase variants SEQ ID NOs: 14, 32, and 44 were produced in shake flask and purified as described in Example 1.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 200 ⁇ M oligonucleotide acceptor, 300 ⁇ M oligonucleotide donor, 0.5 mM ATP, 5 ⁇ M ssRNA ligase, 0.2 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2 and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Table 15.2- ssRNA ligase activity improvements a) Docket Number CX10-269WO4 Table 15.2- ssRNA ligase activity improvements Reaction components % conversion by SEQ ID NO: (nt/aa) Acceptor Donor product 13/14 31/32 43/44 p Activity of SEQ ID NOs: 14, 32, 44, and 140 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated Donor molecule Activity of shake-flask purified ssRNA ligases [0853] ssRNA ligase variants SEQ ID NOs: 14, 32, 44, and 140 were produced in shake flask and purified as described in Example 1.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 150 ⁇ M oligonucleotide donor, 0.5 mM ATP, 25 ⁇ M ssRNA ligase, 0.2 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl 2 and 1 mM CoCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Table 16.2- ssRNA ligase activity improvements a) 0 Docket Number CX10-269WO4 Table 16.2- ssRNA ligase activity improvements /5Phos/mUmAmU /52FA//i2FA//i2FA/m 2FA i2FA 2FA A ATTTTTTTT A A ATTT Improvements over SEQ ID NO: 140 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0856] The ssRNA ligase of SEQ ID NO: 140 was selected as the parent ligase enzyme.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 17.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • ssRNA Ligase activity relative to SEQ ID NO: 140
  • ssRNA Ligase activity relative to SEQ ID NO: 140 349/350 S310G/K314L + 351/352 K214E/K347I/R358D + d
  • the ssRNA ligase of SEQ ID NO: 246 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 18.1. [0860] Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl 2 , and 1 mM CoCl 2 .
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 18.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the Docket Number CX10-269WO4 indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • ssRNA Ligase activity relative to SEQ ID NO: 246 459/460 N303A + 461/462 S125T + Example 19 Improvements over SEQ ID NO: 400 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0862]
  • the ssRNA ligase of SEQ ID NO: 400 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 19.1.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 19.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • ssRNA Ligase activity relative to SEQ ID NO: 400
  • SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 20 Improvements over SEQ ID NO: 492 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants
  • the ssRNA ligase of SEQ ID NO: 492 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 20.1.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a Docket Number CX10-269WO4 single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 20.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • ssRNA Ligase activity relative to SEQ ID NO: 492 SEQ ID NO: Amino Acid Differences FIOP activity relative to Docket Number CX10-269WO4 Table 20.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 492 565/566 F229A + 567/568 S225P + p Improvements over SEQ ID NO: 520 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0868]
  • the ssRNA ligase of SEQ ID NO: 520 was selected as the parent Ligase enzyme.
  • Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 21.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 520 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 520 (where the percent product may be set as Docket Number CX10-269WO4 the average of replicates or else the highest single sample as appropriate). The results are shown in Table 21.2. Table 21.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 520 SEQ ID NO: Amino Acid Differences FIOP activity relative to SEQ (nt/aa) (Relative to SEQ ID NO: 520) ID NO: 520 xamp e Improvements over SEQ ID NO: 594 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0871] The ssRNA ligase of SEQ ID NO: 594 was selected as the parent Ligase enzyme.
  • Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 22.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes.
  • Post- ligation reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates.
  • the reaction plates are heat sealed, vortexed and centrifuged.
  • the reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes.
  • ssRNA Ligase activity relative to SEQ ID NO: 594
  • SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 23 Improvements over SEQ ID NO: 594 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants
  • the ssRNA ligase of SEQ ID NO: 594 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis Docket Number CX10-269WO4 and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 23.1. [0875] Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 23.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 594 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 594 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 23.2. Table 23.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 594 SEQ ID NO: Amino Acid Differences FIOP activity relative Docket Number CX10-269WO4 Table 23.2.
  • the ssRNA ligase of SEQ ID NO: 634 was selected as the parent Ligase enzyme.
  • Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 24.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 634 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 634 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 24.2. Table 24.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 634
  • SEQ ID NO: Amino Acid Differences FIOP activity relative (nt/aa) (Relative to SEQ ID NO: 634) to SEQ ID NO: 634
  • the ssRNA ligase of SEQ ID NO: 710 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 25.1. [0881] Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 25.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the Docket Number CX10-269WO4 indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes. Post- ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment.
  • This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates.
  • the reaction plates are heat sealed, vortexed and centrifuged.
  • the reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 710 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 710 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 25.2. Table 25.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 710
  • SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 26 Improvements over SEQ ID NO: 738 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants
  • the ssRNA ligase of SEQ ID NO: 738 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis Docket Number CX10-269WO4 and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 26.1.
  • Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 100 ⁇ M oligonucleotide acceptor, 200-300 ⁇ M oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 26.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes. Post- ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment.
  • This treatment involves the addition of 10 – 20 uM of SEQ ID NO: 914 to the reaction plates.
  • the reaction plates are heat sealed, vortexed and centrifuged.
  • the reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 738 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 738 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 26.2. Table 26.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 738
  • SEQ ID NO: Amino Acid Differences FIOP activit relative to Example 27 Docket Number CX10-269WO4 Improvements over SEQ ID NO: 756 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants
  • the ssRNA ligase of SEQ ID NO: 756 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations).
  • the polypeptides encoded by each gene were produced in HTP and prepared as described in Table 27.1. [0887] Reactions were performed in 384-well format 50 ⁇ L BioRad PCR plates. Reactions included 1000 ⁇ M oligonucleotide acceptor, 1200 ⁇ M oligonucleotide donor, 2 mM ATP, ssRNA ligase heat-treated lysate, 1 ⁇ M IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 27.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 756 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 756 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 27.2. Table 27.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 756 SEQ ID NO: Amin A id Diff r n FIOP tivit r l tiv to Docket Number CX10-269WO4 Table 27.2.
  • the ssRNA ligase of SEQ ID NO: 768 was selected as the parent Ligase enzyme.
  • the reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction.
  • the substrates and reaction conditions are summarized in Table 28.1.
  • the reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2.
  • Activity relative to SEQ ID NO: 768 was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 768 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 28.2. Table 28.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 768 SEQ ID NO: Amino Acid Differences FIOP %product relative to Docket Number CX10-269WO4 Table 28.2.
  • ssRNA Ligase activity relative to SEQ ID NO: 768 847/848 Y44R/G272L/D280S ++ 849/850 Y44R/G272L/D279R ++
  • the ssRNA ligase of SEQ ID NO: 844 was selected as the parent Ligase enzyme.

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Abstract

The present disclosure relates to methods of using single-stranded RNA ligase for synthesis of oligonucleotides, including oligonucleotides containing a conjugate moiety. In some embodiments, the present disclosure further provides recombinant single-stranded RNA ligases, polynucleotides encoding the recombinant single-stranded RNA ligases, and compositions of the single-stranded RNA ligases.

Description

Docket Number CX10-269WO4 RNA LIGASE MEDIATED OLIGONUCLEOTIDE SYNTHESIS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No.63/634,859, filed April 16, 2024; U.S. Provisional Application No.63/646,841, filed May 13, 2024; and U.S. Provisional Application No. 63/718,884, filed November 11, 2024; the contents of all of which are incorporated by reference herein. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM [0002] The Sequence Listing concurrently submitted herewith as file name CX10-269WO4_ST26.xml, created on April 16, 2025, with a file size of 3,694,688 bytes, is part of the specification and is incorporated by reference herein. BACKGROUND [0003] Oligonucleotide therapeutics, either as synthetic single-stranded or double stranded polynucleotides, represent a class of drugs that exert their biological effects by modulating gene expression. The oligonucleotides are designed primarily to target pre-mRNA, mRNA, or non-coding RNA (e.g., miRNA) to promote RNA degradation, modulate splicing, interrupt translation, or in some instances activate expression. Small interfering RNAs (siRNA) are a class of oligonucleotide therapeutics that are typically double stranded oligonucleotides that act via RISC (RNA-induced silencing complex) pathway, where the strand complementary to an RNA target, also referred to as the guide strand, targets the RNA for degradation or translation inhibition. Antisense oligonucleotides (ASOs) are single-stranded and designed to bind sequence specifically to a target RNA and modulate protein expression via several different mechanisms. Targets of ASOs include pre-mRNA, mRNA, and non-coding RNA. [0004] siRNA and ASO oligonucleotides are designed to include various modifications to improve in vivo stability, cellular delivery, specificity, and efficacy. Sugar modifications include 2’-O-methyl, 2’-O-ethyl, 2’- O-methoxyethyl, 2’-fluoro, and locked nucleic acid (LNA); modification of internucleoside linkages include phosphorothioate and phosphoramidate morpholino linkages; and nucleobase modifications include 5’- methylcytosine and G-clamp. In addition, conjugating targeting moieties to the oligonucleotide, such as GalNac and lipid groups, can enhance delivery to cells and tissues. [0005] Modified oligonucleotides are generally synthesized chemically by solid-phase synthesis using phosphoramidite chemistry. Disadvantages of chemical synthesis include low efficiency in synthesis of long oligonucleotides, difficulty in scale-up of manufacturing, and toxic chemical waste and solvent consumption, e.g., toluene and acetonitrile. Accordingly, desirable are alternative approaches to chemical synthesis. SUMMARY [0006] The present disclosure provides single-stranded RNA ligase polypeptides and compositions thereof, and methods of using the single-stranded RNA ligases for synthesis of oligonucleotides, including modified oligonucleotides with conjugate moieties. Docket Number CX10-269WO4 [0007] In some embodiments, a method of extending an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor. [0008] In some embodiments, the nucleotide donor, the oligonucleotide(A), or both the nucleotide donor and the oligonucleotide(A) comprise a modified nucleotide. [0009] In some embodiments, the oligonucleotide(A) comprises at least one modified nucleoside, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker. [0010] In some embodiments, the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. [0011] In some embodiments, the reactive group comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. [0012] In some embodiments, the modified nucleoside on the oligonucleotide(A) is at the 5’-terminal nucleotide, an internal nucleotide, or the 3’-terminal nucleotide. [0013] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). [0014] In some embodiments, the conjugate moiety or reactive group is attached to the nucleoside via a linker. In some embodiments, the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). In some embodiments, the linker comprises a cleavable linker. [0015] In some embodiments, the oligonucleotide(A) comprises one or more terminal groups. In some embodiments, the terminal group is at the 5’-terminal nucleotide of the oligonucleotide(A). In some embodiments, the terminal group is a 5’-phosphonate (E- or Z-vinylphosphonate), 4’-amino, 4’-aminoalkyl, abasic nucleotide, or inverted abasic nucleotide. In some embodiments, the oligonucleotide(A) comprises a 5’- OH or a 5’-blocking group that inhibits ligation by the single-stranded RNA ligase. [0016] In some embodiments, the oligonucleotide(A) comprises the formula (I): A1[•A2]m•A3-OH (I) wherein each of A1, A2 and A3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage. [0017] In some embodiments, each of A2 is the same or different nucleoside. In some embodiments, the oligonucleotide(A) comprises a modified nucleoside of at least one or more of A1, A2, or A3. In some embodiments, at least one of A1, A2, or A3 is modified with a conjugate moiety or conjugate reactive group. Docket Number CX10-269WO4 [0018] In some embodiments, one or more of the internucleoside linkage is a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phosphorothioate or a phosphorodithioate. [0019] In some embodiments, the modified nucleoside on the oligonucleotide(A) comprises the formula (II): A-[L]g-[M]h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0. [0020] In some embodiments, the oligonucleotide(A) is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length. [0021] In some embodiments, wherein when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside. In some embodiments, wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. [0022] In some embodiments, the nucleotide donor comprises a nucleotide(D) or an oligonucleotide(D). In some embodiments the nucleotide(D) or oligonucleotide(D) comprises a modified nucleoside. In some embodiments, the modified nucleoside of nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or linker. [0023] In some embodiments, the conjugate moiety on the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. [0024] In some embodiments, reactive group on the nucleotide donor comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. [0025] In some embodiments, the modified nucleoside on the oligonucleotide(D) is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. [0026] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the modified nucleoside. [0027] In some embodiments, the conjugate moiety or the conjugate reactive group is attached to the nucleoside via a linker L. In some embodiments, the linker comprises a cleavable linker. [0028] In some embodiments, the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) Docket Number CX10-269WO4 wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120. [0029] In some embodiments, the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length. [0030] In some embodiments, each of D2 is the same or different nucleoside. In some embodiments, wherein the nucleotide donor comprises a modified nucleoside, at least one or more of D1, D2, or D3 is modified. In some embodiments, at least one of D1, D2, or D3 is modified with a conjugate moiety, reactive group, or linker. [0031] In some embodiments, D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or linker. [0032] In some embodiments, the modified nucleoside on nucleotide(D) or oligonucleotide(D) has the formula (IV): D-[L]q-[M]r (IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0. [0033] In some embodiments, wherein when q is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside. In some embodiments, wherein when q is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. [0034] In some embodiments, the nucleotide(D) or pD includes a 3’-phosphate (e.g., pDp). [0035] In some embodiments, the nucleotide donor (i.e., nucleotide(D) or oligonucleotide(D)) comprises a 3’- blocking group that inhibits ligation by the single-stranded RNA ligase to a 3’-OH group or inhibits incorporation of a nucleotide onto an oligonucleotide by a terminal nucleotidyl transferase. In some embodiments, the reaction with the single-stranded RNA ligase and a 3’-blocked nucleotide(D) or 3’-blocked oligonucleotide(D) results in a 3’-blocked extended oligonucleotide product. In some embodiments, the 3’- blocking group comprises a reversible blocking group. [0036] In some embodiments, wherein the product is a 3’-blocked extended oligonucleotide, the 3’-blocked extended oligonucleotide is separated or removed from the single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase is inactivated. Docket Number CX10-269WO4 [0037] In some embodiments, where the 3’-blocking group is a reversible blocking group, the method further comprises removing or cleaving the 3’-blocking group on 3’-blocked extended oligonucleotide with a deblocking agent to form an unblocked extended oligonucleotide. [0038] In some embodiments, the method further comprises inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent. [0039] In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase. [0040] In some embodiments, the method further comprises one or more cycles of extension with a nucleotide donor; separation of 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivation of the single-stranded RNA ligase; removing or cleaving the reversible 3’-blocking group with a deblocking agent; and separating the unblocked extended oligonucleotide, wherein each cycle uses a new nucleotide donor. [0041] In some embodiments, the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors, such as a mixture of different pD or pDp. [0042] In some embodiments, the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor (e.g., nucleotide(D) or oligonucleotide(D)) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence. In particular, the nucleotide(D) comprises a selected or predetermined nucleotide(D) to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence. [0043] In some embodiments of the method, the single-stranded RNA ligase comprises RNA ligase 1. In some embodiments, the single-stranded RNA ligase comprises a recombinant single-stranded RNA ligase described herein. [0044] In some embodiments of the method, the single-stranded RNA ligase is immobilized on a support medium. In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) and nucleotide donor are provided in solution or aqueous phase. [0045] In some embodiments of the method, the oligonucleotide acceptor (oligonucleotide(A)) is immobilized is attached to a support medium. In some embodiments, the single-stranded RNA ligase and the nucleotide donor are provided in solution or aqueous phase. [0046] In some embodiments of the method, the reaction further comprises a pyrophosphatase for cleaving of pyrophosphate product. [0047] In some embodiments of the method, the reaction further comprises an ATP recycling system. [0048] In some embodiments of the method, the suitable reaction conditions comprises one or more of an NTP, a divalent metal ion, and buffer. [0049] In some embodiments of the method, the suitable reaction conditions comprises a reaction temperature of 5-60 ℃. Docket Number CX10-269WO4 [0050] In some embodiments of the method, the suitable reaction conditions comprise a reaction pH of about 5-8. [0051] In another aspect, the present disclosure provides a recombinant single-stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. [0052] In some embodiments, the amino acid sequence of the recombinant single-stranded RNA ligase comprises the sequence comprising amino acid residue 12 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. [0053] In some embodiments, the single-stranded RNA ligase is the single-stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1. [0054] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0055] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. Docket Number CX10-269WO4 [0056] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0057] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0058] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0059] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0060] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. Docket Number CX10-269WO4 [0061] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0062] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0063] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0064] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. Docket Number CX10-269WO4 [0065] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0066] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0067] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0068] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0069] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0070] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237/323, 151/205/314, 151/212/345/347, 92/214/347/358, 162/204/205/310/314/358, 236/314, 151/345/347, 214/347/358, 151/212/358, 204/283/314/358, 236/237/358/362, 151/199/204/231/236/323, 231/236/237/358/362, 162/314/358, 92/151/347/358, 151/236, 212/345/347, 115/314, 345/347/358, 314, 358/362, 115/358, 310/314, 214/347/358, 237/314, 345/347, 151/310/323/343/347, 151/358, 347/358, 93/358/362, 231/236/237/320/358/362, 151/230/345/347/358, 135/231/236/237/358, 310/314/358/362, 151/230/347/358, 231/237/323, 135/358/362, 283/314/358/362, 199/205, 92/151/230/345/347/358, 314/358/362, 199/237, or 199/204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0071] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0072] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0073] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0074] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 254, 240, 118, 347, 167, 281, 303, 205, or 50, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. Docket Number CX10-269WO4 [0075] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 49/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0076] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0077] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0078] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0079] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0080] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/279/280/302, 246/280, 272/276/279/280/302, 272/280, 272/280/302, 276/279/280/302, 276/280, 279/280/302, or 280, wherein the amino acid positions are Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0081] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0082] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/359/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0083] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0084] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0085] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177/254/359, 115/177/359, 115/254, 115/254/350/354/359, 115/254/359, 115/350, 144/145/260, 144/145/260/341, 144/145/260/341/366/371, 144/260/341/366, 144/260/366/369, 145/260/263, 145/260/341, 145/260/341/366/369, 145/341, 145/341/371, 145/371, 177, 177/254, 177/254/281/359, 177/254/354/359, 177/254/359, 177/359, 254, 254/281/354/359, 254/350, 254/354/359, 254/359, 260/366, 341, or 359, wherein Docket Number CX10-269WO4 the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0086] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0087] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0088] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0089] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0090] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0091] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0092] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference Docket Number CX10-269WO4 sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0093] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0094] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0095] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0096] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0097] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0098] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the Docket Number CX10-269WO4 reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0099] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0100] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526. [0101] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0102] In some embodiments, the recombinant single stranded RNA ligase comprising an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244- 912, and 934-1526. [0103] In some embodiments, the recombinant single stranded RNA ligase exhibits single stranded RNA ligase activity and at least an improved property as compared to a reference single stranded RNA ligase. [0104] In some embodiments, the recombinant single stranded RNA ligase exhibits an improved property selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), as compared to a reference single stranded RNA ligase. Docket Number CX10-269WO4 [0105] In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, or 768. In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or an amino acid sequence corresponding to SEQ ID NO: 14. [0106] In some embodiments, the recombinant single-stranded RNA ligase further comprises a fusion protein. [0107] In some embodiments, the recombinant single stranded RNA ligase is provided as a purified preparation. [0108] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding any of the recombinant single stranded RNA ligase disclosed herein. [0109] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. [0110] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. [0111] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. [0112] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon- optimized for expression of the encoded recombinant single stranded RNA ligase. [0113] In some embodiments, polynucleotide sequence encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933- Docket Number CX10-269WO4 1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523. [0114] In some embodiments, the polynucleotide sequence of the recombinant polynucleotide encoding the single stranded RNA ligase comprises nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or a polynucleotide sequence comprising SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499. [0115] In a further aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding any of the single stranded RNA ligases described herein. In some embodiments, the expression vector comprises a control sequence operably linked to the recombinant polynucleotide. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter. [0116] In a further aspect, the present disclosure provides a host cell comprising an expression vector for expression or production of the recombinant single stranded RNA ligase. In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a bacterial cell, fungal cell, insect cell, or mammalian cell. [0117] In a further aspect, the present disclosure provides a method of producing a recombinant single stranded RNA ligase, the method comprising culturing a host cell described herein under suitable culture conditions such that the encoded recombinant single stranded RNA ligase is produced. In some embodiments, the method further comprises recovering the recombinant single stranded RNA ligase polypeptide from the culture and/or host cell. In some embodiments, the method further comprises purifying the recombinant single stranded RNA ligase polypeptide. [0118] In another aspect, the present disclosure provides a composition comprising a recombinant single stranded RNA ligase. In some embodiments, the composition further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and/or one or more polynucleotide substrates for the single stranded RNA ligase. [0119] In some embodiments, the composition further comprises at least an oligonucleotide acceptor and/or a nucleotide donor (e.g., nucleotide(D) or oligonucleotide(D)). In some embodiments, the oligonucleotide donor comprises a modified oligonucleotide donor. In some embodiments, the nucleotide donor nucleotide(D) comprises a modified nucleotide(D). In some embodiments, the nucleotide donor oligonucleotide(D) comprises a modified oligonucleotide(D). [0120] In another aspect, the present disclosure further provides a kit comprising a recombinant single stranded RNA ligase described herein. [0121] In some embodiments, the kit further comprises one or more of a buffer, nucleotide cofactor, divalent metal, ligation enhancer, and/or one or more polynucleotide substrates for the single stranded RNA ligase. DETAILED DESCRIPTION Docket Number CX10-269WO4 [0122] The present disclosure provides recombinant single-stranded RNA ligases and methods of synthesizing oligonucleotide using the recombinant single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligases are used to produce modified oligonucleotide, particularly modified oligonucleotides having a conjugate moiety or a reactive group. Abbreviations and Definitions [0123] In reference to the present disclosure, the technical and scientific terms used in the descriptions herein will have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings. [0124] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a polypeptide” includes more than one polypeptide. [0125] Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Thus, as used herein, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates). [0126] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” [0127] “About” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value. [0128] “EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze. [0129] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains. [0130] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein. [0131] “Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). [0132] “Amino acids” and “amino acid” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glycine (Gly or G), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), Docket Number CX10-269WO4 threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about α-carbon (Cα). For example, whereas “Ala” designates alanine without specifying the configuration about the α-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the α-carbon and lower case letters designate amino acids in the D-configuration about the α-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention. [0133] “Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art). [0134] “RNA ligase” refers to enzymes that covalently joins the 5’-phosphoryl termini of RNA or DNA to the 3’-hydroxyl termini of RNA or DNA. Families of known RNA ligases include RNA ligase 1, also referred to as single-stranded RNA ligase or ssRNA ligase, which catalyzes the covalent joining of single-stranded 5’- phosphoryl termini of RNA or DNA to single-stranded 3--hydroxyl termini of RNA or DNA. RNA ligase 2, also referred to as double stranded RNA ligase or dsRNA ligase, also catalyzes the covalent joining of a 3’- hydroxyl terminus of RNA to a 5’-phosphorylated RNA or DNA but shows preference for double stranded substrates. In some embodiments, RNA ligases include those enzymes classified in EC 6.5.1.3. It is to be understood that the ligation reaction is not limited to naturally occurring RNA and DNA substrates also includes nucleotide substrates that contain modified nucleotides and/or nucleotide analogs. [0135] “Polynucleotide,” “nucleic acid,” or “oligonucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’-deoxyribonucleosides on an individual basis or on an aggregate basis. When a polynucleotide, nucleic acid, or oligonucleotide sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid. The polynucleotide or nucleic acid may be single-stranded or double-stranded, or may include both single-stranded regions and double-stranded regions. [0136] In some embodiments, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” encompass polynucleotide or nucleic acid or oligonucleotide analogs or modified polynucleotide or nucleic acid or oligonucleotide, which include, among others, nucleosides linked together via other than standard phosphodiester linkages, such as non-standard linkages of phosphoramidates, phosphorothioates, amide Docket Number CX10-269WO4 linkages, etc.; nucleosides with modified and/or synthetic nucleobases, for example inosine, xanthine, hypoxanthine, etc.; nucleosides with modified sugar residues, such as 2’-O-alkyl, 2’-halo, 2,3-dideoxy, 2’- halo-2’-deoxy, β-D-ribo LNA, α-L-ribo-LNA (e.g., locked nucleic acids), etc.; and/or 5’-phosphate analogs, including, among others, phosphorothioate, phosphoacetate, phosphoramidate, monomethylphosphate, methylphosphonate, or phosphonocarboxylate. [0137] “Nucleobase” refers to means an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T). cytosine (C). uracil (U). or guanine (G). A “modified nucleobase” refers to a group of atoms other than unmodified A, T, C, U. or G capable of pairing with at least one unmodified nucleobase. [0138] “Nucleoside” refers to a compound comprising a nucleobase and a sugar moiety. The nucleobases and sugar moiety are each, independently, unmodified or modified. [0139] “Internucleoside linkage” refers to as a linkage that covalently couples two nucleosides together. In the oligonucleotides herein, internucleoside linkages covalently couple adjacent nucleosides together, typically forming a bond between the sugar moieties of the adjacent nucleosides. Non-limiting examples of internucleoside linkages include phosphodiester -O-P(O)2-O- linkages and modified internucleoside linkages, such as phosphorothioate -O-P-(O, S)-O- and phosphorodithioate -O-P(S)2-O-. [0140] “Modified oligonucleotide” refers to an oligonucleotide which contains at least one modified internucleoside linkage and/or a modified nucleoside, or a modified terminal group. [0141] “Modified nucleotide” refers to a nucleotide (e.g., NMP, NDP, NTP) in which at least one of the phosphate is a modified phosphate group and/or a modified nucleoside. [0142] “Modified nucleoside” or “nucleoside modification” refers to a nucleoside modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the nucleobase. The modified nucleoside comprises a modified nucleobase and/or a modified sugar residue. The term “modified nucleoside” may also be used herein interchangeably with the term “nucleoside analogue.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein. Nucleosides with modifications in the nucleobase of the DNA or RNA nucleoside are still generally termed DNA or RNA if they allow Watson-Crick base pairing. [0143] “Modified internucleoside linkage” refers to as a linkage other than a phosphodiester (PO) linkage that covalently connects two nucleosides together. In some embodiments, exemplary modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. Other modified phosphorus-containing internucleoside linkages include phosphotriesters, methylphosphonates, and phosphoramidates (P-NH2). See, e.g., Clave et al., RSC Chem Biol., 20212(1): 94–150). In some embodiments, the modified internucleoside linkage is a non-phosphorus containing internucleoside linkage, including but not limited tomethylenemethylimino (-CH2-N(CH3)-O-CH2), thiodiestcr, thionocarbamate (-O- C(=O)(NH)-S-); siloxane (-O-SiH2-O-); N,N’-dimethylhydrazine (-CH2-N((CH3)-N((CH3)-); MMI (3'-CH2- N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O- Docket Number CX10-269WO4 5'), methoxypropyl, and thioformacetal (3’-S-CH2-O-5'). In some embodiments, internucleoside linkages having a chiral atom can be prepared as a mixture of the stereoisomers, or as separate stereoisomers. [0144] “Phosphorothioate internucleoside linkage” refers to an internucleoside linkage in which one of the oxygen atom in a phosphodiester linkage is replaced with a sulfur atom. In some embodiments, a phosphorothioate linkage may be represented as -O-P(O,S)-O-, wherein one of the non-bridging oxygen atoms is replaced with a sulfur atom. Phosphorothioate internucleoside linkages are chiral (see, for example, Jahns et al.2022, Nucleic Acids Research Vol.50, No 3, 1221-1240), with right-handed (Rp) and left-handed (Sp) isomers. In some embodiments, the Rp diastereomer may be referred to as an R-PS internucleoside linkage or an srP internucleoside linkage. The Sp diastereomer may be referred to as an S-PS internucleoside linkage or ssP internucleoside linkage. In some embodiments, the oligonucleotide comprises one or more srP internucleoside linkages. In some embodiments, the oligonucleotide comprises one or more ssP internucleoside linkages. Where the chirality of a phosphorothioate internucleoside linkage is not specified, that phosphorothioate internucleoside linkage may be either an srP linkage or an ssP linkage. [0145] “Non-bridging phosphorothioate internucleoside linkage” refers to a phosphorothioate internucleoside linkage in which the sulfur atom attached to the phosphorous atom is in place of a non-bridging oxygen atom. [0146] “Non-bridging phosphorodithioate internucleoside linkage” refers to a modified internucleoside linkage which is a non-bridging phosphorodithioate internucleoside linkage. A non-bridging phosphorodithioate internucleoside linkage has two identical sulfur atoms attached to the phosphorous atom, achieved by replacing the non-bridging oxygen atom in the phosphorothioate linkage with a sulfur atom. [0147] “Abasic sugar moiety” refers to a sugar moiety of a nucleoside that is not attached to a nucleobase. In some embodiments, such abasic sugar moieties are referred to as “abasic nucleoside.” [0148] “Inverted nucleoside” refers to a nucleotide having a 3’ to 3’ and/or 5’ to 5’ internucleoside linkage. Similarly, and “inverted sugar moiety” refers to the sugar moiety of an inverted nucleoside or an abasic sugar moiety having a 3’ to 3’ and/or 5’ to 5’ internucleoside linkage. [0149] “LNA nucleoside” or “locked nucleoside” refers to 2'-modified nucleoside which comprises a biradical linking the C2' and C4' of the ribose sugar ring of said nucleoside (also referred to as a "2'- 4' bridge"), which restricts or locks the conformation of the ribose ring. These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature. The locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex. [0150] Non-limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO 00/66604, WO 98/039352, WO 2004/046160, WO 00/047599, WO 2007/134181, WO 2010/077578, WO 2010/036698, WO 2007/090071, WO 2009/006478, WO 2011/156202, WO 2008/154401, WO 2009/067647, WO 2008/150729, Morita et al., Bioorganic & Med. Chem. Lett.2002, 12, 73-76, Seth et al. J. Org. Chem.2010, Vol 75(5) pp. 1569-81, and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667. Docket Number CX10-269WO4 [0151] “Terminal group” as used herein refers to a group located at the first or last nucleoside in a polynucleotide or oligonucleotide. A 5’-terminal group refers to the terminal group bonded to 5′-or 4’-carbon atom of the first nucleoside within a polynucleotide. A 3’-terminal group is a terminal group bonded to 3′- carbon atom of the last nucleoside within a polynucleotide or oligonucleotide. [0152] “5’-blocking group” as used herein refers to a moiety or chemical group that prevents or inhibits attachment of another nucleoside, nucleotide or oligonucleotide to the 5’-terminal nucleoside. In context enzymes active on the 5’-terminal nucleoside, a 5’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide or oligonucleotide to the to the 5’-terminal nucleoside, particularly the 5’-OH of the 5’-terminal nucleoside. [0153] “3’-blocking group” refers to moiety or chemical group that prevents or inhibits attachment off another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside. In context of single-stranded RNA ligase or other enzymes active on the 3’-terminal nucleoside, a 3’-blocking group prevents or inhibits the enzyme(s) from attachment of another nucleoside, nucleotide, or oligonucleotide to the 3’-terminal nucleoside, particularly the 3’-OH of the 3’-terminal nucleoside. [0154] “Reversible blocking group” refers to a blocking group that can be removed or cleaved off to provide a free 3’-OH. In some embodiments, the blocking group is removable with a deblocking agent, which can be a chemical or enzymatic deblocking agent. [0155] “Enzymatically reversible blocking group” refers to a blocking group that is susceptible to removal or cleaving by an enzyme. [0156] “Duplex” and “ds” refer to a double-stranded nucleic acid (e.g., DNA or RNA) molecule comprised of two single-stranded polynucleotides that are complementary in their sequence (e.g., A pairs to T or U, C pairs to G), arranged in an antiparallel 5’ to 3’ orientation, and held together by hydrogen bonds between the nucleobases (e.g., adenine [A], guanine [G], cytosine [C], thymine [T], uridine [U]). [0157] “Complementary” is used herein to describe the structural relationship between nucleotide bases that are capable of forming base pairs with one another. For example, a purine nucleotide base present on a polynucleotide that is complementary to a pyrimidine nucleotide base on a polynucleotide may base pair by forming hydrogen bonds with one another. Complementary nucleotide bases can base pair via Watson/Crick base pairing or in any other manner than forms stable duplexes or other nucleic acid structures. [0158] “Watson/Crick Base-Pairing” refers to a pattern of specific pairs of nucleobases and analogs that bind together through sequence-specific hydrogen-bonds, e.g., A pairs with T or U, and G pairs with C. [0159] “Annealing” or “Hybridization” refers to the base-pairing interactions of one nucleobase polymer (e.g., poly- and oligonucleotides) with another that results in the formation of a double-stranded structure, a triplex structure or a quaternary structure. Annealing or hybridization can occur via Watson-Crick base- pairing interactions, but may be mediated by other hydrogen-bonding interactions, such as Hoogsteen base pairing. In some embodiments, the nucleobase polymer that anneals or hybridizes to another is a single nucleobase polymer while in other embodiments, the nucleobase polymers are separate nucleobase polymers. Docket Number CX10-269WO4 [0160] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refer to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques. [0161] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation. [0162] “Coding sequence” and synonymously “encoding” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein. [0163] “Percent (%) sequence identity” refers to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length “W” in the query sequence, which either match or satisfy some positive-valued threshold score “T,” when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as Docket Number CX10-269WO4 the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided. [0164] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence. [0165] “Comparison window” refers to a conceptual segment of contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence. In some embodiments, the comparison window is at least 15 to 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In some embodiments, the comparison window can be longer than 15-20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows. [0166] “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a recombinant primase, Docket Number CX10-269WO4 can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. In some embodiments, the sequence is tagged (e.g., with a histidine tag). [0167] “Mutation” refers to the alteration of a nucleic acid sequence. In some embodiments, mutations result in changes to the encoded polypeptide sequence (i.e., as compared to the original sequence without the mutation). In some embodiments, the mutation comprises a substitution, such that a different amino acid is produced. In some alternative embodiments, the mutation comprises an addition, such that an amino acid is added (e.g., insertion) to the original polypeptide sequence. In some further embodiments, the mutation comprises a deletion, such that an amino acid is deleted from the original polypeptide sequence. Any number of mutations may be present in a given sequence. [0168] “Amino acid difference” and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. The amino acid positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based. In some instances herein, the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide). In some instances, the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some embodiments, the amino acid difference, e.g., a substitution, is denoted by the abbreviation “nB,” without the identifier for the residue in the reference sequence. In some instances, an amino acid residue difference or substitution may be a deletion and may be denoted by a “-“ where appropriate. In some embodiments, the phrase “an amino acid residue nB” denotes the presence of the amino acid residue in the engineered polypeptide, which may or may not be a substitution in context of a reference polypeptide or amino acid sequence. [0169] In some instances, a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence. In some embodiments, where more than one amino acid can be used in a specific residue position of a polypeptide, the various amino acid residues that can be used are separated by a “/”. The present disclosure includes engineered polypeptide sequences comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions, as well as insertions and deletions of amino acids in the sequence. [0170] “Amino acid substitution set” and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, substitution sets comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to the set of Docket Number CX10-269WO4 amino acid substitutions that is present in any of the variant primase polypeptides listed in any of the Tables in the Examples. In some embodiments, the amino acid sequence comprises at least each of the amino acid substitutions in the referenced substitution set. In the substitution sets, the individual substitutions are separated by a semicolon “;” or slash “/”. [0171] “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively. [0172] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid. [0173] “Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining enzymatic activity and/or retaining the improved properties of an recombinant primase. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. As noted above, deletions are indicated by “-“, and may be present in substitution sets. [0174] “Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide. [0175] “Functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which Docket Number CX10-269WO4 it is being compared (e.g., a full length recombinant primase of the present invention) and that retains substantially all of the activity of the full-length polypeptide. [0176] “Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The recombinant primase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant primase polypeptides provided herein are isolated polypeptides. [0177] “Substantially pure polypeptide” or “purified” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure primase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant primase polypeptides are substantially pure polypeptide compositions. [0178] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the primase are codon optimized for optimal production from the host organism selected for expression. [0179] “Control sequence” refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure. Each control sequence may be native or foreign (e.g., heterologous) to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals. [0180] “Operably linked” or “operatively linked” refers to a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such Docket Number CX10-269WO4 that the control sequence directs or regulates the expression of the polynucleotide of interest, and where appropriate, expression of the encoded polypeptide of interest. [0181] “Promoter” or “promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. [0182] “Suitable reaction conditions” or “suitable conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co- solvents, co-factors, etc.) under which an RNA ligase is capable of attaching a nucleotide donor to a nucleotide acceptor. Exemplary “suitable reaction conditions” are provided herein (see, the Examples). [0183] “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the primase polypeptide on the substrate. [0184] “Culturing” refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid). [0185] “Vector” is a recombinant construct for introducing a polynucleotide of interest into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide or a polypeptide encoded in the polynucleotide. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence. [0186] “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell. [0187] “Produces” refers to the production of proteins and/or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell. [0188] “Heterologous” or “recombinant” refers to the relationship between two or more nucleic acid or polypeptide sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature. [0189] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising a polynucleotide provided herein (e.g., a polynucleotide sequences encoding a recombinant primase). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques, and progeny thereof, as known in the art. Docket Number CX10-269WO4 [0190] “Alkyl” refers to straight or branched chain hydrocarbon groups having the number of carbon atoms designated, for example 1 to 20 carbon atoms (C1-C20), particularly 1 to 12 carbon atoms (C1-C12 or C1-12), and more particularly (C1-C8 or C1-8) carbon atoms. Exemplary “alkyl” includes, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl, and s-pentyl. [0191] “Alkenyl” refers to straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 20 carbon atoms (C2-C20), particularly 2 to 12 carbon atoms (C2-C12 or C2-12), and most particularly 2 to 8 (C2-C8 or C2-8)carbon atoms, having at least one double bond. Exemplary “alkenyl” includes, but are not limited to, vinyl ethenyl, allyl, isopropenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl and 5-hexenyl. [0192] “Alkynyl” refers to a straight or branched chain hydrocarbon having the number of carbon atoms designated, for example 2 to 12 carbon atoms (C2-C12 or C2-12), particularly 2 to 8 carbon atoms (C2-C8 or C2- 8), containing at least one triple bond. Exemplary “alkynyl” includes ethynyl, 1-propynyl, 2-propynyl, 1- butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, 4-hexynyl and 5-hexynyl. [0193] “Alkylene”, “alkenylene” and “alkynylene” refers to a straight or branched chain divalent hydrocarbon radical of the corresponding alkyl, alkenyl, and alkynyl, respectively. The “alkylene”, “alkenylene” and “alkynylene” may be optionally substituted, for example with alkyl, alkyloxy, hydroxyl, carbonyl, carboxyl, halo, nitro, and the like. [0194] “Lower” in reference to substituents refers to a group having between one and six carbon atoms. [0195] “Heteroalkyl,” heteroalkenyl,” and “heteroalkynyl” refers to the corresponding alkyl, alkenyl, and akynyl in which one or more of the carbon atoms is replaced with a heteroatom, such as O, S and N. [0196] “Cycloalkyl” refers to any stable monocyclic or polycyclic system which consists of carbon atoms, any ring of which being saturated. “Cycloalkenyl” refers to any stable monocyclic or polycyclic system which consists of carbon atoms, with at least one ring thereof being partially unsaturated. Examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicycloalkyls and tricycloalkyls (e.g., adamantyl). [0197] “Heterocycloalkyl” or “heterocyclyl” refers to a substituted or unsubstituted 3 to 14 membered, mono- or bicyclic, non-aromatic hydrocarbon, wherein 1 to 3 carbon atoms a (e replaced by a heteroatom. Heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR’-, -PH-, -S(O)-, -S(O)2-, -S(O) NR’-, -S(O)2NR’-, and the like, including combinations thereof, where each R’ is independently hydrogen or lower alkyl. Examples include oxiranyl, oxetanyl, azetidynyl, oxazolyl, thiazolidinyl, thiazolyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyranyl, tetrahydrofuranyl, tetrahydropyranyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, azapanyl, and the like. Docket Number CX10-269WO4 [0198] “Aryl” refers to a six- to fourteen-membered, mono- or bi-carbocyclic ring, wherein the monocyclic ring is aromatic and at least one of the rings in the bicyclic ring is aromatic. Unless stated otherwise, the valency of the group may be located on any atom of any ring within the radical, valency rules permitting. Examples of “aryl” groups include phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, and the like. [0199] “Heteroaryl” refers to an aromatic heterocyclic ring, including both monocyclic and bicyclic ring systems, where at least one carbon atom of one or both of the rings is replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur, or at least two carbon atoms of one or both of the rings are replaced with a heteroatom independently selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl can be a 5 to 6 membered monocyclic, or 7 to 11 membered bicyclic ring systems. Examples of “heteroaryl” groups include pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, quinolyl, and the like. [0200] “Bridged bicyclic” refers to any bicyclic ring system, i.e., carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one bridge. As defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, a bridged bicyclic group has 5 to 12 ring members and 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. Such bridged bicyclic groups include those groups set forth below where each group is attached to the rest of the molecule at any substitutable carbon or nitrogen atom. Unless otherwise specified, a bridged bicyclic group is optionally substituted with one or more substituents as set forth for aliphatic groups. Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally substituted. Exemplary bridged bicyclics include: , [0201] In some embodiments, a locked nucleoside is a bridged bicyclic compound. [0202] “Fused ring” refers a ring system with two or more rings having at least one bond and two atoms in common. A “fused aryl” and a “fused heteroaryl” refer to ring systems having at least one aryl and heteroaryl, respectively, that share at least one bond and two atoms in common with another ring. Docket Number CX10-269WO4 [0203] “Carbonyl” refers to -C(O)-. The carbonyl group may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones. For example, an -C(O)R’, wherein R’ is an alkyl is referred to as an alkylcarbonyl. In some embodiments, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. [0204] “Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine. [0205] “Haloalkyl” refers to an alkyl substituted with 1 or more halogen atoms. Preferably, the alkyl is substituted with 1 to 3 halogen atoms. [0206] “Hydroxy” refers to –OH. [0207] “Oxy” refers to group -O-, which may have various substituents to form different oxy groups, including ethers and esters. In some embodiments, the oxy group is an –OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. [0208] “Acyl” refers to -C(O)R’, where R is hydrogen, or an optionally substituted alkyl, heteroalkyl, cylcoalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl as defined herein. Exemplary acyl groups include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like. [0209] “Alkyloxy” or “alkoxy” refers to –OR’, wherein R’ is an optionally substituted alkyl. [0210] “Aryloxy” refers to –OR’, wherein R’ is an optionally substituted aryl. [0211] “Carboxy” refers to –COO- or COOM, wherein H or a M+ counterion. [0212] “Carbamoyl” refers to -C(O)NR’R’, wherein each R’ is independently selected from H or an optionally substituted alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocylcoalkylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl. [0213] “Cyano” refers to –CN. [0214] “Ester” refers to a group such as -C(=O)OR’, alternatively illustrated as –C(O)OR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocyclolalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. [0215] “Silyl” refers to Si, which may have various substituents, for example –SiR’R’R’, where R’ is as defined in the specification. For example, each R’ is independently selected from alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. As defined herein, any heterocyloalkyl or heteroaryl group present in a silyl group has from 1 to 3 heteroatoms selected independently from O, N, and S. [0216] “Thiol” or “sulfhydryl” refers to –SH. [0217] “Disulfied” refers to -S-S- groups. Docket Number CX10-269WO4 [0218] “Sulfanyl” refers to –SR’, wherein R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. For example, -SR, wherein R is an alkyl is an alkylsulfanyl. [0219] “Sulfonyl” refers to -S(O)2-, which may have various substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones. For example, -S(O)2R’, wherein R’ is an alkyl refers to an alkylsulfonyl. In some embodiments of -S(O)2R’, R’ is selected from an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocyloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. [0220] “Amino” or “amine” refers to the group –NR’R’ or –NR’R’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, heterocycloalkyl, alkyloxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkyloxycarbonyl, sulfanyl, sulfinyl, sulfonyl, and the like. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, and the like. [0221] “Amide” refers to a group such as, -C(=O)NR’R’, wherein each R’ is independently selected from H and an optionally substituted: alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. [0222] “Optional” or “optionally” refers to a described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not. For example, “optionally substituted alkyl” refers to an alkyl group that may or may not be substituted and that the description encompasses both substituted alkyl group and unsubstituted alkyl group. [0223] “Substituted” as used herein means one or more hydrogen atoms of the group is replaced with a substituent atom or group commonly used in pharmaceutical chemistry. Each substituent can be the same or different. Examples of suitable substituents include, but are not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, heterocycloalkyl, heteroaryl, OR’ (e.g., hydroxyl, alkyloxy (e.g., methoxy, ethoxy, and propoxy), aryloxy, heteroaryloxy, arylalkyloxy, ether, ester, carbamate, etc.), hydroxyalkyl, alkyloxycarbonyl, alkyloxyalkyloxy, perhaloalkyl, alkyloxyalkyl, SR’ (e.g., thiol, alkylthio, arylthio, heteroarylthio, arylalkylthio, etc.), S+R’2, S(O)R’, SO2R’, NR’R” (e.g., primary amine (i.e., NH2), secondary amine, tertiary amine, amide, carbamate, urea, etc.), hydrazide, halo, nitrile, nitro, sulfide, sulfoxide, sulfone, sulfonamide, thiol, carboxy, aldehyde, keto, carboxylic acid, ester, amide, imine, and imide, including seleno and thio derivatives thereof, wherein each of the substituents can be optionally further substituted. In embodiments in which a functional group with an aromatic carbon ring is substituted, such substitutions will typically number less than about 10 substitutions, more preferably about 1 to 5, with about 1 or 2 substitutions being preferred. [0224] “Stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. Thus, “stereoisomer thereof” with respect to a compound includes any stereoisomer of the compound and mixtures of stereoisomers, and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposable mirror Docket Number CX10-269WO4 images of one another. A compound may have more than one chiral center such that the compound may exist as either an individual diastereomer or as a mixture of diastereomers. Ligation of Oligonucleotides with single-stranded RNA Ligases [0225] In one aspect, the present disclosure provides a method of ligating an acceptor to a donor substrate of single-stranded RNA ligase to form a ligated oligonucleotide product. In some embodiments, the acceptor or donor, or both the acceptor and donor of the single-stranded RNA ligase substrates are modified to generate a ligated modified oligonucleotide product. [0226] In some embodiments, a method of synthesizing an oligonucleotide comprises reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor to form an extended oligonucleotide. [0227] In some embodiments, the oligonucleotide(A), the nucleotide donor, or both the oligonucleotide(A) and the nucleotide donor comprise a modified nucleotide. [0228] In some embodiments, the single stranded RNA ligase can be used to synthesize, among others, antisense oligonucleotides (ASOs), polynucleotide strands for use in synthesis of double stranded polynucleotides (e.g., siRNA compounds), and guide RNA used in CRISPR or related genome editing technology, and the like. In some embodiments, the single stranded RNA ligase is used to add a nucleotide comprising a conjugate moiety, such as cell targeting moieties (e.g., GalNAc, lipids, steroids, etc.), to an oligonucleotide. Oligonucleotide Acceptor [0229] In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) comprises a free 3’-OH group, or a functional form thereof, at the 3’-terminal nucleotide suitable as an acceptor for or reaction with the nucleotide donor in the single-stranded RNA ligase reaction. [0230] In some embodiments, the oligonucleotide acceptor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotide acceptor is up to 122 nucleotides in length. In some embodiments, the oligonucleotides is up to 150 or more nucleotides in length. In some embodiments, the oligonucleotides is up to 200 or more nucleotides in length. [0231] In some embodiments, the oligonucleotide acceptor is 2 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 3 nucleotides in length. In some embodiments, the oligonucleotide acceptors is 4 nucleotides in length. In some embodiments, the oligonucleotide acceptor is 5 nucleotides in length. In some embodiments, the oligonucleotide is 6 nucleotides in length. In some embodiments, the oligonucleotide is 7 nucleotides in length. In some embodiments, the oligonucleotide is 8 nucleotides in length. In some embodiments, the oligonucleotide is 9 nucleotides in length. In some embodiments, the oligonucleotide is 10 nucleotides in length. Docket Number CX10-269WO4 [0232] In some embodiments, the oligonucleotide acceptor comprises a 5’-OH group. In some embodiments, the presence of a 5’-OH group prevents the oligonucleotide acceptor from acting as a nucleotide donor for another RNA ligase reaction. [0233] In some embodiments, the oligonucleotide acceptor comprises a 5’-blocking group that prevents or inhibits the oligonucleotide acceptor to act as a nucleotide donor in another RNA ligase reaction, as further described herein. In some embodiments, use of an oligonucleotide acceptor with a 5’-OH or a 5’-blocking group in combination with a nucleotide donor having a 3’-blocking group can be used to direct or guide the ligation reaction to generate specific ligated products. [0234] In some embodiments, the oligonucleotide acceptor comprises at least a modified nucleotide. In some embodiments, the modified nucleotide on the oligonucleotide acceptor comprises a modified internucleoside linkage, a modified nucleoside, a 5’-terminal group, or any combinations thereof. [0235] In some embodiments, a modified nucleoside on the oligonucleotide acceptor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide acceptor, as limited by the length of the oligonucleotide acceptor. In some embodiments, all nucleosides of the oligonucleotide acceptor comprises a modified nucleoside. [0236] In some embodiments, the modified 3’-terminal nucleoside of the oligonucleotide acceptor comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, the modified 3’-terminal nucleoside is a 2’-fluoro adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro thymidine, 2’-O-methyl adenosine, 2’-O-methyl guanosine, 2’-O-methyl cytidine, 2’-O-methyl uridine, or 2’-O-methyl thymidine. [0237] In some embodiments, the oligonucleotide acceptor comprises at least one modified nucleoside or modified terminal group, wherein the modified nucleoside or terminal group comprises a conjugate moiety, a reactive group, or a linker. [0238] In some embodiments, the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. [0239] In some embodiment, the reactive group comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. [0240] In some embodiments, the reactive group includes those used in click chemistry, such as copper-free click chemistry, and include, by way example and not limitation, azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups. [0241] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the nucleotide acceptor. In some embodiments, the conjugate moiety or reactive group is attached to the nucleoside via a linker. In some embodiments, the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). Docket Number CX10-269WO4 [0242] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 2’-position of the sugar moiety. [0243] In some embodiments, wherein the nucleoside is an internal nucleoside or a 3’-terminal nucleoside, the conjugate moiety, reactive group, or linker is attached to the nucleobase and/or 2’-position of the sugar moiety. [0244] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the 5’-OH group, a 5’-phosphate group, 4’-carbon of the sugar moiety, or through an abasic nucleoside, or an inverted abasic nucleoside on the 5’-terminal nucleotide. [0245] In some embodiments, the oligonucleotide acceptor comprises one or more terminal groups. In some embodiments, the terminal group is present at the 5’-terminal nucleotide of the nucleotide acceptor. In some embodiments, the terminal group present on the oligonucleotide acceptor is a 5’-phosphonate, (e.g., E or Z vinylphosphonate), 5’-phosphoalkyl (e.g., 5’-phosphomethyl or 5’-phosphoethyl, etc.), 4’-amino, 4’- aminoalkyl, abasic nucleoside, or inverted abasic nucleoside. [0246] In some embodiments, the oligonucleotide acceptor comprises one or more modified internucleoside linkages. In some embodiments, the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or a mixture of Sp and Rp stereoisomer. [0247] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 5’ terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages. [0248] In some embodiments, the oligonucleotide acceptor has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide acceptor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages. [0249] In some embodiments, the oligonucleotide acceptor (oligonucleotide(A)) comprises the formula (I): A1[•A2]m•A3-OH (I) wherein each of A1, A2 and A3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage. [0250] In some embodiments, m is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, when m is 2 or greater, each of A2 is same or different. Docket Number CX10-269WO4 [0251] In some embodiments, at least one or more of A1, A2, or A3 comprises a modified nucleoside. In some embodiments, A1 is modified. In some embodiments, at least one of A2 is modified. In some embodiments, at least 2, 3, 4, 5, 6, or all of A2 is modified. In some embodiments, A3 is modified. In some embodiments, at least one of A1, A2, or A3 is modified with a conjugate moiety, reactive group, or linker. [0252] In some embodiments, A3 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, A3 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine. [0253] In some embodiments, at least one internucleoside linkage “•“ comprises a modified internucleoside linkage. In some embodiments, the internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp or Rp stereoisomer [0254] In some embodiments, the linker on the modified oligonucleotide acceptor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises an C2-C20alkylene or polyethylene linker. Various linkers suitable for the purpose herein are described herein and also known in the art. [0255] In some embodiments, the modified nucleoside on the oligonucleotide acceptor comprises the formula (II): A-[L]g-[M]h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0. [0256] In some embodiments, the oligonucleotide acceptor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length. [0257] In some embodiments, wherein for formula (II) when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside, or wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. Nucleotide Donor or Donor Substrate Docket Number CX10-269WO4 [0258] In some embodiments, the nucleotide donor or donor substrate for the RNA ligase comprises a 5’- phosphate group, or functional form thereof, at the 5’-terminal nucleotide suitable for attachment or ligation to any of the oligonucleotide acceptors described herein. [0259] In some embodiments, the nucleotide donor comprises a single nucleotide donor, also referred to herein as nucleotide(D) or D, or an oligonucleotide donor, also referred to as oligonucleotide(D). [0260] In some embodiments, the oligonucleotide donor is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotide donor is up to 122 nucleotides in length. In some embodiments, the oligonucleotides donor is up to 150 or more nucleotides in length. In some embodiments, the oligonucleotide donor is up to 200 or more nucleotides in length. [0261] In some embodiments, the oligonucleotide donor is 2 nucleotides in length. In some embodiments, the oligonucleotide donor is 3 nucleotides in length. In some embodiments, the oligonucleotide donor is 4 nucleotides in length. In some embodiments, the oligonucleotide donor is 5 nucleotides in length. In some embodiments, the oligonucleotide donor is 6 nucleotides in length. In some embodiments, the oligonucleotide donor is 7 nucleotides in length. In some embodiments, the oligonucleotide donor is 8 nucleotides in length. In some embodiments, the oligonucleotide donor is 9 nucleotides in length. In some embodiments, the oligonucleotide donor is 10 nucleotides in length. [0262] In some embodiments, the nucleotide donor, either as a nucleotide(D) or oligonucleotide(D), comprises a modified nucleotide. In some embodiments, the oligonucleotide(D) comprises at least one modified nucleotide. [0263] In some embodiments, the modified nucleotide on nucleotide(D) comprises a modified nucleoside, a 3’-terminal group, or any combinations thereof. [0264] In some embodiments, the modified nucleotide on an oligonucleotide(D) comprises a modified internucleoside linkage, a modified nucleoside, a 3’-terminal group, or any combinations thereof. [0265] In some embodiments, a modified nucleoside on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide donor, as limited by the length of the oligonucleotide donor. In some embodiments, all nucleosides of an oligonucleotide donor comprises a modified nucleoside. [0266] In some embodiments, a modified 5’-terminal nucleoside of the oligonucleotide donor comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, the modified 5’-terminal nucleoside is a 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O- methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine. [0267] In some embodiments, the modified nucleoside of nucleotide donor nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or a linker. Docket Number CX10-269WO4 [0268] In some embodiments, the conjugate moiety on the modified nucleoside of the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. [0269] In some embodiment, the reactive group on the modified nucleoside comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. In some embodiments, the reactive group used in click chemistry, including copper-free click chemistry, such as azido, alkynyl, dibenzocyclooctynyl, vinyl, trans-cyclooctene, or tetrazine groups. [0270] In some embodiments, the linker on the modified nucleotide donor comprises any suitable linker, for example, linker comprised of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, cyclcoalkyl, heterocycloalkyl, arylene, or heteroarylene based linkers, and the like. In some embodiments the linker comprises a C2-C20alkylene or polyethylene linker. As noted above, various linkers suitable for the modified nucleoside of the nucleotide donor are described herein and also known in the art. [0271] In some embodiments, when the nucleotide donor comprises oligonucleotide(D), the nucleoside modified with a conjugate moiety, reactive group, or linker on the oligonucleotide donor is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. In some embodiments, the modified nucleoside is on multiple internal nucleosides, for example, 2, 3, 4 or more internal nucleosides of the oligonucleotide(D), as limited by the length of the oligonucleotide donor. [0272] In some embodiments, the conjugate moiety, reactive group, or linker is attached to the nucleobase, the sugar moiety of a nucleoside of a nucleotide donor, or a 3’-terminal group. [0273] In some embodiments, where the nucleotide donor comprises nucleotide(D), the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or 3’-position of the sugar moiety, a 3’- terminal group, or any combinations thereof. [0274] In some embodiments, where the nucleotide donor comprises an oligonucleotide(D) , the conjugate moiety, reactive group, or linker can be attached to the nucleobase or the 2’-position of the sugar moiety of the 5’-terminal nucleoside and/or the internal nucleoside of oligonucleotide(D). In some embodiments, the conjugate moiety, reactive group, or linker can be attached to the nucleobase, the 2’- or the 3’-position of the sugar moiety, or a 3’-terminal group, or any combinations thereof, of the 3’-terminal nucleotide. [0275] In some embodiments, the nucleotide donor comprises one or more terminal groups. In some embodiments, the terminal group is present on nucleotide(D), or where the nucleotide donor comprises oligonucleotide(D), at the 3’-terminal nucleotide of nucleotide(D) or oligonucleotide(D). In some embodiments, the terminal group present on the nucleotide donor is a 3’-phosphate, 3’-phosphorothioate, 3’- phosphorodithioate, 3’-phosphonate (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 3-phosphomethyl or 3’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside. [0276] In some embodiments, wherein the nucleotide donor comprises nucleotide(D), the 3’-terminal group comprises a 3’-phosphate group for enhancing the efficiency of ligation of the nucleotide donor to the oligonucleotide acceptor, e.g., pDp, where the prefix p is a 5’-phosphate group and suffix p is a 3’-phosphate Docket Number CX10-269WO4 group. In some embodiments, a 3’-phosphate group can also act as a 3’-blocking group, either on the single nucleotide donor or oligonucleotide donor. [0277] In some embodiments, where the nucleotide donor comprises an oligonucleotide(D), the oligonucleotide(D) comprises one or more modified internucleoside linkages. In some embodiments, the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp isomer, or mixtures thereof. [0278] In some embodiments, the oligonucleotide(D) has at least 1 nucleotide at the 5’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 5’-terminal region with modified internucleoside linkages. [0279] In some embodiments, the oligonucleotide(D) has at least 1 nucleotide at the 3’-terminal region with a modified internucleoside linkage. In some embodiments, the oligonucleotide donor has at least 2, at least 3, at least 4 or at least 5 nucleotides at the 3’-terminal region with modified internucleoside linkages. [0280] In some embodiments, the nucleotide donor comprises a 3’-blocking group. In some embodiments, a 3’-blocking group prevents or inhibits reiterative extensions or attachments of the nucleotide donor to another nucleotide donor. In some embodiments, the 3’-blocking group is present on the 3’-OH group or the nucleobase of the 3’-terminal nucleoside of the nucleotide donor. In some embodiments, the 3’-blocking group is a reversible blocking group, which is removable or capable of being cleaved with a deblocking agent to produce a free 3’-OH. Various reversible 3’-blocking agents are described herein, and known in the art. [0281] In some embodiments, the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120. [0282] In some embodiments, for formula (IIIb), n is 0, 1, 2, 4, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, when n is 2 or greater, each of D2 is same or different nucleoside. [0283] In some embodiments, for formula (IIIa), D comprises a modified nucleoside. In some embodiments, D is modified with a conjugate moiety, reactive group, or a linker. [0284] In some embodiments, for formula (IIIb), at least one or more of D1, D2, or D3 comprises a modified nucleoside. In some embodiments, D1 is modified. In some embodiments, at least one of D2 is modified. In some embodiments, at least 2, 3, 4, 5, 6 or all of D2 is modified. In some embodiments, D3 is modified. In Docket Number CX10-269WO4 some embodiments, at least one of D1, D2, or D3 is modified with a conjugate moiety, reactive group, or linker. [0285] In some embodiments, D1 comprises a modified sugar moiety. In some embodiments, the sugar moiety is modified at the 2’-position. In some embodiments, D1 comprises a 2’-fluoro-adenosine, 2’-fluoro- guanosine, 2’-fluoro cytidine, 2’-fluoro uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine. [0286] In some embodiments, for formula (IIIb) at least one internucleoside linkage “•“ comprises a modified internucleoside linkage. In some embodiments, the modified internucleoside linkage is a phospho containing internucleoside linkage or a non-phospho internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage. In some embodiments, the internucleoside linkage is a phosphorothioate linkage, wherein the phosphorothioate linkage is the Sp or Rp stereoisomer, or a mixture of Sp and Rp stereoisomers. [0287] In some embodiments, the modified nucleoside on the nucleotide donor, e.g., of formula (IIIa) or formula (IIIb), comprises the formula (IV): D-[L]q-[M]r (IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0. [0288] In some embodiments, when q is 1, L is attached to the nucleobase or the sugar moiety, or when q is 0, M is attached to the nucleobase or the sugar moiety. [0289] In some embodiments, when q is 1, L can be attached to the 2’-position or 3’-position of the sugar moiety, as appropriate for D, either for nucleotide(D) or for oligonucleotide(D). For example, for formula (IIIa), L can be attached to 2’-position or 3’-position of the sugar moiety. For formula (IIIb), where D is D1 or D2, L can be attached to the 2’-position of the sugar moiety. Where D is D3, the L can be attached to the 2’- or 3’- position of the sugar moiety. [0290] In some embodiments, D or D3 further comprises one or more 3’-terminal groups. In some embodiments, the terminal group is a 3’-phosphate, 3’-phosphorothioate, 3’-phosphorodithioate, 3’- phosphonate, (e.g., E or Z vinylphosphonate), 3’-phosphoalkyl (e.g., 5-phosphomethyl or 5’-phosphoethyl, etc.), an abasic nucleoside, or inverted abasic nucleoside. [0291] In some embodiments, for formula (IIIa), D of the nucleotide donor comprises a 3’-phosphate group, where the nucleotide donor comprises pDp representing the 5’-phosphate and the 3’-phosphate groups on the nucleoside D. In some embodiments, the pDp is pUp, pTp, pCp, pAp, or pGp. In some embodiments, the Docket Number CX10-269WO4 pDp comprises a 2’-modification. In some embodiments, pDp is pmUp, pmTp, pmCp, pmAp, or pmGp, with “m” representing 2’-O-methyl. In some embodiments, pDp is pfUp, pfTp, pfCp, pfAp, or pfGp, with “f” representing 2’-F. In some embodiments, the nucleobase of nucleoside comprises a nucleobase selected from the list described below. [0292] In some embodiments, for formula (IIIb), the nucleotide donor comprises the formula pD1[-D2]n-D3p, wherein the pD1 represents the 5’-phosphate on the 5-terminal nucleoside D1, and D3p represents the 3’- phosphate on the 3’-terminal nucleoside D3. [0293] In some embodiments, for formula (IIIa), D further comprises a 3’-blocking group. In some embodiments, for formula (IIIb), D3 further comprises a 3’-blocking group. In some embodiments, the 3’- blocking group prevents or inhibits attachment of the nucleotide donor to another nucleotide donor in the RNA ligase reaction, or reiterative extensions or attachments of a nucleotide substrate using a terminal nucleotidyl transferase. In some embodiments, the 3’-blocking group is present on the 3’-OH group or the nucleobase of D or D3. In some embodiments, the 3’-blocking group is a reversible blocking group. In some embodiments, the 3’-blocking is removable or capable of being cleaved with a deblocking agent, as further described herein. Reiterative Extensions or Additions with single-stranded RNA ligase and Optionally Terminal Nucleotidyl Transferase [0294] In some embodiments, the method of synthesizing an oligonucleotide comprises reacting an oligonucleotide acceptor and a nucleotide donor in presence of a single-stranded RNA ligase under reaction conditions suitable for ligation of the nucleotide donor to the oligonucleotide acceptor, wherein the nucleotide donor comprises a 3’-blocking group to form a 3’-blocked extended oligonucleotide. In some embodiments, the presence of the 3’-blocking groups provides for attachment of a single nucleotide donor to the oligonucleotide acceptor. In some embodiments, attachment of 3’-blocked nucleotide donor allows for attaching one nucleotide(D) or one oligonucleotide(D) to the oligonucleotide acceptor. In some embodiments, where the nucleotide nucleotide(D) comprises a conjugate moiety, reactive moiety, and/or a linker, the presence of a 3’-blocking group provides for attachment of one nucleotide(D) comprising a conjugate moiety, reactive moiety, and/or a linker. [0295] In some embodiments, the 3’-blocking group present on the nucleotide donor is a reversible 3’- blocking group, thereby resulting in formation of a reversible 3’-blocked extended oligonucleotide by the single-stranded RNA ligase reaction. In some embodiments, the reversible 3’-blocking agent on the blocked extended oligonucleotide is removable or cleavable with a deblocking agent appropriate for the particular 3’- blocking group. As further discussed below, an exemplary 3’-blocking groups comprises a 3’-phosphate, where the deblocking agent is a phosphatase. [0296] In some embodiments, the method further comprises removing or separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase. [0297] In some embodiments, optionally the method further comprises degrading or inactivating the by- products (e.g., unreacted nucleotide donor, pyrophosphate, etc.) of the single-stranded RNA ligase reaction. Docket Number CX10-269WO4 [0298] In some embodiments, wherein the 3’-blocking group is a reversible 3’-blocking group, the method further comprises removing or cleaving the 3’-blocking group from the 3’-blocked extended oligonucleotide to form an unblocked extended oligonucleotide, i.e., an extended oligonucleotide with a free 3’-OH group. In some embodiments, the removing or cleaving of the 3’-blocking group is carried out with a deblocking agent. In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the unblocked extended oligonucleotide is separated from the deblocking agent. [0299] In some embodiments, the removing or cleaving the 3’-blocking group with the deblocking agent and the degrading or inactivating of the by-products of the single-stranded RNA ligase reaction are done concurrently. [0300] In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single-stranded RNA ligase under suitable reaction conditions for attachment or ligation of the second nucleotide donor to the unblocked extended oligonucleotide. [0301] In some embodiments, the method further comprises one or more cycles of: extension with a nucleotide donor comprising a reversible 3’-blocking agent; separating the 3’-blocked extended oligonucleotide from the