WO2019071023A1

WO2019071023A1 - Compositions and methods for making selenocysteine containing polypeptides

Info

Publication number: WO2019071023A1
Application number: PCT/US2018/054437
Authority: WO
Inventors: Dieter Soll; Takahito Mukai; Kyle HOFFMAN
Original assignee: Yale University
Priority date: 2017-10-04
Filing date: 2018-10-04
Publication date: 2019-04-11

Abstract

Non-naturally occurring tRNASec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNASec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNASec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNASec.

Description

COMPOSITIONS AND METHODS FOR MAKING

SELENOCYSTEINE CONTAINING POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S.S.N.

15/724,678, filed October 4, 2017, which is specifically incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM022854 and GM 122560 awarded by National Institute of Health, DE-FG02- 98ER20311 awarded by the Department of Energy and 0950474 awarded by the National Science Foundation. The government has certain rights in the invention.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing submitted as a text file named

"YU_5714_CIP_2_PCT_ST25.txt," created on October 4, 2018, and having a size of 128,961 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD OF THE INVENTION

The field of the invention generally relates to compositions including tRNAs and methods of using them to manufacture recombinant

selenocysteine containing polypeptides.

BACKGROUND OF THE INVENTION

Selenocysteine, commonly referred to as the twenty-first amino acid, is incorporated into at least 25 human proteins. Natural co-translational incorporation of selenocysteine (Sec) into proteins proceeds by a recoding process so that upon encountering the UGA codon in the messenger RNA the ribosome knows to recognize it as Sec instead of Stop. This process requires three components: (i) the aminoacyl-tRNA carrying selenocysteine, Sec- tRNA^Sec; (ii) the specialized elongation factor, SelB, carrying Sec-tRNA^Sec to the ribosome, and (iii) the SECIS element, an RNA secondary structure of the mRNA just downstream of the UGA codon, that interacts with the

45292464vl 1 SelB»Sec-tRNA^Sec complex (Bock, A, Thanbichler, M, Rother, M & Resch, A (2005), eds Ibba M, Francklyn CS, & Cusack S (Landes Bioscience, Georgetown, TX), pp 320-327; Yoshizawa, S & Bock, A (2009) Biochim Biophys Acta 1790: 1404-1414). Additionally, in order to protect the integrity of this recoding process, Sec-tRNA^Sec is not recognized by the general elongation factor EF-Tu because of the presence of three base pairs that act as antideterminants (Rudinger, J, Hillenbrandt, R, Sprinzl, M & Giege, R (1996) EMBO J 15:650-657). Sec-tRNA^Sec cannot be

accommodated during normal translation because it is not an acceptable substrate for EF-Tu, and the SelB* Sec-tRNA^Sec complex will not decode in- frame UGA codons in absence of the SECIS.

Insertion of selenocysteine into a recombinant protein, for example, substitution of a naturally occurring cysteine residue for selenocysteine, can alter the function of the protein. Substituting one or more naturally occurring Cys residues in the active site of an enzyme with a Sec can increase the activity of this enzyme. Diselenide bonds have very low redox potential. Therefore, replacing disulfide bonds with diselenide or selenocysteine - cysteine bonds can lower dosage, increase half-life, increase stability, reduce toxicity, alter pharmacokinetics, change folding properties, or combinations thereof of the recombinant selenocysteine containing protein relative to a reference protein without selenocysteines, such as a naturally occurring counterpart.

However, due the presence the SECIS element as an integral part of the open reading frame (within the mRNA) encoding the protein that harbors Sec in its sequence, it is not possible to insert Sec into proteins by a standard mutational scheme or in the construction of random mutagenic libraries, and production of Sec proteins is limited to costly and inefficient methods of protein synthesis. Accordingly, there is a need for alternative methods of manufacturing selenocysteine containing polypeptides.

It is an object of the invention to provide compositions and methods for recombinant expression of proteins engineered to include one or more selenocysteine residues without the requirement of a SECIS in the mRNA encoding the protein.

45292464vl 2 It is a further object of the invention to provide non-naturally occurring proteins including one or more selenocysteine residues.

SUMMARY OF THE INVENTION

tRNA^Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. Typically, tRNA^Sec (1) can be recognized by SerRS and by EF- Tu, or variants thereof; and is characterized by one or more of the following elements: (2) when aminoacylated with serine, the Ser-tRNA^Sec can be converted to Sec-tRNA^Sec by SelA, or a variant thereof; (3) when aminoacylated with serine, the Ser-tRNA^Sec can be phosphorylated by PSTK or variant thereof; (4) when aminoacylated with phosphorylated serine, the Sep-tRNA^Sec can serve as a substrate for SepSecS or variant thereof; and combinations thereof. In some embodiments, the Ser-tRNA^Sec is characterized by elements (1) and (2). In some embodiments, the Ser- tRNA^Sec is characterized by elements (1), (3), and (4). In some

embodiments, the Ser-tRNA^Sec is characterized by elements (1), (2), (3), and (4). In some embodiments, the Ser-tRNA^Sec is characterized by elements (1), (2), and (3).

Typically, the tRNA^Sec do not require a SECIS element in an mRNA to be incorporated into a growing polypeptide chain during translation.

Typically, the tRNA^Sec is a non-naturally occurring tRNA^Sec. The non-naturally occurring tRNA^Sec can be a variant of a naturally occurring tRNA. In some embodiments, the tRNA^Sec is includes or consists of a naturally occurring nucleic acid sequence.

In some embodiments, the tRNA^Sec has a naturally occurring tRNA sequence. In some embodiments, the tRNA^Sec is an "allo-tRNA". Allo- tRNAs typically have a 8/4 or 9/3 composition of the 12-bp amino-acid acceptor branch. Naturally and non-naturally occurring allo-tRNAs are provided and can be used in the disclosed compositions and methods. Many naturally occurring allo-tRNA have an anticodon that recognizes a codon inconsistent with the amino acid charged to it. For example, some of the allo-tRNAs charge with serine, but have a leucine anticodon. The non- naturally occurring allo-tRNA typically have one or more insertions,

45292464vl 3 deletions, or substitutions relative to the naturally occurring allo-tRNA. For example, the naturally occurring allo-tRNA can be modified to include a SerRS identity element, to have an anticodon that recognizes or hybridizes to a stop codon, or a combination thereof. In some embodiments, the variants include one more additional modifications that improve the tRNAs activity as a tRNA^Sec, for example, to improve binding to a SelA, or improve binding to a EF-Tu.

Recombinant compositions and method of using tRNA^Sec are also provided. Exemplary tRNAs, isolated nucleic acids encoding the tRNAs, vectors thereof, and host cells expressing the tRNA are also provided. For example, an isolated nucleic acid can include a nucleic acid sequence encoding a tRNA^Sec, wherein the tRNA^Sec is recognized by SerRS and by EF-Tu, or variants thereof, and when aminoacylated with serine the Ser- tRNA is a substrate for SelA or a variant thereof.

Consensus sequences for allo-tRNAs and exemplary naturally and non-naturally occurring allo-tRNA include RNA sequences (and DNA sequence encoding them) of SEQ ID NOS: 19-42, 57, 58, 137-146.

Also provided are non-naturally occurring tRNA^Sec that are chimeric tRNAs including sequence elements from a yeast tRNA, for example tRNA^Ser, in combination with elements from a non- yeast tRNA. Typically, the tRNA^Sec can be a substrate for a SerRS (e.g., yeast SerRS), the Ser- tRNA^Sec can be substrate for SelA (e.g., Aeromonas SelA) and the Sec- tRNA^Sec can bind to eEFla (see, e.g., Figure 19). In some embodiments, the tRNA^Sec include elements, for example, the tRNA acceptor branch alone or in combination with other elements (e.g., identity elements), from, for example, Aeromonas salmonicida (e.g., Aeromonas salmonicida tRNA^Sec). In some embodiments, the non-naturally occurring tRNA^sec has a sequence or is encoded by a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97, 98, 99%, or 100% sequence identity to any one of SEQ ID NO: 151-158.

In some embodiments, the isolated nucleic acid includes a heterologous expression control sequence for expression of the tRNA. In some embodiments, the nucleic acid encoding the tRNA is in an expression

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45292464vl vector. Host cells including the nucleic acid encoding the tRNA are also provided. The host cell can be, for example, a prokaryote, archaeon, or eukaryote. The nucleic acid is incorporated into the genome of the cell or expressed episomally. The host cell can be a genetically recoded organism.

Methods of manufacturing selenocysteine containing polypeptides are also disclosed. The tRNA^Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, the tRNA^Sec is co- expressed in a recombinant expression system, such as E. coli, or yeast, with a SerRS, an EF-Tu or an eEFla, a SelD, a SelA, or a PSTK and a SepSecS, or a combination of SelA, PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the tRNA^Sec to manufacture a selenocyteine containing polypeptide encoded by the mRNA. In some embodiments, one or more components of the translation system is endogenous to the host cell. In some embodiments, one or more components of the translation system is exogenous or heterologous to the host cell.

Nucleic acids encoding selenocysteine containing polypeptides are also disclosed. The nucleic acids encode a polypeptide of interest and include a non-natural tRNA^Sec recognition codon, for example a "stop" codon that hybridizes with the anticodon of the tRNA^Sec, such that a selenocysteine is transferred onto the growing polypeptide chain during translation. The selenocysteine containing polypeptides can be polypeptides that contain selenocysteine in nature, or polypeptides that do not contain selenocysteine in nature. For example, a non-naturally occurring tRNA recognition codon can be substituted for a cysteine codon in the naturally occurring mRNA, which changes the cysteine to a selenocysteine when the nucleic acid encoding the polypeptide is expressed recombinantly with the tRNA^Sec. Substituting one or more naturally occurring Cys residues with a Sec can increase activity, lower dosage, reduce toxicity, improve stability, increase efficacy, increase half-life or combinations thereof of a

selenocysteine containing protein relative to its cysteine containing counterpart.

45292464vl 5 Methods of treating subjects in need thereof with recombinant selenocysteine containing polypeptides prepared using the disclosed compositions and methods are also disclosed. Particularly preferred proteins containing selenocysteine include antibodies and enzymes having altered binding affinity and/or pharmacokinetics.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB are illustrations showing the translation apparatus. The canonical amino acids are charged onto their respective tRNA by their cognate aminoacyl-tRNA synthetase. The aminoacyl-tRNA is then delivered by EF-Tu to the ribosome (Figure 1A). In contrast, the Sec pathway requires several biosynthetic steps. First, tRNA^Sec is misacylated to

Ser-tRNA^Sec by SerRS. While in bacteria Ser-tRNA^Sec is directly converted by SelA to Sec-tRNA^Sec, archaea and eukaryotes employ an additional phosphorylation step by PSTK to form Sep-tRNA^Sec, which is then converted by SepSecS to the final product Sec-tRNA^Sec (Figure IB). Sec-tRNA^Sec is bound by elongation factor SelB and delivered to the ribosome. However, reassignment of the opal codon UGA to a Sec codon is only achieved if SelB also binds to the mRNA SECIS hairpin structure.

Figure 2 is a depiction of the primary and secondary structures of human tRNA^Sec (SEQ ID NO: 3) adapted from Yuan, et al., FEBS Lett.,

584(2):342-349 (2010).

Figures 3A-3C are depictions of the primary and secondary structures of E. coli tRNA^Sec (SEQ ID NO: l) (3A), a non-naturally occurring tRNA^UTu with an E. coli body (tRNA^UTu _op, SEQ ID NO:6; tRNA^Utu _am, SEQ ID NO:7) (3B), and E. coli tRNA^Ser (SEQ ID NO:4) (3C). E. coli tRNA^Ser

(3C) serves as a major scaffold for tRNA^UTu (3B) with the exception of the acceptor stem that originates from E. coli tRNA^Sec (boxed sequence elements). Major EF-Tu recognition elements were retained from tRNA^Ser as well (circled sequence elements). Substitution of the amber anti-codon CUA (tRNA^UTlV) for the opal anti-codon UCA (tRNA^UTu _op) are depicted with arrows and labeling.

Figure 4A and 4B are depictions of the primary and secondary structures of a non-naturally occurring tRNA^UTu with a body derived from M.

45292464vl 6 maripaludis (Figure 4A, tRNA^UtuucA, SEQ ID NO: 16_; tRNA^UTu _op, SEQ ID NO: 13; tRNA^Utuam, SEQ ID NO: 14) and a non-naturally occurring tRNA^UTu with a body derived from E. coli (Figure 4B, tRNA^UtuucA, SEQ ID NO: 12_; tRNA^UTuo_P, SEQ ID NO:9; tRNA^Utuam, SEQ ID NO: 10). Transplanted PSTK identity elements are boxed. "<" identifies potential locations of additional base pairs in the acceptor stem. "Arrow" identifies the location of other possible mutations. Specifically, the < depict one possible insertion of a G-C base pair between the 1^st and 2^nd base pair and a second possible insertion of a G-C pair insertion between the 6^th and 7^th base pair of the acceptor stem. The arrows depict a possible change in the 50:64 base pair (A-U) to a U-A pair, and substitution of the serine anticodon (UGA) with opal (UCA) or amber (CUA) anticodon.

Figure 5 is a depiction of the primary and secondary structures of a non-naturally occurring tRNA^UTuX (SEQ ID NO: 17). Nucleotides that were changed from the original tRNA^UTu (SEQ ID NO: 7) are circled and the amber anticodon is boxed. Specific mutations introduced between tRNA¹⁷¹" and tRNA^UTuX include U8G, G9U, and A27G in the core region; A14U and G15C in the D-arm; deletion of U21 in the D-loop; A52G and U62C in the T-arm; A59C in the T-loop; and the insertion of residues U44 and G48 in the variable arm.

Figures 6A-6F are depictions of concensus primary and secondary structures of (8/4) allo-tRNA (bacteria) (SEQ ID NO: 19) (6A), (9/3) allo- tRNA (bacteria) (SEQ ID NO:20) (6B), and consensus primary and secondary structures of minor serine/histidine/cysteine/selenocysteine tRNA species (8/4) SelC* tRNA^Cys (delta-proteobacteria) (SEQ ID NO:43) (6C), (8/4) tRNAs^Ser (bacteria) (SEQ ID NO:21) (6D), (8/4) tRNAs^His (a- proteobacteria bacteria) (SEQ ID NO:44) (6E), (8/4) tRNAs^Sec (bacteria) (SEQ ID NO:45) (6F). The (8/4) tRNAsSer with Y20 are also classified as (8/4) allo-tRNA.

Figures 7A-7E are primary and secondary structures of tRNA.

Figures 7A and 7B show missense suppressor allo-tRNAs with Ser identity and Leu anticodons: tRNA (8/4-1) (SEQ ID NO:46) (7 A) and tRNA (9/3-1)

(SEQ ID NO:47) (7B). Figures 7C-7E show amber suppressor variants of

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45292464vl three allo-tRNA species tRNA (8/4-2) (SEQ ID NO:48) (7C), tRNA (9/3-2) (SEQ ID NO:49) (7D), and tRNA (9/3-3) (SEQ ID NO:50) (7E). Their presumed amino-acid identities are indicated. Figure 7F is images of the results of an amber suppression experiment in E. coli DH10B using sfGFP as reporter. The amino acids incorporated into sfGFP in response to the amber codon at position 2 by allo-tRNAs are shown. Figure 7G is images of the results of an assay testing suppression of Serl46TAG CAT by allo-tRNAs in E. coli. Pre-cultured cells were spotted onto LB agar plates with various Cm concentrations (0, 34, 100 μg/mL). The plates were incubated overnight at 37 °C.

Figure 8A is an illustration of the cloverleaf "junction" of tRNA. Figure 8B illustrates different junction structures of six amber suppressor variants of six allo-tRNAuAu species. Figures 8C-8H are primary and secondary structures of the six allo-tRNAuAu variants: tRNA (8/4-3) (SEQ ID NO:51) (8C), tRNA (8/4-4) (SEQ ID NO:52) (8D), tRNA (8/4-5) (SEQ ID NO:53) (8E), tRNA (8/4-6) (SEQ ID NO:54) (8F), tRNA (8/4-7) (SEQ ID NO:55) (8G), tRNA (8/4-8) (SEQ ID NO:56) (8H). Figure 81 is an image of the results of an assay measuring suppression of Serl46TAG CAT by allo-tRNAcuA variants with different junction structures. Amber suppressor variants of 8/4-1, 9/3-1 and 9/3-2 were used as positive controls. Fresh cultures of the transformants were spotted onto an agar plate with Cm at a concentration of 34μg/mL. The plates were incubated overnight at 37 °C.

Figure 9A is the primary and secondary structures of SupD tRNA^Ser (SEQ ID NO: 136) (left) and "allo-tRNA^UTu" (SEQ ID NO:57) (right).

Figure 9B is a map of the pSecUAG-A plasmid used for Sec insertion in E. coli. Figures 9C-9E are series of images showing the results of an assay designed to test Sec insertion. Formate dehydrogenase H (FDHH) encoded by the E. colifdhF gene has a catalytic Sec residue and four Cys residues accommodating an iron sulfur cluster (Fe₄S4). The images in Figure 9C show FDHH expression in E. coli AselABC AfdhF (ME6) cells with allo-tRNA^UTu, with or without Aeromonas salmonicida (As) SelA, and with fdhF gene variants having UAG mutations at codon positions 8, 11, 15, 42, and 140.

"Am" indicates the amber UAG codon. FDHH reduces benzyl viologen into a

45292464vl 8 purple dye. The images in Figure 9D show FDHH expression in ME6 cells with either of the allo-tRNA^UTu-As SelA pair and the tRNA^SecUx-£^"c SelA pair and with fdhF gene variants having two to four UAG codons. Ec selA' in the pSecUx-A plasmid map indicates that the AUG start codon was changed to GUG with a short insertion "UAAUU" in front of it. The images in Figure 9E show FDHH expression in ME6 cells carrying either pSecUAG-A or pSecUAG-AD and carrying fdhF gene variants having four or five UAG codons. As selD' in the pSecUAG-AD plasmid map indicates that the AUG start codon was changed to GUG. Figure 9F is a spectrograph showing the results of intact mass spectrometry of the human GPxl(Ser49 and Sec49) mixture obtained from ME6 cells carrying pSecUAG-AD. Three exposed Cys residues of GPxl were modified by 2-mercaptoethanol (2-ME) during purification. The calculated masses are 23,361 Da for GPxl(Ser49) with three 2-ME molecules and 23,421 Da for GPxl(Sec49) with three 2-ME molecules. Figure 9G is a spectrograph showing the intact mass spectrometry of the human GPxl(Ser49 and Sec 49) mixture obtained from ME6 cells carrying pSecUAG-ADT. Dithiothreitol (DTT) was used as the reducing agent. The calculated masses are 23,133 Da for GPxl(Ser49) and 23,193 Da for GPxl(Sec49). Figure 9H is a diagram illustrating putative pathways of selenium transfer to As SelA in engineered E. coli carrying pSecUAG-ADT.

Figure 10A is an illustration showing the development of the D-3b variant based on wildtype (UCUAUCUGGUGAUAGA (SEQ ID NO:59)) of allo- tRNA^UTu. Figure 10B is genetic map showing the development the pSecUAG-AD3T system. Figure IOC is a spectrograph showing the results of intact mass spectrometry of the human GPxl(Ser49 and Sec49) mixture obtained from ME6 cells carrying pSecUAG-AD3T. Dithiothreitol (DTT) was used as the reducing agent. The calculated masses are 23,133 Da for GPxl(Ser49) and 23,193 Da for GPxl(Sec49).

Figures 11A and 11B are genetic maps illustrating the cloning of the

E. coli selA gene into the plasmids carrying the tRNA genes. In order to reduce the SelA expression level, the AUG start codon was changed to GUG and UUG, and a short nucleotide sequence was inserted between the Shine-

45292464vl 9 Dalgarno (SD) sequence and the start codon. In wildtype E. coli, SelB binds to the SECIS-like element in the selAB mRNA for autorepression

(UUAAACGCCCUUCUCCGUGUGAGAGGGCCUUGAUCAGCCAGGUUUCCUAUG (SEQ ID NO:60). However, ME6 strain lacks SelB and has no such regulation. Figure 11C is a series of images showing FDHH expression in ME6 cells expressing either tRNA^SecUx or tRNA^UTuX and carrying the fdhF gene variants having one or two UAG codons. The E. coli selA gene variant that has the 5-nt insertion plus the GUG start codon produced the most suitable concentration of E. coli SelA molecules for both tRNA species.

Figure 12A is a genetic map of the pTrc99A plasmid. Figure 12B is a series of images showing the effects of additional expressions of As SelA and Trypanosoma brucei (Tb) and Homo sapiens (Hs) PSTK species from the pTrc99A plasmid. IPTG was added at a two different concentrations to induce these enzymes from the trc promoter. FDHH expression in E. coli AselABC AfdhF (ME6) cells carrying pSecUAG- A plus one of the pTrc99A plasmids and the fdhF gene variant having mutations at codon positions 8, 11, and 140. Figure 12C is a series of images showing FDHH expression in ME6 cells expressing allo-tRNA^UTu and carrying one of the pTrc99A plasmids and the fdhF gene variant having a mutation at codon position 140. The SepCysS species is derived from Parcubacteria bacterium DG_74_2 bin and was cloned after the PSTK sequences in a dicistonic manner.

Figure 13A is a series of images showing FDHH expression in ME6 cells carrying either pSecUAG-A, pSecUAG-A+Asw/D, or pSecUAG-AD and carrying fdhF gene variants having four or five UAG codons. As selD indicates the wildtype gene carrying the AUG start codon. Figure 13B is series of images showing a repeated comparison of pSecUAG-A and pSecUAG-A+Ass<?/D. Figure 13C is a series of images showing FDHH expression in ME6 cells carrying pSecUAG-AD and carrying fdhF gene variants.

Figures 14A-14F is a series of images showing intact mass spectrometry of the human GPxl(Ser49 and Sec 49) mixture obtained from

ME6 cells carrying either of pSecUAG-AD (14A and 14B), pSecUAG-ADT

(14C and 14D), and pSecUAG-AD3T (14E and 14F). Dithiothreitol (DTT)

45292464vl 10 was used as the reducing agent. Possible peaks for oxidized GPxl proteins are indicated with "+0?" (for one site) and "+20?" (for two sites). Another minor peak (indicated by ?) is a putative formic acid adduct of GPxl.

Figures 15A (SEQ ID NO: 57) and 15B (SEQ ID NO: 58) are illustrations showing the primary and secondary structure of engineered allo- tRNA^UTu variants. A part of Aeromonas tRNA^Sec structure was transplanted to allo-tRNA^UTu. Another allo-tRNA was changed to allo-tRNA^UTu2 by introducing mutations. Figure 15C is a series of images showing the results of an assay testing FDHH expression in ME6 cells expressing As SelD, As SelA and allo-tRNA^UTu variants and carrying the fdhF gene variant having five UAG codons. As SelA was expressed from a low-copy-number plasmid vector using the wildtype AUG start codon or an alternative GUG start codon as indicated. Only the positive control plasmid pSecUAG-AD has additional As SelA expression cassette on this high-copy-number plasmid vector. After the incubation in an anaerobic tent for the dye formation, the agar plate was exposed to the air on the lab bench for dye oxidation and bleaching. The cell spot carrying the D-3b variant and pMW-AsSelA(AUG) remained dark for the longest time under oxygen exposure. Figure 15D is a series of images showing the results of repeating the same experiment as the 14C with the G21 variant. Figure 15E is a bar graphs showing Glutathione peroxidase (GPx) activities of GPxl produced with allo-tRNA^UTulD and allo- tRNA^UTu2D in ME6 cells at 25°C. Each bar represents the average of three independent experiments using different E. coli colonies. Figure 15F is a series of images showing the results of an assay testin gFDHH expression in ME68z cells at 25 °C. As SelA was expressed with a strong promoter (++++) or a weak promoter (+). Allo-tRNAsUTu were expressed with the indicated promoters (PargW >PselC).

Figure 16A and 16B are series of images showing the results of assays testing Sec insertion suing three SelA species and seven allo-tRNA variants. Sh, As, Rx denote Sulfurimonas honglongensis, Aeromonas salmonicida, Rubrobacter xylanophilus, respectively. 2225, 2459, S15 were derived from 9/3-1, 9/3-2, 8/4-1, respectively. Figure 16A shows all of the combinations of allo-tRNA and SelA inserted Sec (from the same agar

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45292464vl plate). The/<i/2 (140Amb) gene variant was used as reporter. Figure 16B shows the two allo-tRNA^UTu species derived from (9/3-1 and 9/3-2) were more active than the five allo-tRNA^UTu species derived from (8/4-1) (from the same agar plate). As SelA was used. The fdhF(3 UAG codons) gene variant was used as reporter.

Figure 17A is an illustration showing A. aeolicus SelA complexed with tRNA^sec. Abbreviations: Aa stands for Aquifex aeolicus; Ec for Escherichia coli; Mt for Moorella thermoacetica; Db for Desulfococcus biacutus; As for A. salmonicida; Psp. for Psychromonas sp. CNPT3; Pp for Photobacterium profundum; Pd for Photobacterium damselae; Td for

Treponema denticola; Rx for Rubrobacter xylanophilus. Figure 17B is a gene/protein diagram of NMC-A and series of images showing the results of a screen for highly active As SelA variants by an NMC-A β-lactamase reporter assay in E. coli C321.AA.opt AselAB. Serial dilutions of cells expressing wildtype or mutant SelA were spotted on ampicillin-containing agar plates and incubated at 30°C. Figure 17C is a series of images showing FDHH expression at 30 °C in ME6 fdhF (5 UAG codons) cells with pSecUAG-D-allo-tRNA^UTulD and pMWcat-AsSelA-(GUG) expressing wildtype or mutant SelA. Sodium selenite was added to a final concentration of 5 μΜ. Figure 17D is a series of images showing FDHH expression level in ME68z fdhF (5 UAG codons and A5ECIS) cells at 37°C was highest when both allo-tRNA^UTu2D and SelA^Evolwere expressed at a moderate level (arabinose (ara) 0.01% and "++", respectively).

Figure 18A is mass spectragram of protein by using pSecUAG- Evol2. Sec incorporation is estimated to be 84%. The peak of 23237.601 might derive from GPxl(Sec49) with a Cys-to-Sec substitution at any of the five Cys positions. Figure 18B is a bar graph showing Glutathione peroxidase (GPx) activities of GPxl produced with SelA^Evo1 variants. Each bar represents the average of three independent experiments using different E. coli colonies.

Figure 19 is a cartoon illustrating the Sec pathway engineered in Saccharomyces cerevisiae. Sc = Saccaromyces cerevisiae; As = Aeromonas salmonicida.

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45292464vl Figure 20A-20F are primary and secondary structures of Aeromonas salmonicida subsp. Pectinolytica 34mel tRNA (Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016)) (20A, SEQ ID NO: 148)), 5ctRNA^Ser (20B, SEQ ID NO: 150)), and 5ctRNA^Sec (20C, SEQ ID NO: 152)),

5ctRNA^Sec-2 (2D, SEQ ID NO: 154)), 5ctRNA^Sec-3 (2E, SEQ ID NO: 156)), 5ctRNA^Sec-4 (2F, SEQ ID NO: 158)).

Figure 21A and 21B is a diagram (21 A) and a structural model (2 IB) of a Gal4 reporter for selenocysteine incorporation.

Figure 22 is a series of images from an assay showing Ser incorporation at Cys positions in Gal4 does not permit growth on media lacking uracil.

Figure 23A is a series of images from an assay showing Sec insertion into gal4-CHAm is required for growth on media lacking uracil. Figure 23B is a series of images from an assay showing suppression of gal4- C21Am. Figure 23C is a series of images from an assay showing suppression of gal4-CHAm/C21Am by tRNA^Sec-3. Figure 23D is a series of images showing important components for Sec biosynthesis and translation in a yeast system. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Transfer RNA or tRNA refers to a set of genetically encoded RNAs that act during protein synthesis as adaptor molecules, matching individual amino acids to their corresponding codon on a messenger RNA (mRNA). In higher eukaryotes such as mammals, there is at least one tRNA for each of the 20 naturally occurring amino acids. In eukaryotes, including mammals, tRNAs are encoded by families of genes that are 73 to 150 base pairs long. tRNAs assume a secondary structure with four base paired stems known as the cloverleaf structure. The tRNA contains a stem and an anticodon. The anticodon is complementary to the codon specifying the tRNA's

corresponding amino acid. The anticodon is in the loop that is opposite of the stem containing the terminal nucleotides. The 3' end of a tRNA is aminoacylated by a tRNA synthetase so that an amino acid is attached to the

45292464vl 13 3 'end of the tRNA. This amino acid is delivered to a growing polypeptide chain as the anticodon sequence of the tRNA reads a codon triplet in an mRNA.

As used herein "suppressor tRNA" refers to a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system. For example, a suppressor tRNA can read through a stop codon.

As used herein, an "anticodon" refers to a unit made up of any combination of 2, 3, 4, and 5 bases (G or A or U or C), typically three nucleotides, that correspond to the three bases of a codon on an mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid or "stop codon." Known "stop codons" include, but are not limited to, the three codon bases, UAA known as ochre, UAG known as amber and UGA known as opal, that do not code for an amino acid but act as signals for the termination of protein synthesis. tRNAs do not decode stop codons naturally, but can and have been engineered to do so. Stop codons are usually recognized by enzymes (release factors) that cleave the polypeptide as opposed to encode an AA via a tRNA. Generally the anticodon loop consists of seven nucleotides. In the 5' to 3' direction the first two positions 32 and 33 precede the anticodon positions 34 to 36 followed by two nucleotides in positions 37 and 38 (Alberts, B., et al. in The Molecular Biology of the Cell, 4^th ed, Garland Science, New York, NY (2002)). The size and nucleotide composition of the anticodon is generally the same as the size of the codon with complementary nucleotide composition. A four base pair codon consists of four bases such as 5'- AUGC-3' and an anticodon for such a codon would complement the codon such that the tRNA contained 5'-GCAU-3' with the anticodon starting at position 34 of the tRNA. A 5 base codon 5' -CGGUA-3' codon is recognized by the 5'-UACCG-3' anticodon (Hohsaka T., et al. Nucleic Acids Res.

29:3646-3651 (2001)). The composition of any such anticodon for 2 (16=any possible combination of 4 nucleotides), 3 (64), 4 (256), and 5 (1024) base codons would follow the same logical composition. The "anticodon" typically starts at position 34 of a canonical tRNA, but may also reside in any position of the "anti-codon stem- loop" such that the resulting tRNA is

14

45292464vl complementary to the "stop codon" of equivalent and complementary base composition.

As used herein, "tRNA^Sec" refers to an unaminoacylated tRNA suitable for carrying selenocysteine. Typically the anticodon sequence of the tRNA^Sec can recognize or hybridize with an mRNA codon specific for, or designed to encode, a selenocysteine amino acid, for example UGA. In E.

coli, the endogenous tRNA^Sec is encoded by the selC gene.

As used herein, "tRNA^Ser" refers to an unaminoacylated tRNA suitable for carrying serine. Typically the anticodon sequence of the tRNA^Ser can recognize or hybridize with an mRNA codon specific for, or designed to encode, a serine amino acid, for example UCU, UCC, UCA, UCG, AGU, or AGC.

As used herein, "tRNA^UTu" refers to a non-naturally occurring, unaminoacylated tRNA^Sec suitable for carrying selenocysteine. Typically the anticodon sequence of the tRNA^UTu can recognize or hybridize with an mRNA codon specific for, or designed to encode, a selenocysteine amino acid.

As used herein, "Sec-tRNA^Sec" refers to aminoacylated tRNA^Sec carrying a selenocysteine amino acid.

As used herein, "Ser-tRNA^Sec" refers to aminoacylated tRNA^Sec carrying a serine amino acid.

As used herein, "Ser-tRNA^Ser" refers to aminoacylated tRNA^Ser carrying a serine amino acid.

As used herein, "Sep-tRNA^Ser" refers to a phosphorylated Ser- tRNA^Sec.

As used herein, "EF-Tu" refers to Elongation Factor Thermo

Unstable, a prokaryotic elongation factor mediates the entry of the

aminoacyl-tRNA into a free site of the ribosome.

As used herein, "SerRS" refers to Seryl-tRNA synthetase (also known as Serine— tRNA ligase) which is a prokaryotic factor that catalyzes the attachment of serine to tRNA^Ser.

As used herein "SECIS" refers to a SElenoCysteine Insertion

Sequence, is an RNA element around 60 nucleotides in length that adopts a stem-loop structure which directs the cell to translate UGA codons as

45292464vl 15 selenocysteines. In bacteria the SECIS can be soon after the UGA codon it affects, while in archaea and eukaryotes, it can be in the 3' or 5' UTR of an mRNA, and can cause multiple UGA codons within the mRNA to code for selenocysteine.

As used herein "SelA" refers to selenocysteine synthase, a prokaryotic pyridoxal 5 -phosphate-containing enzyme which catalyzes the conversion of Ser-tRNA^Sec into a Sec-tRNA^Sec.

As used herein "SelB" refers to selenocysteine- specific elongation factor, a prokaryotic elongation factor for delivery of Sec-tRNA^Sec to the ribosome.

As used herein "PSTK" refers to phosphoseryl-tRNA kinase (also known as 0-phosphoseryl-tRNA^Sec kinase and L-seryl-tRNA^Sec kinase), a kinase that phosphorylates Ser-tRNA^Sec to 0-phosphoseryl-tRNA^Sec, an activated intermediate for selenocysteine biosynthesis.

As used herein "SepSecS" refers to Sep (O-phosphoserine) tRNA:Sec

(selenocysteine) tRNA synthase (also known as O-phosphoseryl-tRNA(Sec) selenium transferase and Sep-tRNA:Sec-tRNA synthase), an eukaryotic and archaeal enzyme that converts 0-phosphoseryl-tRNA^Sec to selenocysteinyl- tRNA^Sec in the presence of a selenium donor.

As used herein "SepCysS" refers to Sep-tRNA:Cys-tRNA synthase, an archaeal/bacterial enzyme that converts 0-phosphoseryl-tRNA^Cys (Sep- tRNA^Cys) into Cys-tRNA^Cys in the presence of a sulfur donor.

As used herein "G-C content" (or guanine-cytosine content) refers to the percentage of nitrogenous bases on a nucleic acid molecule, or fragment, section, or region thereof, that are either guanine or cytosine.

Aminoacyl-tRNA Synthetases ("AARS") are enzymes that charge (acylate) tRNAs with amino acids. These charged aminoacyl-tRNAs then participate in mRNA translation and protein synthesis. The AARS show high specificity for charging a specific tRNA with the appropriate amino acid, for example, tRNA^Val with valine by valyl-tRNA synthetase or tRNA^TrP with tryptophan by tryptophanyl-tRNA synthetase. In general, there is at least one AARS for each of the twenty amino acids.

45292464vl 16 As used herein "translation system" refers to the components necessary to incorporate a naturally occurring amino acid into a growing polypeptide chain (protein). Components of a translation system can include, e.g., ribosomes, tRNAs, synthetases, mRNA and the like. The components described herein can be added to a translation system, in vivo or in vitro. A translation system can be either prokaryotic, e.g., an E. coli cell, or eukaryotic, e.g., a yeast, mammal, plant, or insect or cells thereof.

A "transgenic organism" as used herein, is any organism, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. Suitable transgenic organisms include, but are not limited to, bacteria, cyanobacteria, fungi, plants and animals. The nucleic acids described herein can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring DNA into such organisms are widely known and provided in references such as Sambrook, et al. (2000) Molecular Cloning: A Laboratory Manual, 3^rd ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.

As used herein, the term "eukaryote" or "eukaryotic" refers to organisms or cells or tissues derived therefrom belonging to the phylogenetic domain Eukarya such as animals (e.g., mammals, insects, reptiles, and birds), ciliates, plants (e.g., monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidia, and protists.

As used herein, the term "non-eukaryotic organism" refers to organisms including, but not limited to, organisms of the Eubacteria phylogenetic domain, such as Escherichia coli, Thermus thermophilu , and Bacillus stearothermophilus, or organisms of the Archaea phylogenetic domain such as, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus , Halobacterium such as Haloferax volcanii and

17

45292464vl Halobacterium species NRC-1, Ar chaeo globus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, and Aeuropyrum pernix.

The term "construct" refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences. Genetic constructs used for transgene expression in a host organism include in the 5 '-3' direction, a promoter sequence; a sequence encoding a gene of interest; and a termination sequence. The construct may also include selectable marker gene(s) and other regulatory elements for expression.

The term "gene" refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide, polypeptide, or protein. The term "gene" also refers to a DNA sequence that encodes an RNA product. The term gene as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5' and 3' ends.

The term "orthologous genes" or "orthologs" refer to genes that have a similar nucleic acid sequence because they were separated by a speciation event.

The term polypeptide includes proteins and fragments thereof. The polypeptides can be "exogenous," meaning that they are "heterologous," i.e., foreign to the host cell being utilized, such as human polypeptide produced by a bacterial cell. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S),

Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).

"Cofactor", as used herein, refers to a substance, such as a metallic ion or a coenzyme that must be associated with an enzyme for the enzyme to

45292464vl 18 function. Cofactors work by changing the shape of an enzyme or by actually participating in the enzymatic reaction.

"Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide' s biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.

In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine

(+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);

tryptophan (-0.9); tyrosine (- 1.3); proline (- 1.6); histidine (-3.2); glutamate (-

19

45292464vl 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and cofactors. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

Substitution of like amino acids can also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);

isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within + 2 is preferred, those within + 1 are particularly preferred, and those within + 0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin,

45292464vl 20 His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gin), (He: Leu, Val), (Leu: He, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: lie, Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above. In particular, embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polypeptide of interest.

The term "isolated" is meant to describe a compound of interest (e.g., nucleic acids) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature.

"Isolated" is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. Isolated nucleic acids are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components.

The term "vector" refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors can be expression vectors.

The term "expression vector" refers to a vector that includes one or more expression control sequences

The term "expression control sequence" refers to a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and the like. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

"Transformed," "transgenic," "transfected" and "recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can

45292464vl 21 also be present as an extrachromosomal molecule. Such an

extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non- transformed," "non- transgenic," or "non-recombinant" host refers to a wild- type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

The term "endogenous" with regard to a nucleic acid refers to nucleic acids normally present in the host.

The term "heterologous" refers to elements occurring where they are not normally found. For example, a promoter may be linked to a heterologous nucleic acid sequence, e.g., a sequence that is not normally found operably linked to the promoter. When used herein to describe a promoter element, heterologous means a promoter element that differs from that normally found in the native promoter, either in sequence, species, or number. For example, a heterologous control element in a promoter sequence may be a control/ regulatory element of a different promoter added to enhance promoter control, or an additional control element of the same promoter. The term "heterologous" thus can also encompass "exogenous" and "non-native" elements.

The term "percent (%) sequence identity" is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence

45292464vl 22 D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program' s alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C.

The term "stringent hybridization conditions" as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1X SSC at approximately 65 °C. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2000).

As used herein, the term "low stringency" refers to conditions that permit a polynucleotide or polypeptide to bind to another substance with little or no sequence specificity.

As used herein, the term "purified" and like terms relate to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment.

As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a

45292464vl 23 phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.

As used herein, the terms "recoded organism" and "genomically recoded organism (GRO)" in the context of codons refer to an organism in which the genetic code of the organism has been altered such that a codon has been eliminated from the genetic code by reassignment to a synonymous or nonsynonymous codon.

Unless otherwise indicated, the disclosure encompasses conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd edition (2001); Current Protocols In Molecular Biology [(F. M. Ausubel, et al. eds., (1987)];

Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) Current Protocols in Protein Science (John Wiley & Sons, Inc.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)].

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes VII, published by Oxford University Press, 2000; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Wiley-Interscience., 1999; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; Sambrook and Russell. (2001) Molecular Cloning: A Laboratory Manual 3rd. edition, Cold Spring Harbor Laboratory Press.

II. Compositions

A. tRNA

tRNASec suitable for carrying selenocysteine and facilitating synthesis of selenopeptides without requiring a SECIS in the mRNA encoding the peptide are disclosed. Also disclosed are aminoacylated tRNA^Sec. Using the methods discussed in more detail below, the tRNA^Sec disclosed herein are capable of being aminoacylated to form a Sec-tRNA^Sec which can facilitate insertion of selenocysteine into nascent polypeptide chains. Typically, the tRNA^Sec (1) can be recognized by SerRS and by EF-

45292464vl 24 Tu, or variants thereof; and is characterized by one or more of the following elements: (2) when aminoacylated with serine the non-naturally occurring Ser-tRNA^Sec can be converted to non-naturally occurring Sec-tRNA^Sec by SelA, or variant thereof; (3) when aminoacylated with serine the non- naturally occurring Ser-tRNA^Sec can be phosphorylated by PSTK or variant thereof; (4) when aminoacylated with phosphorylated serine the non- naturally occurring Sep-tRNA^Sec can serve as a substrate for SepSecS or variant thereof; and combinations thereof. In some embodiments, the tRNA^Sec is characterized by elements (1) and (2). In some embodiments, the tRNA^Sec is characterized by elements (1), (3), and (4). In some

embodiments, the tRNA^Sec is characterized by elements (1), (2), (3), and (4). Typically, the non-naturally occurring Sec-tRNA^Sec can be bound by EF-Tu. The Sec can be incorporated into a growing peptide chain at a codon of the mRNA that recognizes the anticodon of the tRNA^Sec. Preferably, EF-Tu does not bind Sep-tRNA^Sec. In some embodiments, EF-Tu is less efficient at incorporating Ser-tRNA^Sec than Sec-tRNA^Sec into the growing peptide chain.

Typically, the tRNA^Sec do not require a SECIS element in an mRNA to be incorporated into a growing polypeptide chain during translation. Typically the anticodon of the tRNASec is recognized or hybridizes to a stop codon. Typically the tRNASec can facilitate incorporation of a Sec into a growing peptide chain without the activity of SelB.

Some consensus and exemplary tRNA^Sec disclosed herein are provided as an RNA sequences, while others are provided as a DNA (e.g., the sequence encoding the tRNA^Sec). The RNA sequence is also an express disclosure of the corresponding DNA sequence wherein the "U" of the RNA are replaced with "T." The DNA sequence is also an express disclosure of the corresponding RNA sequence wherein the "T" of the DNA are replaced with "U."

1. Substrates for EF-Tu

EF-Tu is a prokaryotic elongation factor that mediates the entry of the aminoacyl-tRNA into a free site of the ribosome. Endogenous prokaryotic tRNAs, typically include an antideterminant element, which prevents recognition of a Sec-tRNA^Sec by the elongation factor EF-Tu. In some

45292464vl 25 embodiments, the disclosed tRNA can be a substrate for EF-Tu. Therefore, in some embodiments, the disclosed tRNA is a variant of an endogenous tRNA^Sec that has been modified to inactivate the antideterminant element. The antideterminant element can be modified, mutated, or deleted so that tRNA is an acceptable substrate for EF-Tu. For example the antideterminant element in E. coli tRNA^Sec is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA^Sec (encoded by selC), corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem (Rudinger, et al., EMBO J. , 15(3):650-57 (1996), and can be referred to as C7^» G66/G49* U65/C50^» G64 according the numbering in Schon, et al., Nucleic Acids Res. , 17(18):7159-7165 (1989). Accordingly, in some embodiments, the tRNA^Sec is variant of a naturally occurring tRNA^Sec where the corresponding antideterminant sequence is mutated or deleted such that the tRNA^Sec is a substrate for EF-Tu.

2. Substrate for SelA

SelA refers to L-seryl-tRNA(Sec) selenium transferase, which converts seryl-tRNA^Sec to selenocysteinyl-tRNA^Sec during selenoprotein biosynthesis. SelA utilizes selenophosphate synthesized by selenophosphate synthase (SelD) as the selenium-donor molecule. In some embodiments, the disclosed tRNA^Sec can serve as a substrate SelA. E. coli ecodes a SelA, thus, in some embodiments, the SelA is E. coli SelA.

An exemplary protein sequence for E. coli SelA is:

MTTETRFLYSQLPAIDRLLRDS SFLSLRDTYGHTRWELLRQMLDEAREVI RGSQT LPAWCENWAQEVDARLTKEAQSALRPVINLTGTVLHTNLGRALQAEAAVEAVAQAM RSPVTLEYDLDDAGRGHRDRALAQLLCRITGAEDAC IVNNNAAAVLLMLAATASGK EWVSRGELVE I GGAFRIPDVMRQAGCTLHEVGTTNRTHANDYRQAVNENTALLMK VHTSNYS IQGFTKAIDEAELVALGKELDVPWTDLGSGSLVDLSQYGLPKEPMPQE LIAAGVSLVSFSGDKLLGGPQAGI IVGKKEMIARLQSHPLKRALRADKMTLAALEA TLRLYLHPEALSEKLPTLRLLTRSAEVIQIQAQRLQAP LAAHYGAEFAVQVMPCLS QI GSGSLPVDRLP SAALTFTPHDGRGSHLESLAARWRELPVPVI GRIYDGRLWLDL RCLEDEQRFLEMLLK (SEQ ID NO:77)

An exemplary nucleic acid sequence (cloned from E. coli DH10B genome) encoding E. coli SelA is:

45292464vl 26 ATGACAACCGAAACGCGTTTCCTCTATAGTCAACTTCCGGCTATTGATCGCTTATT GCGCGATAGCTCCTTCCTTTCTTTGCGTGATACTTATGGTCACACCCGCGTGGTGG AATTGTTGCGTCAGATGCTCGACGAAGCGCGAGAAGTGATTCGTGGCAGCCAGACG CTGCCTGCGTGGTGTGAAAACTGGGCGCAAGAAGTCGATGCCCGGTTGACGAAAGA AGCGCAGAGCGCGCTGCGTCCGGTGATCAACCTGACGGGAACCGTGCTGCATACCA ACCTTGGGCGAGCTTTACAGGCGGAAGCCGCGGTGGAAGCCGTTGCGCAGGCTATG CGTTCGCCAGTGACCCTCGAGTATGATCTGGACGACGCCGGACGCGGACATCGCGA TCGGGCGCTGGCGCAGCTGCTGTGCCGTATTACGGGGGCGGAAGATGCCTGTATCG TCAATAACAATGCGGCGGCGGTGTTATTGATGTTGGCGGCCACTGCCAGCGGAAAA GAGGTGGTGGTATCTCGCGGCGAACTGGTGGAGATTGGCGGCGCGTTTCGTATTCC CGATGTTATGCGTCAGGCAGGCTGCACCCTACACGAAGTAGGGACCACCAACCGCA CGCACGCGAATGATTATCGTCAGGCGGTGAATGAAAATACCGCACTGTTGATGAAA GTACATACCAGTAACTACAGCATTCAGGGGTTCACCAAAGCGATAGATGAAGCGGA ACTGGTGGCGCTCGGCAAAGAGCTGGATGTTCCCGTAGTGACTGATTTAGGCAGTG GCTCGCTGGTCGATCTTAGCCAGTACGGTTTGCCGAAAGAGCCAATGCCGCAGGAG TTGATTGCGGCGGGCGTCAGTCTGGTGAGTTTCTCCGGCGACAAGTTGTTAGGCGG GCCGCAGGCAGGAATTATTGTTGGTAAAAAAGAGATGATCGCCCGCCTGCAAAGCC ACCCGCTGAAGCGTGCATTACGCGCGGATAAAATGACCCTCGCGGCGCTGGAAGCC ACGTTGCGTCTTTATTTACACCCTGAAGCTCTGAGTGAAAAATTACCGACCCTGCG CCTGCTTACCCGCAGCGCAGAGGTCATTCAAATCCAGGCACAACGTTTACAGGCCC CCCTTGCCGCACATTACGGCGCGGAGTTTGCGGTACAGGTTATGCCATGTCTTTCG CAGATTGGCAGTGGTTCGCTGCCGGTTGATCGCCTGCCGAGCGCGGCATTAACGTT TACACCCCATGATGGACGCGGTAGCCACCTTGAGTCATTAGCCGCCCGCTGGCGTG AATTGCCAGTGCCGGTGATTGGTCGTATTTATGACGGACGATTGTGGCTGGATTTA CGCTGCCTTGAAGATGAGCAACGGTTTTTGGAGATGTTGTTGAAATGA (SEQ ID

NO:78)

Other organisms also encode a SelA. Thus, in other embodiments, the SelA is not wildtype E. coli SelA. Results show wildtype E. coli SelA does not efficiently convert seryl-tRNA^Sec to selenocysteinyl-tRNA^Sec for at least some of the exemplary allo-tRNA disclosed in more detail below. Thus, in these embodiments, an alternative or variant SelA can be utilized for recombinant selenoprotein preparation.

SelA species which recognize 12-bp type tRNA^Sec molecules have Pro and Ala (and Gly in a metagenome sequence) in the position for the Ile25 of Aquifex aeolicus SelA. Therefore, such SelA species can be used to

27

45292464vl convert seryl-tRNA^Sec to selenocysteinyl-tRNA^Sec. Alternatively, SelA species which recognize 13-bp type tRNA^Sec molecules (such as E. coli) can be engineered to have, for example, Pro, Ala, or Gly in the amino acid position corresponding to position 25 (Ile25) of Aquifex aeolicus SelA.

The amino acid residues involved in the fixation of the SelA N- terminal domain are shown in Figure 17. The crystal structure of Aquifex aeolicus SelA and Thermoanaerobacter tengcongensis tRNA^Sec with a 13-bp amino-acid acceptor branch (PDB accession no. 3wlk) is exemplified. Other preferred residues that in can be imported (e.g., substituted) from SelA species which recognize 12-bp type tRNA^Sec molecules into SelA species which recognize 13-bp type tRNA^Sec molecules include, but are not limited to those corresponding with positions 25, 26, 29, 129, 332, and 333 of Aquifex aeolicus SelA.

The amino acid sequence for Aquifex aeolicus SelA (UniProtKB - 067140 (SELA_AQUAE)) is

MKSLLRQIPQISKWEIFKKKYPEjyWKAAREVAEKYRKEIIEGKRKDLNGFLED VERKIKSLMKPNIKRVINATGWINTNLGRAPLSKDVINFISEIANGYSNLEYNLE EGKRGSRIAHIEKYLNELTGAESSFVVNNNAGAVFLVLNTLAEGKEVI ISRGELVE IGGSFRIPDIMKKSGAILREVGTTNKTKVSDYEGAINQNTALLMKVHKSNFYMEGF VEEVKLEDLVKLGHKYGIPTYYDAGSGLLINLKEFGISVDEPNFRDCISLGIDLVS GSGDKLLGGPQAGIIVGKKNLIEKIKKNPIARALRIDKLTLSGLEMTLKLYFEKRY EDIPVIRMLTQDEKALRQKAKRLEKLLKDIPGLKISVIKDKAKPGGGSLPELELPT YCVAIRHDRLSSQELSRRLRLAEPPIVCRIREDQLLFDMRTVFHEDLKTIKKTLQE

LLSi (SEQ ID NO:79)

The amino acid of 125, Y26, K29, E129, F332, and E333 of Aquifex aeolicus SelA are identified with bold and italics. The corresponding amino acid residues from SelA species that recognize 12-bp type tRNA^Sec molecules are illustrated in Figure 17 or can be identified using sequence aligment, and can be used as a basis for reengineering SelA species that recognize 12-bp type tRNA^Sec to variants that recognize allo-tRNAs.

Exemplary SelA proteins that recognize allo-tRNAs as illustrated in the examples below include, but are not limited to, those from Sulfurimonas honglongensis, Aeromonas salmonicida, and Rubrobacter xylanophilus.

45292464vl 28 An exemplary amino acid sequence for Aeromonas salmonicida SelA is

MPNS SHAPAIAHSHSQPESCPTADDSLPDSLPDSLPQP SQQQARRLPQVEQLLQQP FLTGF IEALSRPLVTQAVRDVLSELRQSEAFRQHGVAPEQIEAL IAKRCQQQLRQR QTRVINATGTLVHTNLGRSPLSRELWDEVRDLNTGYNNLELDLATGKRGGRKGL IA PLLRCLTQAEDSLWNNNAASLFLLLQE IAKGREVIVSRGEQIQI GGGFRI PD I LA LSGAKLVEVGTTNI TTAKDYLDAI TDQTALVLMVHRSNFAIRGFTESPDI GEVARA LPEHWLAVDQGSGLTTEEFAPDETSVRQYIKAGADLVCYSGDKLLGGPQS GI I SG RSDL IKRLEKHPMMRTFRP SRIVYSLLERLL IHKLNKSP I GEGIAQRTLSNPAAMQ ARADQLMAALPGCFVPVPAQLWGGGTLPDEFYPAPALECTDPRPAQQLLDALRKL PVPVIATVRQQKVLLNMATLLPTE IALL IAQLKELLLPTPTTATEEP (SEQ ID

NO:80)

An exemplary nucleic acid sequence (cloned from the Aeromonas salmonicida genome) encoding Aeromonas salmonicida SelA is

ATGCCGAACTCGTCTCACGCGCCAGCCATCGCCCACTCTCACAGTCAGCCCGAATC ATGTCCCACTGCCGACGATTCACTGCCAGATTCACTGCCAGATTCACTGCCACAGC CCAGCCAGCAACAAGCGCGCCGTCTACCGCAAGTGGAACAGCTGCTGCAGCAACCC TTTCTCACCGGTTTTATCGAGGCGCTGAGCCGCCCGCTGGTGACCCAGGCGGTGCG CGATGTCCTGAGCGAATTGCGCCAGAGCGAGGCATTTCGCCAGCATGGGGTTGCCC CCGAGCAAATCGAGGCACTGATTGCCAAGCGTTGCCAGCAGCAGCTGCGCCAACGT CAGACCCGGGTGATCAACGCCACCGGCACCCTGGTGCACACCAATCTGGGGCGCTC GCCGCTAAGTCGCGAGCTGTGGGACGAGGTGCGCGACCTCAACACTGGCTACAACA ATCTGGAACTGGATCTCGCCACCGGCAAGCGCGGCGGGCGCAAGGGGCTGATCGCC CCCCTGCTCCGTTGCCTCACCCAGGCCGAGGATTCGCTGGTGGTCAACAACAACGC CGCTTCGCTCTTCTTGCTGCTGCAGGAGATAGCCAAGGGGCGCGAGGTGATCGTCT CGCGGGGCGAACAGATCCAGATTGGTGGCGGCTTTCGCATTCCCGACATTCTGGCG CTCTCCGGCGCCAAACTGGTGGAGGTGGGCACCACCAATATCACTACCGCCAAAGA TTACCTCGATGCCATCACAGATCAGACCGCGCTGGTGCTGATGGTACACAGATCCA ATTTCGCCATTCGCGGCTTTACCGAATCCCCCGATATTGGCGAGGTGGCCCGCGCC CTGCCCGAGCACGTGGTGCTGGCGGTGGATCAGGGCTCGGGCTTGACCACCGAGGA GTTTGCACCGGACGAAACCTCGGTGCGTCAGTACATCAAGGCGGGGGCGGATCTGG TCTGCTACTCCGGCGACAAGCTGCTGGGTGGCCCGCAATCGGGCATCATCAGCGGC CGCAGCGACCTCATCAAGCGGCTGGAAAAACACCCCATGATGCGCACCTTCCGCCC GAGCCGCATCGTCTACTCCCTGCTGGAACGCCTGCTCATCCACAAGCTCAACAAGT CCCCCATCGGCGAGGGCATCGCCCAGCGCACCTTGAGCAACCCTGCCGCCATGCAG

45292464vl 29 GCCCGCGCCGATCAGCTGATGGCCGCCCTGCCCGGCTGCTTTGTGCCGGTCCCCGC CCAGCTGGTGGTGGGTGGTGGCACCCTGCCGGACGAGTTCTACCCTGCGCCTGCGC TCGAATGCACCGACCCGCGTCCGGCCCAGCAGCTGCTCGATGCCCTGCGGAAACTG CCGGTGCCGGTCATCGCCACCGTGCGCCAGCAGAAGGTGCTGCTCAATATGGCGAC CCTGCTGCCGACCGAGATTGCACTGCTTATCGCCCAACTCAAGGAGTTGCTACTGC CCACTCCGACCACTGCGACCGAGGAGCCCTGA (SEQ ID N0:81)

An exemplary amino acid sequence for Rubrobacter xylanophilus

SelA is

MLDAERQSRLRSLPAVDAVLRGPAAGLAARHGRAAVAAAVREVLEGLRRE IAAGGS PDVSGRAVAEGAARLLSGRGLRRWNATGWLHTNLGRAVLSERAAAAAARAGTSY SNLEYDLSRGRRGSRYDHAVPLLRELTGAEDALWNNCAGATLLALSALAGEEGEG PPEWVSRGQL IE I GGGFRIPEVLELSGAVLREVGTTNRTRLSDYERALSERTRAI LWVHP SNFE IRGFTESAGIAELAGLGPPWADLGSGALLPLGGEPLVQAALRDGAE LALFSGDKLLGGPQAGIAAGS SRLVRRMRRHPLVRALRADKLCLAALEATLRAYLE GRAEEEVPAQRMLREPLEGVEARARRLASALSREVPGLEVGWP SVARSGGGTLPG YE IP SFAARVLGADAEALAARLRAAEPPWGRVHEGALLLDARTLLPGDEEAWEA

LREAARG (SEQ ID NO: 82)

An exemplary nucleic acid sequence encoding Rubrobacter xylanophilus SelA is

ATGCTGGATGCAGAACGTCAGAGCCGTCTGCGTAGCCTGCCTGCAGTTGATGCAGT TCTGCGTGGTCCGGCAGCAGGTCTGGCAGCACGTCATGGTCGTGCAGCAGTTGCAG CAGCAGTTCGTGAAGTTCTGGAAGGTCTGCGTCGTGAAATTGCAGCCGGTGGTAGT CCGGATGTTAGCGGTCGTGCCGTTGCAGAAGGTGCAGCCCGTCTGCTGAGTGGTCG TGGCCTGCGTCGCGTTGTTAATGCAACCGGTGTTGTTCTGCATACCAATCTGGGTC GTGCGGTTCTGAGCGAACGTGCAGCCGCAGCAGCGGCACGTGCAGGCACCAGCTAT AGCAATCTGGAATATGATCTGAGCCGTGGTCGTCGTGGTAGCCGTTATGATCATGC AGTTCCTCTGCTGCGTGAACTGACCGGTGCAGAAGATGCACTGGTTGTTAATAACT GTGCCGGTGCAACCCTGCTGGCACTGAGCGCACTGGCAGGCGAAGAAGGTGAAGGT CCGCCTGAAGTTGTTGTTAGTCGTGGTCAGCTGATTGAAATTGGTGGTGGTTTTCG TATTCCGGAAGTGCTGGAACTGAGTGGTGCCGTTCTGCGCGAAGTTGGTACAACCA ATCGTACCCGTCTGAGCGATTATGAACGTGCACTGAGTGAACGTACCCGTGCAATT CTGTGGGTTCATCCGAGCAATTTTGAAATTCGCGGTTTTACCGAAAGCGCAGGTAT TGCAGAACTGGCTGGTCTGGGTCCTCCGGTTGTTGCAGATCTGGGTAGCGGTGCAC TGCTGCCGCTGGGTGGTGAACCGCTGGTTCAGGCAGCACTGCGTGATGGTGCCGAA CTGGCACTGTTTAGCGGTGATAAACTGCTGGGTGGACCGCAGGCTGGTATTGCCGC

45292464vl 30 AGGTAGCAGCCGTCTGGTTCGTCGTATGCGTCGTCATCCGCTGGTGCGTGCCCTGC GTGCAGATAAACTGTGCCTGGCAGCCCTGGAAGCAACACTGCGTGCATATCTGGAA GGCCGTGCCGAAGAAGAAGTTCCGGCACAGCGTATGCTGCGCGAACCACTGGAAGG TGTTGAAGCACGTGCCCGTCGTCTGGCAAGCGCACTGAGTCGTGAAGTGCCTGGTC TGGAAGTTGGTGTTGTGCCGAGCGTTGCACGTAGCGGTGGTGGCACCCTGCCTGGT TATGAAATTCCGAGCTTTGCAGCACGTGTTCTGGGTGCAGATGCAGAAGCCCTGGC AGCGCGTCTGCGTGCCGCAGAACCGCCTGTTGTGGGTCGTGTTCATGAAGGTGCCC TGCTGCTGGATGCCCGTACCCTGCTGCCAGGTGATGAAGAAGCAGTTGTTGAAGCG CTGCGTGAGGCAGCCCGTGGTTAA (SEQ ID NO:83)

An exemplary amino acid sequence for Sulfurimonas honglongensis

SelA is

MFLLKS IPKVDKF IAKKEFKTLGSALVMSLTKELLSELRENI LNGRVTTFSEDELV KELLQRYTELTKP SLQTL INATGI IVHTNLGRSL IDADAFDRVKELMTNYNNLEFN LESGKRGERYSL I SKSVCSLLGCEDVL IVNNNASAVFL ILNTFARKKEWVSRGEL VE I GGSFRVPDVMKQSGAKLVEVGTTNKTHLYDYEDAI GKKTSMLMKVHKSNYS IE GFS SDVEFGEIVKLACEKGLIDYYDMGSGHLFDLPYGLDEP SVLDFMKLNP SLLSF SGDKLLGSVQAGI IVGKKKYIDMLKKNQLLRMLRVDKLTLALLEESFKAI LLGNKE QIPTARMLFRSTDELREDAMQVQQKLKKNIKTNIVDTKTL I GGGTTPNKT I PSVAL VIESKNIKVKKLQKLFRQKS I I GRIEDDEFLLDFRT IQKTQLQQWDAIDE ITDV

(SEQ ID NO:84)

An exemplary nucleic acid sequence encoding Sulfurimonas honglongensis SelA is

ATGTTCCTGCTGAAAAGCATTCCGAAAGTGGATAAGTTTATCGCCAAGAAAGAGTT TAAAACCCTGGGTAGCGCACTGGTTATGAGCCTGACCAAAGAACTGCTGAGCGAAC TGCGTGAAAACATTCTGAATGGTCGTGTTACCACCTTTAGCGAAGATGAACTGGTT AAAGAGCTGCTGCAGCGTTATACCGAACTGACCAAACCGAGCCTGCAGACCCTGAT TAATGCAACCGGTATTATTGTTCATACCAATCTGGGTCGTAGCCTGATTGATGCAG ATGCATTTGATCGTGTTAAAGAACTGATGACCAACTATAACAACCTGGAATTTAAT CTGGAAAGCGGTAAACGTGGTGAACGCTATAGTCTGATTAGCAAAAGCGTTTGTAG CCTGCTGGGTTGTGAAGATGTTCTGATTGTGAATAATAACGCCAGCGCAGTTTTTC TGATTCTGAACACCTTTGCGCGTAAAAAAGAAGTTGTTGTTAGTCGCGGTGAACTG GTGGAAATTGGTGGTAGCTTTCGTGTTCCGGATGTTATGAAACAGAGCGGTGCAAA AC T G G T T G AAG T T G G C AC C AC C AAT AAAAC C C AT C T G T AT G AT T AT G AAG AT G C C A TCGGTAAAAAAACGAGCATGCTGATGAAAGTGCACAAAAGCAACTATAGCATTGAA GGTTTTAGCAGCGACGTGGAATTTGGCGAAATTGTTAAACTGGCATGTGAAAAAGG

45292464vl 31 CCTGATCGATTATTATGATATGGGTAGCGGTCACCTGTTTGATCTGCCGTATGGTC TGGATGAACCGAGCGTTCTGGACTTTATGAAACTGAATCCGAGTCTGCTGAGCTTT AGCGGTGATAAACTGCTGGGTAGTGTTCAGGCAGGCATTATTGTTGGCAAAAAAAA GTATATCGACATGCTGAAGAAAAACCAGCTGCTGCGTATGCTGCGTGTGGATAAAC TGACCCTGGCACTGCTGGAAGAAAGTTTTAAAGCAATTCTGCTGGGCAACAAAGAG CAGATTCCGACCGCACGTATGCTGTTTCGTAGCACCGATGAACTGCGCGAAGATGC AATGCAGGTTCAGCAGAAACTGAAAAAAAACATCAAGACCAACATCGTGGATACCA AAACACTGATTGGTGGCGGTACAACCCCGAATAAAACCATTCCGAGCGTTGCCCTG GT T AT T G AAAG C AAAAAC AT T AAG G T G AAAAAAC T G C AGAAG C T G T T T C G C C AG AA AAGTATTATTGGTCGCATCGAGGATGATGAATTTCTGCTGGATTTTCGTACGATTC AGAAAACCCAACTGCAGCAGGTTGTTGATGCAATTGATGAAATTACCGACGTGTAA

(SEQ ID NO:85)

In some embodiments, the SelA is a variant SelA that has at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any one of SEQ ID NO:79, 80, 82, or 84.