single-stranded RNA ligase or inactivating the single-stranded RNA ligase; removing or cleaving the 3’-blocking group with a deblocking agent; separating the unblocked extended oligonucleotide from the deblocking agent or inactivating the deblocking agent, wherein each cycle is with a new nucleotide donor. Repeating the cycle allows for step-wise extension of the extended oligonucleotide. In some embodiments, where the nucleotide donor is nucleotide(D), repeating each cycle with new nucleotide(D) allows for step-wise extensions of a single nucleotide with each cycle. In some embodiments, where the nucleotide donor is oligonucleotide(D), repeating each cycle with new oligonucleotide(D) allows for step-wise extensions with oligonucleotide(D). [0302] In some embodiments, the 3’-blocked nucleotide donor is used in combination with a 5’-OH containing or 5’-blocked oligonucleotide acceptor. Use of an oligonucleotide acceptor with a 5’-OH or 5’- blocking group limits or inhibits ligation to the 3’-OH of another oligonucleotide acceptor, thereby limiting ligation to the 3’-blocked nucleotide donor. [0303] In some embodiments, each nucleotide donor is a selected or predetermined nucleotide donor for producing an extended oligonucleotide with a defined nucleotide sequence. [0304] In some embodiments, the use of a 3’-blocked nucleotide donor comprising a conjugate moiety, reactive moiety, or linker, where the 3’-blocking group is a reversible blocking group, allows for sequential or stepwise addition of nucleotides with the conjugate moiety, reactive group, or linker. In some embodiments, the method comprises sequential or stepwise attachment or addition of at least two nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; at least three nucleotide donors comprising a conjugate moiety, reactive moiety, or linker; or at least four nucleotide donors comprising a conjugate moiety, reactive moiety, or linker. In some embodiments, the nucleotide donor in the sequential or stepwise addition of nucleotides is nucleotide(D) comprising a conjugate moiety, reactive moiety, or linker. Docket Number CX10-269WO4 [0305] In some embodiments, the unblocked extended oligonucleotide can serve as a substrate for a terminal nucleotidyl transferase. In some embodiments, the method further comprises reacting the unblocked extended oligonucleotide with a nucleotide substrate in presence of a terminal nucleotidyl transferase under reaction conditions suitable for the extension by or addition of at least one nucleotide to form at least a single nucleotide extended oligonucleotide. In some embodiments, more than one nucleotide is attached or added to the extended oligonucleotide by the terminal nucleotidyl transferase. [0306] In some embodiments, the nucleotide substrate for the terminal nucleotidyl transferase comprises a 3’-blocked nucleotide substrate (i.e., a nucleotide substrate comprising a 3’-blocking group), providing for a single nucleotide addition or attachment to the oligonucleotide or the extended oligonucleotide to form a 3’- blocked single nucleotide extended oligonucleotide. [0307] In some embodiments, the method further comprises removing or cleaving the 3’-blocking group to form an unblocked single nucleotide extended oligonucleotide. In some embodiments, the removing or cleaving of the 3’-blocking group is done with a deblocking agent appropriate for the specific 3’-blocking group. [0308] In some embodiments, the method further comprises degrading the by-products (e.g., unreacted NTP, unreacted 3’-blocked NTP, NDP, NMP, 3’-blocked NMP, etc.) of the terminal nucleotidyl transferase reaction. For example, where the 3’-blocking group is a 3’-phosphate, a phosphatase can be used as the deblocking agent. [0309] In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the deblocking agent separated from the unblocked single nucleotide extended oligonucleotide. In some embodiments, the inactivation and degrading of the by-products is done concurrently. [0310] In some embodiments, the method further comprises repeating one or more cycles of reacting the unblocked extended oligonucleotide with a nucleotide substrate comprising a 3’-blocking group in presence of the terminal nucleotidyl transferase, removing or cleaving the 3’-blocking moiety (i.e., deblocking the 3’- blocked nucleoside) on the extended oligonucleotide with a deblocking agent, and inactivating the deblocking agent or separating the deblocking agent from the unblock extended oligonucleotide, where each cycle is with a new nucleotide substrate. [0311] In some embodiments, each cycle of extension, deblocking, and inactivation/separation is with a new nucleotide substrate for step-wise, sequential attachment or addition of nucleotides to the extended oligonucleotide by the terminal nucleotidyl transferase. [0312] In some embodiments, each nucleotide substrate for the terminal nucleotidyl transferase reaction is a selected or predetermined nucleotide substrate for producing an extended oligonucleotide with a defined nucleotide sequence. In some embodiments, the use of a modified nucleotide substrate (e.g., modified nucleobase and/or 2’-position of the sugar moiety) allows for stepwise, sequential addition of modified nucleotides to the extended oligonucleotide. Reaction Conditions and Process Docket Number CX10-269WO4 [0313] In the embodiments herein, the single-stranded RNA ligase reactions are carried out under suitable conditions for the ligase mediated attachment of the oligonucleotide acceptor to the nucleotide donor. [0314] In some embodiments, the suitable reaction conditions include a nucleotide co-factor used by the single-stranded RNA ligase to catalyze the joining reaction. In some embodiments, the nucleotide cofactor is ATP. In some embodiments, the nucleotide cofactor is about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, or more as appropriate. In some embodiments, the nucleotide cofactor or substrate (e.g., ATP) is present at a concentration of about 0.05-25 mM, 1-20 mM, 2-18 mM, or 5-15 mM. In some embodiments, the nucleotide cofactor or substrate is present at a concentration of about 0.5 mM, 1 mM, 2 mM, 3 mM 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 15 mM, 20 mM, or 25 mM. [0315] In some embodiments, the reaction conditions for the ligation include additional components, such as a divalent metal (e.g., Mg+2), buffer, and/or salts. Exemplary reaction components are provided in the Examples. In some embodiments, the salt in the reaction conditions, include, among others, NaCl, KCl, ammonium salts (e.g., NH4Cl), and acetate salts (e.g., sodium acetate). In some embodiments, the salt is present at 0.5 mM-300 mM, 1 mM-250 mM, 2 mM-200 mM, 5 mM-150 mM, 10 mM-100 mM, 20 mM-80 mM, or 40 mM-60 mM. In some embodiments, the salt is present at about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM. In some embodiments, the salt is present at sufficient concentration to reduce degradation or other undesired products. [0316] In some embodiments, the reaction conditions also include a ligation enhancing reagent, including, among others, DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000). [0317] In some embodiments, the single-stranded RNA ligase reaction is carried out at a suitable temperature and reaction time period for the ligation of the oligonucleotide acceptor and the nucleotide donor. In some embodiments, the RNA ligation reaction temperature is from about 2° C to about 60° C. In some embodiments, the RNA ligation reaction temperature is from 4 °C to 55 °C, 4 °C to 50 °C, 4 °C to 45 °C, or 10 °C to 40 °C. In some embodiments, the RNA ligation reaction temperature is 2 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 37 °C, 40 °C, 45 °C, 50 °C, 55 °C, or 60 °C. In some embodiments, the reaction temperature is chosen based on the thermostability of the single-stranded RNA ligase and/or the efficiency of the ligation at defined temperatures and reaction conditions. [0318] In some embodiments, the single-stranded RNA ligase reaction time can be a sufficient time for ligation of the oligonucleotide acceptor to the nucleotide donor. In some embodiments, the ligation reaction time is from 0.5-72 hr or longer. In some embodiments, the ligation reaction time is 1-72 hr, 2-48 hr, or 2-24 hr. In some embodiments, the ligation reaction time is 0.5, 1, 2, 4, 5, 12, 24, 48, or 72 hr or longer. [0319] In the embodiments, the reaction conditions comprise a suitable pH. The desired pH or desired pH range can be maintained by use of an acid or base, an appropriate buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and/or during the course of the reaction. In some embodiments, the suitable reaction conditions comprise a solution pH from about 4 to about Docket Number CX10-269WO4 10, pH from about 5 to about 10, pH from about 5 to about 9, pH from about 6 to about 9, pH from about 6 to about 8. In some embodiments, the reaction conditions comprise a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. [0320] In some embodiments, the pH of the reaction mixture may change during the reaction. In some embodiments, the pH of the reaction solution is maintained at a desired pH or within a desired pH range, such as by the addition of an acid or a base, before and/or during the course of the reaction. In some embodiments, the pH is controlled by using an appropriate buffer. Suitable buffers to maintain desired pH ranges are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N- morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), and the like. In some embodiments, the buffer concentration is from 1 to 500 mM, 1 to 400 mM, 1 to 300 mM, 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM. In some embodiments, the buffer concentration is about 1 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, or 500 mM. [0321] In some embodiments, the concentration of oligonucleotide acceptor and nucleotide donor can be varied based on, among others, the reaction conditions, the activity of the single-stranded ligase, factors affecting efficiency of the oligonucleotide acceptor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the acceptor, presence of secondary structure, 3’-terminal properties, etc.), and the efficiency of the nucleotide donor as substrate (e.g., nucleotide length, presence of modified nucleosides, presence of modified internucleoside linkages, sequence of the nucleotide donor, presence of secondary structure, 5’-terminal properties, etc.). If the process uses an oligonucleotide bound to a support medium, additional considerations include, among others, density of the oligonucleotide acceptor on the support medium, effect of support medium on the single-stranded RNA ligase reaction, and access of the single-stranded RNA ligase and other enzymes (e.g., pyrophosphatase, terminal nucleotidyl transferase, etc.) to the oligonucleotide acceptor bound to the support medium. [0322] In some embodiments, the oligonucleotide acceptor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, oligonucleotide acceptor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM. In some embodiments, the oligonucleotide acceptor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the nucleotide donor. [0323] In some embodiments, the nucleotide donor is provided at a concentration of about 0.05 to about 25 mM, 0.1-20 mM, 0.1-15 mM, 1-10 mM, 2-8 mM, or 4-6 mM. In some embodiments, nucleotide donor concentration is at about 0.01-1 mM, 0.05-0.9 mM, 0.1-0.8 mM, 0.2-0.7 mM, or 0.3 mM-0.6 mM. In some embodiments, the nucleotide donor concentration is at about 0.01 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM, Docket Number CX10-269WO4 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM or 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or any appropriate concentration for efficient ligation to the oligonucleotide acceptor. [0324] In some embodiments, the ratio of nucleotide donor to oligonucleotide acceptor is 0.1:1, 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 or higher. [0325] In some embodiments, the single stranded RNA ligase is provided at concentrations from about 0.01 g/L to about 50 g/L; about 0.01 to about 0.1 g/L; about 0.05 g/L to about 50 g/L; about 0.1 g/L to about 40 g/L; about 1 g/L to about 40 g/L; about 2 g/L to about 40 g/L; about 5 g/L to about 40 g/L; about 5 g/L to about 30 g/L; about 0.1 g/L to about 10 g/L; about 0.5 g/L to about 10 g/L; about 1 g/L to about 10 g/L; about 0.1 g/L to about 5 g/L; about 0.5 g/L to about 5 g/L; or about 0.1 g/L to about 2 g/L. Oligonucleotide Synthesis Using Oligonucleotide Bound to Support Medium [0326] In some embodiments, the oligonucleotide acceptor is bound to a support medium (e.g., a solid support), and the ligation reaction carried out with the single-stranded RNA ligase and the nucleotide donor in solution to ligate the nucleotide donor to the support bound oligonucleotide acceptor. In some embodiments, the oligonucleotide is conjugated to the support medium via the 5’-terminal nucleotide to provide for a ligase accessible free 3’-OH. [0327] In some embodiments, the single-stranded RNA ligase, any unreacted nucleotide donor, and by- products of the single-stranded RNA ligase reaction are removed or separated from the immobilized extended oligonucleotide. In some embodiments, the extended oligonucleotide is released or cleaved from the substrate medium to yield the extended oligonucleotide in solution. In some embodiments, the releasing agent is a chemical cleavage agent appropriate for the covalent attachment to the substrate medium. In some embodiments, a biological releasing or cleaving agent is used, for example a nuclease that can cleave at a specific site in the oligonucleotide bound to the substrate medium. [0328] In some embodiments, for addition of one nucleotide donor, e.g., nucleotide(D), or for reiterative synthesis using the oligonucleotide acceptor bound to a support medium, the ligation reaction is carried out with the single-stranded RNA ligase and a 3’-blocked nucleotide donor in solution to form a 3’-blocked extended oligonucleotide bound to the support medium. The single-stranded RNA ligase, any unreacted nucleotide donor, and by-products of the single-stranded RNA ligase reaction are removed or separated from the immobilized 3’-blocked extended oligonucleotide. In some embodiments, where the 3’-blocking group is desired in the extended oligonucleotide, for example, a conjugate moiety, reactive group, or linker attached to the 3’-OH group of the sugar moiety, no further extensions are carried out and the extended oligonucleotide is released or cleaved from the support medium. [0329] In some embodiments, for the addition of one nucleotide donor nucleotide(D), where removal or cleavage of the 3’-blocking groups is desired, a reversible 3’-blocking group is used and the support bound 3’- blocked extended oligonucleotide reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide. The unblocked extended oligonucleotide is then released or cleaved from the support medium. In some embodiments, the 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution. Docket Number CX10-269WO4 [0330] In some embodiments, for stepwise, sequential additions or extensions with two or more nucleotide donors, the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide bound to the support medium. In some embodiments, following the deblocking reaction, the deblocking agent is inactivated or the unblocked extended oligonucleotide separated from the deblocking agent. In some embodiments, the by-products of the single-stranded RNA ligase reaction are degraded with a degrading agent. [0331] In some embodiments, for each new nucleotide donor in the sequential additions or extensions, the cycle of reaction with the extended oligonucleotide with RNA ligase and deblocking and associated inactivation/separation of deblocking agent and degrading of by-products is repeated for each nucleotide donor. [0332] In some embodiments, following the addition of the last nucleotide donor in the stepwise, sequential additions, the support bound 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide. The unblocked extended oligonucleotide is then released or cleaved from the support medium. [0333] In some embodiments, the desired 3’-blocked extended oligonucleotide is released or cleaved from the support medium and the deblocking step carried out in solution to yield the final extended oligonucleotide product. [0334] In some embodiments, the final extended oligonucleotide product in solution can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.). Oligonucleotide Synthesis Using Immobilized Enzymes [0335] In some embodiments, the ligation reaction is carried out with a single-stranded RNA ligase bound to a support medium, e.g., immobilized single-stranded RNA ligase. In some embodiments, the oligonucleotide acceptor and nucleotide donor in solution can be contacted with the support-bound single-stranded RNA ligase to ligate the oligonucleotide acceptor to the nucleotide donor. In some embodiments, the substrates in solution are passed through a bed (e.g., a column) of support-bound single-stranded RNA ligase until a desired level of extended product has formed. In some embodiments, the extended oligonucleotide product is removed or separated from the immobilized single-stranded RNA ligase. In some embodiments, the nucleotide donor comprises a 3’-blocked nucleotide donor to form a 3’-blocked extended oligonucleotide in solution. [0336] In some embodiments, for ligation reactions using a nucleotide donor comprising a reversible 3’- blocking group, the method further comprises removing or cleaving the 3’-blocking group with a deblocking agent to form an unblocked extended oligonucleotide in solution. [0337] In some embodiments, the method further comprises inactivating and/or separating the deblocking agent from the unblocked extended oligonucleotide. In some embodiments, where the deblocking agent comprises an enzyme, e.g., phosphatase, the enzyme can be on a support substrate (e.g., immobilized Docket Number CX10-269WO4 deblocking enzyme) to facilitate reaction with the blocked extended oligonucleotide in solution and its separation from the deblocking agent following the deblocking reaction. [0338] In some embodiments, the method further comprises degrading the by-products in the reaction containing the extended oligonucleotide in solution. In some embodiments, the degrading of by-products is achieved concurrently or subsequent to the deblocking of the extended oligonucleotide. In some embodiments, the degrading agent, e.g., an enzyme, is immobilized to a support medium to facilitate degradation of by-products in solution and separation of the extended oligonucleotide from the degrading agent. [0339] In some embodiments, for stepwise, sequential additions or extensions with two or more nucleotide donors, the 3’-blocked extended oligonucleotide in solution is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to form an unblocked extended oligonucleotide, and following the deblocking reaction, the deblocking agent is inactivated or the deblocking agent separated from the unblocked extended oligonucleotide. [0340] In some embodiments, for each new nucleotide donor in the sequential additions or extensions, the cycle of reaction with the extended oligonucleotide with immobilized single-stranded RNA ligase and deblocking and associated inactivation/separation of deblocking agent and degrading of by-products is repeated for each new nucleotide donor. [0341] In some embodiments, following the addition of the last nucleotide donor in the stepwise, sequential additions, the final 3’-blocked extended oligonucleotide is reacted with a deblocking agent to remove or cleave the 3’-blocking agent to from the unblocked extended oligonucleotide. In some embodiments, use of an immobilized single-stranded RNA ligase and an immobilized deblocking agent, e.g., a phosphatase, provides an efficient process for stepwise sequential addition of nucleotide donors involving extension reaction with the immobilized single-stranded RNA ligase (e.g., in a first column or chamber), separation and transport of the reaction solution to an immobilized deblocking agent (e.g., in a second column or chamber) for deblocking and degradation of by-products of the ligation reaction, and separation and transport of the reaction solution from the deblocking reaction to the extension with the immobilized single-stranded RNA ligase (e.g., in the first column or chamber) for reaction with the next nucleotide donor. [0342] In some embodiments, the final unblocked extended oligonucleotide can be purified, for example by chromatography (e.g., ion exchange chromatography, reverse phase chromatography, size exclusion chromatography), including HPLP and UPLC; ultrafiltration; and affinity techniques (e.g., capture tags, hybridization, etc.). [0343] Unlike the process using an oligonucleotide bound to a support medium for the extension reaction, a process using oligonucleotides and nucleotide donors in solution does not require release or cleavage of the desired extended oligonucleotide product from the support medium. Modified Nucleotides [0344] In some embodiments, in the compositions or methods of using single-stranded RNA ligase for oligonucleotide synthesis, the oligonucleotide acceptor, nucleotide donor, or the combination of Docket Number CX10-269WO4 oligonucleotide acceptor and nucleotide donor can have various modifications. In some embodiments, the modifications occur on the nucleobase, the sugar moiety, terminal groups, or any combination thereof. The various modifications that can be used are described below. 2’- and 3’-sugar modifications [0345] In some embodiments, the oligonucleotide acceptor and/or the nucleotide donor comprises a modified nucleoside, wherein the modification is on the sugar moiety of the nucleoside. In some embodiments, the modified sugar moiety is a modified furanosyl sugar moiety, for example ribose or deoxyribose. In some embodiments, the furanosyl sugar moiety is modified or substituted at the 2’, 3’, or a combination of 2’ and 3’ positions. Modifications at the 4’, and/or 5’-positions are described below as “terminal group.” [0346] In some embodiments, the modification is at the 2’-position of the sugar moiety. In some embodiments, substitutions at the 2’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 2’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 2’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, and NH2. In some embodiments, substituent groups at the 2’-position include, among others, O-(C1- C10)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N- alkynyl, O-alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl. In some embodiments, substituent groups at the 2’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, the substitution at the 2’-position is a phosphate (see, e.g., Current Protocols in Nucleic Acid Chemistry, 13.1.1- 13.1.31, John Wiley & Sons (2003). [0347] In some embodiments, the modified 2’-position of the sugar moiety is halo, 2’-O-R’, or 2’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a C1-C4alkyl. In some embodiments, the modified 2’-position is a 2’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 2’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3. In some embodiments, the modified 2’-position is 2’- O-(2-methoxyethyl), 2’-O-allyl, 2’-O-propargyl, 2’-O-ethylamine, 2’-O-cyanoethyl, -2’-O-amine, or 2’-O- acetate ester. [0348] In some embodiments, a modification at the 2’-position comprises a locked nucleoside. In some embodiments, locked nucleosides comprises a biradical linking the C2’ and C4’ of the ribose sugar ring of said nucleoside (also referred to as a “2’- 4’ bridge”), which restricts or locks the conformation of the ribose ring (see, e.g., Obika et al., Tetrahedron Letters, 1997, 38(50):8735–8738; Orum et al., Current Pharmaceutical Design, 2008, 14(11):1138–1142). In some embodiments, the ribose moiety of the locked nucleotide is in the C3’-endo (beta-D) or C2’-endo (alpha-L) conformation. In some embodiments, the bridge is a methylene bridge. In some embodiments, the bridge is an ethylene bridge, also referred to as ENA (see, e.g., Morita et al., Bioorg Med Chem Lett., 2002, 12(1):73-6). Other locked nucleoside are described in International patent publication WO 2121249993, incorporated by reference herein. Docket Number CX10-269WO4 [0349] In some embodiments, other locked nucleosides include, among others, 5’-methyl-LNA, 2’-amino- LNA, alpha-L-LNA, and thio-LNA. Structures of certain locked nucleosides are shown below: where R [0350] In some embodiments, a modification at the 2’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker or a linker, as described herein. [0351] In some embodiments, the modification is at the 3’-position of the sugar moiety. In some embodiments, in view of the effect of a 3’-modification on ligase activity, and use of the 3’-OH group for internucleoside linkage, the 3’-modification is on the 3’-terminal nucleoside of the nucleotide donor. In some embodiments, the modification at the 3’-position are similar to those at the 2’-position. In some embodiments, substitutions at the 3’- position include, among others, halo (e.g., Cl, F, Br, etc.) or -O-alkyl or 3’-alkoxy (e.g., O-methyl, O-ethyl, etc.). In some embodiments, other modifications at the 3’-position include, but are not limited to, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, SCH3, SOCH3, SO2CH3, ONO2, NO2, N3, and NH2. In some embodiments, substituent groups at the 3’-position include, among others, O-(C1-C10)alkoxy, alkoxyalkyl, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, O-alkynyl, S-alkynyl, N-alkynyl, O- alkyl-O-alkyl, alkynyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C1-C10 alkenyl and alkynyl. In some embodiments, substituent groups at the 3’-position include, but are not limited to, alkaryl, aralkyl, O-alkaryl, and O-aralkyl. In some embodiments, In some embodiments, the substitution at the 3’-position is a phosphate. [0352] In some embodiments, the modified 3’-position of the sugar moiety is halo, 3’-O-R’, or 3’-O-COR’, where R’ is an alkyl, alkyloxyalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkylalkyl, heterocyclylalkyl, arylalkyl, or heteroarylalkyl. In some embodiments, R’ is a C1-C4alkyl. In some embodiments, the modified 3’-position is a 3’-O-R’, wherein in R’ is alkyloxyalkyl, alkylamine, cyanoalkyl, or -C(O)-alkyl. In some embodiments, the 3’-position of the sugar moiety of the nucleoside substrate is -O- R’, wherein R’ is -CH3 or -CH2CH3 or -CH2CH2OCH3. In some embodiments, the modified 3’-position is 3’- O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O-ethylamine, 3’-O-cyanoethyl, -3’-O-amine, or 3’-O- acetate ester. Docket Number CX10-269WO4 [0353] In some embodiments, the modifications at the 3’-position is a reversible or cleavable 3’-blocking group. In some embodiments, removal or cleaving of the reversible or cleavable 3’-blocking group results in a free 3’-OH group, which in some embodiments can serve as an acceptor for single-stranded RNA ligase or a terminal nucleotidyl transferase. In some embodiments, exemplary reversible or cleavable 3’-blocking groups include, among others, 3’-O-azidomethyl, 3’-O-(2-methoxyethyl), 3’-O-allyl, 3’-O-propargyl, 3’-O- ethylamine, 3’-O-cyanoethyl, -3’-O-amine, 3’-O-acetate ester, 3’-phosphate, 3’-diphosphate, or 3’- triphosphate. In some embodiments, the 3’-blocking group is paired with the corresponding deblocking agent used in the deblocking or cleavage of the 3’-blocking group. Other reversible or cleavable 3’-blocking groups are described in International patent publication WO2023183569, incorporated by reference herein. [0354] In some embodiments, a modification at the 3’-position comprises a reactive moiety; a conjugate moiety, including a conjugate moiety attached via a linker’ or a linker, as described herein. [0355] In some embodiments, the modified sugar moiety comprises an unlocked nucleoside. In some embodiments, in the unlocked nucleoside, the furanosyl ring is opened to result in the structure below: where B represents the nucleobase. are described in, among others, International patent publication WO2022/098990 and Snead et al., Molecular Therapy-Nucleic Acids, 2013, 2, e103. Modified nucleobases [0356] In some embodiments, the oligonucleotide acceptor and/or nucleotide donor comprises one or more nucleosides comprising a modified nucleobase. In some embodiments, modified nucleobase that is capable of hydrogen bonding to form Watson and Crick type base pairing is selected. In some embodiments, some positions for possible modifications on naturally occurring nucleobases are indicated below: Nucleobase Modification Positions Docket Number CX10-269WO4 Nucleobase Modification Positions [0357] In some embodiments, the nucleobase comprise an inosine nucleoside (i.e., nucleosides comprising a hypoxantine nucleobase). Ln some embodiments, the modified nucleobase is 5-substituted pyrimidines, 6- azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2. N-6 and O-6 substituted purines. In some embodiments, the modified nucleobase is 2-aminopropyladenine.5- hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6- N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine.5-propynyl uracil, 5- propynylcytosine.6-azouracil, 6-azocytosine, 6-azothymine.5-ribosyluracil (pseudouracil), 4-thiouracil.8- halo purine, 8-amino purine, 8-thio purine, 8-thioalkyl purine, 8-hydroxy purine, 8-aza purine, 5- bromocytosine.5-trifluoromethylcytosine, 5-halouracil, 5-halocytosine, 7-methylguanine, 7-methyladenine, 2- F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine.3-deazaguanine, 3-deazaadenine, 6-N- benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N- benzoylcytosine, and 5-methyl 4-N-benzoyluracil. Further modified nucleobases include tricyclic pyrimidines, e.g., 1,3-diazaphenoxazine-2-one.1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1.3- diazaphenoxazine-2-one (G-clamp). Docket Number CX10-269WO4 [0358] In some embodiments, the modified nucleobase includes, among others, nucleobases based on 2,4- dihalotolene and benzimidazole groups. In some embodiments, the modified nucleobase is 4- methylbenzimidazole, 2,4-difluorotoluene, 9-methylimidazo[(4,5)-b]pyridine, 2,4-dibromotoluene, benzimidazole, 5-nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole. In some embodiments, the modified nucleobase is 7-azaindole, and isocarbostyril (see, e.g., Berdis et al., Front. Chem.10:1051525). Other modified nucleobases are described in, among others, patent publication WO2021249993. [0359] In some embodiments, included within modified nucleobase is a nucleobase that does not have a nucleobase, also referred to as an abasic nucleoside. In some embodiments, the abasic nucleoside is present in the internal portion of an oligonucleotide acceptor. In some embodiments, an abasic nucleoside is attached to the 3’- or 5’-terminal end, which is in certain embodiments grouped as a terminal group. [0360] In some embodiments, the modified nucleobase is present on the 5’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, 3’-terminal nucleoside of the oligonucleotide acceptor or oligonucleotide donor, and/or present on the internal nucleosides of the oligonucleotide acceptor or oligonucleotide donor. In some embodiments, the blocks or contiguous stretches of nucleosides in the oligonucleotide acceptor or oligonucleotide donor have modified nucleobases. Terminal groups [0361] In some embodiments, the oligonucleotide acceptor and/or nucleotide donor comprises a terminal group. In some embodiments, the oligonucleotide acceptor comprises a terminal group at the 5’-terminal nucleoside. In some embodiments, the terminal group is attached to the 5’-OH or 4’-carbon atom of the terminal nucleoside. [0362] In some embodiments, the terminal group comprises a C-4’ modification of the 5’-terminal nucleoside, including among others, 4’-thio-C2’ modifications, 4’-aminoalkyl, C4’-guanidino-C2’- modifications, and C4’-O-methyl (see, e.g., Gangopadhyay et al., RNA Biology, 2022, 19:1, 452-467) [0363] In some embodiments, the 5’-terminal group is a 5’-phosphate modification. In some embodiments, the 5'-phosphate modification, includes, among others, 5’-C-methyl, particularly S isomer; 5’-(E or Z)- vinylphosphonate, or 5’-methylenephosphonate. [0364] In some embodiments, the 5’-terminal group comprises an abasic nucleotide attached to the 5’-OH. In some embodiments, the 5’-terminal groups comprises an inverted abasic nucleotide (5’-5’) attached to the 5’-OH of the 5’-end nucleoside. [0365] In some embodiments, the nucleotide donor comprises a 3’-terminal group. In some embodiments, the 3’-terminal group comprises a 3’-phosphate, which can also function as a reversible blocking group. In some embodiments, the 3’-phosphate is modified, such as with 3’-(E or Z)-vinylphosphonate, or 3’- methylenephosphonate. In some embodiments, the 3’-terminal group on the nucleotide donor comprises an abasic nucleoside. In some embodiments, the 3’-terminal group comprises an inverted abasic nucleotide (3’- 3’). Internucleoside linkages Docket Number CX10-269WO4 [0366] In some embodiments, the modified oligonucleotide, e.g., oligonucleotide acceptor and/or oligonucleotide donor, comprises at least one modified, non-naturally occurring internucleoside linkage. In some embodiments, the modified oligonucleotide has 1%, 2%, 5%, 10% 20%, 30%, 40%, 50%, or 60% or more modified internucleoside linkages. In some embodiments, all of the internucleoside linkages are modified internucleoside linkages. [0367] In some embodiments, the modified internucleoside linkage is a phosphorous containing modified internucleoside linkage. Exemplary phosphorous-containing internucleoside linkages include, among others, phosphotriesters, alkylphosphonates (e.g., methyl phosphonate, ethyl phosphonate, etc.), phosphoramidates, phosphorothioate, and phosphorodithioate. [0368] In some embodiments, the modified internucleoside linkage is a non-phosphorous containing internucleoside linkage. Exemplary non-phosphorous containing internucleoside linkages include, among others, methylenemethylimino (-CH2-N(CH3)-O-CH2), thiodiestcr, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); N,N’-dimethylhydrazine (-CH2-N((CH3)-N((CH3)-); MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), methoxypropyl, and thioformacctal (3’-S-CH2-O-5'). In some embodiments, the modified internucleoside linkage is amide linkage, such as those of glycine nucleosides or nucleoside β-amino acids (see, e.g., Banerjee et al., Bioconjugate Chem., 2015, 26, 8, 1737–1742). [0369] In some embodiments, the modified internucleoside linkages provides for a chiral center. For example, a phosphorothioate or alkylphosphonate internucleoside linkage can be in the Rp or Sp stereomeric configuration. In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor have a mixture of stereoisomers in the internucleoside linkage. In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor have greater than 50% of the internucleoside linkages as Rp or Sp configuration. In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor have at least 60%, 70%, 80%, 90%, or greater of Rp or Sp stereomeric configuration. [0370] In some embodiments, the modified internucleoside linkages are present in the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments at least 1 or 2 phosphorothioate internucleoside linkages are present at the 5’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments, the phosphorothioate linkage is a non-bridging phosphorothioate internucleoside linkage. [0371] In some embodiments, the modified internucleoside linkages are present in the 3’-terminal region of the oligonucleotide acceptor or oligonucleotide donor. In some embodiments, at least 1, 2, 3, 4, or 5 modified internucleoside linkages are present at the 3’-terminal region of the oligonucleotide acceptor and/or oligonucleotide donor. In some embodiments, at least 1 or 2 phosphorothioate internucleoside linkages are present at the 3’-terminal region of an oligonucleotide acceptor or oligonucleotide donor. [0372] In some embodiments, the modified internucleoside linkages are present in the internal portions of the oligonucleotide acceptor or oligonucleotide donor. Docket Number CX10-269WO4 [0373] In some embodiments, the oligonucleotide acceptor and/or oligonucleotide donor comprises at least a phosphorothioate internucleoside linkage, where the phosphorothioate linkage is in the Sp configuration, the Rp configuration, or a mixture of Sp and Rp configurations in the population of the oligonucleotide acceptor and/or oligonucleotide donor. Conjugate moiety [0374] In some embodiments, the oligonucleotide acceptor, or nucleotide donor comprises a conjugate moiety. In some embodiments, the nucleotide donor comprises a conjugate moiety that is compatible with single-stranded RNA ligase activity and that does not interfere with the nucleotide donor acting as a substrate for the single-stranded RNA ligase. [0375] In some embodiments, the conjugate moiety (i.e., non-nucleotide moiety) includes, among others, carbohydrates (e.g. GalNAc), lipids, sterols, drug substances, hormones, polymers (e.g., polyethylene glycol, etc.), proteins, peptides, toxins (e.g. bacterial toxins, etc.), vitamins (e.g., folate, tocopherol, retinoic acid, etc.), or combinations thereof. In some embodiments, the conjugate moiety is used to affect the pharmacokinetics of the oligonucleotide and/or oligonucleotide cell targeting. [0376] In some embodiments, the conjugate moiety can be attached to the 5’-terminal nucleotide, the 3’- terminal nucleotide, or in an oligonucleotide an internal nucleotide. In some embodiments, the conjugate moiety is attached the 2’-position of the sugar moiety of a nucleoside, for example, to the 2’-OH. In some embodiments, the conjugate moiety is attached to the 3’-position of the sugar moiety of the nucleoside, for example 3’-OH. In some embodiments, the conjugate moiety is attached to the nucleobase, as discussed above (see, e.g., Biscans et al., Nucleic Acids Res.2019 Feb 20; 47(3): 1082–1096). In some embodiments, the conjugate moiety is attached directly or attached using a linker. [0377] In some embodiments, the conjugate moiety comprises a C6-C22 alkyl, C6-22 alkenyl, or C6-C22 alkynyl. In some embodiments, the conjugate moiety comprises a C6-alkyl, C7-alkyl, C8-alkyl, C9-alkyl, C10- alkyl, C11-alkyl, C12-alkyl, C13-alkyl, C14-alkyl, C15-alkyl, C16-alkyl, C17-alkyl, C18-alkyl, C19-alkyl, C20-alkyl, C21-alkyl, or C22-alkyl. In some embodiments, the conjugate moiety comprises a C6 alkenyl, C7 alkenyl, C8 alkenyl C9 alkenyl, C10 alkenyl, C11-alkenyl, C12-alkenyl, C13-alkenyl, C14-alkenyl, C15-alkenyl, C16-alkenyl, C17-alkenyl, C18-alkenyl, C19-alkenyl, C20-alkenyl, C21-alkenyl, or C22-alkenyl. In some embodiments, the conjugate moiety comprises a C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11-alkynyl, C12- alkynyl, C13-alkynyl, C14-alkynyl, C15-alkynyl, C16-alkynyl, C17-alkynyl, C18-alkynyl, C19-alkynyl, C20- alkynyl, C21-alkynyl, or C22-alkynyl. [0378] In some embodiments, the conjugate moiety comprises a heteroalkyl, heteroalkenyl, or heteroalkynyl. In some embodiments, the heteroalkyl, heteroalkenyl or heteroalkynyl has one or more carbon atoms replaced with a heteroatom, such as O, S, or N. [0379] In some embodiments, the conjugate moiety comprises a cycloalkyl or heterocycloalkyl group. In some embodiments, the cycloalkyl includes, among others, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, and cycloheptyl. In some embodiments, the heterocycloalkyl includes, among others, 1-(1,2,5,6-tetrahydropyridyfh l-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- Docket Number CX10-269WO4 morpholinyl, tctrahydrofuran-2-yl, tctrahydrofuran-3-yl, tetrahydrothicn-2-yl, tetrahydrothien-3-yl, l- piperazinyl, and 2-piperazinyl. [0380] In some embodiments, the conjugate moiety comprises an aryl or heteroaryl moiety. In some embodiments, the aryl group includes, among others, phenyl, naphthyl, indenyl, biphenyl, phenanthrenyl, naphthacenyl, anthracenyl, fluorenyl, indenyl, and azulenyl. In some embodiments, a heteroaryl group includes, among others, pyridyl, furanyl, thienyl, pynolyl, oxazolyl, oxadiazolyl, imidazolyl ihiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isoihiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, isoquinolinyl, and indazolyl. [0381] In some embodiments, the conjugate moiety comprises a cycloalkylalkyl-, heterocycloalkylalkyl-, arylalkyl-, heteroarylalkyl-, cycloalkylheteroalkyl-heterocycloalkylheteroalkyl-, arylheteroalkyl-, heteroarylheteroalkyl-, cycloalkylalkenyl-, heterocycloalkylalkenyl-, arylalkenyl-, heteroarylalkenyl-, cycloalkylheteroalkenyl-heterocycloalkylheteroalkenyl-, arylheteroalkenyl-, or heteroarylheteroalkenyl-. [0382] In some embodiments, the conjugate moiety comprises a lipid or lipophilic moiety, for example a fatty acid. In some embodiments, the fatty acid comprises a saturated fatty acid, unsaturated fatty acid, or a polyunsaturated fatty acid. In some embodiments, the fatty acid comprises caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, elaidic acid, cis-vaccenic acid, trans- vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, eicosapentaenoic acid, decanoic acid, docosahexaenoic acid (DHA), and docosanoic acid (DCA) conjugate moieties (see, e.g., Kubo et al., ACS Chem. Biol., 2021, 16, 150−164; see also, WO2024/040041; incorporated herein by reference). [0383] In some embodiments, the conjugate moiety comprises a sterol. In some embodiments, the sterol comprises cholesterol, alpha-cholesterol, cholesterol ester (e.g., cholesteryl palmitate, etc.), cholesterol sulfate, phytosterol, cholic acid, or lithocholic acid. [0384] In some embodiments, the conjugate moiety comprises a phospholipid. In some embodiments, the phospholipid comprises phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, or a sphingolipid. [0385] In some embodiments, the conjugate moiety comprises a carbohydrate, particularly a carbohydrate moiety acting as a ligand for a cellular receptor for cellular targeting of the oligonucleotide. In some embodiments, the carbohydrate moiety comprises galactose or galactose derivatives. In some embodiments, the carbohydrate moiety is attached to the nucleoside via a linker. In some embodiments, exemplary carbohydrates that can be used include the following. , Docket Number CX10-269WO4 , [0386] In some embodiments, the conjugate moiety is an N-acetylgalactosamine (GalNAc) conjugate moiety. In some embodiments, the oligonucleotide acceptor and/or nucleotide donor may be conjugated to at least one conjugate moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the conjugate moiety is a monovalent, divalent, trivalent or tetravalent, GalNAc. [0387] In some embodiments, the GalNAc moiety has the following structure, where L is a linker, and W is a heteroatom (e.g., O or N). In some embodiments, the W is the 2’-OH of the sugar moiety of a nucleoside. In some embodiments, the L includes a 3’-phosphate at the 3’-terminal nucleoside. An exemplary monovalent GalNAc moiety is wherein the monovalent GalNAc is attached via the linker to the 2’-position of a nucleoside, such as adenine or guanine. These conjugate moieties can be present in contiguous nucleotides in an oligonucleotide (see, e.g., WO2024/040041). Docket Number CX10-269WO4 [0388] In some embodiments, the conjugate moiety is a trivalent GalNAc. Tri-valent N-acetylgalactosamine conjugate moieties are described in, for example, WO 2014/076196, WO 2014/207232 and WO 2014/179620. The term “trivalent GalNAc” refers to a residue comprising three N-acetylgalactosamine moieties, typically attached via a linker. Exemplary trivalent GalNAc conjugate moiety is depicted below:
Docket Number CX10-269WO4 [0389] In some embodiments, the conjugate is GalNAc targeting moiety L96. [0390] In some embodiments, the conjugate moiety comprises a reporter molecule. Examples of reporter molecules include, among others, fluorescent moieties, such as fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxy-fluorescein, 6- carboxyfluorescein or FAM), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X- rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine or TMR), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin and aminomethylcoumarin or AMCA), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDyes (e.g., IRD40, IRD 700, IRD 800). (See, e.g., “The Handbook of Fluorescent Probes and Research Products”, 9th Ed., R.P. Haugland, 2002, Molecular Probes, Inc., Eugene, Oregon) [0391] In some embodiments, the reporter moiety is a chemiluminescent moiety, for example acridinium esters, ruthenium derivatives (e.g., tris(2,2′-bipyridyl) ruthenium), and dioxetanes. [0392] In some embodiments, the conjugate moiety comprises an affinity or capture tag. Exemplary affinity or capture tag includes, among others, biotin, desthiobiotin, digoxigenin, 3-amino-3-deoxydigoxigenin, and a hapten (e.g., dinitrophenol, Alexa Fluor 40, Alexa Fluor 488, dansyl, Lucifer yellow, Oregon Green 488, fluorescein). Docket Number CX10-269WO4 [0393] In some embodiments, the conjugate moiety comprises a peptide. In some embodiments, the peptide comprises a cellular targeting peptide and/or cell penetration peptide (CPP) for enhancing cellular delivery of a conjugate modified oligonucleotide. In some embodiments, the cell penetrating peptide is attached via a linker, including a cleavable linker. Cell penetrating peptides, include among others, TAT, penetratin, MAP, transportan/TP10, VP22, polyarginine, MPG, Pep-1, pVEC, YTA2, YTA4, M918, and CADY. In some embodiments, the conjugate moiety comprises an RGD (Arg-Gly-Asp) peptide. Sequence of some penetrating peptides are described in Copolovici et al., 2014, 8(3):1972–1994 and some are provided below: CPP Peptide Peptide Sequence TAT GRKKRRQRRRPPQ t ti R IKIWF NRRMKWKK , , , g others, patent publications WO24063570, WO24044663, US2024083949, WO24026141, WO23230600, WO23219933, WO23177261, WO23178327, WO23093960, WO23086342, WO23081893, WO23069332, WO23070108, WO23034515, US2023248630, US2023053924, WO23003380, WO23277628, WO23277575, US2022378946, WO22171972, WO22162200, WO2020144233, WO22180242, WO22132520, WO22129926, WO22125673, WO22120276, WO22101193, US2023287086, US2023357334, US2023144488, and US2023048338; incorporated by reference herein. In some embodiments, the peptide can be attached using a thiol group on the 5’-phosphate of a polynucleotide or oligonucleotide. [0395] Exemplary nucleotides/nucleosides with conjugate moieties are shown below:
Docket Number CX10-269WO4 and wherein R1 is H or phosphate group, R2 is H or phosphate group, or blocking group, and R3 is H, OH, fluoro, or -O-methyl. Reactive Moiety [0396] In some embodiments, the modification comprises a reactive group that is conjugated to a nucleoside. In some embodiments, the reactive group is attached to the nucleoside via a linker. In some embodiments, the reactive group is a cyano, azido, alkynyl, amino, carboxyl, sulfhydryl, dibenzocyclooctynyl, vinyl, trans- Docket Number CX10-269WO4 cyclooctene, or tetrazine. In some embodiments, the reactive group is those used for click chemistry, including copper free click chemistry. Exemplary reactive groups are provided below: amino [0397] Other reactive groups used in click chemistry, particularly for nucleic acids, is described in Fantoni et al., Chem. Rev.2021, 121, 7122−7154, incorporated by reference herein. [0398] Exemplary single stranded RNA ligase substrates with a reactive moiety is provided below: Docket Number CX10-269WO4 wherein R1 is H or phosphate; R2 is a blocking group or H;; and R3 is H, -OR, or halo, e.g., F, Br, or Cl. Linker [0399] In some embodiments, as described above, the conjugate moiety or reactive moiety is attached to the nucleoside or the terminal group through a linker. Various linkers are known in the art for conjugating chemical groups to nucleosides and phosphate groups. [0400] In some embodiments, mixtures of linkers are used. In some embodiments, different linker types are connected to form a longer linker or linkers with branched or dendritic structure. For example, an alkylene linker is connected to a polyethylene linker through a functional group, e.g., an amide; an arylene linker is attached to an alkylene linker. As such, different combinations of linker types can be connected to provide for longer linkers and/or branched linkers, for example for attaching multiple conjugate moieties. [0401] In some embodiments, linkers include, among others, substituted or unsubstituted alkylene, heteroalkylene, alkenylene, heteroalkenylene, arylene, heteroarylene, arylalkylene, arylalkenylene, heteroarylalkylene, heteroarylalkenylene, arylheteroalkylene, arylheteroalkenylene, heteroarylheteroalkylene, Docket Number CX10-269WO4 and heteroarylalkenylene. In some embodiments, the linker comprises substituted or unsubstituted C2-C22 alkylene, heteroalkylene, or polyethylene glycol. In some embodiments, the linkers have functional groups for conjugation. [0402] In some embodiments, the linker comprises a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 50 carbon atoms, 1 to 20 carbon atoms, or 1 to 14 carbon atoms, wherein one or more of the carbon atoms in the hydrocarbon chain is optionally replaced by -O-, -NR1- , -NR1-C(=O)-, -C(=O)-NR1, or -S-, and wherein R1 is hydrogen or (C1-C6)alkyl, wherein the hydrocarbon chain, is optionally substituted with one or more (e.g.1, 2, 3, or 4) substituents selected from (C1-C6)alkoxy, (C3-C6)cycloalkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, (C1-C6)alkoxycarbonyl, (C1-C6)alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (=O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy. [0403] In some embodiments, the L is attached to the nucleoside and/or conjugate through -NH-, -O-, -S-, - (C═O)-, -(C═O)-NH-, -NH-(C=O)-, -(C=O)-O-, -NH-(C═O)-NH-, or -NH-(SO2)-. [0404] In some embodiments, the L has the structure below: , Docket Number CX10-269WO4 [0405] In some embodiments, the linker comprises a substituted or unsubstituted polyethylene glycol linker. In some embodiments, the polyethylene glycol linker has the formula: [0406] In some embodiments, n is 2-24. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. [0407] In some embodiments, the polyethylene linker has the structure below: , , [0409] In some embodiments, the linker is a cleavable linker in which the linker can be cleaved, for example to detach a conjugate moiety. Example of a cleavable linker includes, by way of example and not limitation, a disulfide linkage, enzymatically cleavable linkers (e.g., peptide linkers), and photocleavable linkers (see, e.g., Docket Number CX10-269WO4 Hermanson, G., Bioconjugate Techniques, 3rd Ed., 2013, Academic Press; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, 2nd Ed., S.S. Mark ed., 2011, Humana Press). [0410] In some embodiments, bifunctional linkers can be used to attach a conjugate moiety to the linker and attach the linker-conjugate to the nucleoside or vice versa (see, e.g., Hermanson, G., supra; see also Bioconjugation Protocols: Strategies and Methods, In Methods in Molecular Biology, supra). In some embodiments, an activating group can be attached to an atom to activate the atom to form a covalent bond with another reactive group. Examples of synthetic activating groups that can be attached to an oxygen atom include, but are not limited to, acetate, succinate, triflate, and mesylate. When an activating group is attached to an oxygen atom of a carboxylic acid, the activating group can be a group that is derivable from a known coupling reagent. Examples of such coupling reagents include, but are not limited to, N,N′- dicyclohexylcarbodimide (DCC), hydroxybenzotriazole (HOBt), N-(3-dimethylaminopropyl)-N′- ethylcarbonate (EDC), (denzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) or O-benzotriazol-1-yl- N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU). Pyrophosphatase coupled reactions and pyrophosphatases [0411] In some embodiments, the single-stranded RNA ligase reaction further includes a pyrophosphatase for degrading pyrophosphate generated in the single-stranded RNA ligase reaction. In some embodiments, the presence of a pyrophosphatase in the ligase reaction can enhance the forward reaction for attachment of the nucleotide donor to the oligonucleotide acceptor by degrading the pyrophosphate generated in the ligase reaction. [0412] In some embodiments, the pyrophosphatase is present concurrently with the single-stranded RNA ligase. In some embodiments, where the single-stranded RNA ligase is immobilized on a substrate medium, the pyrophosphatase can be free in solution, co-immobilized with the single-stranded RNA ligase on the substrate medium, or immobilized on a substrate medium separately from the single-stranded RNA ligase. In some embodiments, the pyrophosphatase and the single-stranded ligase can be prepared as a fusion protein, and in some embodiments, immobilized on a support medium. [0413] In some embodiments, a pyrophosphatase can also be used in conjunction with the attachment of a nucleotide substrate to the oligonucleotide using a terminal nucleotidyl transferase. In some embodiments, the pyrophosphatase can be free in solution in the terminal nucleotidyl transferase reaction, co-immobilized with the terminal nucleotidyl transferase on the substrate medium, or immobilized on a substrate medium separately from the terminal nucleotidyl transferase. [0414] In some embodiments, a wide variety of pyrophosphatases can be adapted for the coupled reactions. In some embodiments, the pyrophosphatase is a Type 1 group of pyrophosphatases. In some embodiments, the pyrophosphatase is a Type II group of pyrophosphatases. In some embodiments, pyrophosphatases useful in coupled reactions with single-stranded RNA ligase or terminal nucleotidyl transferase reaction are disclosed in U.S. provisional application titled “Uses of Type II Inorganic Pyrophosphatases, filed April 16, 2024, incorporated by reference herein in its entirety. Docket Number CX10-269WO4 ATP Regeneration Systems [0415] In some embodiments, the RNA ligase reaction further includes a nucleotide substrate regeneration system to regenerate ATP used as a co-factor in the RNA ligase reaction. In some embodiments, the ATP regeneration system is used to convert product AMP to ATP. In some embodiments, the regeneration of ATP is used to increase ligated product formation (e.g., product yield) in the single stranded RNA ligase reaction. [0416] In some embodiments, the ATP recycling or regeneration system includes a nucleoside monophosphate kinase for converting AMP to ADP in presence of a phosphate donor. Various nucleoside monophosphate kinases can be used for the conversion of AMP to ADP, including various homologs of nucleoside monophosphate kinases. In some embodiments, more than one nucleoside monophosphate kinase can be used in the regeneration system. In some embodiments, the nucleoside monophosphate kinase is an adenosine monophosphate kinase (e.g., adenylate kinase), cytidine monophosphate (CMP) kinase, uridine monophosphate (UMP) kinase, and/or guanylate-monophosphate (GMP) kinase. [0417] In some embodiments, a nucleoside monophosphate kinase useful in the ATP regeneration reaction is a cytidine monophosphate kinase. Various suitable cytidine monophosphate kinases are known in the art. These include homologs of cytidine monophosphate kinases. In some embodiments, a cytidine monophosphate kinase useful in the regeneration reactions include, among others, the cytidine monophosphate kinase of Thermus thermophilus (Q5SL35), Pyrococcus furiosus (Q8U2L4), Pseudomonas putida (AFO48857.1), Escherichia coli K-12 MG1655 (P0A6I0), Clostridium acetobutylicum (Q97I08), Halobacterium salinarum (Q9HPA5) Bacillus acidicola (WP_066270173), Acetobacter aceti (WP_010667744), Acidithiobacillus thiooxidans (WP_024892761.1), Acidithiobacillus ferrooxidans (WP_064220349.1), Metallosphaera sedula (WP_011921264.1), Amphibacillus xylanus (WP_015009966.1) Thioalkalivibrio denitrificans (WP_077278466.1), Vibrio psychroerythus (Q482G4), Pseudoalteromonas haloplanktis (Q3ILA1), Psychrobacter arcticus (Q4FRL5), Psychromonas ingrahamii (A1SZ01), Pseudomonas syringae (xQ4ZQ97) and Halobacterium salinarum (Q9HPA5). [0418] In some embodiments, a nucleoside monophosphate kinase useful in the ATP regeneration reactions is a uridine monophosphate kinase. Various suitable uridine monophosphate kinases are known in the art. These include homologs of uridine monophosphate kinases. In some embodiments, a uridine monophosphate kinase useful in the regeneration reactions includes, among others, the uridine monophosphate kinase of Pyrococcus furiosus (Q8U122), Thermus thermophilus (P43891), Pseudomonas putida (I7BW46), Escherichia coli K-12 MG1655 (P0A7E9), Aspergillus niger (A2R195), Saccharomyces cerevisiae (P15700), Clostridium acetobutylicum (Q97I64) ATCC 824 PyrH Halobacterium salinarum (Q9HNN8), Picrophilus torridus (WP_048059653), Metallosphaera sedula (WP_012021705), Thermoplasma acidophilum (WP_010900913), Sulfolobus solfataricus (WP_009992427), Acetobacter aceti (WP_042788648), Thioalkalivibrio sp. HK1 (WP_081759172.1), Amphibacillus xylanus (WP_015010200.1), Vibrio psychroerythus (Q485G8), Pseudoalteromonas haloplanktis (Q3IIX6), Psychrobacter arcticus (Q4FRH5), Psychromonas ingrahamii (ABM04676.1), Pseudomonas syringae (Q4ZWS6), and Halobacterium salinarum (Q9HNN8). [0419] In some embodiments, the nucleoside monophosphate kinase useful in the ATP regeneration reactions is a guanosine monophosphate kinase (guanylate kinase). Various suitable guanylate kinases are known in the Docket Number CX10-269WO4 art. These include homologs of guanylate kinases. In some embodiments, a guanylate kinase useful in the regeneration reactions includes, among others, the guanylate kinase of Thermotoga maritima (Q9X215), Thermus thermophilus (Q5SI18), Pseudomonas putida (I7C087), Escherichia coli K-12 (P60546), Aspergillus niger (A2QPV2), Saccharomyces cerevisiae (P15454), Clostridium acetobutylicum (Q97ID0), Acidithiobacillus ferrooxidans (WP_064219869.1), Acidithiobacillus thiooxidans (WP_010637919.1), Bacillus acidicola (WP_066264774.1), Acetobacter aceti (WP_018308252.1), Amphibacillus xylanus (WP_015010280.1), Thioalkalivibrio sulfidiphilus (WP_018953989.1), Vibrio psychroerythus (Q47UB3), Pseudoalteromonas haloplanktis (Q3IJH8), Psychrobacter arcticus (Q4FQY7), Psychromonas ingrahamii (A1T0P1), and Pseudomonas syringae (Q4ZZY8). [0420] In some embodiments, the nucleoside monophosphate kinase useful in the ATP regeneration reactions is an adenosine monophosphate kinase (adenylate kinase). Various suitable adenylate kinases are known in the art. These include homologs of adenylate kinases. In some embodiments, the adenylate kinase is a bacterial, fungal, plant, or animal adenylate kinase. In some embodiments, an adenylate kinase useful in the regeneration reactions includes, among others, adenylate kinases of Thermus thermophilus (Q72125), Pyrococcus furiosus (Q8U207), Pseudomonas putida (17CAA9), Escherichia coli K - 12 W3110 (P69441), Aspergillus niger CBS 513.88 (A2QPN9), Saccharomyces cerevisiae (P07170), Clostridium acetobutylicum (Q97E39), Halobacterium salinarum (Q9HPAT), Acidithiobacillus thiooxidans (WP_024894015.1), Acidithiobacillus ferrooxidans (WP_064218420.1), Bacillus acidicola (WP_066267988.1), Sulfolobus solfataricus (WP_009991241.1), Saccharomyces cerevisiae (P07170), Thermotoga neapolitana (Q8GGL2), Escherichia coli (P69441) and Geobacillus stearothermophilus (WP_049624206.1). In some embodiments, the adenylate kinase is an engineered adenylate kinase described in International patent application No. PCT/US2024/051084, filed October 11, 2024, incorporated herein by reference. [0421] In some embodiments, the ATP regeneration system includes at least an enzyme and a phosphate donor for the conversion of ADP to ATP. In some embodiments, the ATP regeneration system includes, among others, an acetate kinase, adenylate kinase, pyruvate kinase, creatine kinase, or polyphosphate kinase (see, e.g., Endo et al., Adv. Synth. Catal., 2002, 343:521–526; Andexer et al., Chem Bio Chem., 2015, 16:380–386). [0422] In the ATP regeneration system, the phosphate donor for the conversion of ADP to ATP is selected based on the ATP regenerating enzyme employed. By way of example and not limitation, if acetate kinase enzyme is used for conversion of ADP to ATP, the phosphate donor is acetyl-phosphate. If pyruvate kinase is used for the conversion of ADP to ATP, the phosphate donor is phosphoenolpyruvate. If creatine kinase is used for the conversion of ADP to ATP, the phosphate donor is creatine phosphate. If polyphosphate kinase is used for the conversion of ADP to ATP, the phosphate donor is inorganic polyphosphate. [0423] In some embodiments, the ATP regenerating system includes pyruvate kinase and phosphoenolpyruvate. In some embodiments, the ATP regenerating system includes creatine kinase and creatine phosphate. In some embodiments, the ATP regenerating system includes polyphosphate kinase and inorganic polyphosphate. In some embodiments, the ATP regenerating system includes acetate kinase and acetyl phosphate. Docket Number CX10-269WO4 [0424] In some embodiments, the ATP regenerating system includes acetate kinase and acetyl phosphate, where the acetate kinase is an acetate kinase of Escherichia coli str. K-12 substr. MG1655 (NP_416799.1), Corynebacterium jeikeium K411 (WP_011272972.1), Lactococcus cremoris subsp. cremoris KW2 (WP_011835968.1), Lactococcus lactis (WP_004254593.1), Marinitoga sp.38H-ov (WP_165147355.1), Thermotoga sp. KOL6 (WP_101510533.1), Thermosipho melaniensis (WP_012057479.1), Thermotoga sp. RQ7 (WP_041844042.1), and Thermosipho africanus (WP_004102380.1). In some embodiments, the acetate kinase is an engineered acetate kinase described in, among others, International patent application No. PCT/US2024/051118, filed October 11, 2024, incorporated by reference herein. Single-stranded RNA (ssRNA) Ligases, Polynucleotides Encoding the Ligases, and Host Cells Single-stranded RNA (ssRNA) ligase polypeptides [0425] In some embodiments, the ligation reactions are mediated by a single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase comprises a viral single-stranded RNA ligase, a bacterial single-stranded RNA ligase, a fungal single-stranded RNA ligase, or a mammalian single-stranded RNA ligase. [0426] In some embodiments, the single-stranded ligase is a bacteriophage single-stranded RNA ligase. In some embodiments, the bacteriophage single-stranded RNA ligase is single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Phage TS2126, or Rhodothermus phage RM378. [0427] In some embodiments, the single-stranded RNA ligase is a bacterial single-stranded RNA ligase. In some embodiments, the single-stranded RNA ligase is an archael single-stranded RNA ligase (see, e.g., Nucleic Acids Res.2008 Nov; 36(19): 6218–6227). In some embodiments, the single-stranded RNA ligase is a Methanobacterium or Thermococcus single-stranded RNA ligase. [0428] In some embodiments, exemplary bacterial single-stranded RNA ligase is a single-stranded RNA ligase of Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, or Thermovibrio ammonificans HB-1. [0429] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. [0430] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, or to a reference sequence corresponding to SEQ ID NO: 2. Docket Number CX10-269WO4 [0431] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 4. [0432] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 6, or to a reference sequence corresponding to SEQ ID NO: 6. [0433] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 8, or to a reference sequence corresponding to SEQ ID NO: 8. [0434] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 4, or to a reference sequence corresponding to SEQ ID NO: 10. [0435] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 12, or to a reference sequence corresponding to SEQ ID NO: 12. [0436] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 14, or to a reference sequence corresponding to SEQ ID NO: 14. [0437] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 16, or to a reference sequence corresponding to SEQ ID NO: 16. [0438] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Docket Number CX10-269WO4 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 18, or to a reference sequence corresponding to SEQ ID NO: 18. [0439] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 20, or to a reference sequence corresponding to SEQ ID NO: 20. [0440] In some embodiments, the single-stranded RNA ligase comprises one or more amino acid differences relative to the reference sequence corresponding to a sequence from amino acid residues 12 to the carboxyl terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 2-20. In some embodiments, the amino acid differences are based on alignment of the amino acid sequence of the naturally occurring single-stranded RNA ligases, and changing the amino acid residue of one sequence to the different amino acid residue present in the amino acid sequence of another naturally occurring single-stranded RNA ligase sequence, thereby generating an amino acid difference relative to the parent amino acid sequence. [0441] In some embodiments, the recombinant single-stranded RNA ligase comprises an amino acid sequence comprising amino acid residues 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprising SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. [0442] In some embodiments, the single-stranded RNA ligase comprise variants of single-stranded RNA ligase of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1. [0443] In some embodiments, the single-stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. In some embodiments, the sequence corresponding to amino acid residues 12 to the carboxy terminal residue is amino acid residues 12 to 387 of the referenced SEQ ID NO. [0444] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0445] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0446] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0447] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0448] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0449] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 123G/S, 125Q/R/T/V, 127I/L/P/Q/V, 130I, 135I/M, 138A, 139R, 141A/D/G/P/R, 144T, 145Y, 146I, 151E/L, 152D, 156A/S, 157L, 160R, 161V, 162S/W, 165F/K/M/P/T, 166H/P/S, 167I/V, 168I/S, 170R, 171P/R/S, 172P, 173E/N, 174P, 177C/I/L/V, 181V, 185E, 190L, 195T, 196D, 197E/T, 198A, 199L/T, 203V, 204E, 205M/T/V, 207E, 212H/S, 213V, 214A/C/E/S, 217G/P, 220A/P/R/S/V, 221P, 222F/G, 223A/G/N/S/T, 224P, 225G/H/P/R/T, 226A/P/Q/T, 229A/L, 230L, 231I, 236H, 237A/G/R, 238G, 240V/W, 246F/I, 248F/H/M, 252R, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 260I/L/R/T/V, 263D/S, 268A/E/R, 269L, 269F/L, 271G/H/M, 272G/L/R/S, 273A/L/S/Y, 274I, 275E/W, 276A/G/H, 277T, 278L/P/R, 279Q/R, 280S, 281A/I/L/M/V/W, 282L, 283K/L/Q/T, 284F/L, 285A/G/P/R/S, 286P/Q, 288T, 289Q, 291L, 295A/R/T, 297C, 299I/R, 302I, 303A/G, 306F/M, 310A/D/G, 311C/F/G/I, 314E/G/L/P/S, 316R, 320A/G, 323D/E/G/N/T/Y, 324T, 325M/P, 326G/M/T/W, 330H/I, 332G/L, 333E, 334I/N/S, 336C/E/N, 337E/K, 340C, 341A/P/S, 343D/P, 345G/T, 346G/P, 347A/E/G/I/L/M/P/R/S/T, 348A/L/S/T, 349S/V, 350A/C/R, 351F/N/Y, 352G/L/M/N/R/S/V, 353T/V, 354E/H/Q/S/V, 355K, 356L/M/V, 357L/M, 358C/D/G/P/R/T/V, 359D/I, 360L/V, 361I, 362A/E/K/Q/R, 363S/V, 365N, 366W, 368G, 369C/L/V, 371R/S, 372A/L/P, 374F/I/L/V, 376E/T, 377L, 380S, 381K/R, or 384C/S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0450] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V31I, L32M, E33R, R34S/Y, N36D, V38M/T/V, V40L, S41V, Y42A, R43G/T, Y44R, N45Y, A46R, F48W, V49I, F50W, L54T, R56L, V84L, R91D, L92I, R93H/N, G94D, P109G/S, N113G, K115E, L118F/M, Q121K, K122C/I/L/M/S/T/V/Y, G123S, S125Q/R/T/V, P127L/Q/V, E130I, L135I, K138A, N141D, T146I, N151E, K152D, P156A/S, L160R, I161V, S162W, F165F/K/M/P/T, Q166H/P/S, L167I/V, V168I/S, P170R, Y171P/R/S, K173E/N, D174P, F177I/L, I181V, K185E, N196D, K199L, I203V, K204E, I205M/T/V, R207E, R212S, L213V, K214E, L217G/P, I220A/P/R/S/V, E221P, N222F/G, R223A/G/N/S, E224P, S225G/H/P/R/T, I226A/P/Q/T, F229A/L, V230L, L231I, K236H, K237G, E238G, F240V, V246F/I, S248F/H/M, V252R, H254A/D/R/S, F255A/H/M/Q/V/W, Y256L/M/R/V, F258L, T259A/S, Y260I/L/R/T/V, N263D/S, K268A/E/R, F269F/L, N271G/H/M, I272G/L/S, Docket Number CX10-269WO4 F273A/L/S/Y, Q275E, G276A/H, R277T, V278L/P/R, D279Q/R, D280S, I281A/L/V/W, F282L, S283K/L/Q/T, R284L, V285A/G/P/R/S, T286P/Q, E295A/R/T, R297C, K299R, T302I, N303A/G, S310G, L311C/F/G/I, K314G/L/P/S, G320A, K323D/E/T, F324T, E326G/M/T/W, R330H/I, V332L, K333E, R336C, K337E, R341S, F343D/P, K345G/T, L346G/P, K347A/E/G/I/L/M/P/R/S/T, I348A/L/S/T, Q349S, L353T, K354E/H/S/V, A356L/M, V357M, R358C/D/G/P/R/T/V, A360L, K361I, T362A/E/R, G363S, K365N, Y369L, or T380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0451] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0452] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 225G/H/P/R/T, 236H, 237A/G/R, 238G, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 269F/L, 271G/H/M, 272G/L/R/S, 275E/W, 276A/G/H, 281A/I/L/M/V/W, 289Q, 316R, 320A/G, 337E/K, 351Y, 354E/H/S/V, 357L, 358C/D/G/P/R/T/V, 359D/I, 362A/E/R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0453] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N/S/Y, 35L, 35S, 38T/V, 45Y, 48W, 49I, 54T, 66T, 90E/S, 93P, 115S, 118L/M, 121K, 122L/S, 127I/L/V, 162W, 165K, 167V, 170R, 177V, 197E/T, 205M, 213V, 220A/R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A/F/Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A/G, 337E, 351Y, 354E, 357L, 358G, 359D/I, 362E, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0454] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue R34Y, V38T/V, N45Y, F48W, V49I, L54T, L118M, Q121K, G123S, P127L, S162W, F165K, L167V, I205M, L213V, I220A/R, N222G, R223G, S225G, K236H, K237G, H255F?Q, Y256R, F258L, T259S, F269L, I272G, G276H, I281L, G320A/G, K337E, K354E, R358G, T362E, or K365N, or any combinations thereof, wherein the amino acid positions are relative to the Docket Number CX10-269WO4 reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0455] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 121K, 45Y, 41V, 34Y, 269L, or 380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Q121K, N45Y, S41V, R34Y, F269L, or T380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34/45/269, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34Y/45Y/269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set or amino acid residues R34Y/N45Y/F269L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0456] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 162, 236, 237, 320, 337, 358, or 362, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 162W, 236H, 237G, 320A, 337E, 358G, or 362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S162W, K236H, K237G, G320A, K337E, R358G, or T362E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, Docket Number CX10-269WO4 the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 162/236/237/320/337/358/362, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 162W/236H/237G/320A/337E/358G/362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues S162W/K236H/K237G/G320A/K337E/R358G/T362E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0457] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 123, 256, or 320, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 123S, 256R, or 320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue G123S, Y256R, or A320G, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 123/256/320, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 123S/256R/320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues G123S/Y256R/A320G, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0458] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 48, 49, 118, or 220, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: Docket Number CX10-269WO4 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38V, 48W, 49I, 118M, or 220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue T38V, F48W, V49I, L118M, or R220A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38/48/49/118/220, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38V/48W/49I/118M/220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues T38V/F48W/V49I/L118M/R220A, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0459] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 38, 54, 205, or 258, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 38T, 54T, 205M, or 258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue V38T, L54T, I205M, or F258L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 38/54/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 38T/54T/205M/258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence Docket Number CX10-269WO4 corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues V38T/L54T/I205M/F258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0460] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 222, 223, 225, 255, 272, or 276, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127L, 222G, 223G, 225G, 255Q, 272G, or 276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue P127L, N222G, R223G, S225G, F255Q, I272G, or G276H, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127/222/223/225/255/272/276, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127L/222G/223G/225G/255Q/272G/276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues P127L/N222G/R223G/S225G/F255Q/I272G/G276H, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0461] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 165, 259, or 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 165K, 259S, or 281L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue F165K, T259S, or I281L, or any combinations thereof, wherein the Docket Number CX10-269WO4 amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 165/259/281, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 165K/259S/281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues F165K/T259S/I281L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0462] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 127, 238, 255, 359, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 127V, 238G, 255A, 359D, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, E238G, Q255A, I359D, or K381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 127/238/255/359/381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 127V/238G/255A/359D/381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues L127V/E238G/Q255A/I359D/K381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0463] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 35, 170, 271, or 357, or any combinations thereof, Docket Number CX10-269WO4 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 35L, 170R, 271G, or 357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue E35L, P170R, N271G, or V357L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 35/170/271/357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 35L/170R/271G/357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues E35L/P170R/N271G/V357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0464] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 34, 35, 118, 127, 275, 351, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 34S, 35S, 118L, 127I, 275W, 351Y, 374L, or 376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue L127V, N34S, L35S, M118L, V127I, Q275W, N351Y, F374L, or T376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 34/35/118/127/275/351/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of Docket Number CX10-269WO4 the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 34S/35S/118L/127I/275W/351Y/374L/376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues N34S/L35S/M118L/V127I/Q275W/N351Y/F374L/T376E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0465] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 66, 90, 93, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 66T, 90S, 93P, or 197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue A19V, K66T, E90S, R93P, or E197T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 19/66/90/93/197, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 19V/66T/90S/93P/197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues A19V/K66T/E90S/R93P/E197T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0466] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 90, 197, 289, or 316, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 90E, 197E, 289Q, or 316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy Docket Number CX10-269WO4 terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue S90E, T197E, N289Q, or S316R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least a substitution set at amino acid positions 90/197 and/or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least the substitution set, or amino acid residues 90E/197E and/or 289Q/316R wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. In some embodiments, the amino acid sequence of the single stranded RNA ligase comprises at least at least the substitution set, or amino acid residues S90E/T197E and/or N289Q/S316R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0467] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0468] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0469] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0470] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, Docket Number CX10-269WO4 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0471] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence having a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0472] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0473] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0474] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, Docket Number CX10-269WO4 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0475] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 123G/S, 125Q/R/T/V, 127I/L/P/Q/V, 130I, 135I/M, 138A, 139R, 141A/D/G/P/R, 144T, 145Y, 146I, 151E/L, 152D, 156A/S, 157L, 160R, 161V, 162S/W, 165F/K/M/P/T, 166H/P/S, 167I/V, 168I/S, 170R, 171P/R/S, 172P, 173E/N, 174P, 177C/I/L/V, 181V, 185E, 190L, 195T, 196D, 197E/T, 198A, 199L/T, 203V, 204E, 205M/T/V, 207E, 212H/S, 213V, 214A/C/E/S, 217G/P, 220A/P/R/S/V, 221P, 222F/G, 223A/G/N/S/T, 224P, 225G/H/P/R/T, 226A/P/Q/T, 229A/L, 230L, 231I, 236H, 237A/G/R, 238G, 240V/W, 246F/I, 248F/H/M, 252R, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 260I/L/R/T/V, 263D/S, 268A/E/R, 269L, 269F/L, 271G/H/M, 272G/L/R/S, 273A/L/S/Y, 274I, 275E/W, 276A/G/H, 277T, 278L/P/R, 279Q/R, 280S, 281A/I/L/M/V/W, 282L, 283K/L/Q/T, 284F/L, 285A/G/P/R/S, 286P/Q, 288T, 289Q, 291L, 295A/R/T, 297C, 299I/R, 302I, 303A/G, 306F/M, 310A/D/G, 311C/F/G/I, 314E/G/L/P/S, 316R, 320A/G, 323D/E/G/N/T/Y, 324T, 325M/P, 326G/M/T/W, 330H/I, 332G/L, 333E, 334I/N/S, 336C/E/N, 337E/K, 340C, 341A/P/S, 343D/P, 345G/T, 346G/P, 347A/E/G/I/L/M/P/R/S/T, 348A/L/S/T, 349S/V, 350A/C/R, 351F/N/Y, 352G/L/M/N/R/S/V, 353T/V, 354E/H/Q/S/V, 355K, 356L/M/V, 357L/M, 358C/D/G/P/R/T/V, 359D/I, 360L/V, 361I, 362A/E/K/Q/R, 363S/V, 365N, 366W, 368G, 369C/L/V, 371R/S, 372A/L/P, 374F/I/L/V, 376E/T, 377L, 380S, 381K/R, or 384C/S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0476] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0477] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 225G/H/P/R/T, 236H, 237A/G/R, 238G, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 269F/L, 271G/H/M, 272G/L/R/S, 275E/W, 276A/G/H, 281A/I/L/M/V/W, 289Q, 316R, 320A/G, 337E/K, 351Y, 354E/H/S/V, 357L, 358C/D/G/P/R/T/V, 359D/I, 362A/E/R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0478] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34N/S/Y, 35L, 35S, 38T/V, 45Y, 48W, 49I, 54T, 66T, 90E/S, 93P, 115S, 118L/M, 121K, 122L/S, 127I/L/V, 162W, 165K, 167V, 170R, 177V, 197E/T, 205M, 213V, 220A/R, 222G, 223G, 225G, 236H, 237G, 238G, 254R, 255A/F/Q, 256R, 258L, 259S, 269L, 271G, 272G, 275W, 276H, 281L, 289Q, 316R, 320A/G, 337E, 351Y, 354E, 357L, 358G, 359D/I, 362E, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0479] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0480] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 44- 64, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 44-64, Docket Number CX10-269WO4 wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0481] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0482] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 34Y/45Y/269L, 34Y/45Y/173E/297C, 34Y/173E/269L/380S, 173E/269L, 156S/269L/380S, 173E/269L/380S, 34Y/173E, 269L, 34Y/380S, 34Y/269L/380S, or 173E/380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0483] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R34Y/N45Y/F269L, R34Y/N45Y/K173E/R297C, R34Y/K173E/F269L/T380S, K173E/F269L, P156S/F269L/T380S, K173E/F269L/T380S, R34Y/K173E, F269L, R34Y/T380S, R34Y/F269L/T380S, or K173E/T380S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0484] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or to the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0485] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 66- 118, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 66-118, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0486] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, Docket Number CX10-269WO4 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0487] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 207E, 237G, 94D/263D, 220V, 236H, 92I, 91D, 94D, 204E, 185E, 213V, 199L, 152D, 196D, 203V, 141D, 138A, 156A, 93N, or 181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0488] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) R207E, K237G, G94D/N263D, I220V, K236H, L92I, R91D, G94D, K204E, K185E, L213V, K199L, K152D, N196D, I203V, N141D, K138A, P156A, R93N, or I181V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0489] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or to the reference sequence corresponding to SEQ ID NO: 100, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0490] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 120- 216, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 120-216, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0491] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0492] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 283L/337E, 347E, 323D, 354V, Docket Number CX10-269WO4 343D, 347P, 118M, 347G, 345G, 347M, 354E, 314G, 345T, 268R/269F, 363S, 356M, 358P, 343P, 358G, 348S, 348L, 358T, 314S, 162W, 324T/330H, 354S, 347L, 346P, 323T, 348T, 160R, 362E, 361I, 341S/349S, 354H, 358D, 347S, 353T, 314P, 369L, 248H, 146I/346G, 332L, 346G, 314L, 170R, 348A, 269F/275E, or 268E/269F, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0493] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) S283L/K337E, K347E, K323D, K354V, F343D, K347P, L118M, K347G, K345G, K347M, K354E, K314G, K345T, K268R/L269F, G363S, A356M, R358P, F343P, R358G, I348S, I348L, R358T, K314S, S162W, F324T/R330H, K354S, K347L, L346P, K323T, I348T, L160R, T362E, K361I, R341S/Q349S, K354H, R358D, K347S, L353T, K314P, Y369L, S248H, T146I/L346G, V332L, L346G, K314L, P170R, I348A, L269F/Q275E, or K268E/L269F, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0494] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or to the reference sequence corresponding to SEQ ID NO: 140, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0495] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 244- 398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 244-398, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0496] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237/323, 151/205/314, 151/212/345/347, 92/214/347/358, 162/204/205/310/314/358, 236/314, 151/345/347, 214/347/358, 151/212/358, 204/283/314/358, 236/237/358/362, 151/199/204/231/236/323, Docket Number CX10-269WO4 231/236/237/358/362, 162/314/358, 92/151/347/358, 151/236, 212/345/347, 115/314, 345/347/358, 314, 358/362, 115/358, 310/314, 214/347/358, 237/314, 345/347, 151/310/323/343/347, 151/358, 347/358, 93/358/362, 231/236/237/320/358/362, 151/230/345/347/358, 135/231/236/237/358, 310/314/358/362, 151/230/347/358, 231/237/323, 135/358/362, 283/314/358/362, 199/205, 92/151/230/345/347/358, 314/358/362, 199/237, or 199/204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0497] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 162W/337E/358C/362E, 162W/236H/237G/320A/337E/358G/362E, 151E/231I/237G/337E, 199L/231I/237G/337E, 231I/237G/314L/337E, 310G/314L/337E, 115E/162W/310G/314S, 199L/237G/337E, 151E/199L/205V/310G/314L, 199L/204E/205V/231I/236H/310G/314L, 320A/337E/358T/362E, 135I/320A/337E/358T/362E, 151E/212S/214E/345T/347L/358D, 135I/337E/358P/362E, 162W/358T/362E, 337E/358C/362E, 337E/358G, 92I/337E, 151E/196D/199L/205V/231I/237G/323E, 151E/214E/347L/358D, 337E, 337E/358P/362E, 162W/358G/362E, 151E/345T/347A/358T, 199L/314L, 199L/205V/231I/237G/323E, 151E/345T/347A/358D, 151E/205V/314L, 151E/212S/345T/347L, 92I/214E/347T/358D, 162W/204E/205V/310G/314S/358T, 337E/358C, 236H/314L, 151E/345T/347P, 337E/358T, 151E/345T/347I, 214E/347P/358D, 151E/212S/358D, 204E/283L/314S/358T, 236H/237G/358T/362E, 151E/199L/204E/231I/236H/323E, 231I/236H/237G/358T/362E, 162W/314G/358T, 92I/151E/347I/358D, 151E/236H, 212S/345T/347I, 115E/314S, 345T/347P/358D, 314L, 358T/362E, 115E/358T, 236H/237G/358P/362E, 162W/358D/362E, 310G/314L, 214E/347I/358D, 237G/314L, 345T/347T, 151E/310G/323E/343D/347T, 151E/358D, 345T/347A/358D, 347A/358D, 93H/358C/362E, 231I/236H/237G/320A/358T/362E, 151E/230L/345T/347A/358D, 135I/231I/236H/237G/358C, 310G/314S/358D/362E, 151E/230L/347A/358D, 231I/237G/323E, 135I/358T/362E, 347I/358D, 283L/314S/358D/362E, 199L/205V, 214E/347R/358D, 92I/151E/230L/345T/347L/358D, 314S/358D/362E, 310G/314S, 199L/237G, or 199L/204E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0498] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) S162W/K337E/R358C/T362E, S162W/K236H/K237G/G320A/K337E/R358G/T362E, N151E/L231I/K237G/K337E, K199L/L231I/K237G/K337E, L231I/K237G/K314L/K337E, S310G/K314L/K337E, K115E/S162W/S310G/K314S, K199L/K237G/K337E, N151E/K199L/I205V/S310G/K314L, K199L/K204E/I205V/L231I/K236H/S310G/K314L, G320A/K337E/R358T/T362E, L135I/G320A/K337E/R358T/T362E, N151E/R212S/K214E/K345T/K347L/R358D, L135I/K337E/R358P/T362E, S162W/R358T/T362E, K337E/R358C/T362E, K337E/R358G, L92I/K337E, N151E/N196D/K199L/I205V/L231I/K237G/K323E, N151E/K214E/K347L/R358D, K337E, K337E/R358P/T362E, S162W/R358G/T362E, N151E/K345T/K347A/R358T, K199L/K314L, K199L/I205V/L231I/K237G/K323E, N151E/K345T/K347A/R358D, N151E/I205V/K314L, Docket Number CX10-269WO4 N151E/R212S/K345T/K347L, L92I/K214E/K347T/R358D, S162W/K204E/I205V/S310G/K314S/R358T, K337E/R358C, K236H/K314L, N151E/K345T/K347P, K337E/R358T, N151E/K345T/K347I, K214E/K347P/R358D, N151E/R212S/R358D, K204E/S283L/K314S/R358T, K236H/K237G/R358T/T362E, N151E/K199L/K204E/L231I/K236H/K323E, L231I/K236H/K237G/R358T/T362E, S162W/K314G/R358T, L92I/N151E/K347I/R358D, N151E/K236H, R212S/K345T/K347I, K115E/K314S, K345T/K347P/R358D, K314L, R358T/T362E, K115E/R358T, K236H/K237G/R358P/T362E, S162W/R358D/T362E, S310G/K314L, K214E/K347I/R358D, K237G/K314L, K345T/K347T, N151E/S310G/K323E/F343D/K347T, N151E/R358D, K345T/K347A/R358D, K347A/R358D, R93H/R358C/T362E, L231I/K236H/K237G/G320A/R358T/T362E, N151E/V230L/K345T/K347A/R358D, L135I/L231I/K236H/K237G/R358C, S310G/K314S/R358D/T362E, N151E/V230L/K347A/R358D, L231I/K237G/K323E, L135I/R358T/T362E, K347I/R358D, S283L/K314S/R358D/T362E, K199L/I205V, K214E/K347R/R358D, L92I/N151E/V230L/K345T/K347L/R358D, K314S/R358D/T362E, S310G/K314S, K199L/K237G, or K199L/K204E, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0499] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or to the reference sequence corresponding to SEQ ID NO: 246, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0500] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 400- 490, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400-490, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0501] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0502] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 255F, 255M, 109G, 256R, 260I, Docket Number CX10-269WO4 260R, 273Y, 260V, 46R, 252R, 161V/162S, 311I/320G, 123S, 320G/326G, 174P, 256M, 162S/167V, 32M, 125V, 311C/320G, 162S/166H, 320G, 320G/326M, 260L, 256V, 283Q, 330I, 278L, 311F/320G, 320G/326T, 303A, 125T, 283T, 320G/326W, 281A, 333E/337K, 281W, 283K, 277T, 260T, 254R, 278R, 311G/320G, 255W, 256L, or 173N, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0503] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) H255F, H255M, P109G, Y256R, Y260I, Y260R, F273Y, Y260V, A46R, V252R, I161V/W162S, L311I/A320G, G123S, A320G/E326G, D174P, Y256M, W162S/L167V, L32M, S125V, L311C/A320G, W162S/Q166H, A320G, A320G/E326M, Y260L, Y256V, S283Q, R330I, V278L, L311F/A320G, A320G/E326T, N303A, S125T, S283T, A320G/E326W, I281A, K333E/E337K, I281W, S283K, R277T, Y260T, H254R, V278R, L311G/A320G, H255W, Y256L, or K173N, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0504] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0505] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 492- 510, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 492-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0506] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0507] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 123S/256R/320G, 260I, 256R/260I, 256R, 260I/281V, 320G, 123S/260I, 123S/320G, 256R/281V, or 260I/273Y, wherein the amino Docket Number CX10-269WO4 acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0508] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G123S/Y256R/A320G, Y260I, Y256R/Y260I, Y256R, Y260I/I281V, A320G, G123S/Y260I, G123S/A320G, Y256R/I281V, or Y260I/F273Y, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0509] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or to the reference sequence corresponding to SEQ ID NO: 492, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0510] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 512- 584, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 512-584, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0511] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0512] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 217P, 220A, 221P, 220S, 220R, 220P, 229L, 246F, 171S, 285R, 217G, 285G, 286Q, 165P, 285P, 226A, 168S, 177L, 226P, 285A, 223N, 224P, 118M/123G, 285S, 248M, 268A, 286P, 229A, 225P, 171P, 165T, 171R, 226Q, 84L, 226T, 284L, or 225T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0513] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L217P, I220A, E221P, I220S, Docket Number CX10-269WO4 I220R, I220P, F229L, V246F, Y171S, V285R, L217G, V285G, T286Q, F165P, V285P, I226A, V168S, F177L, I226P, V285A, R223N, E224P, L118M/S123G, V285S, S248M, K268A, T286P, F229A, S225P, Y171P, F165T, Y171R, I226Q, V84L, I226T, R284L, or S225T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0514] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0515] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 586- 602, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 586-602, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0516] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0517] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 254R, 240V, 118M, 347P, 167V, 281V, 303G, 205T, or 50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0518] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue H254R, F240V, L118M, K347P, L167V, I281V, N303G, I205T, or F50W, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0519] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Docket Number CX10-269WO4 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or to the reference sequence corresponding to SEQ ID NO: 594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0520] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 604- 700, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 604-700, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0521] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 49/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0522] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 118M/220A, 118M/220A/303G/K347L, 347L, 118M/220A/254S/347L, 220A, 38V/220A, 220A/254S/303G/347L, 220A/254S, 220A/303G/347L, 48W/220A/347L, 49I/220A, 217G/220A/347L, 38V/220A/254S, 49I/118M/220A/254S/347L, 49I/220A/254S, 38V/48W/49I/118M/220A, 49I/217G/220A/254S, 49I/220A/347L, 49I/347L, 225G, 280S, 118F, 279R, 165K, 273A, 272S, 273S, 166P, 281A, 248F/357M, 222F, 223S, 281L, 358R, 271H, 168I, 276A, 225R, 36D, 34S, 40L, 263S, 130I, 225H, 356L, 273L, 259S, 358V, or 167I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0523] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) L118M/R220A, L118M/R220A/N303G/K347L, K347L, L118M/R220A/H254S/K347L, R220A, T38V/R220A, R220A/H254S/N303G/K347L, R220A/H254S, R220A/N303G/K347L, F48W/R220A/K347L, V49I/R220A, L217G/R220A/K347L, T38V/R220A/H254S, V49I/L118M/R220A/H254S/K347L, V49I/R220A/H254S, T38V/F48W/V49I/L118M/R220A, V49I/L217G/R220A/H254S, V49I/R220A/K347L, V49I/K347L, S225G, D280S, L118F, D279R, F165K, F273A, I272S, F273S, Q166P, I281A, S248F/V357M, N222F, R223S, Docket Number CX10-269WO4 I281L, G358R, N271H, V168I, G276A, S225R, N36D, Y34S, V40L, N263S, E130I, S225H, A356L, F273L, T259S, G358V, or V167I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0524] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or to the reference sequence corresponding to SEQ ID NO: 634, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0525] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 702- 736, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 702-736, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0526] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0527] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 38M/54T/127V/205M/254A, 165M/255A/258L/259A, 205M/254A/258L, 33R/38T/127V/165M/254A, 38T/54T/205M/258L, 127V, 127V/165M/258L, 127V/205M/254A/255A/258L/259A, 205M/258L, 33R/38T/254A/255A, 127V/254A/258L/259A, 127V/165M, 38T/127V/165M/259A, 38M/127V/258L/259A, 38M/254D/255V, 127V/205M/254A/258L/259A, 127V/165M/258L/259A, or 165M/205M/258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0528] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) V38M/L54T/P127V/I205M/H254A, F165M/F255A/F258L/T259A, I205M/H254A/F258L, E33R/V38T/P127V/F165M/H254A, V38T/L54T/I205M/F258L, P127V, P127V/F165M/F258L, Docket Number CX10-269WO4 P127V/I205M/H254A/F255A/F258L/T259A, I205M/F258L, E33R/V38T/H254A/F255A, P127V/H254A/F258L/T259A, P127V/F165M, V38T/P127V/F165M/T259A, V38M/P127V/F258L/T259A, V38M/H254D/F255V, P127V/I205M/H254A/F258L/T259A, P127V/F165M/F258L/T259A, or F165M/I205M/F258L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0529] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or to the reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0530] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 738- 754, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 738-754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0531] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0532] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 127L/222G/223G/225G/255Q/272G/276H, 127L/255Q/259S, 276H, 255Q/259S, 127L/255Q, 127L/162S/223G/255Q, 127L/162S/255Q/259S/272G/276H, 127L/255M, or 259S/272L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0533] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) P127L/N222G/R223G/S225G/F255Q/I272G/G276H, P127L/F255Q/T259S, G276H, F255Q/T259S, P127L/F255Q, P127L/W162S/R223G/F255Q, P127L/W162S/F255Q/T259S/I272G/G276H, P127L/F255M, or T259S/I272L, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0534] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or to the reference sequence corresponding to SEQ ID NO: 738, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0535] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 756- 760, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 756-760, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0536] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0537] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 165K/259S/281L, 127P, or 259S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0538] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) F165K/T259S/I281L, L127P, or T259S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0539] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or to the reference sequence corresponding to SEQ ID NO: 756, wherein the amino acid sequence comprises one or more substitutions Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0540] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 762- 810, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 762-810, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0541] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0542] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 177I, 362R, 44R, 365N, 46R, 279Q/281I, 303A, 165F/166S, 281I/282L, 302I, 362A, 360L, 295R, 246I, 127Q, 299R, 125Q, 238G, 295T, 56L, 109S, 125R, 38V, 295A, or 31I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0543] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) F177I, E362R, Y44R, K365N, A46R, D279Q/L281I, N303A, K165F/Q166S, L281I/F282L, T302I, E362A, A360L, E295R, V246I, L127Q, K299R, S125Q, E238G, E295T, R56L, P109S, S125R, T38V, E295A, or V31I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0544] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or to the reference sequence corresponding to SEQ ID NO: 768, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0545] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 812- 880, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 812-880, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0546] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/279/280/302, 246/280, 272/276/279/280/302, 272/280, 272/280/302, 276/279/280/302, 276/280, 279/280/302, or 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0547] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) 44R/118F, 44R/118F/246I/272L/276G, 44R/118F/246I/280S, 44R/118F/246I/280S/302I, 44R/118F/272L/276G, 44R/246I, 44R/246I/272L/276G/302I, 44R/246I/276G/279R/280S, 44R/246I/276G/280S, 44R/272L/276G/280S, 44R/272L/279R, 44R/272L/280S, 44R/276G, 44R/276G/279R, 44R/276G/280S/295R/302I, 44R/280S, 44R/302I, 118F/246I/276G/280S/302I, 118F/246I/280S/295R, 118F/272L/276G, 118F/280S/302I, 246I/272L/276G/279R/280S/302I, 246I/272L/279R/280S, 246I/276G, 246I/276G/302I, 246I/279R/280S, 246I/279R/280S/302I, 246I/280S, 272L/276G/279R/280S/302I, 272L/280S, 272L/280S/302I, 276G/279R/280S/302I, 276G/280S, 279R/280S/302I, or 280S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0548] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) Y44R/M118F, Y44R/M118F/V246I/G272L/H276G, Y44R/M118F/V246I/D280S, Y44R/M118F/V246I/D280S/T302I, Y44R/M118F/G272L/H276G, Y44R/V246I, Y44R/V246I/G272L/H276G/T302I, Y44R/V246I/H276G/D279R/D280S, Y44R/V246I/H276G/D280S, Y44R/G272L/H276G/D280S, Y44R/G272L/D279R, Y44R/G272L/D280S, Y44R/H276G, Y44R/H276G/D279R, Y44R/H276G/D280S/E295R/T302I, Y44R/D280S, Y44R/T302I, M118F/V246I/H276G/D280S/T302I, M118F/V246I/D280S/E295R, M118F/G272L/H276G, M118F/D280S/T302I, V246I/G272L/H276G/D279R/D280S/T302I, V246I/G272L/D279R/D280S, V246I/H276G, V246I/H276G/T302I, V246I/D279R/D280S, V246I/D279R/D280S/T302I, V246I/D280S, G272L/H276G/D279R/D280S/T302I, G272L/D280S, G272L/D280S/T302I, H276G/D279R/D280S/T302I, H276G/D280S, D279R/D280S/T302I, or D280S, wherein the amino acid positions are relative to the Docket Number CX10-269WO4 reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0549] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or to the reference sequence corresponding to SEQ ID NO: 844, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0550] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 882- 912, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 882-912, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0551] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0552] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 42A, 43G/T, 122C/I/L/M/S/T/V/Y, 271G/M, 272G, 278P, or 279Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0553] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue Y42A, R43G/T, K122C/I/L/M/S/T/V/Y, N271G/M, L272G, V278P, or D279Q, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0554] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or to the reference sequence corresponding to SEQ ID NO: 882, wherein the amino acid sequence comprises one or more substitutions Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0555] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 934- 990, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 934-990, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0556] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/359/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0557] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 38V/127V/238G/284F, 38V/127V/255A, 38V/127V/255A/359D, 38V/127V/284F, 38V/238G/255A, 38V/238G/255A/359D, 38V/238G/255A/359D/381R, 38V/238G/284F/359D, 127V, 127V/212H/238G/284F/359D, 127V/238G/255A, 127V/238G/255A/359D/381R, 127V/238G/284F/359D, 127V/238G/284F/359D/381R, 127V/238G/359D, 127V/255A/359D/381R, 127V/255A/381R, 238G, 238G/255A, 238G/255A/359D, 238G/255A/381R, 238G/284F, 238G/359D, 255A, 255A/284F, 255A/359D, 255A/359D/362K/381R, 359D, or 381R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0558] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) T38V/L127V/E238G/R284F, T38V/L127V/Q255A, T38V/L127V/Q255A/I359D, T38V/L127V/R284F, T38V/E238G/Q255A, T38V/E238G/Q255A/I359D, T38V/E238G/Q255A/I359D/K381R, T38V/E238G/R284F/I359D, L127V, L127V/R212H/E238G/R284F/I359D, L127V/E238G/Q255A, L127V/E238G/Q255A/I359D/K381R, L127V/E238G/R284F/I359D, L127V/E238G/R284F/I359D/K381R, L127V/E238G/I359D, L127V/Q255A/I359D/K381R, L127V/Q255A/K381R, E238G, E238G/Q255A, E238G/Q255A/I359D, E238G/Q255A/K381R, E238G/R284F, E238G/I359D, Q255A, Q255A/R284F, Q255A/I359D, Q255A/I359D/E362K/K381R, I359D, or K381R, wherein the amino acid positions are relative to the Docket Number CX10-269WO4 reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0559] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or to the reference sequence corresponding to SEQ ID NO: 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0560] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 992- 1094, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 992-1094, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0561] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0562] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 34K/35L/38V, 34K/38V/170R, 34K/135M/170R/271G/357L, 34K/170R/271G, 34K/271G, 35L/38V/170R/357L, 35L/38V/271G/357L, 35L/170R/271G/357L, 38V/170R, 38V/170R/271G, 38V/170R/357L, 56M/135M/170R/357L, 56M/135M/271G/357L, 135M/170R/271G, 170R, 170R/271G/272R, 170R/271G/272R/357L, 170R/271G/357L, 170R/357L, 254R/260R, 254R/281M, 271G, or 357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0563] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) Y34K/E35L/T38V, Y34K/T38V/P170R, Y34K/L135M/P170R/N271G/V357L, Y34K/P170R/N271G, Y34K/N271G, E35L/T38V/P170R/V357L, E35L/T38V/N271G/V357L, E35L/P170R/N271G/V357L, T38V/P170R, T38V/P170R/N271G, T38V/P170R/V357L, R56M/L135M/P170R/V357L, R56M/L135M/N271G/V357L, L135M/P170R/N271G, P170R, P170R/N271G/L272R, P170R/N271G/L272R/V357L, Docket Number CX10-269WO4 P170R/N271G/V357L, P170R/V357L, H254R/Y260R, H254R/L281M, N271G, or V357L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0564] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0565] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 9D/369V, 34K/38V/357L, 34K/135M/170R/271G/357L, 34K/271G, 56M/135M/170R/357L, 56M/135M/271G/357L, 113D, 115S, 141G, 144T, 145Y, 177C, 177V, 214A, 214C, 214S, 254R/260R, 254R/281M, 260R, 274I, 340C, 341A, 350A, 350C, 350R, 354Q/359I, 359I/360V, 359I/362Q, 368G, 369C, 369L, 371R, 377L/381K, 381K/384C, or 381K/384S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0566] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G9D/Y369V, Y34K/T38V/V357L, Y34K/L135M/P170R/N271G/V357L, Y34K/N271G, R56M/L135M/P170R/V357L, R56M/L135M/N271G/V357L, N113D, K115S, N141G, N144T, F145Y, F177C, F177V, K214A, K214C, K214S, H254R/Y260R, H254R/L281M, Y260R, L274I, G340C, R341A, E350A, E350C, E350R, E354Q/D359I, D359I/A360V, D359I/E362Q, S368G, Y369C, Y369L, G371R, M377L/R381K, R381K/E384C, or R381K/E384S, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0567] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO: 992, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0568] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1096-1206, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: Docket Number CX10-269WO4 1096-1206, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0569] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177/254/359, 115/177/359, 115/254, 115/254/350/354/359, 115/254/359, 115/350, 144/145/260, 144/145/260/341, 144/145/260/341/366/371, 144/260/341/366, 144/260/366/369, 145/260/263, 145/260/341, 145/260/341/366/369, 145/341, 145/341/371, 145/371, 177, 177/254, 177/254/281/359, 177/254/354/359, 177/254/359, 177/359, 254, 254/281/354/359, 254/350, 254/354/359, 254/359, 260/366, 341, or 359, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0570] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 113D, 113D/115S, 113D/115S/177V/254R/281M, 113D/115S/177V/254R/281M/350A/359I, 113D/115S/177V/254R/350R, 113D/115S/177V/254R/359I, 113D/115S/177V/281M/359I, 113D/115S/254R, 113D/115S/254R/281M, 113D/115S/254R/350R, 113D/115S/254R/359I, 113D/115S/350R, 113D/115S/359I, 113D/177V/254R/350R, 113D/177V/281M/359I, 113D/177V/350A/354Q/359I, 113D/254R, 113D/254R/281M/350A/359I, 113D/254R/281M/359I, 113D/254R/350A/354Q/359I, 113D/254R/354Q/359I, 113D/254R/359I, 113D/359I, 115S/177V, 115S/177V/254R, 115S/177V/254R/359I, 115S/177V/359I, 115S/254R, 115S/254R/350A/354Q/359I, 115S/254R/359I, 115S/350R, 144T/145Y/260R, 144T/145Y/260R/341A, 144T/145Y/260R/341A/366W/371S, 144T/260R/341A/366W, 144T/260R/366W/369L, 145Y/260R/263D, 145Y/260R/341A, 145Y/260R/341P/366W/369L, 145Y/341A, 145Y/341A/371S, 145Y/371S, 177V, 177V/254R, 177V/254R/281M/359I, 177V/254R/354Q/359I, 177V/254R/359I, 177V/359I, 254R, 254R/281M/354Q/359I, 254R/350R, 254R/354Q/359I, 254R/359I, 260R/366W, 341A, or 359I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0571] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) N113D, N113D/K115S, N113D/K115S/F177V/H254R/L281M, N113D/K115S/F177V/H254R/L281M/E350A/D359I, N113D/K115S/F177V/H254R/E350R, N113D/K115S/F177V/H254R/D359I, N113D/K115S/F177V/L281M/D359I, N113D/K115S/H254R, N113D/K115S/H254R/L281M, N113D/K115S/H254R/E350R, N113D/K115S/H254R/D359I, N113D/K115S/E350R, N113D/K115S/D359I, N113D/F177V/H254R/E350R, N113D/F177V/L281M/D359I, N113D/F177V/E350A/E354Q/D359I, N113D/H254R, N113D/H254R/L281M/E350A/D359I, N113D/H254R/L281M/D359I, Docket Number CX10-269WO4 N113D/H254R/E350A/E354Q/D359I, N113D/H254R/E354Q/D359I, N113D/H254R/D359I, N113D/D359I, K115S/F177V, K115S/F177V/H254R, K115S/F177V/H254R/D359I, K115S/F177V/D359I, K115S/H254R, K115S/H254R/E350A/E354Q/D359I, K115S/H254R/D359I, K115S/E350R, N144T/F145Y/Y260R, N144T/F145Y/Y260R/R341A, N144T/F145Y/Y260R/R341A/Y366W/G371S, N144T/Y260R/R341A/Y366W, N144T/Y260R/Y366W/Y369L, F145Y/Y260R/N263D, F145Y/Y260R/R341A, F145Y/Y260R/R341P/Y366W/Y369L, F145Y/R341A, F145Y/R341A/G371S, F145Y/G371S, F177V, F177V/H254R, F177V/H254R/L281M/D359I, F177V/H254R/E354Q/D359I, F177V/H254R/D359I, F177V/D359I, H254R, H254R/L281M/E354Q/D359I, H254R/E350R, H254R/E354Q/D359I, H254R/D359I, Y260R/Y366W, R341A, or D359I, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0572] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or to the reference sequence corresponding to SEQ ID NO: 1104, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0573] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1208-1254, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1208-1254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0574] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0575] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 27F, 27W, 27Y, 34G, 34N, 34R, 34S, 35A, 35S, 122A, 127I, 255S, 259N, 275W, 349V, 351F, 351Y, 354S, 356V, 363V, 374I, 374L, 374V, or 376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. Docket Number CX10-269WO4 [0576] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K27F, K27W, K27Y, Y34G, Y34N, Y34R, Y34S, L35A, L35S, L122A, V127I, A255S, S259N, Q275W, Q349V, N351F, N351Y, E354S, A356V, G363V, F374I, F374L, F374V, or T376E, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0577] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or to the reference sequence corresponding to SEQ ID NO: 1222, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0578] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0579] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0580] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 27W/127I/374I/376E, 27W/127I/374L, 27W/275W/356V, 27W/351Y, 34S/35S/118L/127I/275W/351Y/374L/376E, 122A, 127I/275W/374V, or 275W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0581] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K27W/V127I/F374I/T376E, K27W/V127I/F374L, K27W/Q275W/A356V, K27W/N351Y, N34S/L35S/M118L/V127I/Q275W/N351Y/F374L/T376E, L122A, V127I/Q275W/F374V, or Q275W, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0582] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0583] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 21A, 66A, 69A, 151L, or 199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0584] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K21A, K66A, S69A, N151L, or K199T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0585] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0586] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 25G, 54I, 54L, 54V, 65Q, 65S, 66A, 66P, 66Q, 66R, 66T, 90S, 93P, or 151L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0587] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue A19V, N25G, T54I, T54L, T54V, R65Q, R65S, K66A, K66P, K66Q, K66R, K66T, E90S, R93P, or N151L, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0588] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. Docket Number CX10-269WO4 [0589] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 21A, 25W, 65P, 65S, 66T, 69C, 88W, 91L, 93A, 93H, 93P, 97L, 97P, 97V, 157L, 190L, 195T, 197T, or 198A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0590] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K21A, N25W, R65P, R65S, K66T, S69C, K88W, R91L, R93A, R93H, R93P, I97L, I97P, I97V, I157L, Y190L, E195T, E197T, or I198A, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0591] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0592] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1308-1364 and 1396-1436, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1308-1364 and 1396-1436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0593] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0594] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 19V, 19V/21A/65Q/66T/90S/93P, 19V/21A/65S, 19V/21A/65S/66A, 19V/21A/65S/66Q/93P, 19V/21A/65S/66T, 19V/21A/65S/93P, 19V/21A/65S/190L, 19V/21A/65S/197T, 19V/21A/66Q, 19V/21A/190L, 19V/65Q/66A/197T, 19V/65S, 19V/65S/66A, 19V/65S/66A/93A, 19V/65S/66Q, 19V/65S/66Q/90S/93P/190L, 19V/65S/66Q/190L, Docket Number CX10-269WO4 19V/65S/66T, 19V/65S/66T/190L, 19V/66A, 19V/66Q, 19V/66T/90S/93P/197T, 25G, 25G/54I, 25G/122A, 65S, 65S/66T, or 66Q, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0595] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) A19V, A19V/K21A/R65Q/K66T/E90S/R93P, A19V/K21A/R65S, A19V/K21A/R65S/K66A, A19V/K21A/R65S/K66Q/R93P, A19V/K21A/R65S/K66T, A19V/K21A/R65S/R93P, A19V/K21A/R65S/Y190L, A19V/K21A/R65S/E197T, A19V/K21A/K66Q, A19V/K21A/Y190L, A19V/R65Q/K66A/E197T, A19V/R65S, A19V/R65S/K66A, A19V/R65S/K66A/R93A, A19V/R65S/K66Q, A19V/R65S/K66Q/E90S/R93P/Y190L, A19V/R65S/K66Q/Y190L, A19V/R65S/K66T, A19V/R65S/K66T/Y190L, A19V/K66A, A19V/K66Q, A19V/K66T/E90S/R93P/E197T, N25G, N25G/T54I, N25G/L122A, R65S, R65S/K66T, or K66Q, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0596] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0597] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237A, 240W, 246I, 289Q, 306F, 306M, 310A, 310D, 323G, 323N, 323Y, 330I, 334I, 334N, 334S, 336E, 336N, 351N/352S, 351N/352V, 351N/353V, or 351N/358V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0598] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237A, F240W, V246I, N289Q, T306F, T306M, S310A, S310D, K323G, K323N, K323Y, R330I, K334I, K334N, K334S, R336E, R336N, Y351N/Y352S, Y351N/Y352V, Y351N/L353V, or Y351N/G358V, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0599] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. Docket Number CX10-269WO4 [0600] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237R, 240W, 288T, 291L, 299I, 306M, 314E, 316R, 325M, 325P, 332G, 336E, 351N/352G, 351N/352L, 351N/352M, 351N/352N, 351N/352R, 351N/352V, 351N/353V, 351N/355K, 372A/374F/376T, 372L/374F/376T, or 372P/374F/376T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0601] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237R, F240W, E288T, V291L, K299I, T306M, K314E, S316R, D325M, D325P, V332G, R336E, Y351N/Y352G, Y351N/Y352L, Y351N/Y352M, Y351N/Y352N, Y351N/Y352R, Y351N/Y352V, Y351N/L353V, Y351N/E355K, T372A/L374F/E376T, T372L/L374F/E376T, or T372P/L374F/E376T, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0602] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or to the reference sequence corresponding to SEQ ID NO: 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0603] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0604] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0605] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 21A/54I, 21A/65Q/66K/151L, 21A/90E/93R/122A, 21A/93R, 54I/58M, 65Q/93R, 65Q/151L, 66K/90E/151L/190L/197E, 90E/93R/122A, Docket Number CX10-269WO4 90E/190L/197E, 90E/197E, 93R, or 93R/151L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0606] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) K21A/T54I, K21A/R65Q/T66K/N151L, K21A/S90E/P93R/L122A, K21A/P93R, T54I/L58M, R65Q/P93R, R65Q/N151L, T66K/S90E/N151L/Y190L/T197E, S90E/P93R/L122A, S90E/Y190L/T197E, S90E/T197E, P93R, or P93R/N151L, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0607] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or to the reference sequence corresponding to SEQ ID NO: 1474, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0608] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0609] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0610] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set or amino acid residue(s) 237A, 240W, or 289Q/316R, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0611] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set, or amino acid residue(s) G237A, F240W, or N289Q/S316R, wherein the amino acid positions are relative to the reference sequence corresponding to Docket Number CX10-269WO4 residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0612] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or to the reference sequence corresponding to SEQ ID NO: 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0613] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0614] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0615] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 27A, 30R, 36R, 40I, 139R, 141A, 141P, 141R, 172P, or 223T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0616] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue K27A, K30R, N36R, V40I, N139R, N141A, N141P, N141R, D172P, or G223T, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0617] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, Docket Number CX10-269WO4 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0618] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0619] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0620] In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0621] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. Docket Number CX10-269WO4 [0622] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526. [0623] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0624] In some embodiments, the recombinant single stranded RNA ligase comprises an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244- 912, and 934-1526. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions, insertions, and/or deletions. In some embodiments, the amino acid sequence of the recombinant single stranded RNA ligase optionally includes 1, 2, 3, 4, or 5 substitutions. [0625] In some embodiments, the recombinant single stranded RNA ligase has single stranded RNA ligase activity. In some embodiments, the recombinant single stranded RNA ligase has single stranded RNA ligase activity and exhibits at least an improved property as compared to a reference single stranded RNA ligase. [0626] In some embodiments, the recombinant single stranded RNA ligase exhibits increased ligase activity as compared to a reference single stranded RNA ligase. In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity as compared to the reference single stranded RNA ligase. Exemplary increases in ligase activity is provided in the Examples. [0627] In some embodiments, the recombinant single stranded RNA ligase exhibits increased activity on modified oligonucleotide substrates. In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with polynucleotide or oligonucleotide acceptor having a 3’-terminal modified nucleoside as compared to the reference single stranded RNA ligase. In some embodiments, the recombinant single stranded RNA ligase has increased activity with an oligonucleotide acceptor having a 3’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro- Docket Number CX10-269WO4 guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl- guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl-thymidine. [0628] In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a nucleotide donor having a 5’-terminal modified nucleoside compared to the reference single stranded RNA ligase. In some embodiments, the recombinant single stranded RNA ligase has increased activity with an nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O- methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl- thymidine. [0629] In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a polynucleotide or oligonucleotide donor having a 5’-terminal modified nucleoside compared to the reference single stranded RNA ligase. In some embodiments, the recombinant single stranded RNA ligase has increased activity with an nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro- uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O- methyl-uridine, 2’-O-methyl-thymidine. [0630] In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity with a nucleotide donor pNp having a modified nucleoside compared to the reference single stranded RNA ligase. In some embodiments, the recombinant single stranded RNA ligase has increased activity with an nucleotide donor pNp having a modified nucleoside of 2’- fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O- methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, 2’-O-methyl- thymidine. [0631] In some embodiments, the recombinant single stranded RNA ligase has at least 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more fold ligase activity as compared to the reference single stranded RNA ligase with oligonucleotide acceptor having a 3’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’- fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro-thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O- methyl-cytidine, 2’-O-methyl-uridine, or 2’-O-methyl-thymidine, and a nucleotide donor having a 5’-terminal nucleoside of 2’-fluoro-adenosine, 2’-fluoro-guanosine, 2’-fluoro-cytidine, 2’-fluoro-uridine, 2’-fluoro- thymidine, 2’-O-methyl-adenosine, 2’-O-methyl-guanosine, 2’-O-methyl-cytidine, 2’-O-methyl-uridine, or 2’- O-methyl-thymidine. [0632] In some embodiments, the recombinant single stranded RNA ligase has increased activity with nucleotide donors of a single nucleotide, or short oligonucleotide donor of 2, 3 or 4 nucleotides in length as compared to the reference single stranded RNA ligase. In some embodiments, the single nucleotide donor, or the short oligonucleotide donor comprises at least a modified 5’-terminal nucleoside. Exemplary modified single stranded RNA ligase acceptors and donors are provided in the Examples. Docket Number CX10-269WO4 [0633] In some embodiments, the single stranded RNA ligase acceptor and donor for comparative activity are provided in Tables 4.1, 5.1, 6.1, 7.1, 8.1, 10.1, 11.1, 12.1, 13.1, 14.1, 14.2, 15.1, 15.2, 16.1, 16.2, 17.1, 18.1, 19.1, 20.1, 21.1, 22.1, 23.1, 24.1, 25.1, 26.1, 27.1, 28.1, 29.1, 32.1, 37.1, 38.1, 39.1, 40.1, 42.1, and 43.1. List of donors and acceptors for the single stranded RNA ligase reaction is listed in the Examples. [0634] In some embodiments, the single stranded RNA ligase exhibits increased thermostability. In some embodiments, the single stranded RNA ligase exhibits increased thermostability at 58 °C as compared to a reference single stranded RNA ligase. In some embodiments, the comparison is made at a defined temperature for 30 min, 1 hr, 2 hr, 3 hr, or 4 hr or more. [0635] In some embodiments, the recombinant single stranded RNA ligase exhibits an improved property selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), as compared to a reference single stranded RNA ligase. [0636] In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. In some embodiments, the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or an amino acid sequence corresponding to SEQ ID NO: 14. [0637] In some embodiments, the single-stranded RNA ligase is provided as a fusion protein. In some embodiments, the single-stranded RNA ligase described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection, purification, immobilization on a support medium, or fusion to another protein. In some embodiments, the fusion protein of the single-stranded RNA ligase comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the single stranded RNA ligase comprises one or more lysine residues inserted or fused to RNA ligase polypeptide, e.g., 1-25 lysine residues. In some embodiments, the single-stranded RNA ligase is fused to a polylysine (e.g., 2-25 or 2-10 lysine residues), for example, for conjugation to a support medium via the amino group of the polylysine. In some embodiments, the fusion protein of the single-stranded RNA ligase comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion protein of the single-stranded RNA ligase comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to cell localization signals (e.g., secretion signals). In some embodiments, the fusion is to the amino (N-) terminus of single-stranded RNA ligase polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the single-stranded RNA ligase polypeptide. In some embodiments, the fusion is selected or designed to preserve the activity of the single-stranded RNA ligase. [0638] In some embodiments, the single-stranded RNA ligase described herein is an isolated composition. In some embodiments, the single-stranded RNA ligase polypeptide is purified. In some embodiments, the Docket Number CX10-269WO4 recombinant phosphatase is provided in solution, as a lyophilizate, or immobilized on a substrate or support medium, as further discussed herein. [0639] In some embodiments, the present disclosure further provides functional fragments or biologically active fragments of single-stranded RNA ligase described herein. Thus, for each and every embodiment herein of a single-stranded RNA ligase described herein, a functional fragment or biologically active fragment of the single-stranded RNA ligase is provided herewith. In some embodiments, a functional fragment or biologically active fragments of a single-stranded RNA ligase comprises at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the single-stranded RNA ligase polypeptide from which it was derived (i.e., the parent single-stranded RNA ligase). In some embodiments, functional fragments or biologically active fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the phosphatase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50 amino acids, less than 55 amino acids, less than 60 amino acids, less than 65 amino acids, or less than 70 amino acids. [0640] In some embodiments, a functional fragment of a single-stranded RNA ligase herein comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the single-stranded RNA ligase. In some embodiments, the functional fragment will be truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids. [0641] In some embodiments, the functional fragments or biologically active fragments of the recombinant single stranded RNA ligase described herein include at least a mutation or mutation set in the amino acid sequence of the recombinant single stranded RNA ligase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the recombinant single stranded RNA ligase displays the enhanced or improved property associated with the mutation or mutation set in the parent single stranded RNA ligase or parent recombinant single stranded RNA ligase. Polynucleotides encoding single-stranded RNA ligases, expression vectors, and host cells [0642] In another aspect, the present disclosure provides recombinant polynucleotides encoding the single- stranded RNA ligases described herein. In some embodiments, the recombinant polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide construct capable of expressing the single-stranded RNA ligases. [0643] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode a recombinant single stranded RNA ligase of the present disclosure. Thus, the present disclosure provides methods and compositions for the production of each and every possible variation of Docket Number CX10-269WO4 polynucleotides that could be made that encode the single-stranded RNA ligases described herein by selecting combinations based on the possible codon choices, and all such variations of polynucleotides are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples and in the accompanying Sequence Listing. [0644] In some embodiments, the codons are preferably optimized for utilization by the chosen host cell for protein production. In some embodiments, preferred codons in bacterial cells are used for expression in bacterial cells. In some embodiments, preferred codons in fungal cells are used for expression in fungal cells. In some embodiments, preferred codons in insect cells are used for expression in insect cells. In some embodiments, preferred codons in mammalian cells are used for expression in mammalian cells. In some embodiments, codon optimized polynucleotides encoding a recombinant single stranded RNA ligase polypeptide described herein contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full length coding region. [0645] Accordingly, in some embodiments, a recombinant polynucleotide of the present disclosure encodes a single-stranded RNA ligase described herein. In some embodiments, the polynucleotide sequence of the recombinant polynucleotide is codon optimized. [0646] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. [0647] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising the single stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1. [0648] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to a sequence from nucleotide residue 34 to the 3’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19, or to a reference sequence corresponding to an odd numbered SEQ ID NO. of SEQ ID NOs: 1- 19, wherein the polynucleotide sequence encodes a single-stranded RNA ligase. [0649] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising the sequence from nucleotide residue 34 to the 3’-terminal nucleotide of an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19, or comprising an odd numbered SEQ ID NO. of SEQ ID NOs: 1-19. [0650] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, Docket Number CX10-269WO4 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244- 912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0651] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0652] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0653] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. Docket Number CX10-269WO4 [0654] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0655] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0656] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0657] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 162, 236, 237, 320, 337, 358, or 362, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0658] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 123, 256, or 320, or any combinations thereof, wherein the amino acid Docket Number CX10-269WO4 positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0659] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 38, 48, 49, 118, or 220, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0660] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 38, 54, 205, or 258, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0661] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 127, 222, 223, 225, 255, 272, or 276, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0662] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 165, 259, or 281, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0663] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 127, 238, 255, 359, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0664] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 35, 170, 271, or 357, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0665] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 115, 177, 254, or 359, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. Docket Number CX10-269WO4 [0666] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 34, 35, 118, 127, 275, 351, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0667] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 66, 90, 93, or 197, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0668] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 90, 197, 289, or 316, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0669] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0670] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0671] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0672] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, Docket Number CX10-269WO4 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0673] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0674] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. [0675] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0676] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference Docket Number CX10-269WO4 sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0677] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0678] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 9, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0679] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or to the reference sequence corresponding to SEQ ID NO: 32, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. Docket Number CX10-269WO4 [0680] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 44-64, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 44-64, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0681] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. [0682] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or to the reference sequence corresponding to SEQ ID NO: 44, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0683] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 66-118, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 66-118, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0684] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. [0685] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, Docket Number CX10-269WO4 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or to the reference sequence corresponding to SEQ ID NO: 100, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0686] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 120-216, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 120-216, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0687] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. [0688] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or to the reference sequence corresponding to SEQ ID NO: 140, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0689] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 244-398, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 244-398, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0690] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237/323, 151/205/314, 151/212/345/347, 92/214/347/358, 162/204/205/310/314/358, 236/314, 151/345/347, 214/347/358, 151/212/358, 204/283/314/358, 236/237/358/362, 151/199/204/231/236/323, 231/236/237/358/362, 162/314/358, 92/151/347/358, 151/236, 212/345/347, 115/314, 345/347/358, 314, 358/362, 115/358, 310/314, 214/347/358, 237/314, 345/347, 151/310/323/343/347, 151/358, 347/358, 93/358/362, 231/236/237/320/358/362, 151/230/345/347/358, 135/231/236/237/358, 310/314/358/362, 151/230/347/358, 231/237/323, 135/358/362, 283/314/358/362, 199/205, 92/151/230/345/347/358, 314/358/362, 199/237, or 199/204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. [0691] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or to the reference sequence corresponding to SEQ ID NO: 246, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0692] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 400-490, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 400-490, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0693] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, Docket Number CX10-269WO4 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. [0694] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or to the reference sequence corresponding to SEQ ID NO: 400, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0695] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 492-510, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 492-510, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0696] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. [0697] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or to the reference sequence corresponding to SEQ ID NO: 492, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0698] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 512-584, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 512-584, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0699] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. [0700] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or to the reference sequence corresponding to SEQ ID NO: 520, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0701] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 586-602, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 586-602, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0702] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. [0703] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or to the reference sequence corresponding to SEQ ID NO: 594, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0704] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 604-700, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 604-700, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0705] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 49/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. [0706] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or to the reference sequence corresponding to SEQ ID NO: 634, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0707] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 702-736, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 702-736, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0708] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, or 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. [0709] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or to the reference sequence corresponding to SEQ ID NO: 710, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0710] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 738-754, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 738-754, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0711] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. [0712] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or to the reference sequence corresponding to SEQ ID NO: 738, Docket Number CX10-269WO4 wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0713] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 756-760, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 756-760, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0714] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. [0715] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or to the reference sequence corresponding to SEQ ID NO: 756, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0716] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 762-810, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 762-810, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0717] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are Docket Number CX10-269WO4 relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. [0718] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or to the reference sequence corresponding to SEQ ID NO: 768, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0719] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 812-880, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 812-880, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0720] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/279/280/302, 246/280, 272/276/279/280/302, 272/280, 272/280/302, 276/279/280/302, 276/280, 279/280/302, or 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. [0721] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or to the reference sequence corresponding to SEQ ID NO: 844, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. Docket Number CX10-269WO4 [0722] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 882-912, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 882-912, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0723] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. [0724] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or to the reference sequence corresponding to SEQ ID NO: 882, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0725] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 934-990, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 934-990, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0726] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/359/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. [0727] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or to the reference sequence corresponding to SEQ ID NO: 936, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0728] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 992-1094, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 992-1094, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0729] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0730] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. [0731] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 992, or to the reference sequence corresponding to SEQ ID NO: 992, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0732] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1096-1206, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1096-1206, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0733] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177/254/359, 115/177/359, 115/254, 115/254/350/354/359, 115/254/359, 115/350, 144/145/260, 144/145/260/341, 144/145/260/341/366/371, 144/260/341/366, 144/260/366/369, 145/260/263, 145/260/341, 145/260/341/366/369, 145/341, 145/341/371, 145/371, 177, 177/254, 177/254/281/359, 177/254/354/359, 177/254/359, 177/359, 254, 254/281/354/359, 254/350, 254/354/359, 254/359, 260/366, 341, or 359, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. [0734] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or to the reference sequence corresponding to SEQ ID NO: 1104, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0735] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1208-1254, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1208-1254, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0736] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. [0737] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or to the reference sequence corresponding to SEQ ID NO: 1222, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0738] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1256-1306, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0739] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0740] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino Docket Number CX10-269WO4 acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0741] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0742] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at amino acid position 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. [0743] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or to the reference sequence corresponding to SEQ ID NO: 1264, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0744] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1308-1364 and 1396-1436, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1308-1364 and 1396- 1436, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0745] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. Docket Number CX10-269WO4 [0746] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0747] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. [0748] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or to the reference sequence corresponding to SEQ ID NO: 1344, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0749] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1474-1498, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0750] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. [0751] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Docket Number CX10-269WO4 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or to the reference sequence corresponding to SEQ ID NO: 1474, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0752] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1500-1504, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0753] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. [0754] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or to the reference sequence corresponding to SEQ ID NO: 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0755] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 1506-1526, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0756] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a Docket Number CX10-269WO4 substitution at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. [0757] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution at an amino acid position provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0758] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0759] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position(s) provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0760] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference Docket Number CX10-269WO4 sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0761] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0762] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526. [0763] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even- numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. [0764] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding a recombinant single stranded RNA ligase comprising an amino acid sequence comprising residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934- 1526, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1526. [0765] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, Docket Number CX10-269WO4 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. [0766] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. [0767] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523. [0768] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or a polynucleotide sequence comprising SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499. [0769] As noted above, in some embodiments, the recombinant polynucleotide is codon-optimized for expression of the encoded recombinant single stranded RNA ligase. In some, the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, insect cell, or mammalian cell. [0770] In some embodiments, a recombinant polynucleotide encoding any of the single-stranded RNA ligase herein is manipulated in a variety of ways to facilitate expression of the single-stranded RNA ligase polypeptide. In some embodiments, the recombinant polynucleotide encoding the single-stranded RNA ligase comprises expression vectors where one or more control sequences is present to regulate the expression of the phosphatase encoding polynucleotides and/or encoded polypeptides. In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. [0771] In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727- 3731), as well as the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80:21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained Docket Number CX10-269WO4 from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in insect cells include, but are not limited to, polyhedrin, p10, ELT, OpIE2, and hr5/ie1 promoters. Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken β-actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, and from Gallus β-actin. [0772] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the single-stranded RNA ligase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3- phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast, 1992, 8(6):423-88). Exemplary terminators for insect cells and mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin. [0773] In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is used for translation by the host cell. In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the single-stranded RNA ligase polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- Docket Number CX10-269WO4 phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). Suitable leaders for mammalian host cells include but are not limited to the 5ʹ-UTR element present in orthopoxvirus mRNA. [0774] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA). Any suitable polyadenylation sequence which is functional in the host cell of choice finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3’ UTR sequences for insect and mammalian host cells include, but are not limited to, OpIE2 polyA sequence, D. melanogaster metallothionein (Mt) polyA signal sequence, D. melanogaster alcohol dehydrogenase (adh), SV40 polyA signal sequence, and the 3ʹ-UTRs of α- and β-globin mRNAs harboring sequence elements that increase the stability and translation of mRNA. [0775] In some embodiments, the control sequence, where appropriate, comprises a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5’ end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5’ end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the recombinant polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for insect and mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide. [0776] In some embodiments, the control sequence is a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is referred to as a Docket Number CX10-269WO4 “proenzyme,” “propolypeptide,” or “zymogen.” A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha- factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. [0777] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to the ADH2 system or GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter. [0778] In another aspect, the present disclosure provides a recombinant expression vector comprising a recombinant polynucleotide encoding a single-stranded RNA ligase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together (i.e., operably linked) to produce recombinant expression vectors capable of expressing the encoded single-stranded RNA ligase. [0779] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the single-stranded RNA ligase encoding polynucleotide. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. [0780] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized. [0781] In some embodiment, the recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be Docket Number CX10-269WO4 based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol.7, Article 75). [0782] In some embodiments, the expression vector contains one or more selectable markers, which permit selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. [0783] In another aspect, the present disclosure provides a host cell comprising a recombinant polynucleotide encoding a single-stranded RNA ligase polypeptide described herein, the polynucleotide(s) being operably linked to one or more control sequences for expression of the recombinant phosphatase enzyme(s) in the host cell. Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present invention are known in the art and include but are not limited to, bacterial cells, such as E. coli, B. subtilis, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (ΔfhuA) and BL21). [0784] In another aspect, the present disclosure provides a method of producing the single-stranded RNA ligase, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the single-stranded RNA ligase under conditions suitable for expression of the polypeptide such that the single- stranded RNA ligase is produced. In some embodiments, the method further comprises isolating the single- stranded RNA ligase polypeptides from the culture and/or host cells. In some embodiments, the method further comprises purifying the expressed single-stranded RNA ligase polypeptide, as described herein. [0785] In some embodiments, the single-stranded RNA ligase expressed in a host cell is recovered from the cells and/or the culture medium using any one or more of the known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, salting-out, ultra- centrifugation, and chromatography, such as described herein. [0786] Chromatographic techniques for isolation/purification of the single-stranded RNA ligase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion- exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the single-stranded RNA ligase depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular Docket Number CX10-269WO4 shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the phosphatase. For affinity chromatography purification, an antibody that specifically binds the single-stranded RNA ligase may be used. In some embodiments, an affinity tag, e.g., His-tag, can be introduced into the single-stranded RNA ligase for purposes of isolation/purification. Compositions of single-stranded RNA (ssRNA) ligases [0787] In a further aspect, the present disclosure provides compositions of the RNA ligases disclosed herein. In some embodiments, the composition comprises a recombinant single stranded RNA ligase polypeptide described herein. In some embodiments, the recombinant single stranded RNA ligase polypeptide in the compositions is isolated or purified. In some embodiments, the recombinant single stranded RNA ligase is combined with other components and compounds to provide compositions and formulations comprising the engineered RNA ligase polypeptide as appropriate for different applications and uses. [0788] In some embodiments, the composition further comprises one or more of a buffer, a nucleotide cofactor (e.g., ATP), salt, and/or at least one or more substrates, for example an oligonucleotide or nucleotide substrate with modified nucleotides. In some embodiments, the components in the composition can comprise components described in the reaction conditions for the single stranded RNA ligase. [0789] In some embodiments, the composition comprises a buffer. In some embodiments, the buffer comprises, among others, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2-hydroxymethyl- propane-1,3-diol (Tris), and the like. In some embodiments, the buffer concentration is from 1 to 200 mM, 5 to 200 mM, 1 to 150 mM, 5 to 150 mM, 1 to 100 mM, 5 to 100 mM, 1 to 50 mM, 5 to 50 mM, 1 to 20 mM, 5 to 20 mM, 1 to 10 mM, or 5 to 10 mM. [0790] In some embodiments, composition comprises a salt, including, among others, NaCl, KCl, ammonium salts (e.g., NH4Cl), and acetate salts (e.g., sodium acetate). In some embodiments, the salt is present at 0.5 mM-300 mM, 1 mM-250 mM, 2 mM-200 mM, 5 mM-150 mM, 10 mM-100 mM, 20 mM-80 mM, or 40 mM-60 mM. In some embodiments, the salt is present at about 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 100 mM, 150 mM, 200 mM, 250 mM, or 300 mM. [0791] In some embodiments, the composition comprises nucleotide cofactor concentration of about 0.1-10 mM. In some embodiments, the nucleotide cofactor concentration is about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, or more as appropriate. In some embodiments, the cofactor is present in the form of a salt, such MgCl2, MnCl2, or CoCl2. In some embodiments, the cofactor is a nonnatural or non- preferred cofactor for the recombinant single stranded RNA ligase and/or the second enzyme. In some embodiments, the cofactor is a natural or preferred cofactor for the recombinant single stranded RNA ligase and/or the second enzyme. In some embodiments, the cofactor is a preferred or natural cofactor for a second enzyme and a nonpreferred or nonnatural cofactor for the recombinant single stranded RNA ligase. In some embodiments, the cofactor is present at a higher concentration compared to an in vivo concentration. Docket Number CX10-269WO4 [0792] In some embodiments, the composition further comprises a divalent metal ion. In some embodiments, the divalent metal ion, such as Ni2+, Mg2+, Mn2+, or Co2+. In some embodiments, the cofactor is present in the form of a salt, such MgCl2, MnCl2, or CoCl2. In some embodiments, the divalent metal is present at a concentration of 0.1 mM to 5 mM. [0793] In some embodiments, the composition comprises one or more of EDTA, DTT, and glycerol, such as for storage of the engineered single stranded RNA ligase. In some embodiments, the composition comprises an additive, such as a preservative, a buffer, or cryoprotectant. In some embodiments, the additive comprises a preservative/cryoprotectant, for example, dextran, polyethylene glycol, or glycerol. In some embodiments, the additive comprises a surfactant, for example, polysorbate (e.g., polysorbate 20 or 80). [0794] In some embodiments, the composition comprises a single-stranded RNA ligase and a nucleotide donor. In some embodiments, the nucleotide donor is a nucleotide(D), or an oligonucleotide donor (oligonucleotide(D)). In some embodiments, nucleotide(D) is a modified nucleotide(D), including a modified nucleotide(D) comprising a conjugate moiety, reactive group, or a linker. In some embodiments, the oligonucleotide donor is a modified oligonucleotide donor, including a modified oligonucleotide donor comprising at least a modified internucleoside linkage, a modified nucleobase, a modified sugar moiety, or comprising a conjugate moiety, reactive group, or linker, as described herein. In some embodiments, the nucleotide donor (e.g., nucleotide(D) or oligonucleotide(D) comprises a 3’-blocking group). [0795] In some embodiments, the composition comprises a single stranded RNA ligase and an oligonucleotide acceptor (oligonucleotide(A)). In some embodiments, the oligonucleotide donor is a modified oligonucleotide acceptor, including a modified oligonucleotide acceptor comprising at least a modified internucleoside linkage, a modified nucleobase, a modified sugar moiety, or comprising a conjugate moiety, reactive group, or linker, as described herein. In some embodiments, the oligonucleotide acceptor comprises a 5'-OH or a 5'-blocking group. [0796] In some embodiments, the composition further comprises an additive or ligation enhancing agent, including, among others, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000). In some embodiments, the composition comprises 1% to 40% v/v of DMSO. In some embodiments, the composition comprises 0.1 M to 3 M betaine. In some embodiments, the composition comprises 0.5% to 20% w/v of PEG (e.g., PEG6000 or PEG8000). [0797] In some embodiments, the composition further comprises an RNase inhibitor, including, among others, porcine RNase inhibitor, human placental RNase inhibitor, human liver RNase inhibitor, mouse RNase inhibitor, rat RNase inhibitor, and recombinantly expressed RNase inhibitors thereof. [0798] In some embodiments, a recombinant single stranded RNA ligase described herein is provided in solution, as a lyophilizate, or is immobilized on a substrate. In some embodiments, the substrate is a solid substrate, porous substrate, membrane, or particles. The enzyme can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, agarose or derivatives thereof (e.g., cross-linked agarose), and collagen, or solid matrices, such as activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a Docket Number CX10-269WO4 particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate. [0799] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support material. In some embodiments, the enzyme is immobilized on the support material by covalent attachment. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex. [0800] In some embodiments, the composition comprises a recombinant single stranded RNA ligase and another enzyme, such as a pyrophosphatase, such as a Type I or Type II pyrophosphatase, or an enzyme used in ATP recycling system. [0801] In some embodiments, the composition further comprises a buffer. Suitable reaction buffers are well known in the art and include but are not limited to, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine (TEoA), and 2-amino-2- hydroxymethyl-propane-1,3-diol (Tris), and the like. Any suitable buffer may be used. In some embodiments, the buffer is present at a concentration of 10-100 mM, 50-500 mM, or 300 mM- 1 M. Terminal Nucleotidyl Transferase (TnT) [0802] In some embodiments, where terminal nucleotidyl transferases are used to attach or incorporate one or more nucleotides to an oligonucleotide having an accessible 3’-OH group, for example, following attachment of a nucleotide donor to a nucleotide acceptor using the single-stranded RNA ligase, various terminal nucleotidyl transferases can be used in the methods. [0803] In some embodiments, the terminal nucleotidyl transferase is a terminal deoxynucleotidyl transferase (TdT). Various terminal deoxynucleotidyl transferases are described in US2023203553, WO23083997, WO23083999, WO23069865, WO20099451, WO21213903, WO22063835, US2023062303, US2022411840, US10435676, US10752887, US11208637, WO20239737, US10059929, US10760063, US10774316, US11390858, US2022119781, WO22029427, US2022127588, and PCT/US2023/076667; all publications incorporated by reference herein. [0804] In some embodiments, the terminal nucleotidyl transferase comprises a poly(N) polymerase, including variants thereof. In some embodiments, the poly(N) polymerase comprises a poly(U) polymerase (see, e.g., Kwak et al., RNA.2007 Jun; 13(6): 860–867), a poly(A) polymerase, and poly(G) polymerase. Variants of poly(N) polymerase are described in patent publication US2022403434, US20220815490, WO17216472, and WO20077227; all publications incorporated by reference herein [0805] In some embodiments, the terminal nucleotidyl transferase comprises polymerase theta (θ). Various forms of polymerase θ are described in U.S. Patent No.11390856; International patent publication WO2018175436; U.S. patent publication 20160108382; Kent et al., eLife, 2016, 5:e13740; Hogg et al., Docket Number CX10-269WO4 Nucleic Acids Res., 2012, 40(6): 2611–2622; and Tredinnick et al., RNA, 2023, 29:1288-130; all publications incorporated by reference herein. Arrays and Kits [0806] In some embodiments, further provided are arrays comprising the recombinant single-stranded ligases, for example, for screening of nucleotide acceptors and nucleotide donors. In some embodiments, further provided is a kit comprising at least one recombinant single-stranded RNA ligase. In some embodiments, the kit further comprises one or more of a buffer, a nucleotide cofactor (e.g., ATP), and/or one or more nucleotide donors and/or oligonucleotide acceptors. [0807] In some embodiments, the kit further comprises an additive or ligation enhancing agent, including but not limited to, one or more of DMSO, betaine, polyethylene glycol (e.g., PEG 6000, PEG 8000, etc.), bovine serum albumin, Ficoll, and dextran (e.g., Dextran 6000). EXAMPLES [0808] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention. [0809] In the experimental disclosure below, the following abbreviations where relevant apply: ppm (parts per million); M (molar); mM (millimolar), uM and µM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and µg (micrograms); L and l (liter); ml and mL (milliliter); ul, uL, µl, and µL (microliter); cm (centimeters); mm (millimeters); um and µm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); OD (optical density); MW (molecular weight); rpm (rotations per minute); rcf (relative centrifugal force); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); NGS (next-generation sequencing); ds (double stranded); ss (single-stranded); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FPLC (fast protein liquid chromatography); ddH2O (double distilled water); PBS (phosphate buffered saline); BSA (bovine serum albumin); DTT (dithiothreitol); CAM (chloramphenicol); CAT (chloramphenicol acetyltransferase); IPTG (isopropyl β-D-1- thiogalactopyranoside); FIOPC or FIOP (fold improvements over positive control or parent); LB (Luria- Bertani); TB (Terrific-Broth). Abbreviations for modified nucleotides Alias Description ' Docket Number CX10-269WO4 mA 2'-methoxyadenosine mC 2'-methoxycytidine ' Docket Number CX10-269WO4 s o o gonuceo e accepors, onors, an proucs. SEQ ID o m Docket Number CX10-269WO4 SEQ ID NO: Oligo Sequence Docket Number CX10-269WO4 SEQ ID NO: Oligo Sequence Docket Number CX10-269WO4 SEQ ID NO: Oligo Sequence o m Docket Number CX10-269WO4 SEQ ID NO: Oligo Sequence ho Example 1 Shake Flask Expression and Purification of ssRNA ligases Shake-flask expression via IPTG induction [0810] Selected E. coli strains expressing genes of interest were plated onto LB agar plates with 1% glucose and 30 µg/mL chloramphenicol and grown overnight at 37 ºC. A single colony from each culture was Docket Number CX10-269WO4 transferred to 5 mL of LB broth with 1% glucose and 30 µg/mL chloramphenicol. The cultures were grown for 20 h at 30 ℃, 250 rpm, and subcultured at a dilution of approximately 1:50 into 250 mL of Terrific Broth with 30 µg/mL of chloramphenicol, to a final OD600 of about 0.05. The cultures were incubated for approximately 195 min at 30 ℃, 250 rpm, to an OD600 of about 0.6, and then induced with the addition of IPTG at a final concentration of 1 mM. The induced cultures were incubated for 20 h at 30 ℃, 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min. The culture supernatant was discarded, and the pellets were resuspended in 35 mL of 20 mM triethanolamine, pH 7.5. This cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M‐ 110L). The crude lysate was pelleted by centrifugation (11,000 rpm for 60 min at 4 ℃), and the supernatant was then filtered through a 0.2 µm PES membrane to further clarify the lysate. Shake-flask expression via autoinduction [0811] Variants described above were streaked out onto agar plates containing 30 μg/mL chloramphenicol and 0.01% glucose (v/v) for single colonies. Then, a single colony was used to inoculate a 5-mL tube containing Terrific Broth (TB) media (Teknova) and 30 μg/mL chloramphenicol, which was then shaken for 20 hours at 30°C, 250 rpm. A glycerol stock was then prepared by mixing the overnight culture with 50% glycerol at a 2:1 ratio. These cultures grown, were thawed, and 30 µL from each glycerol stock was used to inoculate 1 L shake flasks containing: 160 mL Terrific Broth (TB) media (Teknova), 30 μg/mL chloramphenicol, 0.03% (v/v) lactose and 0.075% (v/v) glucose. The shake flasks were grown at 32 °C for 18 hours at 250 rpm. Following this incubation period, the cultures were centrifuged at 4,000 rpm for 10 min at 4 °C. The culture supernatant was discarded, and the pellets were resuspended in 30 mL of 50 mM Tris-HCl, pH 7.5. The cell suspension was chilled in an ice bath and lysed using a Microfluidizer cell disruptor (Microfluidics M-110L). The crude lysate was pelleted by centrifugation (10,000 rpm for 90 min at 4 °C) in an Avanti J-series centrifuge and JLA-16.250 fixed rotor (Beckman Coulter). The supernatant was then filtered through a 0.2 μm PES membrane to further clarify the lysate. The clarified lysates were then supplemented with 20 mM imidazole and 500 mM NaCl. Purification of ssRNA ligases from Shake Flask Lysates [0812] ssRNA ligase lysates were supplemented with 1/10th volume of SF elution buffer (50 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, 0.02% v/v Triton X-100 reagent) per well. Lysates were then purified using an AKTA Pure purification system and a 5 mL HisTrap FF column (GE Healthcare) using run parameters provided below. The SF wash buffer comprised 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 0.02% v/v Triton X-100 reagent. Table 1. Purification Parameters Docket Number CX10-269WO4 Table 1. Purification Parameters Equilibration volume 5 column volumes (CV) = 25 mL [0813] Eluti oled, then dialyzed overnight in dialysis buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol) in a 3.5K Slide-A-Lyzer™ dialysis cassette (Thermo Fisher) for buffer exchange. ssRNA ligase concentrations in the preparations were measured by absorption at 280 nm. Example 2 Capillary Electrophoresis (CE) Analysis of Oligonucleotides Sample preparation for reaction analysis using CE [0814] For analysis of the reaction samples, capillary electrophoresis was performed using an ABI 3500xl Genetic Analyzer (ThermoFisher). Reactions (1 µL) were quenched by the addition of 99 μL of 1 mM aqueous EDTA. Quenched reactions were diluted in water to 1.25 nM FAM-labeled oligonucleotide, and a 2- μL aliquot of this solution was transferred to a new 96-well MicroAmp Optical PCR plate or 384-well MicroAmp Optical PCR plate containing 18 μL Hi-Di™ Formamide (ThermoFisher) containing an appropriate size standard (LIZ or Alexa633). The ABI3500xl was configured with POP6 polymer, 50 cm capillaries, and a 55 °C oven temperature. Pre-run settings were 18KV for 50 sec. Injection was 10KV for 2 sec, and the run settings were 19KV for 620 sec. FAM-labeled oligo substrates and products were identified by their mobility relative to the sizing ladder. Example 3 Gene Acquisition and Expression of Wild-type Single-stranded RNA ligases (ssRNA ligase) [0815] Synthetic genes encoding an N-terminal 6-histidine tagged version of multiple wild-type (WT) ssRNA ligase enzymes were cloned into the pCK110900 vector system (See e.g., US Pat. No.9,714,437, which is hereby incorporated by reference in its entirety) and subsequently expressed in an E. coli strain derived from W3110. [0816] Cells transformed with the ssRNA ligase expression constructs were grown at shake-flask scale as described in Example 1. Cells were then lysed, purified, and dialyzed into storage buffer (20 mM Tris-HCl, pH 7.4, 100 mM KCl, 0.1 mM EDTA, and 50% glycerol). After overnight dialysis, protein samples were removed, and enzyme concentrations were measured by absorption at 280 nm using a NanoDrop™ 1000 spectrophotometer. Soluble protein concentrations are summarized in Table 3.1 below, showing a fold improvement in soluble protein production following shake-flask purification relative to the enzyme isolated from Escherichia phage vB_EcoM_VR25 (SEQ ID NO: 2). Docket Number CX10-269WO4 Table 3.1 Soluble Enzyme Production of Variants Relative to SEQ ID NO: 2 SEQ ID NO: Source organism of Relative Soluble Enzyme Production (nt/ ) RNA li n n (R l tiv t SEQ ID NO: 2) xamp e Activity of SEQ ID NO: 2-8 in a Ligation with a Methoxy-modified RNA donor Activity of shake-flask purified ssRNA ligases [0817] ssRNA ligase variants SEQ ID NO: 2, 4, 6, and 8 were produced in shake flask and purified as described in Example 1. [0818] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 100 μM oligonucleotide donor, 1 mM ATP, 10 mM DTT, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 50 mM TRIS-HCl (pH 7.5), and 10 mM MgCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 4.1 Reaction temperature (C)- 37; Reaction Time (min)- 60 min; Reaction volume (µL)- 1; ssRNA ligase [0819] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 4.2. Table 4.2 Docket Number CX10-269WO4 Table 4.2 3/4 6.9 +++ 5/6 62 +++ D Activity of SEQ ID NOs: 4-6 and SEQ ID NOs: 10-20 in a ligation with a methoxy-modified RNA donor Activity of shake-flask purified ssRNA ligases [0820] ssRNA ligase variants SEQ ID NO: 4, 6, 10, 12, 14, 16, 18, and 20 were produced in shake flask and purified as described in Example 1. [0821] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 1 mM ATP, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 5.1 [0822] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 5.2. Table 5.2 E ID N R l i i f : Docket Number CX10-269WO4 Table 5.2 Levels of increased activity were determined for relative % conversion in reference to the activity of SEQ ID Activity of SEQ ID NO: 6, 10, 14 and 18 in a ligation with a methoxy-modified RNA donor [0823] ssRNA ligase variants SEQ ID NO: 6, 10, 14, and 18 were produced in shake flask and purified as described in Example 1. [0824] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions included 50 μM oligonucleotide acceptor, 100 μM oligonucleotide donor, 200 μM ATP, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 6.1 Reaction temperature (C)- 40; Reaction Time (min)- 995 minutes; Reaction volume (µL)- 1; ssRNA ligase or [0825] The structure of /5Phos/rC/3p-alkyne-1/ is below: Docket Number CX10-269WO4 [0826] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product(s) divided by the sum of the total peak area. The results are shown in Table 6.2. Table 6.2 SEQ ID NO: ssRNA ligase % conversion with % conversion with % conversion with A 0, xamp e Activity of SEQ ID NO: 6 with ATP recycling system [0827] ssRNA ligase variant SEQ ID NO: 6 was produced in shake flask and purified as described in Example 1. A reaction with sub-stoichiometric ATP and an enzymatic ATP-recycle using adenylate kinase and acetate kinase enzymes to convert ligase reaction product AMP to ATP was compared to a reaction with excess ATP. The effect of each reaction condition on the generation of reaction by-products was compared. [0828] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reaction 1 with excess ATP: Reactions included 100 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 250 μM ATP, 6.2 μM ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 10 mM MgCl2. Reaction 2 with recycle enzymes: Reactions included 100 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 0.005 μM ATP, 10 mM lithium acetyl phosphate, 6.2 μM ssRNA ligase solution, 1 uM acetate kinase (SEQ ID NO: 24), 1 uM adenylate kinase (SEQ ID NO: 26), 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), and 10 mM MgCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for LCMS analysis as described in Example 2. Table 7.1 Reaction tem erature (C)- 30 Reaction Time (min)- 120 minutes Reaction volume ( L)- 1 ssRNA li ase [0829] Ratio of % conversion product to byproduct was calculated as the percent product divided by the percent total byproduct. Percent of product or by product was determined by the sum of the peak(s) of interest divided by the sum of the total peak area. The results are shown in Table 7.2. Docket Number CX10-269WO4 Table 7.2 Relative ratio of % Improvements over SEQ ID NO: 14 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule [0830] The ssRNA ligase of SEQ ID NO: 14 was selected as the parent ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in shake flask and purified as described in Example 1. [0831] Prior to the reaction, purified enzymes were preincubated at 58 °C for 1 hour in 200 μL BioRad PCR plates. Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions included 50 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 0.5 mM ATP, 5 μM ssRNA ligase, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) heat-treated ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 6.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 8.1 [0832] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product(s) divided by the sum of the total peak area. The results are shown in Table 8.2. Table 8.2- ssRNA Ligase activity relative to SEQ ID NO: 14 E ID N A i A i Diff FI P A i i 1 4 Docket Number CX10-269WO4 Table 8.2- ssRNA Ligase activity relative to SEQ ID NO: 14 37/38 R34Y + 39/40 F269L + Ligase expression and lysate processing for high throughput (HTP) Screening High Throughput (HTP) Growth of Ligase Enzyme and Variants [0833] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose and 30 µg/mL chloramphenicol. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom NUNC™ (Thermo-Scientific) plates filled with 180 µl/well LB medium supplemented with 1% glucose and 30 µg/mL chloramphenicol. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner). Overnight growth samples (20 µL) were transferred into Costar 96-well deep plates filled with 380 µL of Terrific Broth supplemented with 30 µg/mL chloramphenicol. The plates were incubated for 120 minutes in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner) until the OD600 reached between 0.4-0.8. The cells were then induced with 40 µL of 10 mM IPTG in sterile water and incubated overnight for 18-20 hours in a shaker (250 rpm, 30 C, and 85% relative humidity; Kuhner). The cells were pelleted (4,000 rpm for 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to analysis. Thermal lysis of HTP Cell Pellets with Lysozyme [0834] For lysis, 400 µL lysis buffer containing 50 mM triethanolamine buffer, pH 7.5, and 0.2 g/L lysozyme were added to the cell pellet in each well. The cells were shaken vigorously at room temperature for 60 minutes on a bench top shaker. A 100-uL aliquot of the re-suspended cells was transferred to a 96-well format 200 μL BioRad PCR plate, then briefly spun-down prior to 1 h heat treatment at 60 °C (unless otherwise indicated). Following heat-treatment, the cell debris was pelleted by centrifugation (4,000 rpm at 4 ˚C for 10 minutes), and clear supernatants were then used in biocatalytic reactions to determine their activity levels. Example 10 Improvements over SEQ ID NO: 32 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0835] The ssRNA ligase of SEQ ID NO: 32 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis Docket Number CX10-269WO4 and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 10.1. [0836] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 50 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 0.5 mM ATP, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 10.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 10.1 Reaction Conditions Lysate concentration (vol %): 25; Pre-incubation Temp: 64 °C; Pre-incubation Time (min): 60.0; Reaction D [0837] Activity relative to SEQ ID NO: 32 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 32 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 10.2. Table 10.2- ssRNA Ligase activity relative to SEQ ID NO: 32 SEQ ID NO: Amin A id Diff r n FIOP tivit 1 R l tiv to Docket Number CX10-269WO4 Example 11 Improvements over SEQ ID NO: 44 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0838] SEQ ID NO: 44 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 11.1. [0839] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 50 μM oligonucleotide acceptor, 100 μM oligonucleotide donor, 0.5 mM ATP, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 200 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 11.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 11.1 Reaction conditions n D [0840] Activity relative to SEQ ID NO: 44 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 44 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 11.2. Table 11.2- ssRNA Ligase activity relative to SEQ ID NO: 44 SEQ ID NO: Amino Acid Differences FIOP activit 1 Docket Number CX10-269WO4 Table 11.2- ssRNA Ligase activity relative to SEQ ID NO: 44 81/82 K237G + 4 4D N2 D Example 12 Improvements over SEQ ID NO: 100 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0841] The ssRNA ligase of SEQ ID NO: 100 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 12.1. [0842] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200 μM oligonucleotide donor, 0.5 mM ATP, 25 vol % ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 12.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 12.1 Reaction conditions Lysate concentration (vol %): 25; Pre-incubation Temp: 60 °C; Pre-incubation Time (min): 60.0; Reaction Q ; variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 100 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 12.2. Table 12.2- ssRNA Ligase activity relative to SEQ ID NO: 100 SEQ ID NO: Amino Acid Differences FIOP Activity 1 0 Docket Number CX10-269WO4 Table 12.2- ssRNA Ligase activity relative to SEQ ID NO: 100 179/180 L160R + 181/182 T362E + Example 13 Activity of SEQ ID NOs: 14, 32, and 44 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule Activity of shake-flask purified ssRNA ligases [0844] The ssRNA ligase variants SEQ ID NOs: 14, 32, and 44 were produced in shake flask and purified as described in Example 1. [0845] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 150 μM oligonucleotide donor, 0.5 mM ATP, 25 μM ssRNA ligase, 0.2 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2 and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 13.1 Docket Number CX10-269WO4 [0846] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 13.2. Table 13.2- ssRNA ligase activity improvements Reaction components % conversion by SEQ ID NO: (nt/aa) 4 + xamp e Activity of SEQ ID NOs: 14, 32, and 44 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule Activity of shake-flask purified ssRNA ligases [0847] The ssRNA ligase variants SEQ ID NOs: 14, 32, and 44 were produced in shake flask and purified as described in Example 1. [0848] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 150 μM oligonucleotide donor, 0.5 mM ATP, 6.25 μM ssRNA ligase, 0.2 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2 and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 14.1 Reaction temperature - 50 ℃; Reaction Time (min)- 180 min; Reaction volume (µL)- 1; ssRNA ligase s [0849] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 14.2. Table 14.2- ssRNA ligase activity improvements a) Docket Number CX10-269WO4 Table 14.2- ssRNA ligase activity improvements Acceptor Donor product 13/14 31/32 43/44 SEQ ID NO: 217 /5Phos/mAmUmA SEQ ID NO: 226 ++ ++ +++ p Activity of SEQ ID NOs: 14, 32, and 44 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule Activity of shake-flask purified ssRNA ligases [0850] The ssRNA ligase variants SEQ ID NOs: 14, 32, and 44 were produced in shake flask and purified as described in Example 1. [0851] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 200 μM oligonucleotide acceptor, 300 μM oligonucleotide donor, 0.5 mM ATP, 5 μM ssRNA ligase, 0.2 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2 and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 15.1 Reaction temperature - 50 ℃; Reaction Time (min)- 180 min; Reaction volume (µL)- 1; ssRNA ligase [0852] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 15.2. Table 15.2- ssRNA ligase activity improvements a) Docket Number CX10-269WO4 Table 15.2- ssRNA ligase activity improvements Reaction components % conversion by SEQ ID NO: (nt/aa) Acceptor Donor product 13/14 31/32 43/44 p Activity of SEQ ID NOs: 14, 32, 44, and 140 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated Donor molecule Activity of shake-flask purified ssRNA ligases [0853] ssRNA ligase variants SEQ ID NOs: 14, 32, 44, and 140 were produced in shake flask and purified as described in Example 1. [0854] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 150 μM oligonucleotide donor, 0.5 mM ATP, 25 μM ssRNA ligase, 0.2 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2 and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 16.1 Reaction temperature – 50 ℃; Reaction Time (min)- 180 min; Reaction volume (µL)- 1; ssRNA ligase [0855] Percent conversion was calculated as the percent product of the variant, defined as the sum of the area of product divided by the sum of the total peak area. The relative percent conversion was measured as the % product of the variant relative to the % product of the reference. The results are shown in Table 16.2. Table 16.2- ssRNA ligase activity improvements a) 0 Docket Number CX10-269WO4 Table 16.2- ssRNA ligase activity improvements /5Phos/mUmAmU /52FA//i2FA//i2FA/m 2FA i2FA 2FA A ATTTTTTTT A A ATTT Improvements over SEQ ID NO: 140 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0856] The ssRNA ligase of SEQ ID NO: 140 was selected as the parent ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 17.1. [0857] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 17.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 17.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction [0858] Activity relative to SEQ ID NO: 140 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 140 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 17.2. Docket Number CX10-269WO4 Table 17.2. ssRNA Ligase activity relative to SEQ ID NO: 140 SEQ ID Amino Acid Differences FIOP activity 1 relative NO: (nt/aa) (Relative to SEQ ID NO: 140) to SEQ ID NO: 140 Docket Number CX10-269WO4 Table 17.2. ssRNA Ligase activity relative to SEQ ID NO: 140 349/350 S310G/K314L + 351/352 K214E/K347I/R358D + d Example 18 Improvements over SEQ ID NO: 246 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0859] The ssRNA ligase of SEQ ID NO: 246 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 18.1. [0860] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 18.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the Docket Number CX10-269WO4 indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 18.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction y y p p variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 246 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 18.2. Table 18.2. ssRNA Ligase activity relative to SEQ ID NO: 246 SEQ ID NO: Amino Acid Differences FIOP activity relative to Docket Number CX10-269WO4 Table 18.2. ssRNA Ligase activity relative to SEQ ID NO: 246 459/460 N303A + 461/462 S125T + Example 19 Improvements over SEQ ID NO: 400 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0862] The ssRNA ligase of SEQ ID NO: 400 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 19.1. [0863] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 19.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 19.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 45; Reaction variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 400 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 19.2. Table 19.2. ssRNA Ligase activity relative to SEQ ID NO: 400 SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 20 Improvements over SEQ ID NO: 492 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0865] The ssRNA ligase of SEQ ID NO: 492 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 20.1. [0866] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a Docket Number CX10-269WO4 single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 20.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 20.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 55; Pre-incubation Time (min): 60; Reaction [086 ] ct v ty re at ve to S Q O: 9 ( ct v ty O ) was ca cu ated as t e percent product o t e variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 492 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 20.2. Table 20.2. ssRNA Ligase activity relative to SEQ ID NO: 492 SEQ ID NO: Amino Acid Differences FIOP activity relative to Docket Number CX10-269WO4 Table 20.2. ssRNA Ligase activity relative to SEQ ID NO: 492 565/566 F229A + 567/568 S225P + p Improvements over SEQ ID NO: 520 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0868] The ssRNA ligase of SEQ ID NO: 520 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 21.1. [0869] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 21.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 21.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction 0; [0870] Activity relative to SEQ ID NO: 520 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 520 (where the percent product may be set as Docket Number CX10-269WO4 the average of replicates or else the highest single sample as appropriate). The results are shown in Table 21.2. Table 21.2. ssRNA Ligase activity relative to SEQ ID NO: 520 SEQ ID NO: Amino Acid Differences FIOP activity relative to SEQ (nt/aa) (Relative to SEQ ID NO: 520) ID NO: 520 xamp e Improvements over SEQ ID NO: 594 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0871] The ssRNA ligase of SEQ ID NO: 594 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 22.1. [0872] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 22.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes. Post- ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. The samples are then held at 10 °C until they are removed for quenching and CE workup. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 22.1 Reaction conditions Lysate concentration (vol %) 25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction ° 0; : variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 594 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 22.2. Table 22.2. ssRNA Ligase activity relative to SEQ ID NO: 594 SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 23 Improvements over SEQ ID NO: 594 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0874] The ssRNA ligase of SEQ ID NO: 594 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis Docket Number CX10-269WO4 and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 23.1. [0875] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 23.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 23.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction 0; l/; [0876] Activity relative to SEQ ID NO: 594 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 594 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 23.2. Table 23.2. ssRNA Ligase activity relative to SEQ ID NO: 594 SEQ ID NO: Amino Acid Differences FIOP activity relative Docket Number CX10-269WO4 Table 23.2. ssRNA Ligase activity relative to SEQ ID NO: 594 677/678 S225R + 679/680 N36D + a p e Improvements over SEQ ID NO: 634 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0877] The ssRNA ligase of SEQ ID NO: 634 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 24.1. [0878] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 24.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 24.1 Reaction conditions L sate concentration (vol %) 10 Pre-incubation Tem (°C): 60 Pre-incubation Time (min): 60 Reaction 0; ; Docket Number CX10-269WO4 [0879] Activity relative to SEQ ID NO: 634 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 634 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 24.2. Table 24.2. ssRNA Ligase activity relative to SEQ ID NO: 634 SEQ ID NO: Amino Acid Differences FIOP activity relative (nt/aa) (Relative to SEQ ID NO: 634) to SEQ ID NO: 634 Example 25 Improvements over SEQ ID NO: 710 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0880] The ssRNA ligase of SEQ ID NO: 710 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 25.1. [0881] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 25.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the Docket Number CX10-269WO4 indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes. Post- ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 25.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction 0; [0882] Activity relative to SEQ ID NO: 710 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 710 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 25.2. Table 25.2. ssRNA Ligase activity relative to SEQ ID NO: 710 SEQ ID NO: Amino Acid Differences FIOP activity relative to Example 26 Improvements over SEQ ID NO: 738 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0883] The ssRNA ligase of SEQ ID NO: 738 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis Docket Number CX10-269WO4 and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 26.1. [0884] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor, 200-300 μM oligonucleotide donor, 0.5 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 26.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time followed by a heat inactivation step at 95 °C for 2 minutes. Post- ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 10 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 26.1 Reaction conditions Lysate concentration (vol %) 5; Pre-incubation Temp (°C): 60; Pre-incubation Time (min): 60; Reaction : [0885] Activity relative to SEQ ID NO: 738 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 738 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 26.2. Table 26.2. ssRNA Ligase activity relative to SEQ ID NO: 738 SEQ ID NO: Amino Acid Differences FIOP activit relative to Example 27 Docket Number CX10-269WO4 Improvements over SEQ ID NO: 756 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0886] The ssRNA ligase of SEQ ID NO: 756 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 27.1. [0887] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 1000 μM oligonucleotide acceptor, 1200 μM oligonucleotide donor, 2 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 5 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 27.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 27.1 Reaction conditions Lysate concentration (vol %): 6.25 ;Pre-incubation temperature (°C): 75 ;Pre-incubation time (min): 60 [0888] Activity relative to SEQ ID NO: 756 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 756 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 27.2. Table 27.2. ssRNA Ligase activity relative to SEQ ID NO: 756 SEQ ID NO: Amin A id Diff r n FIOP tivit r l tiv to Docket Number CX10-269WO4 Table 27.2. ssRNA Ligase activity relative to SEQ ID NO: 756 777/778 L281I/F282L ++ 779/780 T302I ++ Example 28 Improvements over SEQ ID NO: 768 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0889] The ssRNA ligase of SEQ ID NO: 768 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 28.1. [0890] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 1000 μM oligonucleotide acceptor, 1200 μM oligonucleotide donor, 2 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 28.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 28.1 Reaction conditions Lysate concentration (vol %): 6.25; Lysis preincubation temp (°C): 58; Lysis preincubation time (min): 60; ° ' t ct [0891] Activity relative to SEQ ID NO: 768 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 768 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 28.2. Table 28.2. ssRNA Ligase activity relative to SEQ ID NO: 768 SEQ ID NO: Amino Acid Differences FIOP %product relative to Docket Number CX10-269WO4 Table 28.2. ssRNA Ligase activity relative to SEQ ID NO: 768 847/848 Y44R/G272L/D280S ++ 849/850 Y44R/G272L/D279R ++ Example 29 Improvements over SEQ ID NO: 844 in the Ligation of an Acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0892] The ssRNA ligase of SEQ ID NO: 844 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 29.1. [0893] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 1000 μM oligonucleotide acceptor, 1200 μM oligonucleotide donor, 2 mM ATP, ssRNA ligase heat-treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 29.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 29.1 Reaction conditions Lysate concentration (vol %): 6.25; Lysis preincubation temp (°C): 58; Lysis preincubation time (min): 60; ° d /; ed [0894] Product ratio relative to SEQ ID NO: 844 was calculated as the percent product of the variant, defined as the sum of the area of (product 3 +product 4)/ (product 1 + product 2) divided by the corresponding ratio for SEQ ID NO: 844 (where the ratio product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 29.2. Table 29.2. ssRNA Ligase activity relative to SEQ ID NO: 844 SEQ ID NO: Amino Acid Differences FIOP product ratio relative Example 30 Docket Number CX10-269WO4 Improvements over SEQ ID NO: 882 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0895] The ssRNA ligase of SEQ ID NO: 882 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0896] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 30.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2.
Docket Number CX10-269WO4 Table 30.1 Reaction conditions Lysate concentration (vol %): 3.125; Pre-incubation Temp (°C): 58; Pre-incubation Time (min): 60; ° e Y; [0897] Activity relative to SEQ ID NO: 882 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 882 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 30.2. Table 30.2. activity relative to SEQ ID NO: 882 SEQ ID NO: Amino Acid Differences FIOP activit relative to Docket Number CX10-269WO4 Table 30.2. activity relative to SEQ ID NO: 882 951/952 T38V/E238G/Q255A/I359D/K381R ++ 953/954 T38V/L127V/E238G/R284F ++ d Example 31 Improvements over SEQ ID NO: 936 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0898] The ssRNA ligase of SEQ ID NO: 936 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0899] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 31.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in Docket Number CX10-269WO4 a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 31.1 Reaction conditions Lysate concentration (vol %): 3.125; Pre-incubation Temp (°C): 58; Pre-incubation Time (min): 60; e [0900] Activity relative to SEQ ID NO: 936 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 936 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 31.2. Table 31.2. Activity relative to SEQ ID NO: 936 SE ID NO A i A id Diff FIOP ti it l ti to Docket Number CX10-269WO4 Table 31.2. Activity relative to SEQ ID NO: 936 1003/1004 H254R/Y260R ++ 1005/1006 L135M/P170R/N271G ++ d Example 32 Improvements over SEQ ID NO: 936 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0901] The ssRNA ligase of SEQ ID NO: 936 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0902] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 32.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP was dispensed, the reaction plates were heat sealed, vortexed and centrifuged. The reaction plate was then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 32.1 Reaction conditions Lysate concentration (vol %) 3.125; Pre-incubation Temp (°C): 58; Pre-incubation Time (min): 60; ° e [0903] Activity relative to SEQ ID NO: 936 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 936 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 32.2. Table 32.2. Activity relative to SEQ ID NO: 936 SEQ ID NO: Amino Acid Differences FIOP activity relative Docket Number CX10-269WO4 Table 32.2. Activity relative to SEQ ID NO: 936 1075/1076 E350R + 1077/1078 N141G + d xamp e Improvements over SEQ ID NO: 992 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0904] The ssRNA ligase of SEQ ID NO: 992 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0905] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 33.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2.
Docket Number CX10-269WO4 Table 33.1 Reaction conditions Lysate concentration (vol %): 3.125; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): ° me or [0906] Activity relative to SEQ ID NO: 992 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 992 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 33.2. Table 33.2. Activity relative to SEQ ID NO: 992 SEQ ID NO: Amino Acid Differences FIOP activity relative Docket Number CX10-269WO4 Table 33.2. Activity relative to SEQ ID NO: 992 1137/1138 H254R ++ 1139/1140 K115S/F177V/H254R ++ d Example 34 Improvements over SEQ ID NO: 1104 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0907] The ssRNA ligase of SEQ ID NO: 1104 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0908] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- Docket Number CX10-269WO4 treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 34.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 34.1 Reaction conditions Lysate concentration (vol %): 1.56; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): 60;Reaction or [0909] Activity relative to SEQ ID NO: 1104 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1104 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 34.2. Table 34.2. Activity relative to SEQ ID NO: 1104 SEQ ID NO: Amino Acid Differences FIOP activit relative to Docket Number CX10-269WO4 Table 34.2. Activity relative to SEQ ID NO: 1104 1227/1228 K27F ++ 1229/1230 F374V ++ ed Example 35 Improvements over SEQ ID NO: 1222 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0910] The ssRNA ligase of SEQ ID NO: 1222 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0911] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 35.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 35.1 Reaction conditions Lysate concentration (vol %): 6.25; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): 60;Reaction ° [0912] Activity relative to SEQ ID NO: 1222 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1222 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 35.2. Table 35.2. Activity relative to SEQ ID NO: 1222 SEQ ID NO: Amino Acid Differences FIOP activity relative to ed Example 36 Improvements over SEQ ID NO: 1222 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants Docket Number CX10-269WO4 [0913] The ssRNA ligase of SEQ ID NO: 1222 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0914] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 36.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 36.1 Reaction conditions Lysate concentration (vol %): 6.25; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): 60;Reaction or 6- [0915] Activity relative to SEQ ID NO: 1222 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1222 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 36.2. Docket Number CX10-269WO4 Table 36.2. Activity relative to SEQ ID NO: 1222 SEQ ID NO: Amino Acid Differences FIOP activity relative to (nt/aa) (Relative to SEQ ID NO: 1222) SEQ ID NO: 1222 ed p Improvements over SEQ ID NO: 1222 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0916] The ssRNA ligase of SEQ ID NO: 1222 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0917] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 37.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 37.1 Reaction conditions Lysate concentration (vol %) 625; Pre-incubation Temp (°C): 73; Pre-incubation Time (min): 60; Reaction Docket Number CX10-269WO4 [0918] Activity relative to SEQ ID NO: 1222 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1222 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 37.2. Table 37.2. Activity relative to SEQ ID NO: 1222 SEQ ID NO: Amino Acid Differences FIOP activity relative to (nt/aa) (Relative to SEQ ID NO: 1222) SEQ ID NO: 1222 Example 38 Improvements over SEQ ID NO: 1264 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0919] The ssRNA ligase of SEQ ID NO: 1264 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0920] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 38.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Docket Number CX10-269WO4 Table 38.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 58; Pre-incubation Time (min): 60; Reaction ° O: [ ] c v y rea ve o Q : ( c v y ) was cacuae as e percen pro uc o e variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 38.2. Table 38.2. Activity relative to SEQ ID NO: 1264 SEQ ID NO: Amino Acid Differences FIOP activity relative to Docket Number CX10-269WO4 Table 38.2. Activity relative to SEQ ID NO: 1264 1363/1364 K66Q + Levels of increased activity were determined for FIOP activity relative to SEQ ID NO: 1264 and are defined Improvements over SEQ ID NO: 1222 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0922] The ssRNA ligase of SEQ ID NO: 1222 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0923] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 39.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 39.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 58; Pre-incubation Time (min): 60; Reaction e Docket Number CX10-269WO4 [0924] Activity relative to SEQ ID NO: 1222 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1222 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 39.2. Table 39.2. Activity relative to SEQ ID NO: 1222 SEQ ID NO: Amino Acid Differences FIOP activity relative to (nt/aa) (Relative to SEQ ID NO: 1222) SEQ ID NO: 1222 ed Example 40 Improvements over SEQ ID NO: 1264 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0925] The ssRNA ligase of SEQ ID NO: 1264 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0926] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized Docket Number CX10-269WO4 in Table 40.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 40.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 73; Pre-incubation Time (min): 60; Reaction [09 ] ct v ty re at ve to S Q O: 6 ( ct v ty O ) was ca cu ated as t e percent product o t e variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 40.2. Table 40.2. Activity relative to SEQ ID NO: 1264 SEQ ID NO: Amino Acid Differences FIOP activity relative to ed Docket Number CX10-269WO4 Example 41 Improvements over SEQ ID NO: 1264 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0928] The ssRNA ligase of SEQ ID NO: 1264 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0929] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 41.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 41.1 Reaction conditions Lysate concentration (vol %): 6.25; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): 60;Reaction or [0930] Activity relative to SEQ ID NO: 1264 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with Docket Number CX10-269WO4 the percent product observed by the reaction with SEQ ID NO: 1264 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 41.2. Table 41.2. Activity relative to SEQ ID NO: 1264 SEQ ID NO: Amino Acid Differences FIOP activity relative to (nt/aa) (Relative to SEQ ID NO: 1264) SEQ ID NO: 1264 ed Example 42 Improvements over SEQ ID NO: 1344 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0931] The ssRNA ligase of SEQ ID NO: 1344 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0932] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution Docket Number CX10-269WO4 was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 42.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 42.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 73; Pre-incubation Time (min): 60; Reaction e [0933] Activity relative to SEQ ID NO: 1344 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1344 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 42.2. Table 42.2. Activity relative to SEQ ID NO: 1344 SEQ ID NO: Amino Acid Differences FIOP activity relative to ed Example 43 Improvements over SEQ ID NO: 1474 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants Docket Number CX10-269WO4 [0934] The ssRNA ligase of SEQ ID NO: 1474 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0935] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 43.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. Post-ligation, reactions were subjected to “Alkaline Phosphate Treatment” or AP Treatment. This treatment involves the addition of 5 – 20 uM of SEQ ID NO: 914 to the reaction plates. After the AP is dispensed, the reaction plates are heat sealed, vortexed and centrifuged. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2. Table 43.1 Reaction conditions Lysate concentration (vol %) 6.25; Pre-incubation Temp (°C): 73; Pre-incubation Time (min): 60; Reaction e [0936] Activity relative to SEQ ID NO: 1474 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1474 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 43.2. Table 43.2. Activity relative to SEQ ID NO: 1474 to Docket Number CX10-269WO4 Table 43.2. Activity relative to SEQ ID NO: 1474 Levels of increased activity were determined for FIOP activity relative to SEQ ID NO: 1474 and are defined Improvements over SEQ ID NO: 1500 in the ligation of an acceptor (oligo)nucleotide with free 3’-OH and a 5’-monophosphorylated donor molecule HTP Screening for Improved Ligase Variants [0937] The ssRNA ligase of SEQ ID NO: 1500 was selected as the parent Ligase enzyme. Libraries of engineered genes were produced from the parent gene using various techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in HTP and prepared as described in Table 9.1. [0938] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100- 1000 μM oligonucleotide acceptor, 200-2000 μM oligonucleotide donor, 1-4 mM ATP, ssRNA ligase heat- treated lysate, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 10 mM MgCl2, and 1 mM CoCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre- mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The substrates and reaction conditions are summarized in Table 44.1. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction followed by a heat inactivation step at 95 °C for 2 minutes. The reaction plate is then incubated in a thermal cycler for 15-30 minutes at 50 °C, followed by a heat inactivation step at 95 °C for 2 minutes. Reactions were quenched and processed for CE analysis as described in Example 2.
Docket Number CX10-269WO4 Table 44.1 Reaction conditions Lysate concentration (vol %): 6.25; Pre-incubation Temp (°C): 58;Pre-incubation Time (min): 60;Reaction ° or [0939] Activity relative to SEQ ID NO: 1500 (Activity FIOP) was calculated as the percent product of the variant, defined as the sum of the area of products divided by the sum of the total peak area, compared with the percent product observed by the reaction with SEQ ID NO: 1500 (where the percent product may be set as the average of replicates or else the highest single sample as appropriate). The results are shown in Table 44.2. Table 44.2. Activity relative to SEQ ID NO: 1500 SEQ ID NO: Amino Acid Differences FIOP activity relative to ed Example 45 Reduction of measured by-products in ligation reactions with added NaCl using variant SEQ ID NO: 936 Docket Number CX10-269WO4 [0940] ssRNA ligase variant SEQ ID NO: 936 was produced in shake flask and purified as described in Example 1. Ligation reactions included 50, 125, or 250 mM NaCl. The ratio of the desired ligation product to undesired by-products was compared to a control reaction with no added NaCl. [0941] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions included 475 μM oligonucleotide acceptor (unlabelled), 25 μM oligonucleotide acceptor (labelled), 550 μM oligonucleotide donor, 1.25 mM ATP, 5 μM ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2 and 10 mM MgCl2. Reactions were supplemented with 0, 50, 125 or 250 mM NaCl. The reactions were set up as follows(i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for CE analysis as described in Example 2. Table 45.1 Reaction temperature (C)- 30; Reaction Time (min)- 180 minutes; Reaction volume (µL)- 1; - 6/; p- [0942] Percent product or by-product was determined by the sum of the peak(s) of interest divided by the sum of the total peak area multiplied by 100. Th ratio of % conversion product to % byproduct was calculated as the percent product divided by the percent total by-product. The value was then divided by the value of the control reaction (no NaCl) to determine the fold improvement. The results are shown in Table 45.2. Table 45.2. Ligation reactions with SED ID no: 936 and NaCl Docket Number CX10-269WO4 Example 46 Activity of SEQ ID NO: 1222 with LNA, DNA, RNA and 5’a-thiophosphate modifications [0943] ssRNA ligase variant SEQ ID NO: 1222 was produced in shake flask and purified as described in Example 1. The variant was assayed with substrates comprising LNA, DNA, RNA and α-thiophosphate modifications at the 5' and 3' positions of the ligation junction. [0944] Reactions were performed in 384-well format 50 μL BioRad PCR plates. Reactions included 100 μM oligonucleotide acceptor (labelled), 900 μM oligonucleotide acceptor (unlabelled), 1200 μM oligonucleotide donor, 4 mM ATP, 5 μM ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2,10 mM MgCl2 and 100 mM NaCl. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for LCMS analysis as described in Example 2. Table 46.1 Condition 1: Reaction temperature (C)- 50; Reaction Time (min)- 60; Reaction volume (µL)- 0.4. e s/, e Docket Number CX10-269WO4 Table 46.1 Reaction 8: Oligonucleotide acceptors-/56- FAM/TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTmUmUmU+G, TTTTTTTTmUmUmU+G; T T T [0945] Percent product was determined by the sum of the peak of interest divided by the sum of the total peak area multiplied by 100. The results are shown in Table 46.2. Table 46.2. Percent conversion in ligation reactions of SEQ ID NO: 1222 with LNA, DNA, RNA and 5'-α−thiophosphate modifications : Example 47 Activity of SEQ ID NO: 936 with ATP recycling systems and effect on ratio of product to by-product Docket Number CX10-269WO4 [0946] ssRNA ligase variant SEQ ID NO: 936 was produced in shake flask and purified as described in Example 1. Reactions with sub-stoichiometric ATP and an enzymatic ATP-recycle system using adenylate kinase (SEQ ID NO: 26) and either acetate kinase (SEQ ID NO: 24), pyruvate kinase (Sigma- Aldrich, Sigma-Aldrich P/N P7768), or creatine kinase (Roche, Sigma-Aldrich P/N CK-RO) were evaluated. The ratio of product to by-product in the reactions with ATP recycle were compared to a control reaction. [0947] Reactions were performed in 96-well format 200 μL BioRad PCR plates. Reactions with acetate kinase included 25 μM labeled oligonucleotide acceptor, 475 μM unlabeled oligonucleotide acceptor, 550 μM oligonucleotide donor, 1 μM ATP, 0.625-2.5 mM lithium acetyl phosphate, 5 μM ssRNA ligase solution, 1 uM acetate kinase (SEQ ID NO: 24), 1 uM adenylate kinase (SEQ ID NO: 26), 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2 and 5 mM MgCl2. Reactions with pyruvate kinase included 25 μM labeled oligonucleotide acceptor, 475 μM unlabeled oligonucleotide acceptor, 550 μM oligonucleotide donor, 1 μM ATP, 0.625-2.5 mM phosphoenol pyruvate, 5 μM ssRNA ligase solution, 0.1 mg/mL pyruvate kinase solution, 1 uM adenylate kinase (SEQ ID NO: 26), 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2 and 5 mM MgCl2. Reactions with creatine kinase included 25 μM labeled oligonucleotide acceptor, 475 μM unlabeled oligonucleotide acceptor, 550 μM oligonucleotide donor, 1 μM ATP, 0.625-2.5 mM creatine phosphate, 5 μM ssRNA ligase solution, 0.1 mg/mL creatine kinase solution, 1 uM adenylate kinase (SEQ ID NO: 26), 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2 and 5 mM MgCl2. The control reaction included 25 μM labeled oligonucleotide acceptor, 475 μM unlabeled oligonucleotide acceptor, 550 μM oligonucleotide donor, 5 mM ATP, 5 μM ssRNA ligase solution, 1 μM IPP (SEQ ID NO: 22), 100 mM triethanolamine (pH 7.8), 1 mM CoCl2 and 5 mM MgCl2. The reactions were set up as follows: (i) all reaction components, except for ssRNA ligase, were pre-mixed in a single solution, and were aliquoted into each well of the 96-well plates (ii) ssRNA ligase solution was then added into the wells to initiate the reaction. The reaction plate was heat-sealed with a peelable aluminum seal and incubated in a thermocycler at the indicated temperature and reaction time, then held at 4 °C until the reaction was quenched. Reactions were quenched and processed for LCMS analysis as described in Example 2. Table 47.1 Reaction temperature (C)- 30; Reaction Time (min)- 180 minutes; Reaction volume (µL)- 1. - Docket Number CX10-269WO4 [0948] Percent of product or by product was determined by the sum of the peak(s) of interest divided by the sum of the total peak area. Ratio of % conversion product to % byproduct was calculated as the percent product divided by the percent total byproduct. The value was then divided by the value of the control reaction (no recycle enzyme with excess ATP) to determine the fold improvement. The results are shown in Table 47.2 Table 47.2. Ligation reactions with SED ID NO: 936 measuring the relative ratios of product to by- product in reactions with ATP recycle enzymes relative to a control reaction to [0949] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed. [0950] For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date. .