In some embodiments, the SelA had one or more the mutations discussed below (see, e.g., Table 5, and the discussion thereof), or the corresponding mutation in another species or organism.

Typically the disclosed SelA or variant SelA can convert seryl- tRNA^Sec, 0-phosphoseryl-tRNA^Sec or both to selenocysteinyl-tRNA^Sec for one or more of the disclosed tRNA^Sec.

3. Substrates for PSTK

PSTK is a kinase in archaeal and eukaryotic systems that phosphorylates Ser-tRNA^Sec to 0-phosphoseryl-tRNA^Sec, an activated intermediate for selenocysteine biosynthesis. Accordingly, in some embodiments, once aminoacylated with serine, the non- naturally occurring tRNA can serve as a substrate for a PSTK, or variant thereof. The enzyme activity of PSTK is strictly tRNA^Sec-dependent. PSTK does not hydrolyze ATP in the absence of tRNA nor in the presence of Ser-tRNA^Ser. The binding of tRNA^Ser, however, promotes ATP hydrolysis (R. Lynn Sherrer, et al., Nucleic Acids Res. , 36(4): 1247-1259 (2008)). This indicates that tRNA^Sec might play an important role in positioning the Ser moiety for initiating phosphoryl transfer. Compared to aminoacyl-tRNA synthetases, PSTK has approximately 20-fold higher affinity toward its substrate, Ser-tRNA^Sec (Km

32

45292464vl = 40 nM) (R. Lynn Sherrer, et al., Nucleic Acids Res. , 36(4): 1247-1259 (2008)), which may compensate for the low abundance of tRNA^Sec in vivo. The concentration of tRNA^Sec in vivo is at least 10-fold lower than tRNA^Ser in tRNA^Sec-rich tissues such as liver, kidney and testes in rat (Diamond, et al., /. Biol. Chem. , 268: 14215-14223 (1993)).

The crystal structure of Methanocaldococcus jannaschii PSTK (MjPSTK) places archaeal PSTK identity elements (G2:C71 and the

C3:G70) (Sherrer, et al., Nucleic Acids Res, 36: 1871-1880 (2008)). within contact of the protein dimer interface. The second base pair in the acceptor stem is highly conserved as C2:G71 in eukaryotic tRNA^Sec, and mutation of G2:C71 to C2:G71 in archaeal tRNA^Sec resulted in a Ser-tRNA^Sec variant that is phosphorylated inefficiently (Sherrer, et al., Nucleic Acids Res, 36: 1871- 1880 (2008). The A5-U68 base pair in Methanococcus maripaludis tRNA^Ser has some antideterminant properties for PSTK (Sherrer, et al., NAR, 36(6): 1871-1880 (2008)). Moreover, the eukaryotic PSTK has been reported to recognize the unusual D-arm of tRNA^Sec as the major identity element for phosphorylation (Wu and Gross EMBO J. , 13:241-248 (1994)).

Accordingly, in some embodiments, the disclosed tRNAs include residues in the acceptor stem, the D-arm, or combinations thereof that are needed for the tRNA to serve as a substrate for a PSTK.

4. Substrate for SepSecS

The conversion of phosphoseryl-tRNA^Sec (Sep-tRNA^Sec) to selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) is the last step of Sec biosynthesis in both archaea and eukaryotes, and it is catalyzed by tetratmeric O- phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). It is believed that one SepSecS homodimer interacts with the sugar-phosphate backbone of both the acceptor- ΤΨΟ and the variable arms of tRNA^Sec, while the other homodimer interacts specifically with the tip of the acceptor arm through interaction between the conserved Arg398 and the discriminator base G73 of human tRNA^Sec.

The co-crystal structure of SepSecS and tRNA^Sec also shows that the

9 bp acceptor stem of tRNA^Sec is probably important for recognition by the enzyme (Palioura, S, Sherrer, RL, Steitz, TA, Soil, D & Simonovic, M

45292464vl 33 (2009) Science 325:321-325). According to structural analysis, the acceptor- T- variable arm elbow region of tRNA^Sec (including bases G50, G51, C64, C65 in the human tRNA^Sec that are recognized by SepSecS) may be important for recognition by SepSecS. Accordingly, in some embodiments, the disclosed tRNAs include residues in the acceptor-T^FC, the variable arms of tRNA^Sec, the tip of the acceptor arm, or combinations thereof needed for the tRNA to serve as a substrate for SepSecS. In some embodiments, the G50, G51, C64, C65 elements of human tRNA^Sec are present in the tRNASec.

The SepSecS enzyme itself can also be mutated to engineer enzyme variants that accept a substrate somewhat less ideal than naturally occurring tRNA^Sec. It is believed that His30, Arg33, Lys38 in SepSecS form key interactions with the protomer and G50, U51, C64 and C65 of the tRNA. Therefore, mutation of some of these residues could result in a SepSecS variant that is better able to recognize one of the tRNASec. The formed Sec- tRNA^Sec can be screened in the formate dehydrogenase-benzyl viologen assay [e.g., (Yuan, J, Palioura, S, Salazar, JC, Su, D, O'Donoghue, P, Hohn, MJ, Cardoso, AM, Whitman, WB & Soil, D (2006), Proc Natl Acad Sci USA 103: 18923-18927; Palioura, S, Sherrer, RL, Steitz, TA, Soil, D &

Simonovic, M (2009) Science 325:321-325)]. Other assays include standard Wolfson assay [e.g., (Yuan, J, Palioura, S, Salazar, JC, Su, D, O'Donoghue, P, Hohn, MJ, Cardoso, AM, Whitman, WB & Soil, D (2006) Proc Natl Acad Sci USA 103: 18923-18927; Palioura, S, Sherrer, RL, Steitz, TA, Soil, D & Simonovic, M (2009) Science 325:321-325)], labeling with [75Se]selenite in the presence of selenophosphate synthase (SelD) [e.g., (Yuan, J, Palioura, S, Salazar, JC, Su, D, O'Donoghue, P, Hohn, MJ, Cardoso, AM, Whitman, WB & Soil, D (2006) Proc Natl Acad Sci USA 103: 18923-18927)], and using [14C] or [3H]serine in the initial charging reaction.

In some embodiments, a SepCysS is used instead of SepSecS.

SepCysS is a key PLP-dependent enzyme in Cys-tRNA formation in methanogens. It converts Sep-tRNA^Cys into Cys-tRNA^Cys using

thiophosphate as sulfur donor. The enzyme's crystal structure is established

(Fukunaga, R & Yokoyama, S (2007) Nat Struct Mol Biol 14:272-279.) and

34

45292464vl its mechanism (Liu, Y., Dos Santos, P.C., Zhu, X., Orlando, R., Dean, D.R., Soil, D. and Yuan, J. (2012) /. Biol. Chem. 287, 5426-5433) is different from that of SepSecS (Palioura, S, Sherrer, RL, Steitz, TA, Soil, D & Simonovic, M (2009) Science 325:321-325.)· The length of the acceptor stem of its tRNA substrates is not critical and acceptor helices between 7-9 bp are acceptable. Therefore, this enzyme's active site can be engineered to allow selenophosphate (instead of thiophosphate) to participate in the reaction.

5. Primary Structure

tRNAs can be described according to their primary structure (i.e., the sequence from 5' to 3') as well as their secondary structure. The secondary structure of tRNA is typically referred to as a "cloverleaf ', which assumes a 3D L-shaped tertiary structure through coaxial stacking of the helices.

Figure 2 illustrates a typical human tRNA^Sec, which includes an acceptor arm, a D-arm, an anticodon arm, a variable arm, and a T^FC-arm.

In some embodiments the tRNA^Sec shares sequence identity or sequence homology with a naturally occurring tRNA, for example a naturally occurring tRNA^Sec, or a naturally occurring tRNA^Ser.

a. Variants of Naturally Occurring tRNA^Sec The non-naturally occurring tRNA^Sec disclosed herein can be a variant of a naturally occurring tRNA^Sec. The naturally occurring tRNA^Sec can be from a prokaryote, including but not limited to E. coli, an archaea, including, but not limited to, M. maripaludis and M. jannaschii, or a eukaryote including, but not limited to human.

In some embodiments, the non-naturally occurring tRNA^Sec is a variant of an E. coli tRNA^Sec, for example,

GGAAGAUCGUCGUCUCCGGUGAGGCGGCUGGACUUCAAAUCCAGUUGGGGC CGCCA GCGGUCCCGGGCAGGUUCGACUCCUGUGAUCUUCCGCCA (SEQ ID NO: l), which is depicted in Figure 3 (left panel).

In some embodiments, the non-naturally occurring tRNA^Sec is a variant of an M. maripaludis tRNA^Sec, for example,

GGCACGGGGUGCUUAUCUUGGUAGAUGAGGGCGGACUUCAGAUCCGUCGAGUUCCG UUGGAAUUCGGGGUUCGAUUCCCCCCCUGCGCCGCCA (SEQ ID NO:2).

45292464vl 35 In some embodiments, the non-naturally occurring tRNA^Sec is a variant of a human tRNA^Sec, for example,

GCCCGGAUGAUCCUCAGUGGUCUGGGGUGCAGGCUUCAAACCUGUAGCUGUCUAGG GACAGAGUGGUUCAAUUCCACCUUUCGGGCGCCA (SEQ ID NO:3), which is depicted in Figure 2.

An exemplary variant of E. coli tRNA^Sec is tRNA^SecUX _am, described in Thyer, et al., /. Am. Chem. Soc , 137:46-49 (2015) (SEQ ID NO: 18), wherein the circled region of Figure 3B was mutated in tRNA^Sec (e.g., SEQ ID NO: l) to enable recognition by EF-Tu. The EF-Tu recognition region is very similar between tRNA^UTu and tRNA^SecUX; residues G7, U64, G65, and C66 are shared between the two. Residues 50 and 49 are different between tRNA^SecUX and tRNA^UTu (discussed in more detail below).

b. Variants of Naturally Occurring tRNA^Ser The non-naturally occurring tRNASec disclosed herein can be a variant of a naturally occurring tRNA^Ser. The naturally occurring tRNA^Ser can be from a prokaryote, including but not limited to E. coli, an archaea, including, but not limited to, M. maripaludis and M. jannaschii, or a eukaryote including, but not limited to human.

In some embodiments, the non-naturally occurring tRNA^Sec is a variant of an E. coli tRNA^Ser, for example,

GGAAGUGUGGCCGAGCGGUUGAAGGCACCGGUCUUGAAAACCGGCGACCCGAAAGG GUUCCAGAGUUCGAAUCUCUGCGCUUCCGCCA (SEQ ID NO:4), depicted in Figure 3C.

In some embodiments, the non-naturally occurring tRNA^Sec is a variant of an M. maripaludis tRNA^Ser, for example,

GCAGAGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUGAAAUCCGGUUCUC CACUG GGGAGCGGGGGUUCAAAUCCCUCCCUCUGCGCCA (SEQ ID NO:5).

c. Chimeric tRNA^Sec

The non-naturally occurring tRNA^Sec disclosed herein can also be a chimeric tRNA including sequences from two or more naturally occurring tRNAs. Some embodiments, the non-naturally occurring tRNA includes sequences from a naturally occurring tRNA^Sec and a naturally occurring tRNA^Ser. The chimeric tRNA can include nucleic acid sequences or features,

45292464vl 36 for example an antideterminant element, from a prokaryote, including but not limited to E. coli, an archaea, including, but not limited to, M. maripaludis and M. jannaschii, or a eukaryote including, but not limited to, human.

Examples of non-naturally occurring tRNA^Sec that are chimeric tRNAs including sequence elements from E. coli include, but are not limited to

GGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAG GGUUCCAGAGUUCGAAUCUCUGCAUCUUCCGCCA (SEQ ID NO:6; E. coli tRNA^UTu-opal), as depicted in Figure 3B;

GGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAG GGUUCCAGAGUUCGAAUCUCUGCAUCUUCCGCCA (SEQ ID NO:7; E. coli tRNA^UTu-amber), as depicted in Figure 3B; and

GGAAGAUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAG GGUUCCAGAGUUCGAAUCUCUGCAUCUUCCGCCA (SEQ ID NO:8; E. coli tRNA^UTu-ochre).

Other examples of non-naturally occurring tRNA^Sec that are chimeric tRNAs including sequence elements from E. coli include, but are not limited to

GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUCAAAACCGGCGACCCGAAAGG GUUCCAGAGUUCGAAUCUCUGCGGUGCCGCCA (SEQ ID NO:9; E. coli tRNA^UTu-opal), as depicted in Figure 4B;

GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUCUAAAACCGGCGACCCGAAAGG GUUCCAGAGUUCGAAUCUCUGCGGUGCCGCCA (SEQ ID NO: 10; E. coli tRNA^UTu-amber), as depicted in Figure 4B; and

GGCACUGUGGCCGAGCGGUUGAAGGCACCGGUCUUUAAAACCGGCGACCCGAAAGG GUUCCAGAGUUCGAAUCUCUGCGGUGCCGCCA (SEQ ID NO: l l ; E. coli tRNA^UTu-ochre), which are non-naturally occurring chimeras of E. coli tRNA^Ser with PSTK identity elements.

In some embodiments, the non-naturally occurring tRNA^sec is a variant of tRNA^UTu, for example, SEQ ID NO:7:

G¹GAAG⁵A^5aUGUGG¹⁰CCGAGCGGU²⁰UGAAGGCACCGG³⁰UCUCC¾AAAC⁴⁰CGGCGA CCCGAAAGGGUUCCA⁵⁰GAGUUCGAAU⁶⁰CUCUGCAU^{67 a}CUU⁷⁰CCGCCA (SEQ ID

37

45292464vl NO:7; E. coli tRNA^UTu-amber) (wherein the anticodon is bolded and in italics),

or the opal or ochre equivalent thereof (e.g., SEQ ID NO:6 or 8). In some embodiments, the non-naturally occurring tRNA^sec has at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97, 98, or 99% sequence identity to SEQ ID NO: 6, 7, or 8.

The anticodon is in italics. tRNA positional markers (e.g., 1, 5, 5a, 10, 20, 30, 40, 50, 60, 67a, 70 according to the numbering of Sprinzl, et al., Nucleic Acids Research, 26(1): 148-153 (1998)) are provided in superscript and are not part of the tRNA sequence.

An exemplary tRNA^UTu variant is UTuX

G¹GAAG⁵A^5aUGGUG¹⁰CCGUCCGGU²⁰GAAGGCGCCGG³⁰UCUCC¾AAAC⁴⁰CGGUCGA CCCGAAAGGGUUCGCA⁵⁰GGGUUCGACU⁶⁰CCCUGCAU^{67 a}CUU⁷⁰CCGCCA (SEQ ID NO:17; E. coli scaffold, tRNA^UTuX- amber, and depected in Figure 5A), or an opal or ochre equivalent thereof.

Examples of non-naturally occurring tRNA^Sec that are chimeric tRNAs including sequence elements from M. maripaludis include, but are not limited to,

GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUCAAAUCCGGUUCUCCACUG GGGAGCGGGGGUUCAAAUCCCUCCCGCGCCGCCA (SEQ ID NO: 13; M.

maripaludis tRNA^UTu-opal), as depicted in Figure 4A;

GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUCUAAAUCCGGUUCUCCACUG

GGGAGCGGGGGUUCAAAUCCCUCCCGCGCCGCCA (SEQ ID NO: 14; M.

maripaludis tRNA^UTu- amber), as depicted in Figure 4A;

GGCGCGGUGGUUGAGCUUGGCCAAAGGCGCCGGACUUUAAAUCCGGUUCUCCACUG

GGGAGCGGGGGUUCAAAUCCCUCCCGCGCCGCCA (SEQ ID NO: 15; M.

maripaludis tRNA^UTu-ochre).

In some embodiments, the non-naturally occurring tRNA^Sec are chimeric tRNAs that include sequence elements from a yeast tRNA in combination with elements from a non-yeast tRNA. Typically the tRNA^Sec can be a substrate for a SerRS and a SelA and bind to eEFla.

For example, in some embodiments, the chimeric tRNA is a variant of a yeast tRNA^Ser such as:

45292464vl 38 GGCAACTTGGCCGAGTGGTTAAGGCGAAAGATTCTAAATCTTTTGGGCTTTGCCCG CGCAGGTTCGAGTCCTGCAGTTGTCG (SEQ ID NO: 149, DNA) GGCAACUUGGCCGAGUGGUUAAGGCGAAAGAUUCUAAAUCUUUUGGGCUUUGCCCG CGCAGGUUCGAGUCCUGCAGUUGUCG (SEQ ID NO: 150, RNA, Figure 20B)

In some embodiments, the tRNA^Sec includes elements, for example, in the tRNA acceptor branch and in combination with other elements (e.g., identity elements, from Aeromonas salmonicida). The anticodon of tRNA^Ser is not an identity element for aminoacylation by SerRS.

An Aeromonas salmonicida tRNA^Sec is

GGCAACTTGGCCGAGTGGTTAAGGCGAAAGATTAGAAATCTTTTGGGCTTTGCCCG CGCAGGTTCGAGTCCTGCAGTTGTCG (SEQ ID NO: 147, DNA) GGCAACUUGGCCGAGUGGUUAAGGCGAAAGAUUAGAAAUCUUUUGGGCUUUGCCCG CGCAGGUUCGAGUCCUGCAGUUGUCG (SEQ ID NO: 148, RNA, Figure 20A)

The Examples below illustrate that an efficient nonsense suppressor was designed using Saccharomyces cerevisiae tRNA^Ser (5ciRNA^Ser) for Sec incorporation at amber (UAG) codons. To generate an amber suppressor tRNA^Sec species from 5ciRNA^Ser, the anticodon was switched from AGA to CUA and AsiRNA^Sec identity elements were introduced.

Four tRNA^Sec variants were tested in the Examples below, each having mutations that may influence tRNA stability and AsSelA recognition. Thus, in some embodiments, the tRNA^Sec is a yeast tRNA^Ser with one or more of the mutations illustrated in Figures 21B-21F. Exemplary yeast tRNA^Sec include,

GGCAACTTCGCCGTCTGGTGGCGGCGAAAGATTCTAAATCTTTTGGGCTTTGCCCG GGCAGGTTCGATTCCTGCAGTTGTCG (SEQ ID NO:151, DNA)

GGCAACUUCGCCGUCUGGUGGCGGCGAAAGAUUCUAAAUCUUUUGGGCUUUGCCCG GGCAGGUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 152, RNA, Figure 20C) GGCAACTACGCCGCCTGGTGGCGGCGAAAGATTCTAAATCTTTTGGGCTTTGCCCG GGCAGGTTCGATTCCTGCAGTTGTCG (SEQ ID NO: 153, DNA) GGCAACUACGCCGCCUGGUGGCGGCGAAAGAUUCUAAAUCUUUUGGGCUUUGCCCG GGCAGGUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 154, RNA, Figure 20D) GGCAACTATGCCGCCTGGTGGCGGCGAAAGATTCTAAATCTTTTGGGCTTTGCCCG GGCAGGTTCGATTCCTGCAGTTGTCG (SEQ ID NO: 155, DNA) and

45292464vl 39 GGCAACUAUGCCGCCUGGUGGCGGCGAAAGAUUCUAAAUCUUUUGGGCUUUGCCCG GGCAGGUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 156, RNA, Figure 20E) GGCAACTATGCCGTCTGGTGGCGGCGAAAGATTCTAAATCTTTTGGGCTTTGCCCG GGCAGGTTCGATTCCTGCAGTTGTCG (SEQ ID NO: 157, DNA) GGCAACUAUGCCGUCUGGUGGCGGCGAAAGAUUCUAAAUCUUUUGGGCUUUGCCCG GGCAGGUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 158, RNA, Figure 20F) or the opal or ochre equivalent thereof. In some embodiments, the non-naturally occurring tRNA^Sec has a sequence or is encoded by a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 97, 98, or 99% sequence identity to any one of SEQ ID NO: 147-158.

d. Allo-tRNA

In some embodiments, the tRNA^Sec is a naturally occurring tRNA or a non-naturally occurring variant thereof. Thus, in some embodiments, the tRNA^Sec includes or consists of a naturally occurring nucleic acid sequence. In other embodiments, the non-naturally occurring tRNA^Sec includes or consists of a naturally occurring nucleic acid sequence with one or more insertions, deletions or subsitutions.

In some embodiments, the tRNA^Sec is an "allo-tRNA." Allo-tRNA are structurally similar to tRNA^Sec as they have a long V-arm and longer anticodon and acceptor stems compared to canonical tRNAs. Moreover, the D-stem-loop of allo-tRNAs resembles that of tRNA^Sec with its long stem and tetraloop. Allo-tRNAs typically have a 8/4 or 9/3 composition of the 12-bp amino-acid acceptor branch. As discussed in more detail in the examples below, naturally occurring allo-tRNA have been identified in, for example, Clostridia, Proteobacteria, and Acidobacteria.

Naturally occurring allo-tRNAs typically have a long V-arm and many have an identity of the discriminator base (G73 or U73) which are important for aminoacylation by seryl-tRNA synthetase (SerRS), though at least one was found to charge with alanine. Many naturally occurring allo- tRNA have an anticodon that recognizes a codon incosistant with the amino acid charged to it. For example, some of the allo-tRNAs charge with serine, but have a leucine anticodon.

45292464vl 40 Non-naturally occurring variants of naturally occurring allo-tRNAs are also provided. The non-naturally occurring allo-tRNA typically have one or more insertions, deletions, or substitutions relative to the naturally occurring allo-tRNA. Thus in some embodiments, the only change(s) in a non-naturally occurring tRNA^Sec is substitution of the naturally-occurring anticodon with an alternative anticodon, preferable an anticodon that recognizes a stop codon.

In some embodiments, the naturally occurring allo-tRNA can be additionally or alternatively modified to include a SerRS identity element.

In some embodiments, the variants include one more additional or alternative modifications that improve the tRNAs activity as a tRNA^Sec, for example, to improve binding to SelA, or improve binding to a EF-Tu.

i. Exemplary Consensus Allo-tRNA Exemplary consensus primary sequences and secondary structures for allo-tRNA are provided. Exemplary consensus structures are depicted in Figures 6A and 6B, and 6D-6F. For the sequences provided below, N denotes A, G, T/U, or C; R denotes A or G; Y denotes T/U or C; K denotes G or T/U; and W denotes A or T/U. The anticodon is in bold and italics.

8/4 allo-tRNA, Figure 6A

GGRGRRNRNNNNNNNNNGGYNNNNNNNNNNGNYUNN2VAANCNNNNNNNNNNNNNNN NNNNNNNNRNRGUYCRANYCYNYYNYYCYCCNCCA (SEQ ID NO: 19)

Typically, an Acceptor Stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-80 respectively;

a D-arm can be formed by base pairing between nucleotides 11-16 with nucleotides 26-21 respectively;

an anti-codon arm can be formed by base pairing between nucleotides 27-32 with nucleotides 45-40 respectively;

a V-arm can be formed by base pairing between nucleotides 47-53 with nucleotides 64-58 respectively;

a T-arm can be formed by base pairing between nucleotides 65-68 with nucleotides 79-76 respectively;

or a combination thereof.

41

45292464vl 9/3 allo-tRNA (Figure 6B)

GGRRNNNNNNNNNNNNNYGGNNNNNNNNNNNRNYUNN2VAANYNNNNNNNNNNNNNN NNNNNNNNNNNNNRGGUUCRAYUCCYNNNNNYYCCRCCA (SEQ ID NO:20)

Typically, an Acceptor Stem can be formed by base pairing between nucleotides 1-9 with nucleotides 91-83 respectively;

a D-arm can be formed by base pairing between nucleotides 12-17 with nucleotides 27-22 respectively;

an anti-codon arm can be formed by base pairing between nucleotides 28-33 with nucleotides 46-41 respectively;

a V-arm can be formed by base pairing between nucleotides 48-56 with nucleotides 69-61 respectively;

a T-arm can be formed by base pairing between nucleotides 70-72 with nucleotides 82-80 respectively;

or a combination thereof.

8/4 tRNASer (bacteria) (Figure 6D)

GNNNNRYNANNNNNNNNGGYNNNNNNNNNNGNYYNN2VAANCNNNNNNNNNNNNNNN NNNNNNNNNNNGNUCRANNCNNNNNYNNNNCGCCA (SEQ ID NO:21)

or a combination thereof.

ii. Exemplary allo-tRNA

The following table provides exemplary allo-tRNA sequences.

Results show that SEQ ID NOS:24-30 can be charged with serine by SerRS. SEQ ID NO:27 cannot be charged with serine, however, its discriminator

42

45292464vl base can be substituted to a nucleotide or nucleotides that are recognized by

Exemplary Allo-tRNA

45292464vl 43 UAG CUA (9/3) G 3300004 Wetland microbial GGAGGGTGGT

074.u:Ga communities from the CGCTGTTGGT

0055518 San Francisco Bay, GCAGCGGGCG

_301460 California, USA, that GGCC TAGAAC

185 impact long-term CCGCTGGAGC carbon sequestration - CTCACCGGGC

White_ThreeSqA_Dl TAAGGTTCGA

TTCCTCCACC

CTCCGCCA

(SEQ ID

NO:24)

CAG CUG (9/3) G 3300002 Forest soil microbial GGAGAGGGCA

954.u:JG communities from AGAGTGACGG

120281J4 Harvard Forest TTCACTCACC

4786_31 LTER, USA - PH CGTCTCAGAA

0361524 H12_0 (Forest soil ACGGGTAACG microbial TCTATCCGGG communities from CGTTGGGTTC

Harvard Forest AATTCCCGCC

LTER, USA - PH CTCTCCG

H12_0, (SEQ ID

ASSEMBLY_DATE NO:25)

=20140709)

UUA UAA (9/3) G 3300002 Switchgrass GGGGTGGGGT

459.u:JG rhizosphere microbial TCCGGCTGGT

124751J2 communities from GCCGGTCGCG

9686_33 Kellogg Biological GGCT TTAAAC

7535576 Station, Michigan, CCGTCAGGAC

USA - S6 (KBS GCTGCGACGC

Switchgrass S6, GTAAGGTTCG

ASSEMBLY_DATE ATTCCTCCCC

=20140130) ACTCCG (SEQ

ID NO:26)

45292464vl 44 UUA UAA (9/3) A 3300000 Soil microbial GGGCGGGGGT

000.u:GP communities from TCCGTCTGGT

CYDRA Great Prairies - GACGGTCGCG

FT_c328 Wisconsin Native GGCT2TAAAC

587791 Prairie soil CCGTCAGGAC

GCTGTGCAGG

CGTTAGGTTC

GATTCCTCCC

CCGTCCA

(SEQ ID

NO:27)

UAA UUA (9/3) G 3300002 Oil polluted marine GGAGGGGAAC

225.u:JG microbial TTCTATCTGG

I24723J2 communities from TGATAGACGG

6617_31 Coal Oil Point, Santa GAACT TAAAA

3779256 Barbara, California, TTCCTTGAAA

USA - Santa Barbara TGCCTCGCCG

Oil Seep Sample 6 CATTGGGTTC

(Crude oil GATTCCCTTC metagenome 6, CCCTCCGCCA

ASSEMBLY_DATE (SEQ ID

=20131204) NO:28)

45292464vl 45 CAA UUG (9/3) G 3300003 Arabidopsis thaliana GGAGGGCGGC

396.u:JG rhizosphere microbial TGCTGCTGGT

I26137J5 communities from the GCAGCGGGTG

0245_30 Joint Genome GACTCAAAAT

9810974 Institute, USA, that CCACTGGAGC affect carbon cycling CTGTCGGGGC

- Inoculated plant M3 TAGGGTTCGA

PM (Arabidopsis TTCCCCCGCC thaliana rhizosphere CTCCG (SEQ microbial ID NO:29) communities from the

Joint Genome

Institute, USA, that

affect carbon cycling

- Inoculated plant M3

PM,

ASSEMBLY_DATE

=20140903)

CAA UUG (9/3) G 3300003 Bog forest soil GGAGAGTAGA

218.u:JG microbial TTTCATGCGG

I26339J4 communities from TTATGAAATG

6600_30 Calvert Island, CGTCTCAAAA

1272239 British Columbia, ACGCAGAGGG

Canada - GGCTACACAC

ECP12_OMl (Bog CCCCAGGGTT

Forest metaG CAACTCCCCT

ECP120M1 , ACTCTCCG

AS S EMB LY_D ATE (SEQ ID

=20140815) NO:30)

45292464vl 46 Preferred non-naturally occurring allo-tRNA^Sec include

>allo-tRNA^UTu (also referred to as "2225")

GGAGGGGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCTC GCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:31)

GGAGGGGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO:57, RNA) and variants thereof.

Other allo-tRNA^Sec include, for example,

>allo-tRNA^UTu (Ac-3U variant)

GGAGGTTGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCT CGCCGCATTGGGTTCGATTCCCTTCTCCTCCGCCA (SEQ ID NO:32, DNA) GGAGGUUGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCU CGCCGCAUUGGGUUCGAUUCCCUUCUCCUCCGCCA (SEQ ID NO: 137, RNA)

>allo-tRNA^UTu (Ac-bU variant)

GGAGGTGGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCT CGCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:33, DNA) GGAGGUGGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCU CGCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO: 138, RNA)

>allo-tRNA^UTu (D-3b variant) (also referred to as allo- tRNA^UTulD)

GGAGGGGAACTTCTGTCTGGTGGCAGACGGGAACTCrAAATTCCTTGAAATGCCTC GCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:34, DNA) GGAGGGGAACUUCUGUCUGGUGGCAGACGGGAACUCUAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO: 139, RNA)

>allo-tRNA^UTu2

GGACGGGGGTTCCGTCTGGTGACGGTCGCGGGCTCTAAACCCGTCAGGACGCTGTG CAGGCGTTAGGTTCGATTCCTCCCCCGTCCGCCA (SEQ ID NO:35, DNA) GGACGGGGGUUCCGUCUGGUGACGGUCGCGGGCUCUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCGCCA (SEQ ID NO:58, RNA)

>allo-tRNA^UTu2 (G21 variant) (also referred to as allo- tRNA^UTu2D)

GGACGGGGGTTCCGTCTGGTGGCGGTCGCGGGCTCTAAACCCGTCAGGACGCTGTG CAGGCGTTAGGTTCGATTCCTCCCCCGTCCGCCA (SEQ ID NO:36, DNA)

45292464vl 47 GGACGGGGGUUCCGUCUGGUGGCGGUCGCGGGCUCUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCGCCA (SEQ ID NO: 140, RNA)

>2459

GGAGTGGGGTTCCGGCTGGTGCCGGTCGCGGGCTCTAAACCCGTCAGGACGCTGCG ACGCGTAAGGTTCGATTCCTCCCCACTCCGCCA (SEQ ID NO:37, DNA) GGAGUGGGGUUCCGGCUGGUGCCGGUCGCGGGCUCUAAACCCGUCAGGACGCUGCG ACGCGUAAGGUUCGAUUCCUCCCCACUCCGCCA (SEQ ID NO:141, RNA)

>S15 UU variant

GGAGGGCATTTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:38, DNA) GGAGGGCAUUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 142, RNA)

>S15 CU variant

GGAGGGCACTTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:39, DNA) GGAGGGCACUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 143, RNA)

>S15 UC variant

GGAGGGCATCTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:40, DNA) GGAGGGCAUCUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 144, RNA)

>S15 AA variant

GGAGGGCAAATTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:41, DNA) GGAGGGCAAAUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 145, RNA)

>S15 AU variant

GGAGGGCAATTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:42, DNA) GGAGGGCAAUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 146, RNA)

45292464vl 48 In some embodiments, the non-naturally occurring allo-tRNA^Sec is a variant of allo-tRNA^UTu encoded by a sequence at least 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:31.

In some embodiments, the variant has a sequence or is encoded by a sequence with at least 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NO:22-42, 57, 58, 137-146.

In some embodiments, the variant includes, for example,

transplanting one or more features of Aeromonas tRNA^Sec such as a bulged pyrimidine at position 5 or 5a in the 7-bp acceptor stem, U14:G21 wobble base pair in the D-stem of As tRNA^Sec or a combination thereof to a disclosed tRNA^Sec. Thus mutations are designed to improve binding to Aeromonas SelA.

e. 8/4 SelC* tRNA (Figure 6C)

The Examples below also describe the identification of SelC* tRNAs which were named after the selC gene, which encodes tRNA^Sec in E. coli. SelC* tRNA^Cys isoacceptors have an U73 discriminator base and cysteine GCA or opal UCA anticodons. U73 and GCA are the most important identity elements for CysRS, and certain CysRS forms are known to cysteinylate tRNA^CysucA.

A consensus sequence for SelC* tRNA^Cys is

RGGGGCAAYGGYGCUGGGCRCCCCNYGGNCUKCAAANCCRYNGGCYYNGYCUNNNN ARCNRGGAGGAGGUUCGAUUCCCCUUGCCCCYUCCA (SEQ ID NO:43)

Typically, an Acceptor Stem can be formed by base pairing between nucleotides 1-8 with nucleotides 91-84 respectively;

a D-arm can be formed by base pairing between nucleotides 12-16 with nucleotides 25-21 respectively;

an anti-codon arm can be formed by base pairing between nucleotides 27-32 with nucleotides 46-45 and 43-40 respectively;

a V-arm can be formed by base pairing between nucleotides 48-53 with nucleotides 66-61 respectively;

45292464vl 49 a T-arm can be formed by base pairing between nucleotides 68-69 and 71-72 with nucleotides 83-80 respectively;

or a combination thereof.

f. Other tRNA Consensus Sequences

8/4 tRNAHis (alpha-proteobacteria) (Figure 6E)

NCYRRNNANGNUGUAANGGUNGCAYNYNNNRYUGtJGANYNNNNNGGAYNRGGUUCR RNYCCYNUNNYYRGNACCA (SEQ ID NO:44)

Typically, an Acceptor Stem can be formed by base pairing between nucleotides 1-8 with nucleotides 71-64 respectively;

a D-arm can be formed by base pairing between nucleotides 11-14 with nucleotides 25-22 respectively;

an anti-codon arm can be formed by base pairing between nucleotides 27-31 with nucleotides 43-39 respectively;

a T-arm can be formed by base pairing between nucleotides 49-52 with nucleotides 63-60 respectively;

or a combination thereof.

8/4 tRNASec (bacteria) (Figure 6F)

GGRANNNNNNNNGNYCYGGUGRNCNNNNCGGNCUIVCAAANCCGNNUNNNNNNNNNN NNNNNNNNNNNNNNGGYGGUUCGAYUCCYCCNNNUYCCGCCA (SEQ ID NO:45) Typically, an Acceptor Stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-94 respectively;

a V-arm can be formed by base pairing between nucleotides 47-56 with nucleotides 70-61 respectively;

a T-arm can be formed by base pairing between nucleotides 71-72 and 74-75 with nucleotides 86-83 respectively;

or a combination thereof.

g. Variants and Modifications

Any of the disclosed tRNA can be further modified. Modifications can include single and combined exchanges (i.e., substitutions), one or more

45292464vl 50 insertions, one or more deletions, and combinations thereof, of nucleotides in various regions of the tRNA. In some embodiments the modifications alter the variant relative to tRNA^UTu to (i) more closely resemble the features of tRNA^Sec that contribute to binding of SelA than tRNA^UTu. The mechanism by which SelA discriminates between tRNA^Ser and tRNA^Sec is described in Itoh, et al., Science, 340:75-78 (2013). In some embodiments, the important tRNA^UTu features that (ii) provide thermodynamic binding specificity for EF- Tu [Schrader, et al., /. Mol. Biol. , 386: 1255-1264 (2009)], (iii) contribute to reducing the incompatibility between tRNA^Sec and EF-Tu [Rudinger, et al., EMBO J. , 15:650-657 (1996)], or the combination thereof are left intact. In some embodiments, the variant exhibits (i), (ii), (iii), or a combination thereof, most preferably (i), (ii), and (iii).

In some embodiments, the tRNA exhibits reduced misincorporation Ser in vivo, in vitro, or a combination thereof relative another tRNA^Sec. In some embodiments, the tRNA exhibits better interaction with SelA (e.g., tighter binding), while retaining robust Ser-tRNA formation by SerRS.

Nucleotide positions within a tRNA sequence can also be identified according to the primary sequence or based the nucleotide numbering established in Sprinzl, et al., Nucleic Acids Research, 26(1): 148-153 (1998). As illustrated in text, figures, and sequences provide herein, this numbering system coordinates the relative locations of nucleotides and base pairs between two or more tRNA that may differ in the total number of nucleotides due to insertions and/or deletions. Thus nucleotides in any of the disclosed tRNA can be characterized based the nucleotide numbering from the terminal 5' nucleotide, or the nucleotide(s) at the base position(s) identified according to the Sprinzl numbering system.

tRNA elements that can be important for selenocysteine insertion, are illustrated with reference tRNA^UTu in Figure 3B. In this figure, the acceptor stem of the tRNA is highlighted as originating from tRNA^Sec, and is important for recognition by the enzyme SelA. The circled region in Figure 3 originates from tRNA^Ser, and is important both for recognition by the enzyme EF-Tu and for its lack of recognition by the enzyme SelB. Thus in some

45292464vl 51 embodiments, the tRNA (e.g., the aminoacylated tRNA) is recognized by SelA and EF-Tu, and optionally is not recognized by selB.

Some of the tRNA disclosed herein feature an anticodon that recognize a codon encoding an amino acid, some feature an anticodon that recognizes a stop codon, and some feature an "NNN" anticodon. The anticodon in any of the disclosed sequences can be substituted with any other anticodon. Anticodons are typically the reverse complement of the codon. Codons are illustrated in Table 2. Thus, each of the disclosed tRNAs are expressly disclosed having every anticodon, preferably an anticodon that recognizes a stop codon. In some embodiments, the anticodon-codon interaction includes basepairing of one or more unnatural nucleobases. Thus, in some embodiments, the anticodon includes one or more unnatural bases. Table 2: Universal Genetic Code Chart: Messenger RNA Codons and Amino Acids for Which They Code.

A non-naturally occurring tRNA^Sec tRNA can have a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any one or more of SEQ ID NOS:l-63 or 137-158. In some embodiments, the non-

45292464vl 52 naturally occurring tRNA^Sec is characterized by one or more of the following elements: (1) the non-naturally occurring tRNA^Sec can be recognized by SerRS and by EF-Tu, or variants thereof; (2) when aminoacylated with serine the non-naturally occurring Ser-tRNA^Sec can be converted to non- naturally occurring Sec-tRNA^Sec by SelA or variant thereof; (3) when aminoacylated with serine the non-naturally occurring Ser-tRNA^Sec can be phosphorylated by PSTK or variant thereof; (4) when aminoacylated with phosphorylated serine the non-naturally occurring Sep-tRNA^Sec can serve as a substrate for SepSecS or variant thereof.

6. Secondary Structure

The tRNAs disclosed herein typically include an acceptor arm, a D- arm, an anticodon arm, a variable arm, and a T^FC-arm, as described in more detail below.

a. Acceptor Arm

The non-naturally occurring tRNA^Sec disclosed herein includes an acceptor arm. The acceptor arm is the end of a tRNA molecule to which an amino acid becomes bound. It contains both the 5' and 3' ends of the tRNA. The 3'-terminal sequence of cytidine-cytidine-adenosine (CCA) overhangs the end, and the terminal A is the site of 'acceptance' of the amino acid.

The acceptor stem refers to the 5' and 3' sequences to the acceptor arm that form duplex RNA. The acceptor stem can be separate from the CCA overhang by one or more nucleotides, for example one or more guanine. In some embodiments, one or more nucleotides that separate the acceptor stem and the overhang are referred to as the discriminator base(s). For some tRNAs, the discriminator base preceding the CCA sequence at the 3' end is important for aminoacylation. The discriminator base can influence the stability of the base pair of the acceptor arm onto which it is stacked which can affect the energetic cost of opening the base pair and modulate the structure of the tRNA near the site of aminoacylation. For some aminoacyl- tRNA synthetases and other proteins that interact with tRNA, these factors could be important for specific recognition and/or formation of the transition state during catalysis (Lee et al., PNAS, 90(15):7149-52 (1993)). In some

45292464vl 53 embodiments, the acceptor stem and the CCA sequence are separated by a single guanine discriminator base.

The acceptor stem of the non-naturally occurring tRNA^Sec disclosed herein typically include 4 to 12, preferably 5 to 11, more preferably 6 to 10, most preferably 7 to 9 base pairs of duplex RNA. In some embodiments, the acceptor stem is 7, 8, or 9 base pairs of duplex RNA.

The acceptor stem can be high in G-C content. For example, in some embodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the acceptor stem. In some embodiments the G-C is lower, for example, 10%, 20%, 30%, or 40%. In some embodiments, the G-C content is between about 30% and 40%.

The 5 ' and 3 ' sequences of the tRNA that form the acceptor stem typically form a RNA duplex by Waston-Crick base pairing. The 5' and 3' sequences of the tRNA that form the acceptor stem are typically substantially complementary. Preferably, the 5' and 3' sequences of the tRNA that form the acceptor stem bind to or hybridize to each other under conditions of high stringency and specificity. In some embodiments, 5' sequence of the tRNA that forms the acceptor stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3' sequence of the tRNA that forms the acceptor stem. In some embodiments the 5' and 3' sequences of the tRNA that form the acceptor stem are 100% complementary.

b. D-arm

The non-naturally occurring tRNA^Sec disclosed herein include a D- arm. The D-arm is typically composed of a D stem of duplex RNA and a D loop of non-duplex RNA. The D stem refers to the two segments of the tRNA primary sequence in the D-arm that form duplex RNA. The D stem of the non-naturally occurring tRNA^Sec typically include 2 to 8, preferably 3 to 7, more preferably 4 to 6, base pairs of duplex RNA. In some embodiments, the D stem is 4, 5, or 6 base pairs of duplex RNA.

The D stem can be high in G-C content. For example, in some embodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the D stem.

45292464vl 54 The two segments of the tRNA that form the D stem typically form a RNA duplex by Waston-Crick base pairing. The two segments of the tRNA that form the D stem are typically substantially complementary. Preferably, the 5 ' and 3 ' sequences of the tRNA that form the acceptor stem bind to or hybridize to each other under conditions of high stringency and specificity. In some embodiments, 5' segment of the tRNA that forms the D stem is between 25% and 50% complementary to the 3' segment of the tRNA that forms the D stem. In some embodiments the 5' segment of the tRNA that forms the D stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3' sequence of the tRNA that forms the D stem. In some embodiments the 5' and 3' sequences of the tRNA that form the D stem are 100% complementary.

The D loop refers to the part of the D-arm that does not form duplex RNA. The D loop's main function is that of recognition. The D loop can contain the base dihydrouracil. It is widely believed that it will act as a recognition site for aminoacyl-tRNA synthetase, which is an enzyme involved in the aminoacylation of the tRNA molecule. The D-loop can have between 3 and 15 nucleotides inclusive, preferably between 4 and 12 nucleotides inclusive. In some embodiments the D-loop has 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides.

c. Anticodon Arm

The non-naturally occurring tRNA^Sec disclosed herein include an anticodon arm. The anticodon arm is typically composed of an anticodon stem of duplex RNA and an anticodon loop of non-duplex RNA. The anticodon stem refers to the two segments of the tRNA primary sequence in the anticodon arm that form duplex RNA. The anticodon stem of the non- naturally occurring tRNA^Sec disclosed herein typically include 2 to 8, preferably 3 to 7, more preferably 4 to 6, base pairs of duplex RNA. In some embodiments, the anticodon stem is 4, 5, or 6 base pairs of duplex RNA.

The anticodon stem can be high in G-C content. For example, in some embodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the anticodon stem.

45292464vl 55 The two segments of the tRNA that form the anticodon stem typically form a RNA duplex by Waston-Crick base pairing. The two segments of the tRNA that form the anticodon stem are typically substantially

complementary. Preferably, the 5' and 3' sequences of the tRNA that form the anticodon stem bind to or hybridize to each other under conditions of high stringency and specificity. In some embodiments the 5 ' segment of the tRNA that forms the anticodon stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3' sequence of the tRNA that forms the anticodon stem. In some embodiments the 5' and 3' sequences of the tRNA that form the anticodon stem are 100% complementary.

The anticodon loop refers to the part of the anticodon -arm that does not form duplex RNA. The anticodon loop's main function is to present the anticodon sequence which can hybridize to the target codon in the mRNA sequence of interest. The anticodon sequence can be any three nucleotide sequence that binds by complementary base pairing to the target codon sequence in the mRNA of interest. In some embodiments, the anticodon pairs specifically with only one codon. Some anticodon sequences can pair with more than one codon (i.e., wobble base pairing). In some embodiments, the first nucleotide of the anticodon is inosine or pseudouridine, which can hydrogen bond to more than one base in the corresponding codon position.

In some embodiments, the anticodon hybridizes to a "stop" codon such as UAA, UAG, or UGA, preferably UAG (amber) or UGA (opal). Accordingly, in some embodiments the sequence of the anticodon is UUA, CUA, UCA, preferably CUA (amber) or UCA (opal) (in the 5' to 3' direction). The anticodon loop can have between 5 and 11 nucleotides inclusive, preferably about 7 nucleotides. In some embodiments the anticodon-loop has 5, 7, or 9 nucleotides. Typically, the three nucleotide anticodon sequence is flanked by an equal number of nucleotides both 5 ' and 3' of the anticodon sequence within the anticodon loop.

Although in some embodiments, the anticodon is one that recognizes a stop codon, all other possible anticodons (e.g., those that recognize an amino acid codon) are also specifically disclosed for all tRNA disclosed herein. Thus, for example, in some embodiments, a non-naturally occurring

45292464vl 56 tRNA includes the sequence of any one of SEQ ID NO: 1-63, or a variant there with at least 80% sequence identity, wherein the anti-codon is substituted with an alternative anti-codon. In addition of the standard A, C, G, U bases the anticodon and/or the corresponding codon of the mRNA of interest may also contain unnatural nucleotide bases. Suitable basepairing to create additional codon-anticodon interaction is described in, for example, Bain, et al., Nature, 356:537-539 (1992), and Malyshev, et al., Nature, 509:385-388 (2014), and supplemental information associated therewith, and include, but are not limited to d5SICS and dNaM(d5SICS-dNaM).

d. Variable arm

The non-naturally occurring tRNA^Sec disclosed herein typically include a variable arm. The variable arm is typically composed of a variable stem of duplex RNA and a variable loop of non-duplex RNA. The variable stem refers to the two segments of the tRNA primary sequence in the variable arm that form duplex RNA. The variable stem of the non-naturally occurring tRNA^Sec typically includes 2 to 8, preferably 3 to 7, more preferably 4 to 6, base pairs of duplex RNA. In some embodiments, the variable stem is 4, 5, or 6 base pairs of duplex RNA. In some embodiments the variable stem has 9, 10, 11, or more base pairs of duplex RNA.

The variable stem can be high in G-C content. For example, in some embodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the variable stem.

The two segments of the tRNA that form the variable stem typically form a RNA duplex by Waston-Crick base pairing. The two segments of the tRNA that form the anticodon stem are typically substantially

complementary. Preferably, the 5' and 3' sequences of the tRNA that form the variable stem bind to or hybridize to each other under conditions of high stringency and specificity. In some embodiments the 5' segment of the tRNA that forms the variable stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3' sequence of the tRNA that forms the variable stem. In some embodiments the 5' and 3' sequences of the tRNA that form the variable stem are 100% complementary.

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45292464vl The variable loop refers to the part of the variable -arm that does not form duplex RNA. The variable loop can have between 3 and 7 nucleotides inclusive, preferably between 4 and 6 nucleotides inclusive. In some embodiments the variable loop has 3, 4, 5, 6, or 7 nucleotides.

The non-naturally occurring tRNA^Sec disclosed herein includes a TFC-arm (also referred to herein as a T-arm). The T-arm is the region on the tRNA molecule that acts as a recognition site for the ribosome, and allows a tRNA-ribosome complex to form during the process of protein biosynthesis. The T-arm is typically composed of a T stem of duplex RNA and a T loop of non-duplex RNA. The T stem refers to the two segments of the tRNA primary sequence in the T-arm that form duplex RNA. The T stem of the non-naturally occurring tRNA^Sec typically includes 2 to 8, preferably 3 to 7, more preferably 4 to 6, base pairs of duplex RNA. In some

embodiments, the T stem is 3, 4, or 5 base pairs of duplex RNA.

The T stem can be high in G-C content. For example, in some embodiments, the G-C content is 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides of the T stem.

The two segments of the tRNA that form the T stem typically form a RNA duplex by Waston-Crick base pairing. The two segments of the tRNA that form the T stem are typically substantially complementary. Preferably, the 5 ' and 3 ' sequences of the tRNA that form the acceptor stem bind to or hybridize to each other under conditions of high stringency and specificity. In some embodiments, 5' segment of the tRNA that forms the T stem is equal to or greater than 50% complementary to the 3 ' segment of the tRNA that forms the T stem. In some embodiments the 5' segment of the tRNA that forms the T stem is 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more complementary to the 3 ' sequence of the tRNA that forms the T stem. In some embodiments the 5' and 3' sequences of the tRNA that form the T stem are 100% complementary.

The T loop refers to the part of the T-arm that does not form duplex

RNA. In some embodiments the T-loop includes thymidine, pseudouridine, residues, or combinations thereof. The T-loop can have between 3 and 15

45292464vl 58 nucleotides inclusive, preferably between 4 and 12 nucleotides inclusive. In some embodiments the D-loop has 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides,

f. Linker nucleotides

The five arms of the tRNA can be linked directly, or can be separated by one or more linker or spacer nucleotides to ensure the tRNA assumes the proper secondary structure. For example, the acceptor arm and the D-arm can separated by 0, 1, 2, 3, or more nucleotides; the D-arm and the anticodon arm can be separated by 0, 1, 2, 3, or more nucleotides; the anticodon arm and the variable arm can be separated by 0, 1, 2, 3, or more nucleotides; the variable arm and the T-arm can be separated by 0, 1, 2, 3, or more nucleotides ; and the T-arm and the acceptor arm can be separated by 0, 1, 2, 3, or more nucleotides.

B. mRNA and Polypeptides of Interest

As discussed in more detail below, the tRNA^Sec disclosed herein can be used in combination with an mRNA to manufacture selenocysteine containing polypeptides and proteins. The mRNA does not require, and preferably does not include, a SECIS element. The mRNA, which encodes a polypeptide of interest, includes one or more codons that is recognized by the anticodon of the Sec-tRNA^Sec, referred to herein as an "tRNA^Sec recognition codon," such that tRNA catalyzes the attachment of a selenocysteine amino acid to the growing polypeptide chain during translation.

For example, if the tRNA^Sec recognition codon is a stop codon, such as UGA, the mRNA will contain at least one UGA codon where a selenocysteine will be added to the growing polypeptide chain during translation. The tRNA^Sec recognition codon can be added to or inserted into any mRNA to add a codon encoding selenocysteine at any desired location in the amino acid sequence. The tRNA^Sec recognition codon can be substituted for any existing codon in the mRNA sequence so that any one or more amino acids from a reference polypeptide sequence is substituted with

selenocysteine during translation. For example, as discussed in more detail below, in some embodiments, one or more codons encoding cysteine in a reference sequence are substituted with a tRNA^Sec recognition sequence so

45292464vl 59 that the one or more cysteines are replaced with selenocysteine during translation.

Various types of mutagenesis can be used to modify the sequence of a nucleic acid encoding the mRNA of interest to generate the tRNA^Sec recognition codon. They include but are not limited to site-directed, random point mutagenesis, homologous recombination (DNA shuffling), mutagenesis using uracil containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, and mutagenesis using gapped duplex DNA or the like. Additional suitable methods include point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis and double-strand break repair.

In some embodiments, the coding sequence, excluding the tRNA^Sec recognition site as discussed above, is further altered for optimal expression (also referred to herein as "codon optimized") in an expression system of interest. Methods for modifying coding sequences to achieve optimal expression are known in the art.

C. Isolated Nucleic Acid Molecules

tRNA^Sec and nucleic acids encoding tRNA^Sec are disclosed. Also disclosed are mRNAs, cDNAs and other nucleic acids encoding proteins of interest that are engineered such that a tRNA^Sec , such as the tRNA^Sec disclosed herein, "reads" at least one codon of the mRNA during translation of the protein encoded by the mRNA. As used herein, "isolated nucleic acid" refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a genome, including nucleic acids that normally flank one or both sides of the nucleic acid in the genome. The term

"isolated" as used herein with respect to nucleic acids also includes the combination with any non-naturally-occurring nucleic acid sequence, since such non-naturally-occurring sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule or an

RNA molecule, provided one of the nucleic acid sequences normally found

45292464vl 60 immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a DNA molecule or RNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA, or RNA, or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule or RNA molecule that is part of a hybrid or fusion nucleic acid. A nucleic acid existing among hundreds to millions of other nucleic acids within, for example, a cDNA library or a genomic library, or a gel slice containing a genomic DNA restriction digest, is not to be considered an isolated nucleic acid.

Nucleic acids encoding the tRNA^Sec and mRNA disclosed herein may be optimized for expression in the expression host of choice. In the case of nucleic acids encoding expressed polypeptides, codons may be substituted with alternative codons encoding the same amino acid to account for differences in codon usage between the organism from which the nucleic acid sequence is derived and the expression host. In this manner, the nucleic acids may be synthesized using expression host-preferred codons.

Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence, for example, a sequence encoding the disclosed tRNA^Sec and mRNA. Nucleic acids can be DNA, RNA, nucleic acid analogs, or combinations thereof. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone. Such modification can improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine or 5- bromo-2'-deoxycytidine for deoxycytidine. Modifications of the sugar moiety can include modification of the 2' hydroxyl of the ribose sugar to form 2'-0-methyl or 2'-0-allyl sugars. The deoxyribose phosphate

45292464vl 61 backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7: 187- 195; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a

phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

D. Methods for producing isolated nucleic acid molecules

Isolated nucleic acid molecules can be produced by standard techniques, including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid encoding a non-naturally occurring tRNA^Sec. PCR is a technique in which target nucleic acids are enzymatically amplified. Typically, sequence information from the ends of the region of interest or beyond can be employed to design oligonucleotide primers that are identical in sequence to opposite strands of the template to be amplified. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Primers typically are 14 to 40 nucleotides in length, but can range from 10 nucleotides to hundreds of nucleotides in length. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, ed. by Dieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.

When using RNA as a source of template, reverse transcriptase can be used to synthesize a complementary DNA (cDNA) strand. Ligase chain reaction, strand displacement amplification, self-sustained sequence replication or nucleic acid sequence-based amplification also can be used to obtain isolated nucleic acids. See, for example, Lewis (1992) Genetic Engineering News 12: 1; Guatelli et al. (1990) Proc. Natl. Acad. Set USA 87: 1874-1878; and Weiss (1991) Science 254: 1292-1293.

62

45292464vl Isolated nucleic acids can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides (e.g., using phosphoramidite technology for automated DNA synthesis in the 3' to 5' direction). For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase can be used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per

oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids can also obtained by mutagenesis. Nucleic acids can be mutated using standard techniques, including oligonucleotide-directed mutagenesis and/or site-directed mutagenesis through PCR. See, Short Protocols in Molecular Biology. Chapter 8, Green Publishing Associates and John Wiley & Sons, edited by Ausubel et al, 1992. Examples of nucleic acid amino acid positions relative to a reference sequence that can be modified include those described herein.

E. Vectors and host cells

Vectors encoding tRNA^Sec and polypeptides manufactured using the tRNA^Sec as well as other compontents of the translation system including but not limited to SerRS, EF-Tu, SelA, SelD, PSTK, and SepSecS are also provided. Nucleic acids, such as those described above, can be inserted into vectors for expression in cells. As used herein, a "vector" is a replicon, such as a plasmid, phage, virus or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.

Vectors can be expression vectors. An "expression vector" is a vector that includes one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.

Nucleic acids in vectors can be operably linked to one or more expression control sequences. Operably linked means the disclosed sequences are incorporated into a genetic construct so that expression control sequences effectively control expression of a sequence of interest. Examples

45292464vl 63 of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II).

A "promoter" as used herein is a DNA regulatory region capable of initiating transcription of a gene of interest. Some promoters are

"constitutive," and direct transcription in the absence of regulatory influences. Some promoters are "tissue specific," and initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are "inducible," and achieve gene transcription under the influence of an inducer. Induction can occur, e.g., as the result of a physiologic response, a response to outside signals, or as the result of artificial manipulation. Some promoters respond to the presence of tetracycline; "rtTA" is a reverse tetracycline controlled transactivator. Such promoters are well known to those of skill in the art.

To bring a coding sequence under the control of a promoter, it is advantageous to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is "operably linked" and "under the control" of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

Likewise, although tRNA^Sec sequences do not encode a protein, control sequence can be operably linked to a sequence encoding a tRNA^Sec, to control expression of the tRNA^Sec in a host cell. Methods of recombinant expression of tRNA from vectors is known in the art, see for example, Ponchon and Dardel, Nature Methods, 4(7):571-6 (2007); Masson and

64

45292464vl Miller, J.H., Gene, 47: 179-183 (1986); Meinnel, et al., Nucleic Acids Res., 16:8095-6 (1988); Tisne, et al., RNA, 6: 1403-1412 (2000).

F. Host Cells

Host cell including the nucleic acids disclosed herein are also provided. Prokaryotes useful as host cells include, but are not limited to, gram negative or gram positive organisms such as E. coli or Bacilli. In a prokaryotic host cell, a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide. Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include lactamase and the lactose promoter system.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. To construct an expression vector using pBR322, an appropriate promoter and a DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, T7 expression vectors from Invitrogen, pET vectors from Novagen and pALTER® vectors and PinPoint® vectors from Promega Corporation.

In a prokaryotic host cell, a polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed recombinant polypeptide. Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include lactamase and the lactose promoter system.

In some embodiments, the host cells are E. coli. The E. coli strain can be a selA, selB, selC, deletion strain, or combinations thereof. For

45292464vl 65 example, the E. coli can be a selA, selB, and selC deletion strain, or a selB and selC deletion strain. Examples of suitable E. coli strains include, but are not limited to, MH5 and ME6.

Yeasts useful as host cells include, but are not limited to, those from the genus Saccharomyces, Pichia, K. Actinomycetes and Kluyveromyces. Yeast vectors will often contain an origin of replication sequence, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, (1980)) or other glycolytic enzymes (Holland et al., Biochem. 17:4900, (1978)) such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,

phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Fleer et al., Gene, 107:285-195 (1991), in Li, et al., Lett Appl Microbiol. 40(5):347-52 (2005), Jansen, et al., Gene 344:43-51 (2005) and Daly and Hearn, /. Mol. Recognit. 18(2): 119-38 (2005). A yeast promoter is, for example, the ADH1 promoter (Ruohonen, et al., J Biotechnol. 1995 May 1;39(3): 193-203), or a constitutively active version thereof (e.g., the first 700bp). Some embodiments include a terminator, such as the rpl41b terminator resulted in the highest GFP expression out of over 5300 yeast promoters tested (Yamaishi, et al., ACS Synth. Biol. , 2013, 2 (6), pp 337-347). Other suitable promoters, terminators, and vectors for yeast and yeast transformation protocols are well known in the art.

In some embodiments, the host cells are eukaryotic cells. For example, mammalian and insect host cell culture systems well known in the art can also be employed to express non-naturally occurring tRNA^Sec and mRNA for producing proteins or polypeptides containing selenocysteine.

Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human

45292464vl 66 cytomegalovirus. DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell, e.g., SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication. Exemplary expression vectors for use in mammalian host cells are well known in the art.

Mammalian or insect host cell culture systems well known in the art can also be employed to express ribosomes (or a ribosomal rRNA thereof), tRNAs, synthetases or a combination thereof for producing proteins or polypeptides containing one or more dipeptides, non-standard-, non-natural-, or non-oc-amino acids. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell, e.g., SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication. Exemplary expression vectors for use in mammalian host cells are well known in the art.

The host organism can be a genomically recoded organism "GRO." Typically, the GRO is a bacterial strain, for example, an E. coli bacterial strain, wherein a codon has been replaced by a synonymous codon. Because there are 64 possible 3-base codons, but only 20 canonical amino acids (plus stop codons), some amino acids are coded for by 2, 3, 4, or 6 different codons (referred to herein as "synonymous codons"). In a GRO, most or all of the iterations of a particular codon are replaced with a synonymous codon. The precursor strain of the GRO is recoded such that at a least one codon is completely absent from the genome. Removal of a codon from the precursor

GRO allows reintroduction of the deleted codon in, for example, a heterologous mRNA of interest. As discussed in more detail below, the

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45292464vl reintroduced codon is typically dedicated to a non-standard amino acid, which in the presence of the appropriate translation machinery, can be incorporated in the nascent peptide chain during translation of the mRNA.

Different organisms often show particular preferences for one of the several codons that encode the same amino acid, and some codons are considered rare or infrequent. Preferably, the replaced codon is one that is rare or infrequent in the genome. The replaced codon can be one that codes for an amino acid (i.e., a sense codon) or a translation termination codon (i.e., a stop codon). GRO that are suitable for use as host or parental strains for the disclosed systems and methods are known in the art, or can be constructed using known methods. See, for example, Isaacs, et al., Science, 333, 348-53 (2011), Lajoie, et al., Science 342, 357-60 (2013), Lajoie, et al., Science, 342, 361-363 (2013).

Preferably, the replaced codon is one that codes for a rare stop codon. In a particular embodiment, the GRO is one in which all instances of the UAG (TAG) codon have been removed and replaced by another stop codon (e.g., TAA, TGA), and preferably wherein release factor 1 (RF1; terminates translation at UAG and UAA) has also been deleted, eliminating translational termination at UAG codons (Lajoie, et al., Science 342, 357-60 (2013)). In a particular embodiment, the host or precursor GRO is C321.A A [321

UAG→UAA conversions and deletion of prfA (encodes RF1)] (genome sequence at GenBank accession CP006698). This GRO allows the reintroduction of UAG codons in a heterologous mRNA, along with orthogonal translation machinery (i.e., aminoacyl-tRNA synthetases (aaRSs) and tRNAs as discussed in more detail below), to permit efficient and site specific incorporation of non-standard amino acids into protein encoded by the recoded gene of interest. That is, UAG has been transformed from a nonsense codon (terminates translation) to a sense codon (incorporates amino acid of choice), provided the appropriate translation machinery is present. UAG is a preferred codon for recoding because it is the rarest codon in Escherichia coli MG1655 (321 known instances) and a rich collection of translation machinery capable of incorporating non-standard amino acids has

45292464vl 68 been developed for UAG (Liu and Schultz, Annu. Rev. Biochem. , 79:413-44 (2010)).

Stop codons include TAG (UAG), TAA (UAA), and TGA (UGA). Although recoding to UAG (TAG) is discussed in more detail above, it will be appreciated that either of the other stop codons (or any sense codon) can be recoded using the same strategy. Accordingly, in some embodiments, a sense codon is reassigned, e.g., AGG or AGA to CGG, CGA, CGC, or CGG (arginine), e.g., as the principles can be extended to any set of synonymous or even non-synonymous codons, that are coding or non-coding. Similarly, the cognate translation machinery can be removed/mutated/deleted to remove natural codon function (UAG - RF1, UGA - RF2). The orthogonal translation system, particularly the antisense codon of the tRNA, can be designed to match the reassigned codon.

GRO can have two, three, or more codons replaced with a synonymous or non-synonymous codon. Such GRO allow for reintroduction of the two, three, or more deleted codons in one or more recoded genes of interest, each dedicated to a different non-standard amino acid. Such GRO can be used in combination with the appropriate orthogonal translation machinery to produce polypeptides having two, three, or more different non- standard amino acids.

Another host cell system for the use of codons containing unnatural bases is E. coli expressing Phaeodactylum tricornutum nucleotide triphosphate transporters as reported (Malyshev, et al., Nature, 509:385-388 (2014)).

III. Methods for Manufacturing Proteins Containing Selenocysteine A. Expression of Selenocysteine Containing Polypeptides

Generally, the canonical amino acids are charged onto their respective tRNA by their cognate aminoacyl-tRNA synthetase. The aminoacyl-tRNA is then delivered by EF-Tu to the ribosome (Figure 1A). In contrast, the endogenous Sec pathway requires several biosynthetic steps.