Claims

Docket Number CX10-269WO4 CLAIMS WHAT IS CLAIMED IS: 1. A method of synthesizing an oligonucleotide, comprising reacting a nucleotide donor and an oligonucleotide acceptor (oligonucleotide(A)) in presence of a single strand RNA ligase under reaction conditions suitable for the ligation of the nucleotide donor to the oligonucleotide acceptor. 2. The method of claim 1, wherein the nucleotide donor, the oligonucleotide acceptor, or both the nucleotide donor and the oligonucleotide(A) comprise a modified nucleotide. 3. The method of claim 1 or 2, wherein the oligonucleotide(A) comprises at least one modified nucleoside, wherein the modified nucleoside comprises a conjugate moiety, reactive group, or linker. 4. The method of claim 3, wherein the conjugate moiety comprises carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. 5. The method of claim 3, wherein the reactive group comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. 6. The method of claim 3, wherein the modified nucleoside on the oligonucleotide(A) is at the 5’-terminal nucleotide, an internal nucleotide, or the 3’-terminal nucleotide. 7. The method of any one of claims 3-5, wherein the conjugate moiety, reactive group, or linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). 8. The method of any one of claims 3-7, wherein the conjugate moiety or the reactive group is attached to the nucleoside via a linker L. 9. The method of claim 8, wherein the linker is attached to the nucleobase or the sugar moiety of the nucleoside on the oligonucleotide(A). 10. The method of any one of claims 1-9, wherein the oligonucleotide acceptor comprises one or more terminal groups. 11. The method of claim 10, wherein the terminal group is at the 5’-terminal nucleotide of the oligonucleotide acceptor. 12. The method of claim 11, wherein the terminal group is a 5’-phosphonate (E or Z vinylphosphonate), 4’-amino, 4’-aminoalkyl, abasic nucleotide, or inverted abasic nucleotide. 13. The method of any one of claims 1-12, wherein the oligonucleotide(A) comprises a 5’-OH or a 5’-blocking group that inhibits ligation by the single stranded RNA ligase. 14. The method of any one of claims 1-13, wherein the oligonucleotide acceptor comprises at least one modified internucleoside linkage. 15. The method of claim 14, wherein the internucleoside linkage comprises a phosphorothioate or phosphorodithioate internucleoside linkage. Docket Number CX10-269WO4 16. The method of claim 15, wherein the phosphorothioate comprises a mixture of Rp and Sp stereoisomers, or is a Rp stereoisomer or an Sp stereoisomer. 17. The method of claim 1, wherein the oligonucleotide(A) comprises the formula (I): A1 [•A 2 ] m •A 3 -OH (I) wherein each of A1, A2 and A3 is a nucleoside; m is 0-120; OH is at the 3’-position of the sugar moiety; and “•“ is an internucleoside linkage. 18. The method of claim 17, wherein m is 2 or greater, each of A2 is the same or different nucleoside. 19. The method of claim 17 or 18, wherein when the oligonucleotide(A) comprises a modified nucleotide of at least one or more of A1, A2, or A3. 20. The method of claim 19, wherein at least one of A1, A2, or A3 is modified with a conjugate moiety, reactive group, or linker. 21. The method of any one of claims 17-20, wherein one or more of the internucleoside linkage “•“ comprises a modified internucleoside linkage. 22. The method of claim 21, wherein the modified internucleoside linkage comprises a phosphorothioate or a phosphorodithioate. 23. The method of claim 20, wherein modified nucleoside on the oligonucleotide(A) comprises the formula (II): A-[L]g-[M]h (II) wherein A is a nucleoside; L is a linker; g is 0 or 1; M is a conjugate moiety or reactive group; and h is 0-4; wherein g and h are not simultaneously 0. 24. The method of any one of claims 1-23, wherein the oligonucleotide acceptor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length. 25. The method of claim 23 or 24, wherein when g is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside, or wherein when g is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. Docket Number CX10-269WO4 26. The method of any one of claims 1-25, wherein the nucleotide donor comprises a nucleotide(D) or an oligonucleotide(D). 27. The method of claim 26, wherein the nucleotide(D) or oligonucleotide(D) comprises a modified nucleoside. 28. The method of claim 27, wherein the modified nucleoside of nucleotide(D) or oligonucleotide(D) comprises a conjugate moiety, a reactive group, or linker. 29. The method of claim 28, wherein the conjugate moiety on the nucleotide donor comprises a carbohydrate, lipid or lipophilic group, sterol, drug compound, hormone, polymer, proteins, peptides, toxins, vitamins, or combinations thereof. 30. The method of claim 28, wherein the reactive group on the nucleotide donor comprises an amino, -CN (cyano), N3 (azido), akynyl, bicyclo[6.1.0]nonyne (BCN), dibenzocyclooctynyl, cyano, tetrazinyl, or vinyl group. 31. The method of any one of claims 27-30, wherein the modified nucleoside on the oligonucleotide(D) is at the 5’-terminal nucleoside, an internal nucleoside, or the 3’-terminal nucleoside. 32. The method of any one of claims 28-31, wherein the conjugate moiety, reactive group or linker is attached to the nucleobase or the sugar moiety of the nucleoside. 33. The method of any one of claims 28-32, wherein the conjugate moiety or the reactive group is attached to the nucleoside via a linker (L). 34. The method of any one of claims 1-33, wherein the nucleotide donor comprises the formula (IIIa) or (IIIb): pD; or (IIIa) pD1[•D2]n•D3 (IIIb) wherein p is a 5’-phosphate group; each of D, D1, D2, and D3 is a nucleoside; “•“ is an internucleoside linkage; and n is 0-120. 35. The method of any one of claims 1-34, wherein the nucleotide donor is 2, 3, 4, 5, or 6 or more up to 122 nucleotides in length. 36. The method of claim 34 or 35, wherein for formula (IIIb), when n is 2 or greater, each of D2 is the same or different nucleoside. 37. The method of any one of claims 34-36, wherein the nucleotide donor comprises a modified nucleoside of at least one or more of D1, D2, or D3. 38. The method of claim 37, wherein at least one of DO, D2, or D3 is modified with a conjugate moiety or conjugate reactive group. Docket Number CX10-269WO4 39. The method of any one of claims 34-38, wherein for formula (IIIb) at least one internucleoside linkage “•“ comprises a modified internucleoside linkage. 40. The method of claim 34, for formula (IIIa), D comprises a modified nucleoside. 41. The method of claim 40, wherein D is modified with a conjugate moiety or conjugate reactive group. 42. The method of any one of claims 37-41, wherein the modified nucleoside on nucleotide(D) or oligonucleotide(D) has the formula (IV): D-[L]q-[M]r (IV) wherein D is a nucleoside; L is a linker; q is 0 or 1; M is a conjugate moiety or reactive group; and r is 0-4; wherein q and r are not simultaneously 0. 43. The method of claim 42, wherein when q is 1, L is attached to the nucleobase or the sugar moiety of the nucleoside; or wherein when q is 0, M is attached to the nucleobase or the sugar moiety of the nucleoside. 44. The method of any one of claims 1-43, wherein the nucleotide donor comprises a 3’-blocking group that inhibits ligation by the single stranded RNA ligase to a 3’-OH group of another nucleotide acceptor to produce a 3’-blocked extended oligonucleotide. 45. The method of claim 44, wherein the 3’-blocking group comprises a reversible 3’-blocking group to produce a reversible 3’-blocked extended oligonucleotide. 46. The method of claim 45, further comprising separating the 3’-blocked extended oligonucleotide from the single stranded RNA ligase or inactivating the single stranded RNA ligase. 47. The method of claim 46, further comprising removing or cleaving the 3’-blocking group with a deblocking agent to form an unblocked extended oligonucleotide. 48. The method of claim 47, further comprising inactivating the deblocking agent or removing or separating the unblocked extended oligonucleotide from the deblocking agent. 49. The method of claim 48, further comprising reacting the unblocked extended oligonucleotide with a second nucleotide donor in presence of the single stranded RNA ligase. 50. The method of claim 49, wherein the second nucleotide donor comprises a 3’-blocking group. 51. The method of any one of claims 45-48, further comprising one or more cycles of: extension with a nucleotide donor; separating the 3’-blocked extended oligonucleotide from the single stranded RNA Docket Number CX10-269WO4 ligase or inactivating the single stranded RNA ligase; removing or cleaving the reversible 3’-blocking group with a deblocking agent; and separating the unblocked extended oligonucleotide, wherein each cycle uses a new nucleotide donor. 52. The method of claim 51, wherein the nucleotide donor for at least one cycle comprises a mixture of different nucleotide donors. 53. The method of claim 51, wherein the nucleotide donor for each cycle comprises a selected or predetermined nucleotide donor nucleotide(D) or oligonucleotide(D)to form an extended oligonucleotide, wherein at least the extended portion of the oligonucleotide has a defined nucleotide sequence. 54. The method of claim 53, wherein the selected or predetermined nucleotide donor for each cycle is nucleotide(D). 55. The method of any one of claims 1-54, wherein the single stranded RNA ligase comprises RNA ligase 1. 56. The method of any one of claims 1-55, wherein the single stranded RNA ligase comprises a recombinant single stranded RNA ligase of any one of claims 64-125. 57. The method of any one of claims 1-56, wherein the single stranded RNA ligase is immobilized on a support medium and the oligonucleotide acceptor (oligonucleotide(A)) and nucleotide donor are provided in solution or aqueous phase. 58. The method of any one of claims 1-56, wherein the oligonucleotide acceptor is immobilized is attached to a support medium, and the single stranded RNA ligase and the nucleotide donor are provided in solution or aqueous phase. 59. The method of any one of claims 1-58, wherein the reaction with the single stranded RNA ligase further comprises a pyrophosphatase. 60. The method of any one of claims 1-59, wherein the reaction with the single stranded RNA ligase further comprises an ATP recycling system. 61. The method of any one of claims 1-60, wherein the suitable reaction conditions comprises one or more of cofactor ATP, a divalent metal ion, and a buffer. 62. The method of any one of claims 1-61, wherein the suitable reaction conditions comprise a reaction temperature of 5-60 ℃. 63. The method of any one of claims 1-62, wherein the suitable reaction conditions comprise a reaction pH of about 5-8. 64. A recombinant single stranded RNA ligase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from residue 12 to the carboxy terminal of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. Docket Number CX10-269WO4 65. The recombinant single stranded RNA ligase of claim 64, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises amino acid residues 12 to the carboxy terminus of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20, or comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. 66. The recombinant single stranded RNA ligase of claim 64 or 65, wherein the single stranded RNA ligase is the single stranded RNA ligase or RNA ligase 1 of Escherichia phage T4, Citrobacter phage Merlin, Escherichia phage vB_EcoM_VR25, Serratia phage PS2, Meiothermus luteus, Thermus arciformis, Balnearium lithotrophicum, Phage TS2126, Rhodothermus phage RM378, or Thermovibrio ammonificans HB-1. 67. The recombinant single stranded RNA ligase of claim 64, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 14, 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 68. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 69. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 14, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to the sequence from residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. Docket Number CX10-269WO4 70. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or to the reference sequence corresponding to SEQ ID NO: SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 71. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 72. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 73. The recombinant single stranded RNA ligase of any one of claims 67-72, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 123G/S, 125Q/R/T/V, 127I/L/P/Q/V, 130I, 135I/M, 138A, 139R, 141A/D/G/P/R, 144T, 145Y, 146I, 151E/L, 152D, 156A/S, 157L, 160R, 161V, 162S/W, 165F/K/M/P/T, 166H/P/S, 167I/V, 168I/S, 170R, 171P/R/S, 172P, 173E/N, 174P, 177C/I/L/V, 181V, 185E, 190L, 195T, 196D, 197E/T, 198A, 199L/T, 203V, 204E, 205M/T/V, 207E, 212H/S, 213V, 214A/C/E/S, Docket Number CX10-269WO4 217G/P, 220A/P/R/S/V, 221P, 222F/G, 223A/G/N/S/T, 224P, 225G/H/P/R/T, 226A/P/Q/T, 229A/L, 230L, 231I, 236H, 237A/G/R, 238G, 240V/W, 246F/I, 248F/H/M, 252R, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 260I/L/R/T/V, 263D/S, 268A/E/R, 269L, 269F/L, 271G/H/M, 272G/L/R/S, 273A/L/S/Y, 274I, 275E/W, 276A/G/H, 277T, 278L/P/R, 279Q/R, 280S, 281A/I/L/M/V/W, 282L, 283K/L/Q/T, 284F/L, 285A/G/P/R/S, 286P/Q, 288T, 289Q, 291L, 295A/R/T, 297C, 299I/R, 302I, 303A/G, 306F/M, 310A/D/G, 311C/F/G/I, 314E/G/L/P/S, 316R, 320A/G, 323D/E/G/N/T/Y, 324T, 325M/P, 326G/M/T/W, 330H/I, 332G/L, 333E, 334I/N/S, 336C/E/N, 337E/K, 340C, 341A/P/S, 343D/P, 345G/T, 346G/P, 347A/E/G/I/L/M/P/R/S/T, 348A/L/S/T, 349S/V, 350A/C/R, 351F/N/Y, 352G/L/M/N/R/S/V, 353T/V, 354E/H/Q/S/V, 355K, 356L/M/V, 357L/M, 358C/D/G/P/R/T/V, 359D/I, 360L/V, 361I, 362A/E/K/Q/R, 363S/V, 365N, 366W, 368G, 369C/L/V, 371R/S, 372A/L/P, 374F/I/L/V, 376E/T, 377L, 380S, 381K/R, or 384C/S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 74. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 75. The recombinant single stranded RNA ligase of any one of claims 67-71 or 74, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 225G/H/P/R/T, 236H, 237A/G/R, 238G, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 269F/L, 271G/H/M, 272G/L/R/S, 275E/W, 276A/G/H, 281A/I/L/M/V/W, 289Q, 316R, 320A/G, 337E/K, 351Y, 354E/H/S/V, 357L, 358C/D/G/P/R/T/V, 359D/I, 362A/E/R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 76. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 121, 45, 41, 34, 269, or 380, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 77. The recombinant single stranded RNA ligase of any one of claims 67-71 or 76, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino Docket Number CX10-269WO4 acid residue 121K, 45Y, 41V, 34Y, 269L, or 380S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 78. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 79. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 80. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, or relative to the reference sequence corresponding to SEQ ID NO: 14. 81. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 82. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence Docket Number CX10-269WO4 corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 83. The recombinant single stranded RNA ligase of claims 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 9, 19, 21, 25, 27, 30, 31, 32, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 54, 56, 58, 65, 66, 69, 84, 88, 90, 91, 92, 93, 94, 97, 109, 113, 115, 118, 121, 122, 123, 125, 127, 130, 135, 138, 139, 141, 144, 145, 146, 151, 152, 156, 157, 160, 161, 162, 165, 166, 167, 168, 170, 171, 172, 173, 174, 177, 181, 185, 190, 195, 196, 197, 198, 199, 203, 204, 205, 207, 212, 213, 214, 217, 220, 221, 222, 223, 224, 225, 226, 229, 230, 231, 236, 237, 238, 240, 246, 248, 252, 254, 255, 256, 258, 259, 260, 263, 268, 269, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 289, 291, 295, 297, 299, 302, 303, 306, 310, 311, 314, 316, 320, 323, 324, 325, 326, 330, 332, 333, 334, 336, 337, 340, 341, 343, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 365, 366, 368, 369, 371, 372, 374, 376, 377, 380, 381, or 384, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 84. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 9D, 19V, 21A, 25G/W, 27A/F/W/Y, 30R, 31I, 32M, 33R, 34G/K/N/R/S/Y, 35A/L/S, 36D/R, 38M/T/V, 40I/L, 41V, 42A, 43G/T, 44R, 45Y, 46R, 48W, 49I, 50W, 54I/L/T/V, 56L/M, 58M, 65P/Q/S, 66A/K/P/Q/R/T, 69A/C, 84L, 88W, 90E/S, 91D/L, 92I, 93A/H/N/P/R, 94D, 97L/P/V, 109G/S, 113D/G, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 123G/S, 125Q/R/T/V, 127I/L/P/Q/V, 130I, 135I/M, 138A, 139R, 141A/D/G/P/R, 144T, 145Y, 146I, 151E/L, 152D, 156A/S, 157L, 160R, 161V, 162S/W, 165F/K/M/P/T, 166H/P/S, 167I/V, 168I/S, 170R, 171P/R/S, 172P, 173E/N, 174P, 177C/I/L/V, 181V, 185E, 190L, 195T, 196D, 197E/T, 198A, 199L/T, 203V, 204E, 205M/T/V, 207E, 212H/S, 213V, 214A/C/E/S, 217G/P, 220A/P/R/S/V, 221P, 222F/G, 223A/G/N/S/T, 224P, 225G/H/P/R/T, 226A/P/Q/T, 229A/L, 230L, 231I, 236H, 237A/G/R, 238G, 240V/W, 246F/I, 248F/H/M, 252R, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 260I/L/R/T/V, 263D/S, 268A/E/R, 269L, 269F/L, 271G/H/M, 272G/L/R/S, 273A/L/S/Y, 274I, 275E/W, 276A/G/H, 277T, 278L/P/R, 279Q/R, 280S, 281A/I/L/M/V/W, 282L, 283K/L/Q/T, 284F/L, 285A/G/P/R/S, 286P/Q, 288T, 289Q, 291L, 295A/R/T, 297C, 299I/R, 302I, 303A/G, 306F/M, 310A/D/G, 311C/F/G/I, 314E/G/L/P/S, 316R, 320A/G, 323D/E/G/N/T/Y, 324T, 325M/P, 326G/M/T/W, 330H/I, 332G/L, 333E, 334I/N/S, 336C/E/N, 337E/K, 340C, 341A/P/S, 343D/P, 345G/T, Docket Number CX10-269WO4 346G/P, 347A/E/G/I/L/M/P/R/S/T, 348A/L/S/T, 349S/V, 350A/C/R, 351F/N/Y, 352G/L/M/N/R/S/V, 353T/V, 354E/H/Q/S/V, 355K, 356L/M/V, 357L/M, 358C/D/G/P/R/T/V, 359D/I, 360L/V, 361I, 362A/E/K/Q/R, 363S/V, 365N, 366W, 368G, 369C/L/V, 371R/S, 372A/L/P, 374F/I/L/V, 376E/T, 377L, 380S, 381K/R, or 384C/S, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 85. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 34, 35, 38, 45, 48, 49, 54, 66, 90, 93, 115, 118, 121, 122, 123, 127, 162, 165, 167, 170, 177, 197, 205, 213, 220, 222, 223, 225, 236, 237, 238, 254, 255, 256, 258, 259, 269, 271, 272, 275, 276, 281, 289, 316, 320, 337, 351, 354, 357, 358, 359, 362, 365, 374, 376, or 381, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 86. The recombinant single stranded RNA ligase of any one of claims 81-83, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or amino acid residue 19V, 34G/K/N/R/S/Y, 35A/L/S, 38M/T/V, 45Y, 48W, 49I, 54I/L/T/V, 66A/K/P/Q/R/T, 90E/S, 93A/H/N/P/R, 115E/S, 118F/L/M, 121K, 122A/C/I/L/M/S/T/V/Y, 127I/L/P/Q/V, 162S/W, 165F/K/M/P/T, 167I/V, 170R, 177C/I/L/V, 197E/T, 205M/T/V, 213V, 220A/P/R/S/V, 222F/G, 223A/G/N/S/T, 225G/H/P/R/T, 236H, 237A/G/R, 238G, 254A/D/R/S, 255A/F/M/Q/S/V/W, 256L/M/R/V, 258L, 259A/N/S, 269F/L, 271G/H/M, 272G/L/R/S, 275E/W, 276A/G/H, 281A/I/L/M/V/W, 289Q, 316R, 320A/G, 337E/K, 351Y, 354E/H/S/V, 357L, 358C/D/G/P/R/T/V, 359D/I, 362A/E/R, 365N, 374L, 376E, or 381R, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 87. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/45/269, 34/45/173/297, 34/173/269/380, 173/269, 156/269/380, 173/269/380, 34/173, 269, 34/380, 34/269/380, or 173/380, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, or relative to the reference sequence corresponding to SEQ ID NO: 32. Docket Number CX10-269WO4 88. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 207, 237, 94/263, 220, 236, 92, 91, 94, 204, 185, 213, 199, 152, 196, 203, 141, 138, 156, 93, or 181, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 44, or relative to the reference sequence corresponding to SEQ ID NO: 44. 89. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 283/337, 347, 323, 354, 343, 118, 345, 314, 268/269, 363, 356, 358, 348, 162, 324/330, 346, 160, 362, 361, 341/349, 353, 369, 248, 146/346, 332, 170, or 269/275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 100, or relative to the reference sequence corresponding to SEQ ID NO: 100. 90. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 162/337/358/362, 162/236/237/320/337/358/362, 151/231/237/337, 199/231/237/337, 231/237/314/337, 310/314/337, 115/162/310/314, 199/237/337, 151/199/205/310/314, 199/204/205/231/236/310/314, 320/337/358/362, 135/320/337/358/362, 151/212/214/345/347/358, 135/337/358/362, 162/358/362, 337/358/362, 337/358, 92/337, 151/196/199/205/231/237/323, 151/214/347/358, 337, 151/345/347/358, 199/314, 199/205/231/237/323, 151/205/314, 151/212/345/347, 92/214/347/358, 162/204/205/310/314/358, 236/314, 151/345/347, 214/347/358, 151/212/358, 204/283/314/358, 236/237/358/362, 151/199/204/231/236/323, 231/236/237/358/362, 162/314/358, 92/151/347/358, 151/236, 212/345/347, 115/314, 345/347/358, 314, 358/362, 115/358, 310/314, 214/347/358, 237/314, 345/347, 151/310/323/343/347, 151/358, 347/358, 93/358/362, 231/236/237/320/358/362, 151/230/345/347/358, 135/231/236/237/358, 310/314/358/362, 151/230/347/358, 231/237/323, 135/358/362, 283/314/358/362, 199/205, 92/151/230/345/347/358, 314/358/362, 199/237, or 199/204, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 140, or relative to the reference sequence corresponding to SEQ ID NO: 140. 91. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 255, 109, 256, 260, 273, 46, 252, 161/162, 311/320, 123, 320/326, 174, 162/167, 32, 125, 162/166, 320, 283, 330, 278, 303, 281, 333/337, 277, 254, or 173, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 246, or relative to the reference sequence corresponding to SEQ ID NO: 246. 92. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 123/256/320, 260, 256/260, 256, 260/281, 320, 123/260, 123/320, 256/281, or 260/273, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the Docket Number CX10-269WO4 carboxy terminal of SEQ ID NO: 400, or relative to the reference sequence corresponding to SEQ ID NO: 400. 93. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 217, 220, 221, 229, 246, 171, 285, 286, 165, 226, 168, 177, 223, 224, 118/123, 248, 268, 225, 84, or 284, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 492, or relative to the reference sequence corresponding to SEQ ID NO: 492. 94. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 254, 240, 118, 347, 167, 281, 303, 205, or 50, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 520, or relative to the reference sequence corresponding to SEQ ID NO: 520. 95. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 118/220, 118/220/303/347, 347, 118/220/254/347, 220, 38/220, 220/254/303/347, 220/254, 220/303/347, 48/220/347, 49/220, 217/220/347, 38/220/254, 49/118/220/254/347, 49/220/254, 38/48/49/118/220, 49/217/220/254, 49/220/347, 49/347, 225, 280, 118, 279, 165, 273, 272, 166, 281, 248/357, 222, 223, 358, 271, 168, 276, 36, 34, 40, 263, 130, 356, 259, or 167, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 594, or relative to the reference sequence corresponding to SEQ ID NO: 594. 96. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/54/127/205/254, 165/255/258/259, 205/254/258, 33/38/127/165/254, 38/54/205/258, 127, 127/165/258, 127/205/254/255/258/259, 205/258, 33/38/254/255, 127/254/258/259, 127/165, 38/127/165/259, 38/127/258/259, 38/254/255, 127/205/254/258/259, 127/165/258/259, 165/205/258, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 634, or relative to the reference sequence corresponding to SEQ ID NO: 634. 97. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 127/222/223/225/255/272/276, 127/255/259, 276, 255/259, 127/255, 127/162/223/255, 127/162/255/259/272/276, or 259/272, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 710, or relative to the reference sequence corresponding to SEQ ID NO: 710. 98. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at Docket Number CX10-269WO4 amino acid position(s) 165/259/281, 127, or 259, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 738, or relative to the reference sequence corresponding to SEQ ID NO: 738. 99. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 177, 362, 44, 365, 46, 279/281, 303, 165/166, 281/282, 302, 360, 295, 246, 127, 299, 125, 238, 56, 109, 38, or 31, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 756, or relative to the reference sequence corresponding to SEQ ID NO: 756. 100. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 44/118, 44/118/246/272/276, 44/118/246/280, 44/118/246/280/302, 44/118/272/276, 44/246, 44/246/272/276/302, 44/246/276/279/280, 44/246/276/280, 44/272/276/280, 44/272/279, 44/272/280, 44/276, 44/276/279, 44/276/280/295/302, 44/280, 44/302, 118/246/276/280/302, 118/246/280/295, 118/272/276, 118/280/302, 246/272/276/279/280/302, 246/272/279/280, 246/276, 246/276/302, 246/279/280, 246/279/280/302, 246/280, 272/276/279/280/302, 272/280, 272/280/302, 276/279/280/302, 276/280, 279/280/302, or 280, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 768, or relative to the reference sequence corresponding to SEQ ID NO: 768. 101. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 42, 43, 122, 271, 272, 278, or 279, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 844, or relative to the reference sequence corresponding to SEQ ID NO: 844. 102. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 38/127/238/284, 38/127/255, 38/127/255/359, 38/127/284, 38/238/255, 38/238/255/359, 38/238/255/359/381, 38/238/284/359, 127, 127/212/238/284/359, 127/238/255, 127/238/255/359/381, 127/238/284/359, 127/238/284/359/381, 127/238/359, 127/255/359/381, 127/255/381, 238, 238/255, 238/255/359, 238/255/381, 238/284, 238/359, 255, 255/284, 255/359, 255/359/362/381, 359, or 381, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 882, or relative to the reference sequence corresponding to SEQ ID NO: 882. 103. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 34/35/38, 34/38/170, 34/135/170/271/357, 34/170/271, 34/271, 35/38/170/357, 35/38/271/357, 35/170/271/357, 38/170, 38/170/271, 38/170/357, 56/135/170/357, 56/135/271/357, 135/170/271, 170, 170/271/272, 170/271/272/357, 170/271/357, 170/357, 254/260, 254/281, 271, or 357, Docket Number CX10-269WO4 wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. 104. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 9/369, 34/38/357, 34/135/170/271/357, 34/271, 56/135/170/357, 56/135/271/357, 113, 115, 141, 144, 145, 177, 214, 254/260, 254/281, 260, 274, 340, 341, 350, 354/359, 359/360, 359/362, 368, 369, 371, 377/381, or 381/384, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 936, or relative to the reference sequence corresponding to SEQ ID NO: 936. 105. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 113, 113/115, 113/115/177/254/281, 113/115/177/254/281/350/359, 113/115/177/254/350, 113/115/177/254/359, 113/115/177/281/359, 113/115/254, 113/115/254/281, 113/115/254/350, 113/115/254/359, 113/115/350, 113/115/359, 113/177/254/350, 113/177/281/359, 113/177/350/354/359, 113/254, 113/254/281/350/359, 113/254/281/359, 113/254/350/354/359, 113/254/354/359, 113/254/359, 113/359, 115/177, 115/177/254, 115/177/254/359, 115/177/359, 115/254, 115/254/350/354/359, 115/254/359, 115/350, 144/145/260, 144/145/260/341, 144/145/260/341/366/371, 144/260/341/366, 144/260/366/369, 145/260/263, 145/260/341, 145/260/341/366/369, 145/341, 145/341/371, 145/371, 177, 177/254, 177/254/281/359, 177/254/354/359, 177/254/359, 177/359, 254, 254/281/354/359, 254/350, 254/354/359, 254/359, 260/366, 341, or 359, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 992, or relative to the reference sequence corresponding to SEQ ID NO: 992. 106. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 27, 34, 35, 122, 127, 255, 259, 275, 349, 351, 354, 356, 363, 374, or 376, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1104, or relative to the reference sequence corresponding to SEQ ID NO: 1104. 107. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 27/127/374, 27/127/374/376, 27/275/356, 27/351, 34/35/118/127/275/351/374/376, 122, 127/275/374, or 275, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. 108. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 21, 66, 69, 151, or 199, or any combinations thereof, wherein the amino acid positions are relative to Docket Number CX10-269WO4 the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. 109. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position 19, 25, 54, 65, 66, 90, 93, or 151, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. 110. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution at amino acid position(s) 21, 25, 65, 66, 69, 88, 91, 93, 97, 157, 190, 195, 197, or 198, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1222, or relative to the reference sequence corresponding to SEQ ID NO: 1222. 111. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 19, 19/21/65, 19/21/65/66, 19/21/65/66/90/93, 19/21/65/66/93, 19/21/65/93, 19/21/65/190, 19/21/65/197, 19/21/66, 19/21/190, 19/65, 19/65/66, 19/65/66/90/93/190, 19/65/66/93, 19/65/66/190, 19/65/66/197, 19/66, 19/66/90/93/197, 25, 25/54, 25/122, 65, 65/66, or 66, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. 112. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 246, 289, 306, 310, 323, 330, 334, 336, 351/352, 351/353, or 351/358, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. 113. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, 288, 291, 299, 306, 314, 316, 325, 332, 336, 351/352, 351/353, 351/355, or 372/374/376, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1264, or relative to the reference sequence corresponding to SEQ ID NO: 1264. 114. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 21/54, 21/65/66/151, 21/90/93/122, 21/93, 54/58, 65/93, 65/151, 66/90/151/190/197, 90/93/122, 90/190/197, 90/197, 93, or 93/151, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1344, or relative to the reference sequence corresponding to SEQ ID NO: 1344. Docket Number CX10-269WO4 115. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set at amino acid position(s) 237, 240, or 289/316, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1474, or relative to the reference sequence corresponding to SEQ ID NO: 1474. 116. The recombinant single stranded RNA ligase of claim 81 or 82, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution set at amino acid position 27, 30, 36, 40, 139, 141, 172, or 223, or any combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 1500, or relative to the reference sequence corresponding to SEQ ID NO: 1500. 117. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least one substitution provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: of 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 118. The recombinant single stranded RNA ligase of any one of claims 67-71, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprises at least a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 119. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to a substitution or substitution set provided in Tables 8.2, 10.2, 11.2, 12.2, 17.2, 18.2, 19.2, 20.2, 21.2, 22.2, 22.3, 24.2, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31.2, 32.2, 33.2, 34.2, 35.2, 36.2, 37.2, 38.2, 39.2, 40.2, 41.2, 42.2, 43.2, and 44.2, wherein the amino acid positions are relative to the reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or relative to the reference sequence corresponding to SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. Docket Number CX10-269WO4 120. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of an even-numbered SEQ ID NO. of SEQ ID NOs: 32- 216, 244-912, and 934-1524, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524. 121. The recombinant single stranded RNA ligase of claim 67, comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOs: 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 122. The recombinant single stranded RNA ligase of claim 67, wherein the amino acid sequence of the recombinant single stranded RNA ligase comprising residues 12 to the carboxy terminal of an even- numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524, or comprising an even-numbered SEQ ID NO. of SEQ ID NOs: 32-216, 244-912, and 934-1524. 123. The recombinant single stranded RNA ligase of any one of claims 67-122, having single stranded RNA ligase activity and exhibits at least an improved property as compared to a reference single stranded RNA ligase, wherein the improved property is selected from i) increased expression in a host cell, ii) increased single stranded RNA ligase activity, iii) increased single stranded ligase activity with modified oligonucleotide substrates, and iv) increased thermostability, or any combination of i), ii), iii) and iv), wherein the reference single stranded RNA ligase has an amino acid sequence corresponding to residues 12 to the carboxy terminal of SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500, or an amino acid sequence corresponding to SEQ ID NO: 14, 32, 44, 100, 140, 246, 400, 492, 520, 594, 634, 710, 738, 756, 768, 844, 882, 936, 992, 1104, 1222, 1264, 1344, 1474, or 1500. 124. The recombinant single stranded RNA ligase of any one of claims 64-123, wherein the recombinant single stranded RNA ligase further comprises a fusion polypeptide. 125. The recombinant single stranded RNA ligase of any one of claims 64-124, wherein the recombinant single stranded RNA ligase is a purified preparation. 126. A recombinant polynucleotide comprising a polynucleotide sequence encoding a single stranded RNA ligase of any one of claims 64-124. 127. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to the sequence from nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, Docket Number CX10-269WO4 13, 15, 17, or 19, or to a reference nucleotide sequence corresponding to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. 128. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises nucleotide residues 34 to the 3’-terminal of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19, or comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19. 129. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, or to a reference polynucleotide sequence corresponding to SEQ ID NO: 31, 43, 99, 139, 245, 399, 491, 519, 593, 633, 709, 737, 755, 767, 843, 881, 935, 991, 1103, 1221, 1263, 1343, 1473, or 1499, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. 130. The recombinant polynucleotide of claim 126, wherein the polynucleotide sequence comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, or to a reference polynucleotide sequence corresponding to an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523, wherein the recombinant polynucleotide encodes a single stranded RNA ligase. 131. The recombinant polynucleotide of any one of claims 126-130, wherein the polynucleotide sequence is codon-optimized for expression of the encoded recombinant single stranded RNA ligase. 132. The recombinant polynucleotide of claim 126, comprising a polynucleotide sequence comprising nucleotide residues 34 to 1161 of an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243- 911, and 933-1523, or a polynucleotide sequence comprising an odd-numbered SEQ ID NO. of SEQ ID NOs: 31-215, 243-911, and 933-1523. 133. An expression vector comprising a recombinant polynucleotide of any one of claims 126- 132. 134. The expression vector of claim 133, wherein the polynucleotide is operably linked to a control sequence. 135. A host cell comprising an expression vector of claim 133 or 134. 136. The host cell of claim 135, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell. 137. A method of producing a recombinant single stranded RNA ligase polypeptide in a host cell, comprising culturing a host cell of any one of claims 135 or 136, under suitable culture conditions such that the recombinant single stranded RNA ligase is produced. Docket Number CX10-269WO4 138. The method of claim 137, further comprising recovering the recombinant single stranded RNA ligase polypeptide from the culture and/or host cells. 139. The method of claim 137 or 138, further comprising purifying the recombinant single stranded RNA ligase polypeptide. 140. A composition comprising a single stranded RNA ligase of any one of claims 64-125. 141. A kit comprising at least an RNA ligase of any one of claims 64-125. 142. The kit of claim 141, further comprising one or more of a buffer, nucleotide cofactor, a ligation enhancer, and a single stranded RNA ligase substrate.
PCT/US2025/025017 2024-04-16 2025-04-16 Rna ligase mediated oligonucleotide synthesis Pending WO2025221925A1 (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
US20180127822A1 (en) * 2010-04-21 2018-05-10 Pierce Biotechnology, Inc. Modified nucleotides methods and kits
US20240035025A1 (en) * 2021-04-27 2024-02-01 Factor Bioscience Inc. Circular rna

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180127822A1 (en) * 2010-04-21 2018-05-10 Pierce Biotechnology, Inc. Modified nucleotides methods and kits
US20240035025A1 (en) * 2021-04-27 2024-02-01 Factor Bioscience Inc. Circular rna

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