First, tRNA^Sec is misacylated to Ser-tRNA^Sec by SerRS. While in bacteria

Ser-tRNA^Sec is directly converted by SelA to Sec-tRNA^Sec, archaea and eukaryotes employ an additional phosphorylation step by PSTK to form Sep-

45292464vl 69 tRNA^Sec, which is then converted by SepSecS to the final product Sec- tRNA^Sec Figure IB. Sec-tRNA^Sec is bound by elongation factor SelB and delivered to the ribosome. However, reassignment of the opal codon UGA to a Sec codon is only achieved if SelB also binds to the mRNA SECIS hairpin structure.

The compositions disclosed herein can be used to prepare polypeptides including one or more selenocysteine residues from mRNA that does not contain an SECIS element. The tRNA^Sec disclosed herein is recognized by SerRS and misacylated to form the intermediate Ser-tRNA^Sec. Next the Ser-tRNA^Sec is converted to Sec-tRNA^Sec by SelA in prokaryotic system or hybrid systems, or PSTK and SepSecS in archaeal, eukaryotic, or hybrid systems. Finally, the Sec-tRNA^Sec is delivered to the ribosome by EF-Tu, where the anticodon of the Sec-tRNA^Sec recognizes the codon engineered to encode a Sec amino acid, and transfers the Sec onto the growing polypeptide chain. Accordingly, the non- naturally occurring tRNA^Sec disclosed herein are typically recognized by SerRS, or a variant thereof, and when aminoacylated with serine the Ser-tRNA can (1) be a substrate for SelA or a variant thereof; or (2) be a substrate for PSTK and when aminoacylated with phosphorylated serine the Sep-tRNA can serve as a substrate for SepSecS or a variant thereof, and (3) when aminoacylated, the non-naturally occurring Sec-tRNA^Sec is recognized by EF-Tu.

As discussed in more detail below, recombinant proteins including selenocysteine can be prepared using in vitro transcription/translation or in vivo expression systems. The system can be of prokaryotic, eukaryotic, or archaeal origin or combinations thereof. For example, the system can be hybrid system including selenocysteine biogenesis and translation factors from prokaryotic, eukaryotic, archaeal origin, or combinations thereof.

In some embodiments, the system is an in vivo prokaryotic expression including an E. coli strain in which the endogenous genes encoding selB, selC, or selA, selB, selC are deleted or mutated to reduce or eliminate expression of endogenous SelA, SelB, SelC or combinations thereof. The selB, selC, or selA, selB, selC mutant strains can be engineered to express a non-naturally occurring tRNA^Sec, as well as a PSTK and a

45292464vl 70 SepSecS. In some embodiments recombinant SelA is expressed. The PSTK or SepSecS can of eukaryotic or archaeal origin, or a variant thereof. For example, in one embodiment, the PSTK is a M. maripaludis PSTK and the SepSecS is a M. jannaschii SepSecS.

In some embodiments, SelA, PSTK and SepSecS are all expressed in the expression system.

SelD refers to selenide, water dikinase, which synthesizes selenophosphate utilized by SelA from selenide and ATP.

An exemplary protein sequence for E. coli SelD is:

MSENSIRLTQYSHGAGCGCKISPKVLETILHSEQAKFVDPNLLVGNETRDDAAVYD LGNGTSVISTTDFFMPIVDNPFDFGRIAATNAISDIFAMGGKPIMAIAILGWPINK LSPEIAREVTEGGRYACRQAGIALAGGHSIDAPEPIFGLAVTGIVPTERVKKNSTA QAGCKLFLTKPLGIGVLTTAEKKSLLKPEHQGLATEVMCRMNIAGASFANIEGVKA MTDVTGFGLLGHLSEMCQGAGVQARVDYEAIPKLPGVEEYIKLGAVPGGTERNFAS YGHLMGEMPREVRDLLCDPQTSGGLLLAVMPEAENEVKATAAEFGIELTAIGELVP ARGGRAMVEIR (SEQ ID NO:86)

Other organisms also encode a SelD. Thus, in other embodiments, the SelD is not from E. coli. Thus, in these embodiments, an alternative SelD is utilized for recombinant selenoprotein preparation. Exemplary alternative SelD proteins include, but are not limited to, SelD from

Aeromonas salmonicida.

An amino acid sequence for Aeromonas salmonicida SelD is

MSSIRLTQYSHGAGCGCKISPKVLDTILKSQIPGFDDPTLWGNSSKDDAAWDIG NGQGIVSTTDFFMPIVDDPFTFGRIAATNAISDIYAMGGKPIVAIAILGWP INTLA PEVAQQVIDGGRQVCHEAGISLAGGHSIDAPEPIFGLAVTGIVPLNAIKQNDTAQA GDILYLTKPLGIGILTTAQKKGKLKPEHEQLAPNAMCTLNKIGQRFAELPGVHAMT DVTGFGLAGHLLEMCEGSGVCATLDFKALPLLDEVDYYLSEGCVPGGTLRNFDSYG AKLGAMDERTRNIMCDPQTSGGLLVAVGKESEAELLAIATQAGLTLSPIGQLKAYT

GNQFIEVIQ (SEQ ID NO:87)

A nucleic acid sequence encoding Aeromonas salmonicida SelD (cloned from the Aeromonas salmonicida genome. The AUG start codon was changed to GUG)

GTGTCTTCCATTCGTCTGACCCAATACAGCCACGGGGCTGGCTGCGGCTGCAAAAT TTCTCCCAAGGTGCTCGACACCATTCTCAAGAGCCAGATCCCGGGCTTTGACGACC

71

45292464vl CGACCCTGGTGGTTGGCAACAGCAGCAAGGATGACGCGGCCGTGGTCGATATCGGC AACGGTCAGGGCATTGTTTCCACCACCGACTTCTTCATGCCCATCGTCGATGATCC CTTTACCTTTGGCCGCATCGCGGCCACCAACGCCATCAGCGACATCTACGCCATGG GCGGCAAGCCCATCGTTGCCATTGCCATCCTTGGCTGGCCCATCAACACCCTAGCC CCGGAAGTGGCCCAGCAGGTGATAGATGGCGGCCGCCAGGTGTGCCATGAAGCGGG CATATCCTTGGCTGGCGGCCACAGTATCGATGCCCCCGAGCCCATCTTCGGTCTTG CTGTGACCGGTATAGTGCCGCTCAATGCCATCAAGCAGAACGACACGGCCCAGGCG GGTGACATCCTCTACCTGACCAAGCCCCTCGGTATCGGCATCCTCACCACGGCCCA GAAGAAGGGCAAATTGAAGCCAGAGCATGAGCAGCTGGCCCCCAACGCCATGTGCA CCCTCAACAAGATTGGCCAGCGCTTTGCCGAACTGCCCGGCGTGCACGCCATGACG GATGTGACCGGGTTTGGCCTGGCGGGACACCTGCTTGAGATGTGCGAAGGCTCAGG GGTGTGTGCCACCCTCGATTTCAAGGCGCTGCCACTGCTCGACGAAGTAGATTACT ACCTGTCCGAGGGCTGCGTACCGGGCGGTACCCTGCGCAACTTCGATTCCTATGGC GCCAAGCTCGGTGCCATGGATGAACGCACCCGCAACATCATGTGCGATCCGCAGAC CAGCGGCGGCTTGCTGGTTGCCGTCGGTAAAGAAAGTGAAGCCGAGCTCCTTGCTA TCGCGACACAAGCGGGGCTGACCCTCTCCCCCATAGGCCAGCTGAAAGCCTATACC GGAAACCAGTTTATCGAGGTTATCCAATGA (SEQ ID NO:88)

In some embodiments selenocysteine biogenesis and translation factors are mutated to improve their specificity or activity for tRNA^Sec. In the recombinant tRNA^Sec biosynthetic pathway disclosed herein tRNA^Sec is first misacylated to Ser-tRNA^Sec by SerRS, and subsequently converted to Sec-tRNA^Sec by SelA, or PSTK and SepSecS, or combinations thereof. Accordingly, if the SelA, or PSTK and SepSecS, enzymes are not 100% efficient at converting Ser-tRNA^Sec to Sec-tRNA^Sec, the system may incorporate Sec or Ser at the desired position. Additionally, in some embodiments, recognition of the non-naturally occurring Sec-tRNA^Sec by EF-Tu, is less efficient than EF-Tu recognition of other naturally occurring aminoacyl-tRNAs. Mutating the EF-Tu, SerRS, SelA, PSTK, SepSecS, or combinations thereof can improve the efficiency or recognition of the enzyme for the non-naturally occurring tRNA^Sec, the non-naturally occurring Sec-tRNA^Sec, or various intermediates thereof. In some embodiment, the EF-Tu, SerRS, SelA, PSTK, SepSecS, SelD or combinations thereof are variants of a naturally occurring protein.

45292464vl 72 In some embodiments, the variant mRNA can include or consist of replacing of the AUG start codon with GUG or UUG and optionally a UAAUU inserted in front of it. Replacing AUG with GUG or UUG can reduce the expression of the encoded protein. The corresponding DNA sequence encoding the variants are also expressly provided.

It is understood that if the tRNA^Sec recognition codon of the mRNA of interest is one of the three mRNA stop codons (UAG, UAA, or UGA) translation of some of the mRNA of interest will terminate at each of the tRNA^Sec recognition codons, resulting in a heterogeneous mixture of full- length and truncated proteins. The experimental results presented in the examples below show that allo-tRNA such as allo-tRNA^UTu insert a larger number of Sec amino acids into a nascent protein chain than other tRNA^Sec including, for example, tRNA^SecUx. Thus in some embodiments, an allo- tRNA^Sec can generate a higher yield (e.g., a higher amount) of the desired protein, particularly when the protein contains multiple Sec residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) compared to other tRNA^Sec including, for example, tRNA^SecUx.

Additionally or alternatively, in some embodiments, the

selenocysteine containing protein is expressed in a system that has been modified or mutated to reduce or eliminate expression of one or more translation release factors. A release factor is a protein that allows for the termination of translation by recognizing the termination codon or stop codon in an mRNA sequence. Prokaryotic release factors include RFl, RF2 and RF3; and eukaryotic release factors include eRFl and eRF3.

Deletion of one or more release factors may result in "read-through" of the intended stop codon. Accordingly, some of recombinant proteins expressed in a system with one or more release factors may include one or more additional amino acids at the C-terminal end of the protein.

Selenoproteins have widely been lost in the fungal kingdom, with the exception of five species recently discovered that have selenoprotein genes along with genes required for Sec biosynthesis (Mariotti et al., Biorxiv,

2018). Despite S. cerevisiae lacking Sec utilizing traits, selenium can accumulate intracellularly at high concentrations, largely in the form of

45292464vl 73 selenomethionine (SeMet). When adding low doses of sodium selenite to medium during early logarithmic growth phase, selenium levels in S.

cerevisiae can reach 2354 ^ig/g (de Leon, et a!., JAppl Microbiol 2002, 92, 602-610). However, selenium is toxic to yeast in high concentrations. The trans-sulfuration pathway converts SeMet to Sec, and as a consequence of free Sec production, cysteinyl-tRNA synthetase (CysRS) misincorporates Sec at Cys codons, which causes protein aggregation within the cell (Plateau, et al., Sci Rep 2017, 7, 44761, doi: 10.1038/srep44761). In Sec utilizing organisms, Sec is hydroiyzed by selenocysteine lyase to dehydroalanine and selenide, which is the form of selenium, used for tRNA-dependent Sec biosynthesis on tRNA^Sec.

The Examples below show that through metabolic engineering, the Sec biosynthesis pathway can be reconstituted in S, cerevisiae for production of selenoproteins. There are several advantages to using a yeast system. For example, not all selenoproteins can be efficiently synthesized in bacteria, and eukaryotic selenoproteins would lack post-translational modifications when produced in the currently used bacterial systems. Moreover, expression of selenoproteins in S. cerevisiae allows for systems biology studies of interaction networks when implemented in yeast two-hybrid and synthetic genetic array screens.

The design and expression of a functional Cys/Sec-specific reporter and a Sec translation system in Saccharomyces cerevisiae are exemplified in Example 12 below. Suppression of up to two amber codons in replace of important Cys residues in Gal4 indicates that Sec-tRNA^Sec is being efficiently produced at high enough levels to incorporate two Sec residues into the same polypeptide, while competing with endogenous translation release factors. In addition to these findings, this work also shows that eukaryotic elongation factor, eEFla, can bind Sec-tRNA^Sec and deliver it to the ribosome in a non-canonical manner that does not require an mRNA structural element (selenocysteine insertion sequence; SECIS) or a specialized elongation factor for Sec (SelB or EFSec) (Squires, et al.,

IUBMB Life 2008, 60, 232-235, doi: 10.1002/iub.38, Donovan, et al.,

Antioxid Redox Signal 2010, 12, 881-892, doi: 10.1089/ars.2009.2878). This

74

45292464vl work allows for the production of selenoproteins in yeast using Aeromonas pathway components that were used for efficient selenoprotein production in bacteria (Mukai, et al., Angew Chem Int Ed Engl 2018, 57, 7215-7219, doi: 10.1002/anie.201713215). Producing selenoproteins in S. cerevisiae can facilitate systems biology studies of protein-protein interactions and can be a method to cost-effectively produce medically or industrially relevant selenoproteins that require synthesis in a eukaryotic system.

In some embodiments, selenocysteine-protein production in yeast includes expression in yeast of a non-naturally occurring yeast tRNA^Sec alone or preferably in combination with a SelA (e.g., AsSelA), and preferably a SelD (e.g., AsSelD) compatible therewith. Typically, the yeast also expresses its own SerRS and eEFla. In some embodiments, an SCL (e.g., MmSCL) is also expressed.

The protein of interest can be purified from the truncated proteins and other contaminants using standard methods of protein purification as discussed in more detail below.

1. In vitro Transcription/Translation

In one embodiment, the genes encoding a tRNA^Sec, mRNA encoding the protein of interest, mRNA encoding EF-Tu, SerRS, SelA, PSTK, SepSecS, SelD or combinations thereof are synthesized in vitro prior to or along with transcription and translation of the protein of interest. The synthesis of protein from a DNA sequence in vitro takes two steps. The first is transcription of an RNA copy and the second is the translation of a protein.

In vitro protein synthesis does not depend on having a polyadenylated mRNA, but if having a poly(A) tail is important for some other purpose a vector may be used that has a stretch of about 100 A residues incorporated into the polylinker region. That way, the poly(A) tail is "built in" by the synthetic method.

Eukaryotic ribosomes read RNAs more efficiently if they have a 5' methyl guanosine cap. RNA caps can be incorporated by initiation of transcription using a capped base analogue, or adding a cap in a separate in vitro reaction post-transcriptionally.

45292464vl 75 The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases. Various approaches to in vitro protein synthesis are known in the art and include translation of purified RNA, as well as "linked" and "coupled" transcription:translation. In vitro translation systems can be eukaryotic or prokaryotic cell-free systems.

Combined transcription/translation systems are available, in which both phage RNA polymerases (such as T7 or SP6) and eukaryotic ribosomes are present. One example of a kit is the TNT® system from Promega Corporation.

Other suitable in vitro transcription/translation systems include, but are not limited to, the rabbit reticulocyte system, the E. coli S-30

transcription-translation system, and the wheat germ based translational system.

2. In vivo Methods Transcription/Translation

a. Extrachromosomal Expression

Host cells can be genetically engineered (e.g., transformed, transduced or transfected) with the vectors encoding tRNA^Sec, a nucleic acid encoding the protein of interest, EF-Tu, SerRS, SelA, PSTK, SepSecS, SelD or combinations, which can be, for example, a cloning vector or an expression vector. In some embodiments, two or more of tRNA^Sec, EF-Tu,

SerRS, SelA, PSTK, SepSecS, and SelD are expressed from the same vector.

The vector can be, for example, in the form of a plasmid, a bacterium, a virus, a naked polynucleotide, or a conjugated polynucleotide. The vectors are introduced into cells and/or microorganisms by standard methods including electroporation (From et al., Proc. Natl. Acad. Sci. USA 82, 5824

(1985), infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327, 70-73 (1987)). Methods of expressing recombinant proteins in various recombinant expression systems including bacteria, yeast, insect, and mammalian cells are known in

45292464vl 76 the art, see for example Current Protocols in Protein Science (Print ISSN: 1934-3655 Online ISSN: 1934-3663, Last updated Jan. 2012). Plasmids can be high copy number or low copy number plasmids. In some embodiments, a low copy number plasmid generates between about 1 and about 20 copies per cell (e.g., approximately 5-8 copies per cell). In some embodiments, a high copy number plasmid generates at least about 100, 500, 1,000 or more copies per cell (e.g., approximately 100 to about 1,000 copies per cell).

Kits are commercially available for the purification of plasmids from bacteria, (see, e.g., GFX™ Micro Plasmid Prep Kit from GE Healthcare; Strataprep® Plasmid Miniprep Kit and StrataPrep® EF Plasmid Midiprep Kit from Stratagene; GenElute™ HP Plasmid Midiprep and Maxiprep Kits from Sigma- Aldrich, and, Qiagen plasmid prep kits and QIAfilter™ kits from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect cells or

incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or

prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.

Useful prokaryotic and eukaryotic systems for expressing and producing polypeptides are well known in the art include, for example, Escherichia coli strains such as BL-21, and cultured mammalian cells such as CHO cells.

In eukaryotic host cells, a number of viral-based expression systems can be utilized to express non-naturally occurring tRNA^Sec and mRNA for producing proteins or polypeptides containing selenocysteine. Viral based expression systems are well known in the art and include, but are not limited to, baculoviral, SV40, retroviral, or vaccinia based viral vectors.

Mammalian cell lines that stably express tRNA and proteins can be produced using expression vectors with appropriate control elements and a

77

45292464vl selectable marker. For example, the eukaryotic expression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B) (see Wong et al. (1985) Science 228:810-815) are suitable for expression of recombinant proteins in, for example, Chinese hamster ovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3 cells, BHK21 cells, MDCK cells, and human vascular endothelial cells (HUVEC). Additional suitable expression systems include the GS Gene Expression System™ available through Lonza Group Ltd.

U6 and HI are exemplary promoters that can be used for expressing bacterial tRNA in mammalian cells.

Following introduction of an expression vector by electroporation, lipofection, calcium phosphate, or calcium chloride co-precipitation, DEAE dextran, or other suitable transfection method, stable cell lines can be selected (e.g., by metabolic selection, or antibiotic resistance to G418, kanamycin, or hygromycin or by metabolic selection using the Glutamine Synthetase-NSO system). The transfected cells can be cultured such that the polypeptide of interest is expressed, and the polypeptide can be recovered from, for example, the cell culture supernatant or from lysed cells.

b. Expression by Genomic Integration

Methods of engineering a microorganism or cell line to incorporate a nucleic acid sequence into its genome are known in the art. Any one or more of tRNA^Sec, EF-Tu, SerRS, SelA, PSTK, SepSecS, SelD or combinations can be expressed from one or more genomic copies. For example, cloning vectors expressing a transposase and containing a nucleic acid sequence of interest between inverted repeats transposable by the transposase can be used to clone the stably insert the gene of interest into a bacterial genome (Barry, Gene, 71:75-84 (1980)). Stably insertion can be obtained using elements derived from transposons including, but not limited to Tn7 (Drahos, et al., Bio/Tech. 4:439-444 (1986)), Tn9 (Joseph-Liauzun, et al., Gene, 85:83-89 (1989)), TnlO (Way, et al., Gene, 32:369-379 (1984)), and Tn5 (Berg, In

Mobile DNA. (Berg, et al., Ed.), pp. 185-210 and 879-926. Washington,

D.C. (1989)). Additional methods for inserting heterologous nucleic acid sequences in E. coli and other gram-negative bacteria include use of

45292464vl 78 specialized lambda phage cloning vectors that can exist stably in the lysogenic state (Silhavy, et al., Experiments with gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984)), homologous recombination (Raibaud, et al., Gene, 29:231-241 (1984)), and transposition (Grinter, et al., Gene, 21:133-143 (1983), and Herrero, et al., /. Bacteriology, 172(11):6557-6567 (1990)).

Methods of engineering other microorganisms or cell lines to incorporate a nucleic acid sequence into its genome are also known in the art. Nucleic acids that are delivered to cells which are to be integrated into the host cell genome can contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non-nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can become integrated into the host genome. Techniques for integration of genetic material into a host genome are also known and include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods needed to promote homologous recombination are known to those of skill in the art.

For example, cloning vectors expressing a transposase and containing a nucleic acid sequence of interest between inverted repeats transposable by the transposase can be used to clone the stably insert the gene of interest into a bacterial genome (Barry, Gene, 71:75-84 (1980)). Stably insertion can be obtained using elements derived from transposons including, but not limited to Tn7 (Drahos, et al., Bio/Tech. 4:439-444 (1986)), Tn9 (Joseph-Liauzun, et al., Gene, 85:83-89 (1989)), TnlO (Way, et al., Gene, 32:369-379 (1984)), and Tn5 (Berg, In Mobile DNA. (Berg, et al., Ed.), pp. 185-210 and 879-926.

Washington, D.C. (1989)). Additional methods for inserting heterologous nucleic acid sequences in E. coli and other gram-negative bacteria include

45292464vl 79 use of specialized lambda phage cloning vectors that can exist stably in the lysogenic state (Silhavy, et al., Experiments with gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984)), homologous recombination (Raibaud, et al., Gene, 29:231-241 (1984)), and transposition (Grinter, et al., Gene, 21:133-143 (1983), and Herrero, et al., /. Bacteriology, 172(11):6557-6567 (1990)).

Integrative plasmids can be used to incorporate nucleic acid sequences into yeast chromosomes. See for example, Taxis and Knop, Bio/Tech., 40(l):73-78 (2006), and Hoslot and Gaillardin, Molecular Biology and Genetic Engineering of Yeasts. CRC Press, Inc. Boca Raton, FL (1992). Methods of incorporating nucleic acid sequence into the genomes of mammalian lines are also well known in the art using, for example, engineered retroviruses such lentiviruses.

B. Purification of Selenocysteine Containing Polypeptides Selenocysteine containing polypeptides can be isolated using, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, selenocysteine containing polypeptides can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, an Fc- containing polypeptide in a cell culture supernatant or a cytoplasmic extract can be isolated using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify selenocysteine containing polypeptides.

Selenocysteine containing polypeptides can additionally be engineered to contain a secretory signal (if there is not a secretory signal already present) that causes the protein to be secreted by the cells in which it is produced.

The secreted proteins can then conveniently be isolated from the cell media.

45292464vl 80 In some embodiments, selenocysteine containing polypeptides are isolated using activated thiol SEPHAROSE®, for example, Activated Thiol SEPHAROSE® 4B. As discussed above, in the recombinant tRNA^Sec biosynthetic pathway disclosed herein non-naturally occurring tRNA^Sec is first misacylated to a non-naturally occurring Ser-tRNA^Sec by SerRS, and subsequently converted to Sec-tRNA^Sec by SelA, or PSTK and SepSecS, or combinations thereof. Accordingly, if the SelA, or PSTK and SepSecS, enzymes are not 100% efficient at converting Ser-tRNA^Sec to Sec-tRNA^Sec, the system may incorporate Sec or Ser at the desired position, leading to a heterogeneous mixture of proteins. Activated thiol SEPHAROSE® can be incorporated into the protein purification process to purify Sec containing proteins from the Ser containing contaminants.

IV. Methods of Using Selenocysteine Containing Polypeptide

The compositions and methods disclosed herein can be used to manufacture polypeptides and proteins with one or more selenocysteine residues. In some embodiments, the mRNA encodes a polypeptide that is a naturally occurring selenocysteine containing polypeptide. In some embodiments, the mRNA encodes a polypeptide that is not a naturally occurring selenocysteine containing polypeptide. A nucleic acid sequence can include a codon that is recognized by the anticodon of a tRNA^Sec disclosed herein, for example a nucleic acid encoding a naturally occurring selenocysteine containing protein, or can be modified to include a codon recognized by the anticodon of a tRNA^Sec. The nucleic acid sequence encoding the polypeptide can also be codon optimized for expression in the desired recombinant expression system. The nucleic acid can be expressed from a vector or incorporated into the genome of the desired expression system.

A. Recombinant Selenocysteine Containing Peptides - Naturally Occurring

The disclosed compositions and methods can be used for recombinant expression of naturally occurring selenocysteine containing peptides, or variants thereof. Selenoproteins exist in all major forms of life, including, eukaryotes, bacteria and archaea. Accordingly, in some embodiments, the

45292464vl 81 mRNA of interest is an mRNA encoding a selenocysteine containing peptide from an eukaryote, a bacteria, or an archaea. The human genome encodes at least 25 naturally occurring selenocysteine containing peptides (Kryukov, et al, Science, 300: 1439-1443 (2003)). Therefore, in some embodiments the mRNA encodes a iodothyronine deiodinase such as DIOl, DI02, DI03; a glutathione peroxidase such as GPX1, GPX2, GPX3, GPX4, or GPX6; a selenoprotein such as SelH, Sell, SelK, SelM, SelN, SelO, SelP, SelR, SelS, SelT, SelV, SelW, or Sell5; selenophosphate synthetase 2 (SPS2); or a thioredoxin reductase such as TXNRD1, TXNRD2, or TXNRD3.

Conditions to be Treated

In some embodiments, recombinant selenocysteine containing polypeptides prepared according to the claimed methods are administered to a subject in an effective amount to treat a disease, or one or more symptoms thereof. As discussed in Riaz and Mehmood, JPMI, 26(02): 120-133 (2012) and Tapiero, et al., Biomedicine & Pharmacotherapy 57: 134-144 (2003), many health effects of low selenium are thought to be due to lack of one or more specific selenocysteine containing proteins. For example, reduction or loss of one or more selenocysteine containing protein in a subject can be associated with increased oxidative stress in the subject. Accordingly, a recombinant selenocysteine containing protein can be administered to subject in an effective amount to increase antioxidant activity, or reduce oxidative stress in the subject. In some embodiments, the recombinant selenocysteine containing protein can be used to treat or prevent an age-related disorder, asthma, diabetes, an infectious disease, a cardiovascular disorder, a cancer, male infertility, pre-eclampsia, a gastrointestinal disorder, thyroid metabolism, or another diseases or condition associated with reduced levels or activity of selenocysteine containing proteins.

B. Recombinant Selenocysteine Containing Peptides - Non- Naturally Occurring

The disclosed compositions and methods can also be used for producing by recombinant expression a selenocysteine containing polypeptide variant of any polypeptide that does not naturally contain selenocysteine.

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45292464vl 1. Insertion of selenocysteine

One or more selenocysteines can be added to the beginning, end, and/or inserted into a polypeptide that does not typically have a

selenocysteine. Adding one or more selenocysteines can change the biochemical and functional properties of the protein, for example, change the redox potential of the protein, increase the half-life of the protein, increase the stability or resistance to degradation, increase the activity of the protein (such as enzymatic activity), alter the pharmacokinetics of the protein, alter the binding affinity (such as the binding affinity of an antibody to antigen or ligand to receptor), change the folding properties of the protein, induce new epitopes onto the protein, or tag the protein for purification.

In some embodiments, the one or more selenocysteines changes the biochemical properties of the protein so it can be easily purified after recombinant expression. In some embodiments, selenocysteine can be added to a protein and used as a purification tag. For example, activated thiol SEPHAROSE®, or an equivalent thereof, can be incorporated into the protein purification process to purify Sec containing proteins from contaminants.

2. Substitution with selenocysteine

In some embodiments, selenocysteine is substitute for one or more naturally occurring cysteines.

Reversible oxidation of thiols to disulfides or sulfenic acid residues controls biological functions in at least three general ways, by chemically altering active site cysteines, by altering macromolecular interactions, and by regulating activity through modification of allosteric Cys (reviewed in Jones, Am. J. Physiol., 295(4):C849-868 (2008)). Half of all enzyme activities are sensitive to either oxidation, reaction with electrophiles, or interaction with metal ions. Enzymes with active-site Cys include caspases, kinases, phosphatases, and proteases. Cys is also a component of active sites of iron- sulfur clusters of electron transfer proteins and an element of zinc fingers in transcription factors and zinc -binding domains of metallothioneins. Cys residues are also conserved in structural proteins such as actin and docking proteins such as 14-3-3. Oxidation of Cys residues in αΠ¾β3 integrin

45292464vl 83 controls platelet activation. Cys-rich regions are present in plasma membrane receptors and ion channels, including the NMD A receptors, EGF receptor, and others. Thus reversible oxidation of active site thiols can provide a common and central "on-off ' mechanism for control of cell functions.

β-Actin contains a conserved Cys, which results in reversible binding of proteins, S-GS-ylation, and crosslinking of actin filaments upon oxidation. Oxidation functions in glucocorticoid receptor translocation into nuclei, and oxidation controls export of yeast AP-1 (Yap-1) from nuclei. Disulfide crosslinks control fluidity of mucus. Such changes in protein structure and interaction due to reversible oxidation can provide a central mechanism for specificity in redox signaling. In addition to containing active site and/or structural thiols, many proteins contain Cys which regulate activity by an allosteric mechanism. This type of regulation can provide a "rheostat" rather than an "on-off switch, thereby providing a means to throttle processes by GS-ylation or S-nitrosylation.

Many naturally occurring selenoproteins with known functions are oxidoreductases which contain catalytic redox-active Sec (Jacob C, et al., Angew. Chem. Int. Ed. Engl , 42:4742-4758 (2003)). Variants of the naturally occurring selenoprotein in which the Sec residues are replaced with Cys residues are typically 100-1,000 times less active (Johansson L, et al, Biochim. Biophys. Acta. , 1726: 1-13 (2005)). Furthermore, analogs of naturally occurring proteins where one or more Cys residues are replaced with Sec can generate analogs that retain the folding of the native peptides, are more potent, and have the same or greater biological activity (Raffa, Life Set , 87(15-16):451-6 (2010)).

Therefore, in some embodiments, the disclosed compositions and methods are used to manufacture recombinant variants or analogs where one or more naturally occurring Cys residues, for example Cys residues in the active site of an enzyme, are replaced with Sec residues. The methods and compositions can be used to generate analogs that retain a folding of the protein similar or the same as the native peptides, but are more potent while having the same or greater biological activity. Substituting one or more naturally occurring Cys residues with a Sec can increase the activity of the

84

45292464vl protein by 2, 5, 10, 100, 250, 500, 1,000 or more -fold over the activity of the protein that does not contain the Sec residue(s). Accordingly, the analogs can be used in therapeutic or research applications at a lower dosage, less frequently, with reduced toxicity, or combinations thereof relative to the naturally occurring protein.

In some embodiments, the disclosed compositions and methods can be used to prepare recombinant polypeptides where one or more cysteines that contributes to the formation of a disulfide bond in the protein is replaced with selenocysteine. Therefore, recombinant proteins having one or more Sec-Sec (diselenide) or Cys-Sec (selenocysteine-cysteine) bonds are disclosed.

A disulfide bond is a covalent bond, usually derived by the coupling of two thiol groups. Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. A disulfide bond can stabilize the folded form of a protein in several ways. For example a disulfide bond can hold two portions of the protein together, favoring a folded topology and contributing to the formation and stability of secondary and tertiary structures. A disulfide bond can also form the center of a hydrophobic core in a folded protein, i.e., local hydrophobic residues may condense around the disulfide bond and onto each other through hydrophobic interactions. In some cases the hydrophobic core is an enzyme's active site, and the disulfide bond is needed for enzymatic efficiency or activity.

A diselenide bond, which is formed between two selenocysteine residues, or a selenocysteine-cysteine bond between a selenocysteine and cysteine can impart similar structural and functional characteristics to the protein as a disulfide bond. Diselenide and selenocysteine-cysteine bonds are infrequent in nature, but have been reported to be in the active site of some enzymes, for example the selenocysteine protein SelL (Shchedrina, et al., PNAS, 104(35): 13919-13924 (2007)). Diselenide bonds have very low redox potential, but in some cases can be reduced by thioredoxin.

Therefore, in some embodiments, the disclosed compositions and methods are used to manufacture recombinant variants where one or more

45292464vl 85 naturally occurring disulfide bonds are replaced with a diselenide or a selenocysteine-cysteine bond.

Replacing disulfide bonds with diselenide or selenocysteine-cysteine bonds can be used to reduce the redox potential of the bond, increase the half-life of the protein, increase the activity of the protein, alter the pharmacokinetics of the protein, for example, increase or decrease the association or dissociation constant, alter the folding and unfolding properties of the protein, or combinations thereof. For example, substituting one or more naturally occurring Cys residues with a Sec can increase the activity of the protein by 2, 5, 10, 100, 250, 500, 1,000 or more -fold over the activity of the protein that does not contain the Sec residue(s). Accordingly, the analogs can be used in therapeutic or research applications at a lower dosage, less frequently, with reduced toxicity, or combinations thereof relative to the naturally occurring protein.

Exemplary proteins where a naturally occurring Cys can be replaced with Sec according to the compositions and methods disclosed herein include, but are not limited to, caspases, kinases, phosphatases, proteases, transcription factors, metallothioneins, structural proteins such as actin and docking proteins such as 14-3-3, integrins such as αΠ¾β3, plasma membrane receptors, ion channels, including the NMDA receptors, EGF receptor, and others.

The disclosed compositions and methods can be particularly useful for preparing recombinant antibodies, antigen binding fragments thereof, fusion proteins including a least one antibody domain (i.e., Ig fusion proteins) with altered properties, and receptor such as T cell receptors or receptor fragments including the binding domains. Antibodies contain interchain disulfide bonds which link the heavy and light chains, disulfide bonds that link two heavy chains, and disulfide bonds that link the two hinge regions. Antibodies also have disulfide bonds within the chains themselves (referred to as intra-chain disulfide bonds). The disclosed compositions and methods can be used to prepare recombinant antibodies where one or more disulfide bonds are replaced with diselenide bonds. The one or more of the inter-chain disulfide bonds which link the heavy and light chains, the

45292464vl 86 disulfide bonds that link two heavy chains, the disulfide bonds that link the two hinge regions, the intra-chain disulfide bonds, or combinations thereof can be replaced with diselenide bonds.

Disulfide bonds in antibodies are important for assembly, stability and dimerization of the antibody. For example, disulfide bonds play a critical role in the stabilization of the immunoglobulin β-sandwich. Under reducing conditions, such as those characteristic of recombinant protein expression systems, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment are misfolded or inactive (Seo, et al., Protein Sci. , 18(2): 259-267 (2009)). Furthermore, stability and homogeneity of therapeutic antibodies are important for safety and efficacy of therapeutic antibodies (McAuley, et al, Protein Sci. , 17(1): 95-106 (2008)). Undesired biochemical, structural, and conformational forms, such as those generated when disulfide bonds are reduced, can lead to loss of efficacy and risk of adverse side effects.

Replacing one or more of the disulfide bonds of an antibody with diselenide or selenocysteine-cysteine bonds according to the disclosed compositions and methods can improve the yield, purity, or combinations thereof, of recombinantly produced antibodies. Replacing one or more of the disulfide bonds of an antibody with diselenide or selenocysteine-cysteine bonds according to the disclosed compositions and methods can also improve stability, increase efficacy, increase half-life, reduce toxicity, alter the pharmacokinetics of the antibody, for example, increase or decrease the association or dissociation constant, or combinations thereof of antibodies, such as therapeutic antibodies.

The antibodies can be xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized, single chain or chimeric antibodies. Antibodies may also be anti-idiotypic antibodies specific for a idiotype of the desired antigen. The term "antibody" is also meant to include both intact molecules as well as fragments thereof that include the antigen-binding site and are capable of binding to a desired epitope. These include Fab and

F(ab')2 fragments which lack the Fc fragment of an intact antibody, and therefore clear more rapidly from the circulation, and may have less non- 87

45292464vl specific tissue binding than an intact antibody (Wahl et al., /. Nuc. Med. 24:316-325 (1983)). Also included are Fv fragments (Hochman, J. et al., Biochemistry, 12: 1130-1135(1973); Sharon, J. et al., Biochemistry, 15: 1591- 1594 (1976)). These various fragments can be produced using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux et al., Meth. Enzymol , 121 :663-69 (1986)).

Antibody "formats" and methods of making recombinant antibodies are known in the art and reviewed in Laffly and Sodoyer, Hum Antibodies, 14(l-2):33-35 (2005). Methods of expressing and purifying antibodies from a recombinant expression system are known in the art, see for example, Knappik and Brundiers, "Recombinant Antibody Expression and

Purification," The Protein Protocols Handbook, Third Edition Edited by: J.M. Walker © Humana Press, a Part of Springer Science + Business Media, LLC (2009).

Therapeutic antibodies that could benefit from replacement of one or more disulfide bonds with a diselenide or selenocysteine-cysteine bond are known in the art and include, but are not limited to, those discussed in Reichert, Mabs,3(l): 76-99 (2011), for example, AIN-457, bapineuzumab, brentuximab vedotin, briakinumab, dalotuzumab, epratuzumab,

farletuzumab, girentuximab (WX-G250), naptumomab estafenatox, necitumumab, obinutuzumab, otelixizumab, pagibaximab, pertuzumab, ramucirumab, REGN88, reslizumab, solanezumab, Tlh, teplizumab, trastuzumab emtansine, tremelimumab, vedolizumab, zalutumumab and zanolimumab.

Other therapeutic antibodies that could benefit from replacement of one or more disulfide bonds with a diselenide bond include antibodies approved for use, in clinical trials, or in development for clinical use which include, but are not limited to, rituximab (Rituxan®,

IDEC/Genentech/Roche) (see for example U.S. Patent No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma;

HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-

CD20 antibody described in U.S. Patent No. 5,500,362, AME-133 (Applied

Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM (Intracel),

45292464vl 88 and PRO70769 (PCT/US2003/040426, entitled "Immunoglobulin Variants and Uses Thereof"), trastuzumab (Herceptin®, Genentech) (see for example U.S. Patent No. 5,677,171), a humanized anti-Her2/neu antibody approved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarge), currently being developed by Genentech; an anti-Her2 antibody described in U.S. Patent No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Patent No. 4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinical trials for a variety of cancers; ABX-EGF (U.S. Patent No. 6,235,883), currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No. 10/172,317), currently being developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Patent No. 5,558,864; Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83); 1CR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(l-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centra de Immunologia Molecular, Cuba (U.S. Patent No. 5,891,996; U.S. Patent No. 6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806 (Ludwig

Institute for Cancer Research, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MRI-1 (IV AX, National Cancer Institute) (PCT WO 0162931A2); and SCIOO (Scancell) (PCT WO 01/88138); alemtuzumab (Campath®,

Millenium), a humanized mAb currently approved for treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67 protein) antibody developed by Celltech/Wyeth, alefacept (Amcvive®), anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®), developed by Centocor/Lilly, basiliximab (Simulect®), developed by

Novartis, palivizumab (Synagis®), developed by Medimmune, infliximab

45292464vl 89 (Remicade®), an anti-TNFalpha antibody developed by Centocor, adalimumab (Humira®), an anti-TNFalpha antibody developed by Abbott, Humicade®, an anti-TNFalpha antibody developed by Celltech, golimumab (CNTO-148), a fully human TNF antibody developed by Centocor, etanercept (Enbrel®), an p75 TNF receptor Fc fusion developed by

Immunex/Amgen, lenercept, an p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL, an anti-CD 147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8 antibody being developed by Abgenix, ABX-MAI, an anti-MUC18 antibody being developed by Abgenix, Pemtumomab (R1549,90Y-muHMFGl), an anti-MUCl in development by Antisoma, Therex (R1550), an anti-MUCl antibody being developed by Antisoma, AngioMab (AS 1405), being developed by Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS 1407) being developed by Antisoma, Antegrene (natalizumab), an anti-alpha-4-beta-l (VLA-4) and alpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, an anti- VLA-1 integrin antibody being developed by Biogen, LTBR mAb, an anti- lymphotoxin beta receptor (LTBR) antibody being developed by Biogen, CAT-152, an anti-TGF-.beta.2 antibody being developed by Cambridge Antibody Technology, ABT 874 (J695), an anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an anti-TGF.beta.l antibody being developed by Cambridge Antibody Technology and Genzyme, CAT-213, an anti-Eotaxinl antibody being developed by Cambridge Antibody

Technology, LyntphoStat-B® an anti-Blys antibody being developed by Cambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-RlmAb, an anti-TRAIL-Rl antibody being developed by Cambridge Antibody Technology and Human Genome Sciences, Inc. Avastin® bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, an anti-HER receptor family antibody being developed by Genentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developed by Genentech. Xolair® (Omalizurnab), an anti-IgE antibody being developed by Genentech, Raptiva® (Efalizurnab), an anti-CDlla antibody being developed by Genentech and Xoma, MLN-02 Antibody

(formerly LDP-02), being developed by Genentech and Millenium

45292464vl 90 Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab and Amgen, HuMax-Inflam, being developed by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody being developed by IDEC Pharmaceuticals, IDEC- 114, an anti-CD80 antibody being developed by IDFC Pharmaceuticals, IDEC- 152, an anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage migration factor (MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, an anti- idiotypic antibody being developed by Imclone, IMC-1C11, an anti-KDR antibody being developed by Imclone, DC 101, an anti-flk-l antibody being developed by Imclone, anti-VE cadherin antibodies being developed by Imclone, CEA-Cide® (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibody being developed by Immunomedics, LymphoCide®

(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics, AFP-Cide, being developed by Immunomedics, MyelomaCide, being developed by Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide, being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex, MDX-070 being developed by Medarex, MDX-018 being developed by Medarex, Osidem® (IDM-I), and anti-Her2 antibody being developed by Medarex and Immuno-Designed Molecules, HuMaxe-CD4, an anti-CD4 antibody being developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Medarex and Genmab, CNTO 148, an anti-TNFa antibody being developed by Medarex and Centocor/J&J. CNTO 1275, an anti-cytokine antibody being developed by Centocor/J&J, MOR101 and MOR102, anti-intercellular adhesion molecule- 1 (ICAM-1) (CD54) antibodies being developed by

MorphoSys, MOR201, an anti-fibroblast growth factor receptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion® (visilizumab), an anti-

45292464vl 91 CD3 antibody being developed by Protein Design Labs, HuZAFO, an anti- gamma interferon antibody being developed by Protein Design Labs, Anti- α5β1 Integrin, being developed by Protein Design Labs, anti-IL-12, being developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody being developed by Xoma, Xolair® (Omalizumab) a humanized anti-IgE antibody developed by Genentech and Novartis, and MLN01, an anti-Beta2 integrin antibody being developed by Xoma. In another embodiment, the therapeutics include KRN330 (Kirin); huA 33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha V133 integrin, Medimmune); volociximab (ανβΐ integrin,

Biogen/PDL); Human mAb 216 (B cell glycosolated epitope, NCI); BiTE MT103 (bispecific CD19x CD3, Medimmune); 4G7x H22 (Bispecific BcellxFcgammaRl, Meclarex/Merck KGa); rM28 (Bispecific CD28 x MAPG, U.S. Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific CD64 x EGFR, Medarex); Catumaxomab (removah) (Bispecific EpCAM x anti-CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI- 507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); THIOMAB (Genentech);

veltuzumab (hA20) (CD20, Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152) (CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX- 060 (CD30, Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle Genetics); SGN-33 (Lintuzumab) (CD33, Seattle Genetics);

Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD 122 (CD40, Novartis); SGN-40 (CD40, Seattle Genetics); Campathlh (Alemtuzumab) (CD52, Genzyme); MDX-1411 (CD70, Medarex); hLLl (EPB-I) (CD74.38,

Immunomedics); Galiximab (IDEC- 144) (CD80, Biogen); MT293

(TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma); ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab

45292464vl 92 (Ticilimumab, CP-675,2) (CTLA4, Pfizer); 1-IGS-ETRl (Mapatumumab) (DR4TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline); AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5, Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist, HGS) ; Cetuximab (Erbitux) (EGFR, Imclone) ; IMC- 11 F8 , (EGFR, Imclone) ; Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen); Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck); edrecolomab (Panorex, 17-1A) (Epcam Glaxo/Centocor); MORAb-003 (folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa); MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex); Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4) (HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma); AMG-479 (IGF-1R, Amgen); anti-IGF-lR R1507 (IGF1-R, Roche); CP 751871 (IGF 1-R, Pfizer); IMC-A12 (IGF1-R, Imclone);

B1111022 Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche); CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute of Cancer Research); hCBE-11 (LT R, Biogen); HuHMFGl (MUCl, Antisoma/NCI); RAV 12 (N-linked carbohydrate epitope, Raven); CAL (parathyroid hormone-related protein (PTH-rP), University of California); CT-011 (PD1, CtireTech); MDX-1106 (ono-4538) (PDL Nileclarox/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa, Imclone); bavituximab

(phosphatidylserine, Peregrine); huJ591 (PSMA, Cornell Research

Foundation); muJ591 (PSMA, Cornell Research Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme); Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin receptor, Salk Institute, INSERN WO

2005/111082); E2.3 (transferrin receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech); HuMV833 (VEGF, Tsukuba Research Lab- WO/2000/034337, University of Texas); IMC-18F1 (VEGFRl, Imclone);

IMC-1121 (VEGFR2, Imclone)

In another embodiment, the recombinant protein is a fusion protein having a least one Cys, preferably at least one Cys-Cys bond. In some

45292464vl 93 embodiments, the fusion protein is a fusion protein containing an antibody domain, for example an Ig fusion protein. A fusion protein typically includes two or more domains, where a first domain including a peptide of interest is fused, directly or indirectly to a second polypeptide. In some embodiments, the second domain includes one or more domains of an Ig heavy chain constant region, preferably having an amino acid sequence corresponding to the hinge, Cm and CH3 regions of a human immunoglobulin Cyl chain. Construction of immunoglobulin fusion proteins is discussed in Current Protocols in Immunology, (ed. Diane Hollenbaugh, Alejandro Aruffo) UNIT 10.19A, Published May 1, 2002, by John Wiley and Sons, Inc.

3. Selenocysteine-containing Polypeptide Conjugates In some embodiments, the addition of one or more selenocysteines can be used to facilitate linkage of second therapeutic, prophylactic or diagnostic agent to the selenocysteine containing polypeptide. Methods of utilizing cysteines as reactive sites for attachment of a second agent, for example, via a disulfide bridge, are known in the art. See for example, Ritter, Pharmaceutical Technology, 42-47 (2012), Miao, et al., Bioconjug. Chem., 19(1): 15-19 (2008); and Dosio, et al., Toxins (Basel), 3(7):848-83 (2011). Accordingly, one or more selenocysteines can be added to a recombinant polypeptide, or substitute for an existing amino acid such as cysteine, to create or replace a reactive site for conjugation of the second agent. The recombinant polypeptide and the second agent can be conjugated via a linker. In a preferred embodiment, the recombinant polypeptide engineered to a contain one or more selenocysteines is an antibody, for example a therapeutic antibody.

In some embodiments, the second agent is a toxin, diagnostic imaging agent, purification ligand or other engineered element that modifies the stability, activity, pharmacokinetics, or other properties of the protein. The second agent can be a small molecule.

In a preferred embodiment, the second agent is a therapeutic agent.

For example, the second agent can be a chemotherapeutic drug. The majority of chemotherapeutic drugs can be divided into: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors,

94

45292464vl and other antitumour agents. All of these drugs affect cell division or DNA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the new tyrosine kinase inhibitors e.g. imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, vincristine, vinblastine, vinorelbine, vindesine, taxol and derivatives thereof, irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide, epipodophyllo toxins, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof.

In some preferred embodiments, recombinant antibody including one or more selenocysteine polypeptides manufactured according to the disclosed methods is conjugated with second therapeutic agent such as a

chemotherapeutic drug.

Conditions to be Treated

As discussed above, substituting one or more naturally occurring Cys residues with a Sec can increase activity, lower dosage, reduce toxicity, improve stability, increase efficacy, increase half-life or combinations thereof of a selenocysteine containing protein relative to its cysteine containing counterpart. Accordingly, therapeutic proteins containing one or more selenocysteine residues can be prepared according to the compositions and methods disclosed herein and administered to a subject in need thereof in an effective amount to reduce or alleviate one or more symptoms of a disease or disorder. Therapeutic proteins such as enzymes and antibodies which contain one or more cysteine residues or disulfide bonds can be replaced with Sec to increase activity, lower dosage, reduce toxicity, improve stability, increase efficacy, increase half-life, or attach a second agent or combinations thereof are discussed above and known in the art, and can be administered to subject to treat diseases or disorders including, but not

45292464vl 95 limited to, infectious diseases, cancers, metabolic disorders autoimmune disorders, inflammatory disorders, and age-related disorders.

C. Administration

The recombinant selenocysteine containing polypeptides disclosed herein can be part of a pharmaceutical composition. The compositions can be administered in a physiologically acceptable carrier to a host. Preferred methods of administration include systemic or direct administration to a cell. The compositions can be administered to a cell or patient, as is generally known in the art for protein therapy applications.

The compositions can be combined in admixture with a

pharmaceutically acceptable carrier vehicle. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides;

proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as Tween^®, Pluronics^® or PEG.

The compositions can be administered parenterally. As used herein, "parenteral administration" is characterized by administering a

pharmaceutical composition through a physical breach of a subject's tissue. Parenteral administration includes administering by injection, through a surgical incision, or through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration includes subcutaneous,

45292464vl 96 intraperitoneal, intravenous, intraarterial, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.

Parenteral formulations can include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Parenteral administration formulations include suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, reconstitutable dry (i.e. powder or granular) formulations, and implantable sustained-release or biodegradable formulations. Such formulations may also include one or more additional ingredients including suspending, stabilizing, or dispersing agents. Parenteral formulations may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. Parenteral formulations may also include dispersing agents, wetting agents, or suspending agents described herein.

Methods for preparing these types of formulations are known. Sterile injectable formulations may be prepared using non-toxic parenterally- acceptable diluents or solvents, such as water, 1,3-butane diol, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other parentally-administrable formulations include microcrystalline forms, liposomal preparations, and biodegradable polymer systems. Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers, and sparingly soluble salts.

Pharmaceutical compositions may be prepared, packaged, or sold in a buccal formulation. Such formulations may be in the form of tablets, powders, aerosols, atomized solutions, suspensions, or lozenges made using known methods, and may contain from about 0.1% to about 20% (w/w) active ingredient with the balance of the formulation containing an orally dissolvable or degradable composition and/or one or more additional

45292464vl 97 ingredients as described herein. Preferably, powdered or aerosolized formulations have an average particle or droplet size ranging from about 0.1 nanometers to about 200 nanometers when dispersed.

As used herein, "additional ingredients" include one or more of the following: excipients, surface active agents, dispersing agents, inert diluents, granulating agents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, preservatives, physiologically degradable compositions (e.g., gelatin), aqueous vehicles, aqueous solvents, oily vehicles and oily solvents, suspending agents, dispersing agents, wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifying agents, antioxidants, antibiotics, antifungal agents, stabilizing agents, and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions are known. Suitable additional ingredients are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Genaro, ed., Easton, Pa. (1985).

Dosages and desired concentrations of the pharmaceutical compositions disclosed herein may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.

Examples

Mukai, et al., "Transfer RNAs with novel cloverleaf structures,"

Nucleic Acids Research, 45(5):2776-2785 (2017), and all of the associated Supplementary Data and materials, doi: 10.1093/nar/gkw898; U.S.

Provisional Application No. 61/506,338; Mukai, et al., Angew Chem Int Ed

Engl. 2018 Jun 11;57(24):7215-7219. doi: 10.1002/anie.201713215, and 39 pages of supplemental material (anie201713215-sup-0001-

45292464vl 98 misc_information.pdf), WO 2013/009868; U.S. Patent No. 9,464,288; and U.S. Published Application Nos. 2017/0002347 and 2018/0105854 are specifically incorporated by reference in their entireties.

Example 1: tRNA^Sec-like tRNAs are widespread in bacteria

Since the discovery of tRNA in the late-1950s (Hoagland, et al., /

Biol Chem, 231:241-257 (1958)), its role as an adaptor molecule during translation of the genetic information has been extensively investigated (RajBhandary, et al., American Society for Microbiology, Washington, DC (1995)). While recent research has focused on the non-translational functions of tRNAs (Keam, et al., Life (Basel), 5: 1638-1651 (2015); Raina, et al., Front Genet, 5:Article 171 (2014); Hamashima, et al., Biomol Concepts, 4:309-318 (2013); Katz, et al., Mol Microbiol, doi:

10.1111/mmi.l3419 (2016)), the rapidly increasing expanse of genomic and metagenomic sequence information has revived the interests in the canonical function of tRNAs (Ling, et al., Nat Rev Microbiol, 13:707-721 (2015)).

Recent studies have identified a number of non-canonical tRNA species that were previously mis-annotated or undetected due to their unusual recognition elements, anticodon sequences, and irregular secondary structures (Katz, et al., Mol Microbiol, doi: 10.1111/mmi.l3419 (2016); Ling, et al., Nat Rev Microbiol, 13:707-721 (2015); Hamashima, et al., Mol Biol Evol, 33:530-540 (2016); Marck, et al., RNA, 8: 1189-1232 (2002); Campbell, et al., Proc Natl Acad Sci U S A, 110:5540-5545 (2013); Borrel, et al., Archaea, 374146 (2014); Ivanova, et al., Science, 344:909-913 (2014); Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016); Miihlhausen, et al., Genome Res, 26:945-955 (2016); Swart, et al., Cell, 166:691-702 (2016)). Interestingly, although the function of many of them remains unknown (Hamashima, et al., Biomol Concepts, 4:309-318 (2013); Katz, et al., Mol Microbiol, doi:

10.1111/mmi.13419 (2016); Hamashima, et al., Mol Biol Evol, 33:530-540 (2016)), some of these non-canonical tRNAs are known to be responsible for changes to the universal meaning of the genetic code (Ling, et al., Nat Rev

Microbiol, 13:707-721 (2015); Campbell, et al., Proc Natl Acad Sci U S A,

110:5540-5545 (2013); Borrel, et al., Archaea, 374146 (2014); Ivanova, et al., Science, 344:909-913 (2014); Mukai, et al., Angew Chem Int Ed Engl,

45292464vl 99 55:5337-5341 (2016); Muhlhausen, et al., Genome Res, 26:945-955 (2016); Swart, et al., Cell, 166:691-702 (2016)). Therefore, proper identification of tRNA genes is important to identify genetic code variations in nature (Campbell, et al., Proc Natl Acad Sci U S A, 110:5540-5545 (2013); Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016)).

All tRNAs fold into an L- shaped tertiary structure which physically links the amino acid moiety attached to one end (amino-acid acceptor branch) to the genetic information of the anticodon sequence on the other end (the anticodon branch) (RajBhandary, et al., American Society for Microbiology, Washington, DC (1995); Katz, et al., Mol Microbiol, doi:

10.1111/mmi.13419 (2016)). The amino-acid acceptor branch consists of a 7-bp acceptor stem and a 5-bp T-stem, and this 12-bp branch is recognized by the elongation factor (EF-Tu), whereas the anticodon branch consists of the D-arm, V-arm, and anticodon arm. The size and structure of tRNAs are normally standardized in a particular genetic code system, as they share the same apparatus such as processing RNases, base modification enzymes, CCA-adding enzyme, EF-Tu, and the ribosome. Although tRNA size reduction is common in the mitochondrial genomes, all prokaryotic and eukaryotic tRNA species are believed to have a 12-bp amino-acid acceptor branch (7/5) with a few exceptions. To date such exceptions are known for selenocysteine (Sec) tRNAs and histidine (His) tRNAs. Most tRNA^His species have an additional guanosine at the 5 '-end (G-l) that produces a non- canonical 7/5 structure (Cooley, et al., Proc Natl Acad Sci U S A, 79:6475- 6479 (1982); Orellana, et al., Mol Cell Biol, 6:525-529 (1986)). In contrast, tRNA^Sec has a 13-bp amino-acid acceptor branch (8/5 or 9/4) (Hubert, et al., RNA, 4: 1029-1033 (1998); Schon, et al., Nucleic Acids Res, 17:7159-7165 (1989)) and are actually longer than the other tRNA species.

Bacterial tRNA^Sec species with 12-bp amino-acid acceptor branches have also been identified (Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016); Cravedi, et al., Genome Biol Evol, 7:2692-2704 (2015)). Among these, tRNA^Sec species with a 12-bp amino-acid acceptor branch composed of an 8-bp acceptor stem and a 4-bp T-stem and a bulge nucleotide at position 51a were identified. The existence of such 8/4 tRNA^Sec structure in

45292464vl 100 two different bacterial phyla (Actinobacteria and Chloroflexi) prompted a search for other previously unidentified or mischaracterized tRNAs with an 8/4 structure. In the results below, a large number of bacterial tRNA sequences with different secondary structures were identified, annotated, and classified, and their translational functions in Escherichia coli evaluated.

Materials and Methods

Identification of tRNA sequences

The false positive sequences of a previous tRNA^Sec search study (Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016)) were re- analyzed, and tRNA^Sec-like sequences with a non-canonical composition of the amino-acid acceptor branch were manually collected with the aid of the ARAGORN server (Laslett, et al., Nucleic Acids Res, 32: 11-16 (2004)) and the Clustal X program (Larkin, et al., Bioinformatics, 23:2947-2948 (2007)). Next, a BLAST search of some soil and sediment metagenome data was performed in the Integrated Microbial Genomes (IMG) system (Markowitz, et al., Nucleic Acids Res, 42:D568-573 (2014)) and the National Center for Biotechnology Information (NCBI) for more allo-tRNA sequences. A number of metagenomic contigs were found to contain up to two allo-tRNA genes and frequently annotated as belonging to Acidobacteria.

The resulting allo-tRNA sequences were classified into several groups, and representative sequences were used as query for BLAST searches of acidobacterial genomes. Acidobacterium strain C40 was renamed in this study as Edaphobacter strain C40, based on the 99.4% 16S rRNA sequence similarity with Edaphobacter modestus Jbg-1^T (Koch, et al., Int J Syst Evol Microbiol, 58: 1114-1122 (2008)). Some of the selC* tRNA sequences were found in the false positive sequences of the tRNA^Sec search. By using them as query for BLAST, most of the selC* tRNA sequences were detected. The resulting selC* tRNA sequences were classified into several groups with the aid of the ARAGORN server (Laslett, et al., Nucleic Acids Res, 32: 11-16 (2004)) and the Clustal X program (Larkin, et al.,

Bioinformatics, 23:2947-2948 (2007)). A few allo-tRNA sequences with a serine anticodon were found in tRNA gene clusters of unknown

bacteriophages in the Macroalgal surface ecosystem from Botany Bay,

45292464vl 101 Sydney, Australia. The secondary structures of all tRNAs were manually predicted in the clusters and found (8/4) tRNA^Ser and (8/4) tRNA^His species. Next, a BLAST search of all metagenomic assembled sequence data was performed in IMG and NCBI for more (8/4) tRNA^Ser and (8/4) tRNA^His sequences. Some allo-tRNA sequences missing nucleotides 9-11 were found in some metatranscriptome reads of the Harvard Forest Long Term

Ecological Research site (Petersham, MA, USA) and from the Peat soil microbial communities from Weissenstadt, Germany. The secondary structures of these tRNAs were predicted BLAST searches of all metatranscriptome datasets were performed in IMG for more read sequences containing even a part of these tRNAs.

Results

The tRNA^Sec search pipeline used previously (Mukai, et al., Angew Chem Int Ed Engl, 55:5337-5341 (2016)) produced tRNA sequences with high similarity to the tRNA^Sec covariance model, but they were considered false positives after further curation. Upon re-analysis a series of tRNA sequences with non-canonical secondary structures were identified. These tRNA genes were classified into two groups: "allo-tRNA" (named after their irregular appearance) and "SelC* tRNA^Cys" (Fig. 6A-6F). While allo-tRNA genes belong to bacteria from Clostridia, Proteobacteria, and Acidobacteria, selC* genes were found in anaerobic bacteria from the phyla Firmicutes, Thermodesulfobacteria, Nitrospirae, and Proteobacteria. Both tRNA groups are structurally similar to tRNA^Sec as they have a long V-arm and longer anticodon and acceptor stems compared to canonical tRNAs (Figs. 6A-6F). Moreover, the D-stem-loop of allo-tRNAs resembles that of tRNA^Sec with its long stem and tetraloop (Fig. 6A-6B). The most striking feature of allo- tRNAs is their 8/4 or 9/3 composition of the 12-bp amino-acid acceptor branch (Fig. 6A-6B), whereas SelC* tRNA^Cys species of certain

δ-proteobacteria may have a modified 8/4 structure with a bulge base A51a (Fig. 6C).

The presence of a long V-arm and the identity of the discriminator base (G73 or U73) in most allo-tRNAs indicate that these tRNAs may be serine tRNA isoacceptors, since these unique elements are important for

102

45292464vl aminoacylation by seryl-tRNA synthetase (SerRS) (Wu, et al., Nucleic Acids Res, 21:5589-5594 (1993); Suzuki, et al., EMBO J, 16: 1122-1134 (1997); Himeno, et al., Nucleic Acids Res, 18:6815-6819 (1990); Tukalo, et al., Biopolymers and Cell, 29:311-323 (2013); Biou, et al., Science, 263: 1404- 1410 (1994)). In addition, SerRS also recognizes not only (7/5) tRNA^Ser but also (8/5, 9/4, and 8/4) tRNA^Sec and even a variant of tRNA^Sec with a 9/3 structure (Mizutani, et al., Mol Biol Rep, 25:211-216 (1998)). SelC* tRNAs were named after the selC gene, which encodes tRNA^Sec in E. coli. SelC* tRNA^Cys isoacceptors have an U73 discriminator base and cysteine GCA or opal UCA anticodons (Fig. 6C). U73 and GCA are the most important identity elements for CysRS (Pallanck, et al., J Biol Chem, 267:7221-7223 (1992); Komatsoulis, et al., Biochemistry, 32:7435-7444 (1993)), and certain CysRS forms are known to cysteinylate tRNA^CysucA (Turanov, et al., Science, 323:259-261 (2009)).

Next metagenomic contigs containing allo-tRNA genes were analyzed and tRNA^Ser and tRNA^His species with 8/4 structure (Fig. 6A-6E) and polycistrons of irregular tRNAs with 7/5 and 8/4 structures were discovered. (8/4) tRNA^Ser was found in bacteriophages, proteobacteria, and bovine rumen bacteria such as Clostridia, while (8/4) tRNA^Hls was found in bacteriophages and an a-proteobac-terium. Interestingly, (8/4) tRNA^Ser and (8/4) tRNA^Hls genes coexist in the same tRNA gene clusters of a- proteobacterial phages. The (8/4) tRNA^Ser species are included in the (8/4) allo-tRNA group or in the Y20-lacking (8/4) allo-tRNA derivative group. The (8/4) tRNA^His species lack G-l and have A73 (or U73), which is characteristic of the (7/5) tRNA^Hls species of a group of a-proteobacteria (41). The polycistrons of irregular tRNAs are discussed below.

Example 2: tRNA have extensive structural plasticity

The structural features that enable these tRNAs to be folded into a standardized tertiary tRNA structure were examined. In most cases, the cloverleaf-like secondary structure of (8/4) and (9/3) tRNAs could not be predicted properly by any of the commonly used RNA folding prediction programs (Laslett, et al., Nucleic Acids Res, 32: 11-16 (2004)). Therefore, the cloverleaf structures of (8/4 and 9/3) allo-tRNAs and SelC* tRNAs were

45292464vl 103 manually curated and predicted using structural alignments guided by tRNA^Sec crystal structures, an approach typically used for predicting tRNA^Sec cloverleaf structures (Itoh, et al., Nucleic Acids Res, 41:6729-6738 (2013)).

Allo-tRNAs are generally similar to archaeal and eukaryotic (9/4) tRNA^Sec (Hubert, et al., RNA, 4: 1029-1033 (1998)), except for the lengths of acceptor- and T-stems. A more detailed analysis of the secondary structure shows that a few nucleotides at junctions may be involved in the tertiary structures of allo-tRNAs. The base at position 48 may be involved in the V- stem structure in most cases (Fig. IB), similar to (9/4) tRNA^Sec (Hubert, et al., RNA, 4: 1029-1033 (1998)). Some (8/4 and 9/3) allo-tRNA and (8/4) tRNA^Ser species lack the nucleotide at position 10 (Fig. 6A-6B) and may require, at least, one or two linker nucleotides between the acceptor arm and D-arm. Such alternative folding was observed in the crystal structure of a pyrrolysine tRNA (tRNA^Pyl), where the nucleotides at positions 25 and 44a form a non-WC base pair (Ambrogelly, et al., Proc Natl Acad Sci U S A, 104:3141-3146 (2007); Nozawa, et al., Nature, 457: 1163-1167 (2009); Mustoe, et al., J Am Chem Soc, 137:3592-3599 (2015)). Thus, tRNAs missing N10 might form similar tertiary arrangement (Fig. 6A-6B).

Rubrobacter tRNA^Sec also lacks nucleotides at positions 9 and 10 and has U44a (Fig. 6E). The unpaired nucleotide at position 45 found in some (9/3) allo-tRNAs (Fig. 6B) might fill the space between the L-shaped tRNA body and the V-stem, which is occupied by the G45-A48 pair in human tRNA^Sec (Itoh, et al., Nucleic Acids Res, 37:6259-6268 (2009)).

The selC* tRNA^Cys species of certain δ-proteobacteria (Fig. 6C) can in theory have three alternative cloverleaf structures, two of which are 7/5. However, the 8/4 structure with a bulge nucleotide at position 51a (Fig. 6E) may be more energetically favorable, since the other two possible structures, 7/5 and 7/5 with the bulge nucleotide, eliminate two and five hydrogen bonds, respectively. In the 8/4 structure, residues at positions 9 and 10 are missing, which might be compensated by G45 (Fig. 6C), as discussed above.

In contrast, the structure of the D-stem-loop is more difficult to predict since

WC and G:U wobble base pairing patterns leads to a sterically unfavorable tri-loop. The D-stem-loop may have either a triloop hairpin structure or a

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45292464vl larger loop with a shorter stem. Thus, the hypothetical three successive WC and G:U wobble base pairs between bases at positions 13-15 and 20a-23 were indicated by dashed lines on the predicted cloverleaf structure (Fig. 6C). Actually, GGG-triloop is not observed in the D-stem-loop of

Archaeo globus fulgidus tRNA^Cys crystal structure (Fukunaga, et al., Nat Struct Mol Biol, 14:272-279 (2007)), while a CGG-triloop may form in the D-stem-loop of Candidatus Methanomassiliicoccus intestinalis tRNA¹^¹ (Borrel, et al., Archaea, 374146 (2014)).

Example 3: Allo-tRNAs have diverse anticodon sequences

Although the (8/4) tRNA^Ser species have anticodons corresponding to serine codons, most of allo-tRNA species have non-serine anticodons (Table 3). In fact, their anticodon sequences are highly diverse and correspond to 35 distinct codons (Table 3). Among them, the UAU, GCG, and GUC anticodons corresponding to the AUA isoleucine (lie) codon, the CGC arginine (Arg) codon, and the GAC aspartic acid (Asp) codon, respectively, are predominant, whereas (8/4) allo-tRNAs with anticodons corresponding to phenylalanine (Phe), valine (Val), His, and lysine (Lys) codons were only found once in the examined metagenomic dataset (Table 3). In contrast, the (9/3) allo-tRNA species have anticodons corresponding to the AGA/AGG Arg codons, the UUA / UUG / CUA / CUG leucine (Leu) codons, and the UAA stop codon (Table 3).

45292464vl 105 Table 3: Anticodon diversity of allo-tRNAs. Possible codon- anticodon interactions are indicated with bars. The numbers of (8/4) and (9/3) allo-tRNA sequences are indicated with red and blue letters, respectively.

Example 4: Allo-tRNAs are fully compatible with a bacterial translation system

Materials and Methods

Plasmids and E. coli strains

Plasmid pGFiB (Normanly, et al., Proc Natl Acad Sci U S A,

83:6548-6552 (1986)) was used for cloning the Desulfococcus biacutus selC* tRNA^Cys sequence and its variants using EcoRI and BamHI sites. The arabinose promoter cassette of pBAD-myc-HisA (Invitrogen) was previously transplanted into pRSFDuetl (Haruna, et al., Nucleic Acids Res, 42:9976- 9983 (2014)) to make pBAD-RSF. The open reading frame (ORF) of the

Desulfomonile tiedjei cysS gene was cloned from the genomic DNA obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and fused directly downstream of the kan marker gene of pBAD-RSF with a weak Shine-Dalgarno sequence of the E. coli prfA gene [TTTACAGGGTGCATTTACGCCT (SEQ ID NO:64)]. The cloned cysS gene was mutated using Infusion (Clontech) to make the cysS variant genes. The multiple cloning site (MCS) of pBAD-RSF was replaced by the MCS and the rrnC terminator sequence of pGFiB with a modification of BamHI site to Bglll site using Infusion to make pBAD-RSF5. Allo-tRNA sequences

45292464vl 106 were cloned into the MCS of pBAD-RSF5 using EcoRI and Bglll sites. Plasmid pBAD-sfGFP (Fan, et al., Nucleic Acids Res, 43:el56 (2015)) was mutated using Infusion to make the sfGFP variant genes. Plasmid pACYC184 was mutated using Infusion to make the cat variant genes. The ORF of the cat marker gene in pACYC184 was replaced using Infusion with the ORF of D. tiedjei selD gene cloned from the genomic DNA to make pACYC-DtselD. The E. coli strain DH10B was used for allo-tRNA experiments. The E. coli WL400 (MC4100 selD204::cat+) (Leinfelder, et al., Proc Natl Acad Sci U S A, 87:543-547 (1990)) cells harboring pACYC- DtselD corresponded to E. coli AselD with D. tiedjei selD.

Mass analysis

sfGFP variants encoding a C-terminal His-tag were purified using nickel-nitrilotriacetic acid agarose (QIAGEN). Purified sfGFP solutions were concentrated by centrifugation using Amicon Ultra 10k (Merck Millipore) and subjected to peptide mass fingerprinting (PMF) analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) performed by the Keck Foundation Biotechnology Resource Laboratory (Yale University).

tRNA sequences for Figures 7A-7E

Figure 7A

GGAGGGCAUCUUCAGUAGGUACUGGACGCCGUCUGAGAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCG (SEQ ID NO:46)

Figure 7B

GGAGGGGAACUUCUAUCUGGUGAUAGACGGGAACUUAAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO:47)

Figure 7C

GGGGAGUAAUGUGCGGUGGUCCGCACCGUAGUCUGCGAAACUAUUGGUUCGUUUAU ACGAAUGGGGUUCAAUUCCCCUGCUCUCCACCA (SEQ ID NO:48)

Figure 7D

GGGGUGGGGUUCCGGCUGGUGCCGGUCGCGGGCUUUAAACCCGUCAGGACGCUGCG ACGCGUAAGGUUCGAUUCCUCCCCACUCCG (SEQ ID NO:49)

107

45292464vl Figure 7E

GGGCGGGGGUUCCGUCUGGUGACGGUCGCGGGCUUUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCA (SEQ ID NO:50)

Results

To investigate whether allo-tRNAs are active translational adaptors, super-folder green fluorescence protein (sfGFP) were used as a reporter in E. coli. The Ser codon at position 2 of wild-type (WT) sfGFP was mutated to either CUC or UUA and the resulting mutants were expressed together with Silvibacterium bohemicum (8/4) allo-tRNAcAG (Llado, et al., Syst Appl Microbiol, 39: 14- 19 (2016)) or (9/3) allo-tRNAuAA in E. coli (Fig. 7A-7B). Interestingly, both tRNAs efficiently inserted Ser in response to the CUC and UUA Leu codons, respectively, as confirmed by the liquid chromatography (LC) coupled with tandem mass spectrometry (LC-MS/MS) and peptide mass fingerprinting (PMF) analyses of purified sfGFP. Furthermore, induction of the allo-tRNA expression from the araBAD promoter led to severe cell growth arrest and ultimately to cell death, which is possibly caused by global mis-incorporation of Ser at Leu codons in the E. coli proteome.

(8/4) and (9/3) allo-tRNAs with a G3:U70 wobble pair, the most important structural element for aminoacylation by alanyl-tRNA synthetase (AlaRS) were also identified (Hou, et al., Nature, 333: 140-145 (1988); McClain, et al., Science, 240:793-796 (1988); Naganuma, et al., Nature, 510:507-511 (2014)). To test whether these allo-tRNAs can be acylated by AlaRS in vivo, three examples were choosen, and their wild-type anticodons (GCG and UUA) replaced with the amber anticodon CUA (Fig. 7C-7E) and mutated the Ser2 codon of sfGFP to an amber stop codon. The mutant sfGFP was then co-expressed with one of the three allo-tRNAs in E. coli. The three amber suppressor tRNAs efficiently translated the amber codon, leading to expression of the full-length sfGFP variant, and producing as much fluorescence as did cells expressing WT sfGFP (Fig. 7F). The sfGFP variants were then purified, and the identity of the amino acid incorporated at position 2 was revealed by LC-MS/MS and PMF analyses (Fig. 3D). The amber codon was mainly translated as Ala and Ser, as judged by the probability

45292464vl 108 scores. Insertion of Asn, Gin, Lys, and possibly Cys, lie, and Glu was also detected. Only the allo-tRNA 9/3-2 variant inserted Ser, probably due to its discriminator base G73.

To further confirm the allo-tRNA-mediated incorporation of Ala and Ser, the chloramphenicol (Cm) acetyltransferase (CAT) gene was used as a reporter since it contains an important catalytic Ser residue at position 146 that only tolerates substitutions with Ala (Lewendon, et al., Biochemistry, 29:2075-2080 (1990)). Replacing Serl46 with Asn, Gin, or Lys produced an inactive CAT, while the Ser 146 Ala CAT mutant retained activity. Then, an amber codon was substituted for Serl46 and the resulting CAT variant was expressed with any of the three allo-tRNA variants. The allo-tRNAs suppressed the amber codon and conferred Cm resistance to the E. coli cells with the Serl46TAG cat gene (Fig. 7G). However, only the allo-tRNA 9/3-2 variant conferred the resistance at a Cm concentration of 100 μg/mL, which corroborates that only this tRNA incorporates Ser. Together, these results clearly demonstrated that these allo-tRNA (both 8/4 and 9/3) sequences were properly folded, processed, aminoacylated, and delivered to the ribosomes by EF-Tu in E. coli.

Example 5: Active and inactive allo-tRNAs are associated with toxin- antitoxin systems

Materials and Methods

In vitro aminoacylation with E. coli aminoacyl-tRNA synthetases

E. coli cells harboring expression plasmids for E. coli threonyl-tRNA synthetase (ThrRS), glycyl-tRNA synthetase (GlyRS) (glyQ and glyS subunits), and histidyl-tRNA synthetase (HisRS) were obtained from the

ASKA collections (Kitagawa, et al., (A Complete Set ofE. coli K-12 ORF

Archive): Unique Resources for Biological Research. DNA Research,

12:291-299 (2006)). Overnight-night cultures for each protein were used to inoculate 1 L of fresh LB media containing chloramphenicol. Cells were grown to an Αβοο of 0.6 and protein overexpression was induced with 0.1 mM IPTG overnight at 25 °C. Cells were harvested by centrifugation and the resulting pellet was lysed with buffer containing 50 mM Tris (pH 8), 300

45292464vl 109 mM NaCl, and protease inhibitor cocktail tablets (cOmplete, Roche). Lysed cells were then centrifuged at 4 °C for 45 min at 18,000 x g. The lysate was loaded on a TALON metal affinity resin (Clontech), and the protein was eluted with varying concentrations of imidazole. The protein-containing fractions were pooled and stored in buffer containing 50 mM HEPES (pH 7.3) and 150 mM NaCl. tRNA genes were cloned into pUC18 using Gibson Assembly (New England Biolabs), and the tRNAs were prepared using in vitro transcription as previously described (Ahel, et al., /. Biol. Chem. , 277:34743-34748 (2002)). Aminoacylation assays were carried out with 5 μΜ tRNA and 0.5 μΜ tRNA synthetase in buffer containing 50 mM Hepes (pH 7.3), 4 mM ATP, 10 mM MgCk, 0.1 mg/mL BSA, 1 mM dithiothreitol, and 20 μΜ [¹⁴C]His (590 cpm/pmol) (PerkinElmer), 100 μΜ [¹⁴C]Gly (146 cpm/pmol) (PerkinElmer) or 25 μΜ [³H]Thr (7779 cpm/mol) (American Radiolabeled Chemicals). Reactions were incubated at 37 °C and after 15 min, 10 μΐ, of the reaction mixture was spotted on Whatman 3MM filters pre-soaked with 5% trichloroacetic acid (TCA). Filters were washed three times with 5% TCA, and the remaining radioactivity was quantified using a scintillation counter.

tRNA Sequences for Figures 8C-8I

Figure 8C

GGAGGGCGUCUCGCUGGCGCGAGAAGCGGUCUUAUAAACCGCAAAUGUCUUGACGG GCAUUGGGGUCCGAUCCCCCCGCCCUCCG (SEQ ID NO:51)

Figure 8D

GGAGGGCGACAGGCCGGUGCCUGGAGCCGACUUAUAAUCGGCGAAUCCUUC GCAGG GGAUAGCGGUUCGACUCCGCCGCCCUCCG (SEQ ID NO:52)

Figure 8E

GGAGGGAGAUCCCGGCUGGUGCCUGGAGCCGACUUAUAAUCGGUCGAUCCC GUUCC GGGGAUCGCGGUUCAAAUCCGCCUCCCUCCGCCA (SEQ ID NO:53)

Figure 8F

GGAGGGUGUCACGCUGGUGCGUGGGCCGGUCUUAUAAACCGGAGAUUCCUUGCCGG GAAUGGAGUUCGAUUCUCCCACCCUCCGCCA (SEQ ID NO:54)

45292464vl 110 Figure 8G

GGGGGACACAACUCGUGGGUGCGAGAGUUGGUCUUAUAAACCAAUGGCGUCGUUGC AGCGACGCAAGGUUCAAUUCCUUUGUCCCCCG (SEQ ID NO:55)

Figure 8H

GGAGGGUGUCUAGCUGGUGCUAGGACCGGCCUUAUAAGCCGGAUUACCUUCCACGG GUAUUGGGGUCCGAUCCCCCCACCCUCCGCCA (SEQ ID NO:56)

Figure 9A

GGAGGGGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO:57)

Results

Although the S. bohemicum allo-tRNA^SerGAc gene is in a metabolic gene cluster, the Edaphobacter strain C40 has an allo-tRNAuAu pseudogene overlapping with the ORF of a transposon-related protein. In soil and sediment metagenomic sequences, allo-tRNA genes are often found in the vicinity of a variety of toxin- antitoxin systems (Wen, et al., Pathog Dis,

70:240-249 (2014)). Among others, allo-tRNAuAu species compose the most abundant allo-tRNA group (Table 3). Interestingly, they have cloverleaf structures slightly different from that of the standard allo-tRNA^Ser, stem- destabilizing mutations as in the Edaphobacter strain C40 allo-tRNAuAu, and a variety of possible five- stem-junction structures (Fig. 8A-8H). To assess their ability to serve in translation, six allo-tRNAuAu species with a potential Ser identity were converted to allo-tRNAcuA and examined their activity in E. coli using the CAT (Serl46TAG) reporter. However, none of the six variants conferred Cm resistance in response to the amber codon, and two of them caused cell death (Fig. 81). Thus, it is possible that most of the allo-tRNAuAu species are not used for translation and instead may be associated with transposable elements or toxin-antitoxin systems.

In addition to 9/3-2 (in Figure 7G), the amber suppressor variants of 8/4-1 and 9/3-1 allo-tRNAs translated the amber codon in E. coli. The 9/3-1 (CUA) is corresponding to allo-tRNA^UTul (in Figure 9A).

>8/4-l (CUA)

GGAGGGCATCTTCAGTAGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:61)

111

45292464vl >9/3-2

GGGGTGGGGTTCCGGCTGGTGCCGGTCGCGGGCTCTAAACCCGTCAGGACGCTGCG ACGCGTAAGGTTCGATTCCTCCCCACTCCGCCA (SEQ ID NO:62)

>9/3-l (CUA) equals to allo-tRNAUTul

Polycistrons formed by allo-tRNA-like sequences and other irregular tRNA sequences were discovered in metatranscriptome sequences of two forest/peat soil metatranscriptome projects. Both might be nonsense or missense suppressor tRNA, and may also be associated with a toxin- antitoxin system. Among the polycistronic tRNA groups (tRNAO, tRNAl, and tRNA2), 8/4 structures were predicted for tRNAccu species containing a G-l and a group of tRNAs containing an extra-loop (E-loop).

Three tRNAl species from the polycistrons were examined; a (8/4) tRNAl with an E-loop and two tRNAl with G-l, one of which has an 8/4 structure. The two G-l tRNAl species with GGU anticodon were thought to be substrates for histidyl-tRNA synthetase (HisRS), whereas the E-loop tRNAl with U73 and GCU anticodon were thought to be a poor substrate for E. coli threonyl-tRNA synthetase (ThrRS) and/or glycyl-tRNA synthetase (GlyRS). The three tRNAs were transcribed in vitro and tested for aminoacylation with E. coli HisRS, GlyRS, and ThrRS. The two G-l tRNAl species were aminoacylated by E. coli HisRS, even more efficiently than E. coli tRNA^Hls, despite the Thr GGU anticodon. The GUG triplet at positions 35-37 (but not the anticodon positions 34-36 in tRNA^Hls) of the two G-l tRNAl species might have recognized by HisRS (Tukalo, et al., Biopolymers and Cell, 29:311-323 (2013); Biou, et al., Science, 263: 1404-1410 (1994); Mizutani, et al., Mol Biol Rep, 25:211-216 (1998)). Interestingly, the two G- 1 tRNAl species did not insert His in response to the ACC Thr codon at position 2 in a sfGFP variant gene in E. coli. Thus, G-l tRNAl species may be charged, but not used for translation. In contrast, the E-loop tRNAl was not aminoacylated either by E. coli GlyRS or ThrRS in vitro. However, it is possible that the organisms encoding these irregular tRNAs encode a GlyRS and ThrRS capable of charging them. Alternatively, another aminoacyl-

112

45292464vl tRNA synthetase or homolog might charge them in a non-canonical manner, similar to the aminoacylation of a tRNA- like small RNA (tRNA°^ther) by a complex of class I and II lysyl-tRNA synthetases in Bacillus cereus (Ataide, et al., EMBO Rep, 6:742-747 (2005)).

Example 6: selC* tRNA^Cys functions in translation

Materials and Methods

Identification and analysis of protein sequences BLASTp search were preformed of all genomic and metagenomic protein sequence data in the IMG and NCBI systems in order to identify cysS, selA, selB and selD genes and their homologs. To manually enlarge a metagenomic contig containing a selC* gene from Wastewater microbial communities from Syncrude, Ft. McMurray, Alberta - Microbes from Suncor tailings pond 6 2012TP6_6, BLAST searches of the raw data (SRR943333) were performed using NCBI short read archive (SRA) BLAST. Likewise, the amino-acid sequences for the selB* and cysS* genes of Desulfonema limicola Jadebusen DSM 2076 were identified by filling the gaps of the partially-sequenced genome using the raw data (SRR058919) in the NCBI SRA database. Sequence alignments and phylogenetic trees (Bootstrap N-J Tree) were made using Clustal X 2.1. The BoxShade Server was also used for the alignments.

selD reporter assay

WL400 [pACYC-DtselD] cells were transformed with pGFiB or one of the tRNA-expressing plasmids together with pBAD-RSF or one of the D. tiedjei c_ys5-expressing plasmids. Their overnight cultures were spotted onto LB agar plates supplemented with 1 μΜ Na2Mo0₄, 1 μΜ Na2Se03, 50 mM sodium formate, 100 μg/mL carbenicillin, 30 μg/mL kanamycin, 100 μg/mL spectinomycin, and 0.1% 1-arabinose and grown anaerobically at 37 °C overnight. These plates were overlaid under anaerobic condition with a 0.75% top agar containing 1 mg/mL benzyl viologen, 250 mM sodium formate, and 25 mM KH2P0₄ (pH 7.0). Within a few seconds or less than a minute, spots on the plates became dark or light purple, depending on the formate dehydrogenase activity of cells.

₄5292₄6₄vl 113 In vitro cysteinylation assay

D. tiedjei selC* tRNA^Cys was prepared by T7 RNA polymerase transcription, purified in preparative 10 % urea PAGE and electroeluted. The tRNA was refolded by heating at 95 °C for 3 min followed by cooling to 65 °C at a rate of 0.5 °C/s. During a 3 min hold at 65 °C, MgCh was added to a final concentration of 10 mM. The refolding was completed by cooling to 30 °C at 0.2 °C/s. tRNA was radiolabeled at the 3'-end using [a-³²P]ATP and E. coli CCA-adding enzyme as previously described (Ledoux, et al., Methods, 44:74-80 (2008)). D. tiedjei cysteinyl-tRNA synthetase (CysRS) with a His- tag was recombinantly obtained using E. coli. Aminoacylation reactions were performed in [50 mM HEPES-NaOH, pH 7.2, 50 mM KC1, 10 mM MgCk, 10 mM ATP, 1 mM cysteine, 1 mM DTT]. At the indicated time points, 10 μΐ_^ aliquots were removed and digested by nuclease PI. 1 μΐ_^ of the quenched reaction was spotted on PEI cellulose plates. [³²P]AMP and

[³²P]AMP-Cys were separated with 100 mM ammonium acetate - 5 % acetic acid. The plates were analyzed using a Phosphorlmager.

Results

selC* genes were found in isolated genomic sequences of Clostridia, Bacilli, Thermodesulfobacteria, Thermodesulfo vibrio, δ-proteobacteria, a composite genome of Smithella, and a few metagenomic contigs, probably derived from Nitrospirae or δ-proteobacteria. Interestingly, selC* tRNA^Cys species belonging to a particular phylum or class show a unique conserved secondary arrangement. Furthermore, complete selenocysteine-inserting apparatus (consisting of the selA, selB, selC and selD genes) was identified in all s<?/C*-containing genomes, whereas incomplete selenocysteine systems were also found in the metagenomic contigs. Strikingly, in two δ- proteobacterial subgroups, Syntrophobacterales and Desulfobacterales , a second copy of selB, referred to here as selB*, was found downstream of the selC* genes. Because selB encodes the Sec-tRNA^Sec-specific elongation factor, it is possible that selC* tRNA^Cys mimics tRNA^Sec, both structurally and functionally, and is recognized by SelB*.

In addition to selB*, in a subgroup of Desulfobacterales, an additional copy of the cysS gene was discovered, which encodes CysRS,

114

45292464vl downstream of the selB* genes (Fig. 5A). This second copy, named cysS*, lacks the region that encodes the anticodon binding domain (ABD) of CysRS. Intriguingly, genomes with cysS* genes appear to always encode selC* tRNA^Cys species containing an A1:U72 base pair and an opal anticodon (UCA).

To assess the ability of selC* tRNA^CysucA to suppress opal codons, a reporter system was developed using the Desulfomonile tiedjei (Dt) selD gene, which has a naturally occurring UGA selenocysteine codon at position 15. The Dt selD gene was expressed together with Desulfococcus biacutus (Db) tRNA^CysucA in an E. coli AselD strain. Only when the UGA codon is translated as either Sec or Cys, functional selenophosphate synthase (SelD) is produced. SelD then catalyzes the synthesis of the selenophosphate needed for the conversion of Ser-tRNA^Sec to Sec-tRNA^Sec by selenocysteine synthase (SelA), which allows synthesis of selenoproteins. The overall suppression efficiency of Db tRNA^CysucA can then be evaluated by monitoring the activity of the Sec-containing formate dehydrogenase H

(FDHH) through the FDHH-catalyzed reduction of benzyl viologen that results in a purple dye (Lacourciere, et al., Proc Natl Acad Sci U S A, 99:9150-9153 (2002)). Expression of Db CysRS* as well as two other CysRS* did not lead to suppression of the selD opal codon.

Because Dt CysRS efficiently aminoacylated Dt selC* tRNA^Cy¾cA in vitro, a series of Dt CysRS variants were created that may recapitulate the activity of CysRS*. Because the main difference between Db CysRS* and Dt CysRS is the presence of an ABD, Dt CysRS mutant lacking the ABD (Dt CysRS AABD) was created. However, Dt CysRS AABD did not efficiently aminoacylate Db tRNA^CysucA as indicated by the light purple color (Fig. 5E). Multiple sequence alignments of canonical CysRS and CysRS* revealed that a highly conserved and critical Gly residue of the canonical CysRS CP1 domain (Liu, et al., RNA, 18:213-221 (2012)) was not conserved in CysRS*. This Gly is responsible for accurate recognition of first base pair of tRNA^Cys acceptor stem (Liu, et al., RNA, 18:213-221 (2012)), which is typically

G1:C72. In Dt CysRS, this Gly residue is part of the "MSGA" motif, whereas the "MSGA" is mutated to "PTVS" in Db CysRS*.

45292464vl 115 Therefore, to test the role of this motif in the activity of Dt CysRS, the "MSGA" sequence was mutated to "PTVS" to construct the Dt CysRS PTVS mutant. Expression of Dt CysRS PTVS and Dt selD in the E. coli AselD strain produced a dark purple color indicating efficient aminoacylation of Db tRNA^CysucA- Furthermore, a Dt CysRS variant containing the AABD and PTVS (AABD/PTVS) was more efficient than the Dt CysRS PTVS as confirmed by the saturated purple color. These results show that CysRS* may have evolved to specifically aminoacylate selC* tRNA^CysucA species with A1 :U72. This hypothesis was explored by using a Db tRNA^CysucA variant in which the A1:U72 base pair was mutated to G1:C72. The CysRS AABD/PTVS was unable to efficiently aminoacylate the G1:C72 Db tRNA^CysucA mutant. Lastly, the 8/4 conformation of Db tRNA^CysucA was experimentally confirmed by employing a previously developed method used to confirm the 9/4 structure of eukaryotic tRNA^Sec (Mizutani, et al., FEBS Lett, 466:359-362 (2000). The 8-bp acceptor stem was important for efficient opal suppression, whereas the bulge structure was dispensable.

In sum, a large number of tRNAs with new secondary structures have been identified. The function of these tRNAs is still uncharted: some of them may be involved in the translation of Ser and His codons, in opal suppression or recoding with Cys, and in mis -translation of diverse codons with Ser or Ala; others may have non-translational roles. The (8/4) tRNACys species may have co-evolved with its dedicated aminoacyl-tRNA synthetase and elongation factor. Future studies should elucidate the biological functions of these non-canonical tRNAs and protein components, and structural studies of these tRNAs will add to the knowledge of the structural plasticity of tRNA. Example 7: Allo-tRNA can incorporate SEC in a nascent protein

Selenocysteine (Sec), the 21^st amino acid, is a fascinating building block of recombinant proteins (Metanis, et al., Angew Chem Int Ed Engl (2017)), because Sec is more active and oxygen-resistant than cysteine (Cys) (Reich, et al., ACS Chem Biol, 11 :821-841 (2016); Marques, et al., Nat Chem

Biol, 13, 544-550 (2017)) and is chemically modifiable (Liu, et al., J Am

Chem Soc, 139, 3430-3437 (2017); Rakauskaite, et al., Chem Commun

(Camb), 51, 8245-8248 (2015)), and because a diselenide bond is more

45292464vl 116 stable than a disulfide bond in proteins (Arai, et al., Angew Chem Int Ed Engl, 56, 5522-5526 (2017)). Furthermore, Sec residues can be chemically converted to another side chain via dehydroalanine intermediate (Liu, et al., / Am Chem Soc, 139, 3430-3437 (2017); Wright, et al., Science, 354 (6312), aagl465 (2016)). Recent advances in the genetic code expansion field allows one to site-specifically insert Sec into recombinant proteins in response to the amber UAG codon in E. coli via the elongation factor Tu (EF-Tu), without relying on the dedicated elongation factor (SelB) and Sec- insertion sequence (SECIS element) (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013); Haruna, et al., Nucleic Acids Res, 42:9976-9983

(2014); Thyer, et al., J Am Chem Soc, 137:46-49 (2015); Miller, et al., FEBS Lett, 589:2194-2199 (2015); Fan, et al., ACS Synth Biol. (2017)). Although wildtype tRNA^Sec species have antideterminants against EF-Tu (Rudinger, et al., EMBO J, 15:650-657 (1996)), a few point mutations in the acceptor stem and the T-stem of tRNA^Sec are sufficient to remove these antideterminants (Thyer, et al., J Am Chem Soc, 137:46-49 (2015); Rudinger, et al., EMBO J, 15:650-657 (1996)). Thus, EF-Tu-compatible variants of E. coli tRNA^Sec enabled the production of bacterial and human selenoproteins using E. coli cells (Aldag, et al., Angew Chem Int Ed Engl, 52:1441-1445 (2013); Haruna, et al., Nucleic Acids Res, 42:9976-9983 (2014); Thyer, et al., J Am Chem

Soc, 137:46-49 (2015); Miller, et al., FEBS Lett, 589:2194-2199 (2015); Fan, et al., ACS Synth Biol. (2017)). However, this Sec-insertion technology has a room for improvement in as far as product yield and the extent of Sec insertion is concerned.

Materials and Methods

Escherichia coli strains

The AselABC AfdhF ME6 strain of Escherichia coli was reported previously (Mukai, et al., Angew Chem Int Ed Engl, 55, 5337-5341 (2016)). E. coli HST08 strain (Clontech) was mainly used for plasmid construction.

The T7 RNAP AselABC AfdhF ME6 strain of Escherichia coli was reported in (Mukai, et al., Angew Chem Int Ed Engl 2016, 55, 5337) E. coli

ME68z strain is ME6 transformed with pBAC8z. E. coli Stellar strain

(Clontech) was mainly used for plasmid construction. E. coli C321.AA.opt

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45292464vl AselAB strain was constructed from C321.AA.opt (Kuznetsov, et al., Genome Biol 2017, 18, 100.) by scar-less knockout of the selAB genes by

recombination.

First, the selAB gene locus was replaced with a tetRA cassette generated by PCR with oligos

#AS950 (5'-ttctccgtgtgagagggccttgatcagccaggtttcct ATGACttaagacccactttcacatttaag-3' (SEQ ID NO:96) and

#AS952 (5'-taattaatcatttccTTATTTTTCCGGAAATAATAA TGCGTCGCGcctaagcacttgtctcctgtttac-3' (SEQ ID NO:97)) using λ-Red-mediated recombination as described, (Lajoie, et al., Science

2013, 342, 357) resulting in strain AS358 (TetR). Next, the tetra cassette was removed using a PCR-generated DNA fragment composed of upstream and downstream regions of selAB

(ctgtgccgtcggatatctcaatttccgccgcgttgcgccaggttggtgtgtcgac cgcccacacttagtcaaactggtcgaaaacctgtttagccgcgaaagttttgctcg caccgaaccgccaaaggcttgatgcgcgatatgtcctcctgacccatctcacgtta caatccgtggttatgttaaacgcccttctccgtgtgagagggccttgatcagccag gtttccgcgacgcattattatttccggaaaaataaggaaatgattaattaagtttt aaaataaattaatacaaaattcttatgaatttaaaaaaagcacattgtttaatgaa tacaatgtgctttttattagattaattttg) (SEQ ID NO:98) by λ-Red- mediated recombination. Tetracycline-sensitive colonies were selected on plates containing fusaric acid as described, (Blank, M. Hensel, R. G.

Gerlach, PLoS One 2011, 6, el5763) except anhydrotetracycline was used in place of chlortetracycline. Excision of tetRA from selAB loci was confirmed by PCR using oligos #AS1113 (5'-ctgtgccgtcggatatctcaatttc-3'

(SEQ ID NO:99)) and #AS1148 (5'-ggcagcttactgaggattttcattcg-3' (SEQ ID NO: 100)).

Construction ofplasmids

The native E. colifdhF gene was cloned into the pACYC184 plasmid by replacing the chloramphenicol acety transferase (cat) gene in a similar manner with a reference (Thyer, et al., J Am Chem Soc, 137:46-49 (2015) The UGA codon and four cysteine codons oifdhF were then mutated to UAG or AGC codons by Infusion (Clontech). The UGA codon was also

45292464vl 118 changed to UCA. The SECIS element was disabled in fdhF gene variants by introducing neutral amino acid mutations by PCR using oligos fdhFam_Fno

(5'- CGTGTCtagCACGGaCCcagcGTaGCcGGatTaCAtCAAagt GTaGGTAATGGCGCAATGAGCAATGC-3' (SEQ ID NO: 101)) and fdhFam_Rno (5'- tCCGTGctaGACACGAGCGCAGCAGTCAACG-3' (SEQ ID NO: 102)).

All tRNA sequences were cloned between the EcoRI and Bglll sites into the pBAD-RSF5 plasmid (Mukai, et al., Nucleic Acids Res, 45:2776- 2785 (2017)). The ORF of Aeromonas salmonicida subsp. pectinolytica 34mel SelA was amplified by PCR from the genomic DNA and cloned together with the EM7 promoter (Thyer, et al., J Am Chem Soc, 137:46-49 (2015)) immediately downstream of the kan marker gene (Mukai, et al., Nucleic Acids Res, 45:2776-2785 (2017)) in pBAD-RSF5 carrying allo- tRNA^UTu to produce pSecUAG-A.

Sequence

>allo-tRNA^UTu (also referred to as allo-tRNA^UTul)

GGAGGGGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCTC GCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:31),

In vivo FDHH activity assay in E. coli

E. coli ME6 strain was transformed with indicated plasmids. The concentrations of the antibiotics were 5 or 7 μg/ml for tetracycline, 30 μg/ml for kanamycin, 34 μg/ml for chloramphenicol and 100 μg/ml for carbenicillin. Overnight cultures of transformed ME6 cells were spotted onto LB agar plates supplemented with 1 μΜ Na2Mo0₄, 1 μΜ Na2Se03 unless otherwise noted, 50 mM sodium formate, 0.1% L-arabinose, and antibiotics and grown anaerobically (90% N₂, 5% ¾, 5% CO2) at the room temperature for two days in an anaerobic tent (Coy Laboratories). When necessary, IPTG at final concentrations of 0.01 and 0.1 mM was also added into the agar plates. After incubation, these agar plates were overlaid with a top agar (0.75%) containing 1 mg/mL benzyl viologen, 250 mM sodium formate, and 25 mM KH₂P0₄ (pH 7.0) in the tent.

Results

E. coli tRNA^Sec and its EF-Tu-compatible variants (UTu, UTuX,

UTu6, and SecUx) (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445

₄5292₄6₄vl 119 (2013) ; Thyer, et al., J Am Chem Soc, 137:46-49 (2015); Miller, et al., FEBS Lett, 589:2194-2199 (2015); Fan, et al., ACS Synth Biol. (2017)) have a non- canonical 13-base pair (13-bp) amino-acid acceptor branch that is one of the important identity elements for E. coli selenocysteine synthase (SelA) that produces Sec-tRNA^Sec (Itoh, et al., Science, 340:75-78 (2013)). Sec-tRNA^Sec is synthesized in two steps in bacteria; seryl-tRNA synthetase (SerRS) attaches serine (Ser) to tRNA^Sec; SelA converts the Ser moiety to Sec by using selenophosphate synthesized by selenophosphate synthase (SelD) (Silva, et al, J Biol Chem, 290:29178-29188 (2015)). While SelB rejects Ser-tRNA^Sec, EF-Tu accepts both Ser-tRNA and Sec-tRNA molecules.

Therefore, the Ser-tRNAs must be quickly converted to Sec-tRNA to prevent Ser-tRNA translating UAG codons via EF-Tu (Thyer, et al., J Am Chem Soc, 137:46-49 (2015)). Although several studies improved the purity and the yield of recombinant selenoproteins (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013); Haruna, et al., Nucleic Acids Res, 42:9976-9983

(2014) ; Thyer, et al., J Am Chem Soc, 137:46-49 (2015); Miller, et al., FEBS Lett, 589:2194-2199 (2015); Fan, et al., ACS Synth Biol. (2017)), these tRNAs with a 13-bp branch may be less compatible than canonical tRNAs having a 12-bp branch in EF-Tu-mediated translation by the ribosome.

To overcome this drawback, experiments were designed to investigate another SelA species which can recognize tRNA^Sec with a 12-bp branch. A close relative of E. coli, Aeromonas salmonicida subsp.

pectinolytica 34mel, has one of such SelA and tRNA^Sec pairs (Mukai, et al., Angew Chem Int Ed Engl, 55, 5337-5341 (2016)). EF-Tu-compatible variants of A. salmonicida tRNA^Sec were designed, however, they were not good amber suppressors, probably because the tRNA^Sec tertiary structure may not be suitable for canonical translation via EF-Tu but is optimized for SelB-mediated codon recoding (Fischer, et al., Nature, 540:80-85 (2016)). Therefore, a search for tRNA^Ser species which may be recognized by A. salmonicida SelA was conducted. Bacterial tRNA^Sec species have a characteristic tRNA elbow structure composed of the D-loop (YGGU) and the T-loop (UUCRAYU) (Y denotes C/U, while R denotes G/A; the two Ys are either C-C or U-U pairs) (Mukai, et al., Angew Chem Int Ed Engl, 55,

45292464vl 120 5337-5341 (2016); Santesmasses, et al., PLoS Comput Biol, 13, el005383 (2017)). This elbow structure is recognized by the N-terminal domain of SelA. The Examples above identified a new group of tRNA^Sec-like tRNAs ("allo-tRNAs" named after their non-canonical cloverleaf structures), some of which were revealed as tRNA^Ser ( Mukai, et al., Nucleic Acids Res, 45:2776-2785 (2017)) (see also Examples above). Among them, one allo- tRNA species (referred to as "9/3-1" and "allo-tRNA^UTu") (Fig. 9A), derived from a metagenomic sequence, has a 12-bp branch and tRNA^Sec-like D- and T- loops and functioned as active tRNA^Ser in E. coli (Mukai, et al., Nucleic Acids Res, 45:2776-2785 (2017)) (see also Examples above).

The E. colifdhF gene encoding formate dehydrogenase H (FDHH) (Fig. 9C), one of the three selenoproteins in E. coli, was used as a reporter gene to check EF-Tu-mediated Sec incorporation (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013); Thyer, et al., J Am Chem Soc, 137:46-49 (2015); Miller, et al., FEBS Lett, 589:2194-2199 (2015)). The UGA codon 140 encoding the catalytic Secl40 residue was changed to UAG (Fig. 9C). Coexpression of the amber suppressor variant of allo-tRNA (9/3-1) (renamed "allo-tRNA^UTu") and A. salmonicida SelA (Fig. 1A) inserted Sec into the UAG 140 position and expressed wildtype FDHH which reduced benzyl viologen into a purple dye (Fig. 9C).

In order to further estimate the activity of allo-tRNA^UTu, four Cys codons were changed at positions 8, 11, 15 and 42 to UAG in fdhF. These four Cys residues are accommodating an important iron sulfur cluster (Fig. IB) (Boyington, et al., Science, 275: 1305-1308 (1997)). A preliminary study revealed that each of the four Cys residues can be separately replaced by Sec without impairing the FDHH activity but cannot be replaced by Ser (Fig. 9C). Thus, the reporter fdhF gene variants have one to five UAG codons which must be translated as Sec; premature translation stop by release factor 1 (RF- 1) and Ser incorporation by Ser-allo-tRNA^UTu make inactive FDHH. The allo-tRNA^UTu and A. salmonicida SelA pair enabled the translation of up to five UAG codons in the reporter fdhF gene variants (Fig. 9C). Thus, the

FDHH variants may have up to five Sec residues. However, premature stop or Ser incorporation was also occurring, since increasing the number of UAG

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45292464vl codons in i &fdhF reading frame decreased the FDHH activity of the cell spots (Fig. 9C).

Example 8: allo-tRNA^UTu improves yield of proteins containing multiple Sec residues relative to other tRNA^sec

Materials and Methods

>tRNA^UTuX

GGAAGATGGTGCCGTCCGGTGAAGGCGCCGGTCTCTAAAACCGGTCGACCCGAAAG GGTTCGCAGGGTTCGACTCCCTGCATCTTCCGCCA (SEQ ID NO:63) >tRNA^SecUx

GGAAGATGGTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCCA GCGGTCCCGGTCAGGTTCGACTCCTTGCATCTTCCGCCA (SEQ ID NO: 18)

Results

Next allo-tRNA^UTu with tRNA^UTuX (Miller, et al., FEBS Lett, 589:2194-2199 (2015)) and tRNA^SecUx (Thyer, et al., J Am Chem Soc,

137:46-49 (2015)). For proper comparison, the two tRNA sequences were first cloned the under the araBAD promoter in the same manner as allo- tRNA^UTu (Mukai, et al., Nucleic Acids Res, 45 :2776-2785 (2017)). The expression level of E. coli SelA for each of the tRNA^UTuX and tRNA^SecUx species were optomized, because excess SelA molecules completely sequestered these tRNA molecules (Fig. 11A- 11C). After the optimization step, it was revealed that tRNA^SecUx is more active than tRNA^UTuX and translated two UAG codons in anfdhF variant gene (Figs. 1 lA-1 1C and 9D). The FDHH activities of cell spots expressing tRNA^SecUx +fdhF(2 UAG codons) and tRNA^UTuX +fdhF(l UAG codon) were comparable (Fig. 1 1 A- 1 1C). Next, the allo-tRNA^UTu + As SelA pair and the tRNA^SecUx + Ec SelA pair were compared. The E. coli pair hardly translated three UAG codons in anfdhF variant gene (Fig. 9D), probably due to the competition with RF-1 (Cheng, et &l., J Biol Chem, 292:5476-5487 (2017)) which terminates translation at UAG and UAA. The FDHH activity of cell spots expressing allo-tRNA^UTu +fdhF( UAG codons) was higher than that of cells expressing tRNA^SecUx +fdhF(2 UAG codons) (Fig. 9D). Thus, allo-tRNA^UTu is apparently the best in terms of the yield of proteins containing multiple

122

45292464vl Sec residues. However, tRNA^SecUx might be superior to allo-tRNA^UTu in terms of the purity of yielded selenoproteins, because 100% pure (no Ser incorporation) recombinant selenoproteins were obtained in some cases under optimal conditions by using tRNA^SecUx (Thyer, et al., J Am Chem Soc, 137:46-49 (2015)).

Example 9: The conversion rate of Ser-allo-tRNA^UTu to Sec-allo- tRNA^UTu can be improved by modifying the translation system

Materials and Methods

Construction ofplasmids

The A. salmonicida 34mel selD gene with its native promoter was amplified by PCR from the same genomic DNA and cloned immediately downstream of the SelA ORF of pSecUAG-A together with an intervening E. coli trp terminator sequence. The AUG start codon of the A. salmonicida selD gene was changed to GUG (designated as As selD') by Infusion to produce pSecUAG-AD. The allo-tRNAUTu sequence of pSecUAG-AD was mutated by Infusion to make allo-tRNAUTu variants. The A. salmonicida SelA expression cassette (but not the Ttrp) was removed from these allo- tRNAUTu variant-carrying plasmids to produce pSecUAG-D series. Thus, the Ttrp is remaining between the kan gene and the As selD ' gene in pSecUAG-D series. The ORF of an Enterobacter cloacae NMC-A variant carrying two UAG codons was cloned between the kan gene and the Ttrp of pSecUAG-D-allo-tRNAUTulD to develop pNMC-A(2xAm). The codon usage of NMC-A was optimized for use in E. coli (see below). The ORF of Treponema denticola Trxl(32UAG) (Kim, et al., Biochem Biophys Res Commun 2015, 461, 648.) was cloned immediately downstream of the As selD' gene together with a linker sequence including a Shine-Dalgarno (SD) sequence (5'-TCACACAGGAAACAGACC-3') (SEQ ID NO:76) in pSecUAG- AD to make pSecUAG-ADT. The allo-tRNAUTu sequence of pSecUAG- ADT was also mutated by Infusion to make allo-tRNAUTu variants. The araC-ParaBAD in pSecUAG-ADT plasmids was replaced with core sequences of YargW and YselC (Mukai, et al., Nucleic Acids Res 2015, 43,

8111.) by PCR using primer sets

(5'-CTACTGTTTCTCCATACCCGTTTG-3' (SEQ ID NO: 103)) and

45292464vl 123 (5'-TTTGAATTCAAACATGCGGCATGAGTATACCCGCTAATGGAGTGCGG GGTAAGTACGCTGGTAACGAATCAGACAATTGAC-3' (SEQ ID NO: 104) ΟΓ

5'-TTTGAATTCAAACGGGGCGCATTATAGCTACTTCCTTGAGTTTCT ACATCCCCCAGATCGGTAACGAATCAGACAATTGAC-3' (SEQ ID NO: 105)) followed by EcoRI digestion and ligation with T4 DNA ligase.

For expressing the wildtype As SelA with Ybla, the kan gene and the As SelA ORF were linked with a linker sequence (5'-TAACCCGCCAGGT TTCCTTAATTGTG-3' (SEQ ID NO: 106)) including a SD sequence and a GUG start codon for As SelA. The pSecUAG-ADT-allo-tRNAUTu2D plasmid was modified to express the As SelAEvol variant (#2.1 variant with a Pro2-to-Thr2 mutation). The ORF of the As SelAEvol variant starts with GTGACG (encoding the start Met codon and the next Thr codon), while its promoter and SD sequences were modified by Infusion; 1) EM7 promoter with the native SD sequence (5' -GAGGAACTAAACC-3' (SEQ ID NO: 107)) to make pSecUAG-Evol2; 2) EM7 promoter with a weaker SD sequence (5'- GCCAGGTTTCCTTAATT-3' (SEQ ID NO: 108)) to make pSecUAG-Evol3; 3) directly linked to the Ybla-kan gene with a linker sequence (5'- TAACCCGCCAGGTTTCCTTAATT-3' (SEQ ID NO: 109)) to make pSecUAG- Evol4. To make pSecUAG-Evoll, the GTG start codon of SelAEvol was changed to ATG. The "A-S-S-A-S" (SEQ ID NO: 110) and "P-Y-R" amino- acid sequences of the two SelAEvol variants are encoded with "GCC-AGT- TCT-GCT-AGT" and "CCT-TAT-CGC" codons, respectively. The E. coli sufS wildtype and C364A (Mihara, et al., J Biochem 2000, 127, 559) genes were cloned between the kan marker and the EM7 promoter of pSecUAG- Evoll/2 (see the plasmid map below). A low-copy-number plasmid pMW219 (NIPPON GENE) was modified to carry a cat gene instead of the kan marker and the multiple cloning site to make pMWcat by Infusion using the following primer sets (5'-ccact ccaagaattgCAAAAAGGCCATCCG TCAGG-3' (SEQ ID NO:65) and 5'-cgtgtgctt ct caaaGAGCGCAACGC AATTAATGTG-3' (SEQ ID NO:66)) and (5'-TTTGAGAAGCACACGGTCAC-3' (SEQ ID NO:67) and 5' -CAATTCTTGGAGTGGTGAATC-3' (SEQ ID NO:68)). The As SelA expression cassette (with the Tirp) was cloned in front of the

124

45292464vl cat gene in pMWcat to make pMWcat-AsSelA(AUG) by Infusion using the following primer sets

(5' -TCCCACAGCCGCCAGTTCCGCTGGCGGCATTTTACCCGA CGCACTTTGCGCCG-3' (SEQ ID NO:69) and

5'-AGGCCCTTTCGTCTTCAAG-3' (SEQ ID NO:70)) and

(5'-aagacgaaagggcctCACGTGTTGACAATTAATCATCG-3' (SEQ ID NO:71) and

5'-ctggcggctgtgggaTCAGGGCTCCTCGGTCGCAG-3' (SEQ ID NO:72)). The AUG start codon for the As SelA gene on pMWcat- AsSelA(AUG) was changed to GUG to make pMWcat-AsSelA(GUG). Note that pMWcat-AsSelA(GUG) used for mutant library construction has a deletion in the stop codon. This deletion was seemingly harmless but corrected by Infusion to make true pMWcat-AsSelA(GUG). The E. coli selA gene with its native promoter was amplified by PCR from a genomic DNA and cloned immediately downstream of the kan marker gene together with an intervening E. coli trp terminator sequence in pBAD-RSF5 carrying tRNAUTuX or tRNASecUx. The start codon of the E. coli selA gene was mutated by Infusion. In particular, pSecUx-A has tRNASecUx and an E. coli selA gene variant carrying a GUG start codon with a short insertion

"UAAUU" in front of it.

The plasmid vector pTrc99A was used for the inducible expression of a few enzymes. For cloning the ORFs of A. salmonicida SelA and phosphoseryl-tRNA kinases (PTSKs) of Trypanosoma brucei (Aeby, et al., Proc Natl Acad Sci U S A, 106:5088-5092 (2009)) and Homo sapiens, they were cloned between CACACAGGAAACAGACC (SEQ ID NO:73) and

TGTTTTGGCGGATGAGAGAAG (SEQ ID NO:74). The codon usage of the human PSTK was partially optimized for use in E. coli (see below). The ORF of Sep-tRNA:Cys-tRNA synthetase (SepCysS) from the Parcubacteria DG_74_2 bin (Mukai, et al., MBio, 8, e00561-00517 (2017)) was cloned after the PSTK ORFs with a short upstream sequence including a ribosome binding site (TTTTAAGAAGGAGATATACAT (SEQ ID NO:75)). The plasmid vector pETDuet-1 (Novagen) was used for the inducible expression of

45292464vl 125 selenoproteins. The ORF of a human GPxl(49UAG) variant having additional N-terminal MetGly sequence (for Ncol site) and a C-terminal Leu- Glu-His-His-His-His-His-His (SEQ ID NO:95) tag (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013)) was transferred from pRSFDuet-1 plasmids into the Ncol and Hindlll sites of pETDuet-1. The ORF of the Grxl(Cl lU/C14S) variant was amplified by PCR using primer pair GRX F and GRX R (Aldag, et al., Angew Chem Int Ed Engl 2013, 52, 1441.) and cloned into pETDuet-1 to develop pET-Grxl(HUAG/14Ser). The ORF of the Mxe GyrA intein variant with the in- frame UAG 384 codon had been cloned in plasmid MXB.RSF (Haruna, et al., Nucleic Acids Res 2014, 42, 9976) The expression cassette of this intein reporter gene was transferred to pET-Duetl by PCR using the following primer sets (5' -ggttttttgctga aaGGAGGAACTATATCCGGATTG-3' (SEQ ID NO: 111) and

5'-GAAAGCGGGCAGTGAGCGCAAC-3' (SEQ ID NO: 112)) and

(5'-tttcagcaaaaaacccctCAAG-3' (SEQ ID NO: 113) and

5'-TCACTGCCCGCTTTCCAGTC-3' (SEQ ID NO: 114)) to develop pET- MXB(384UAG). pMWcat-Se was constructed to express proteins and enzymes putatively involved in Se-transfer (Thyer, et al., J Am Chem Soc 2015, 137, 46; Pryjma, et al., J Bacteriol 2012, 194, 3803; bR. Hatrongjit, K. Packdibamrung, Enzyme and Microbial Technology 2010, 46, 557; cS.

Nelson, Electronic Theses and Dissertations 2014, Paper 4807] as shown in its plasmid map (see below). pBAC8z was constructed by replacing the ORF of the gent marker gene of BAC7gent (Mukai, et al., Nucleic Acids Res 2010, 38, 8188.) with an artificial cistron of Sh ble and sucB(TAA stop codon) by λ-Red-mediated recombination in BAC7 gent-carrying Stellar cells using pKD46 (Datsenko, et al., Proc Natl Acad Sci USA 2000, 97, 6640)

Sequences

>Trypanosoma brucei pstk

ATGACAGTTTGTCTTGTTCTACTAACTGGGCTGCCAGGAGCGGGGAAGACGACACT AGGCAAGGCTCTTAAACAGTTGGGGGATCACATAACCCATGAACTCTCCCTCATAG TCACGGCAGTGGTGGAATTAGATGACTTTATGTGTAACGTCGGTGCGAGTAATGGG TCCCGTGTAGAGAGTACCGTTTTCGATCCAAGTCGGTGGCGAGAGGCGTTCGAAGC GGCTCGTCAGGCAACTCGCCAGGAGTTGGAGCGGTGCCTAATGATGGAGAGGAATA

45292464vl 126 AAGCGGTAATGCACTTGGTTTTTCTGGTGGATCCGCTGCCATATAGGAGTATGAGA GCATCGTACTGGAAAATGTGCAAGGAATTAAGTGCCAAGTGTGCTGAGACTCACTT TCATGATTCATGGGAAGTGCAGAGCATTGTTGTCTTGTTGGAGGTGCGGATGAACA CCCCGGAGGAGGTTTGTCTCCAACGCAATGAGCTCCGCGCCGGAACCCCGCAGTAT ATTCCCCCGTATGTTATTAAGGGGATAAGTGACTCGTTTGACCGTGGTGACCTCAC TGCTGTGCTGCTGGGTACAGACGGAAATATGTGGGCCGTACTTCCCGGGCAGAAGT CGGCACCGTGGCCCGTTCTTTTACTGGTTGATGAAGTGAGATGCTGCGCGTCACCA CCCAATTTGTTGGCCACGCAGTTGCTGGAGCGTATCCGAGGGGAAGACATAATGCG TGAGATGACGGAACAACAAGTAAGTGTTTTTAATTATTACAAGTGCCAAGTGGAAG GGGGGAAGTCGAAGTGTTTGGCGAGTGGAGAAGCACATGACAACGTTAACAACTGT CTTCATCAAGTGGACCTCCACATGCGGGCAGTTGTGGGACATTACATGGTCGAGCG GCAGAGTAGTGGTTCACTGAAGCCAGGCACTGGGCAACGCGTAAGCAAATGTCGGT C G AC C C AC T AC G C G G G AAT T C G C G C AG C AAT C AC G AAG GG AAC G AG AAAC AC AG G A GGATCTTTTTCCGAAGTGCAAGGACTACTGCAGCAGTTACTTTTGGAATTCGAGCA TGCCTTAGTAGATCTTTAA (SEQ ID NO:89)

>Homo sapiens pstk isoform XI

ATGAAAACCGCAGAAAATATTCGTGGCACCGGTTCAGATGGTCCGCGTAAACGTGG TCTGTGTGTTCTGTGTGGTCTGCCTGCAGCAGGTAAAAGCACCTTTGCACGTGCCC TGGCACATCGTCTGCAGCAAGAACAAGGTTGGGCAATTGGTGTTGTTGCATATGAT GATGTTATGCCGGATGCATTTCTGGCAGGCGCACGCGCACGTCCGGCACCGAGTCA GTGGAAACTGCTGCGTCAAGAACTGCTGAAATATCTGGAATATTTCCTGATGGCCG TGATTAATGGTTGTCAGATGAGCGTTCCGCCTAATCGTACCGAAGCAATGTGGGAA GATTTTATCACCTGTCTGAAAGATCAGGACCTGATTTTTAGCGCAGCATTTGAAGC ACAGAGCTGTTATCTGCTGACCAAAACAGCAGTTAGCCGTCCGCTGTTTCTGGTTC TGGATGATAATTTCTATTATCAGAGCATGCGCTATGAGGTTTATCAGCTGGCACGT AAATATAGCCTGGGTTTTTGTCAGCTGTTCCTGGATTGTCCGCTGGAAACCTGTCT GCAGCGTAATGGTCAGCGTCCGCAGGCACTGCCTCCGGAAACCATTCATCTGATGG GT C G T AAAC T G G AAAAAC C GAAT C C G G AAAAAAAT G C C T G G G AAC AT AAT AGC C T G ACCATTCCGAGTCCGGCATGTGCAAGCGAAGCAAGCCTGGAAGTTACCGATCTGCT GCTGACCGCACTGGAAAATCCGGTTAAATATGCCGAAGATAACATGGAACAGAAAG AT AC C GAT CGCATTATTTG C AG C AC C AAC AT T C T G CAT AAAAC C G AT C AG AC C C T G CGTCGTATTGTTAGCCAGACCATGAAAGAAGCAAAAGATGAACAGGTTCTGCCGCA TAATCTGAAACTGCTGGCAGAAGAACT GAAT AAAC TGAAAGCAGAATTCCTTGAGG ATCTGAAACAGGGCAATAAAAAGTATCTGTGTTTTCAGCAGACCATCGATATTCCG

127

45292464vl GATGTGATCAGCTTTTTCCACTATGAGAAAGATAACATCGTGCAGAAATACTTCAG CAAGCAGCATTAA (SEQ ID NO:90)

>Treponema denticola Trxl(32UAG)

ATGATTATGGCAGTATTGGATATTACAAATGCTAATTTTGATGAAACCGTtAAgAC CGCCAAGCCCGTTTTAATTGACTTTTGGGCACCGTGGTAGCCGGGATGCGTACAGC TCAGTCCTGAGCTGCAGGCTGCCGAGGCGGAACTCGGCGACAAGGCTGTGATAGCA CAGTCTAACGTGGATAATGCACGTGAATTGGCAGTAAAATTTAAGTTTATGTCAAT ACCTACCCTCATCGTTTTAAAAGACGGAAAAGAGGTGGACAGGCACACAGGCTATA TGGATAAgAAGAGCCTTGTAAACTTTGTTTCAAAGCATATCTAA (SEQ ID

NO:91)

>pSecUAG-AD plasmid annotation

misc_feature complement(5998..6876)

misc_feature 6906..127

/\abe\=ParaBAD

misc_feature 151..240

/label=allo-tRNA^UTu

misc_feature 262..290

/label=Tm?C

misc_feature 366..523

/labe mfi Tl and T2

misc_feature join(946..1982,1984..2116)

/label=As selD with a GUG start codon

misc_feature complement^ 117..2149)

/label=T7?p

misc_feature complement(2150..3708)

/label=As SelA with an EM7 promoter

45292464vl 128 >pSecUAG-AD

GTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTG CTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTAT CGCAACTCTCTACTGTTTCTCCATACCCGTTTGAATTCggaggggaacttctatct ggtgatagacgggaactctaaattccttgaaatgcctcgccgcattgggttcgatt CCCttCCCCtCCgccaGGATCTAGAGTCGACCTGCAGATCCTTAGCGAAAGCTAAG GATTTTTTTTAGGAATTAACCATGGATCCGAGCAGCCTGATACAGATTAAATCAGA ACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCC CACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTG GGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTC AGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTG AGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGG GTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCA TCCTGACGGATGGCcgcggccGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAA ACCTCAGGCATTTGAGAAGCACACGGTCACACTGCTTCCGGTAGTCAATAAACCGG TAAACCAGCAATAGACATAAGCGGCTATTTAACGACCCTGCCCTGAACCGACGACA AGCTGACGACCGGGTCTCCGCAAGTGGCACTTTTCGGGGAAATGTGCGCGGAACCC CTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATCATTGG ATAACCTCGATAAACTGGTTTCCGGTATAGGCTTTCAGCTGGCCTATGGGGGAGAG GGTCAGCCCCGCTTGTGTCGCGATAGCAAGGAGCTCGGCTTCACTTTCTTTACCGA CGGCAACCAGCAAGCCGCCGCTGGTCTGCGGATCGCACATGATGTTGCGGGTGCGT TCATCCATGGCACCGAGCTTGGCGCCATAGGAATCGAAGTTGCGCAGGGTACCGCC CGGTACGCAGCCCTCGGACAGGTAGTAATCTACTTCGTCGAGCAGTGGCAGCGCCT TGAAATCGAGGGTGGCACACACCCCTGAGCCTTCGCACATCTCAAGCAGGTGTCCC GCCAGGCCAAACCCGGTCACATCCGTCATGGCGTGCACGCCGGGCAGTTCGGCAAA GCGCTGGCCAATCTTGTTGAGGGTGCACATGGCGTTGGGGGCCAGCTGCTCATGCT CTGGCTTCAATTTGCCCTTCTTCTGGGCCGTGGTGAGGATGCCGATACCGAGGGGC TTGGTCAGGTAGAGGATGTCACCCGCCTGGGCCGTGTCGTTCTGCTTGATGGCATT GAGCGGCACTATACCGGTCACAGCAAGACCGAAGATGGGCTCGGGGGCATCGATAC TGTGGCCGCCAGCCAAGGATATGCCCGCTTCATGGCACACCTGGCGGCCGCCATCT ATCACCTGCTGGGCCACTTCCGGGGCTAGGGTGTTGATGGGCCAGCCAAGGATGGC AATGGCAACGATGGGCTTGCCGCCCATGGCGTAGATGTCGCTGATGGCGTTGGTGG CCGCGATGCGGCCAAAGGTAAAGGGATCATCGACGATGGGCATGAAGAAGTCGGTG GTGGAAACAATGCCCTGACCGTTGCCGATATCGACCACGGCCGCGTCATCCTTGCT GCTGTTGCCAACCACCAGGGTCGGGTCGTCAAAGCCCGGGATCTGGCTCTTGAGAA

45292464vl 129 TGGTGTCGAGCACCTTGGGAGAAATTTTGCAGCCGCAGCCAGCCCCGTGGCTGTAT TGGGTCAGACGAATGGAAGACACgattaccccttgtttggctgtttctcaagatga aacagcgtatatcaggcaaaaggagatgaccctgagcgggccattggacagggcat tatgccacaaggactctgcgggttcgaatcacaatagcctgtcgAAAATGCCGCCA GCGGAACTGGCGGCTGTGGGAtcagggctcctcggtcgcagtggtcggagtgggca gtagcaactccttgagttgggcgataagcagtgcaatctcggtcggcagcagggtc gccatattgagcagcaccttctgctggcgcacggtggcgatgaccggcaccggcag tttccgcagggcatcgagcagctgctgggccggacgcgggtcggtgcattcgagcg caggcgcagggtagaactcgtccggcagggtgccaccacccaccaccagctgggcg gggaccggcacaaagcagccgggcagggcggccatcagctgatcggcgcgggcctg catggcggcagggttgctcaaggtgcgctgggcgatgccctcgccgatgggggact tgttgagcttgtggatgagcaggcgttccagcagggagtagacgatgcggctcggg cggaaggtgcgcatcatggggtgtttttccagccgcttgatgaggtcgctgcggcc gctgatgatgcccgattgcgggccacccagcagcttgtcgccggagtagcagacca gatccgcccccgccttgatgtactgacgcaccgaggtttcgtccggtgcaaactcc tcggtggtcaagcccgagccctgatccaccgccagcaccacgtgctcgggcagggc gcgggccacctcgccaatatcgggggattcggtaaagccgcgaatggcgaaattgg atctgtgtaccatcagcaccagcgcggtctgatctgtgatggcatcgaggtaatct ttggcggtagtgatattggtggtgcccacctccaccagtttggcgccggagagcgc cagaatgtcgggaatgcgaaagccgccaccaatctggatctgttcgccccgcgaga cgatcacctcgcgccccttggctatctcctgcagcagcaagaagagcgaagcggcg ttgttgttgaccaccagcgaatcctcggcctgggtgaggcaacggagcaggggggc gatcagecccttgcgcccgccgcgcttgccggtggcgagatccagttccagattgt tgtagccagtgttgaggtcgcgcacctcgtcccacagctcgcgacttagcggcgag cgccccagattggtgtgcaccagggtgccggtggcgttgatcacccgggtctgacg ttggcgcagctgctgctggcaacgcttggcaatcagtgcctcgatttgctcggggg caaccccatgctggcgaaatgcctcgctctggcgcaattcgctcaggacatcgcgc accgcctgggtcaccagcgggcggctcagcgcctcgataaaaccggtgagaaaggg ttgctgcagcagctgttccacttgcggtagacggcgcgcttgttgctggctgggct gtggcagtgaatctggcagtgaatctggcagtgaatcgtcggcagtgggacatgat tcgggctgactgtgagagtgggcgatggctggcgcgtgagacgagttcggcatggt ttagttcctcaccttgtcgtattatactatgccgatatactatgccgatgattaat tgtcaacacgtgTTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAAT TTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGA AGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTG CGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATA

45292464vl 130 AGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAA AAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCAT CAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGA CGAAATACGCGGTCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCG GCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTT CTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCA TCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCA GTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTT TCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCT GATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTT GGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATACTCT TCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATAC ATATTTGAATGTATTTAGAAAAATAAACAAATAGGCATGCAGCGCTCTTCCGCTTC CTCGCTCACTGACTCGCTACGCTCGGTCGTTCGACTGCGGCGAGCGGTGTCAGCTC ACTCAAAAGCGGTAATACGGTTATCCACAGAATCAGGGGATAAAGCCGGAAAGAAC ATGTGAGCAAAAAGCAAAGCACCGGAAGAAGCCAACGCCGCAGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGCCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGG GAAGCGTGGCGCTTTCTCATAGCTCACGCTGTTGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGC CTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCAC TGGCAGCAGCCATTGGTAACTGATTTAGAGGACTTTGTCTTGAAGTTATGCACCTG TTAAGGCTAAACTGAAAGAACAGATTTTGGTGAGTGCGGTCCTCCAACCCACTTAC CTTGGTTCAAAGAGTTGGTAGCTCAGCGAACCTTGAGAAAACCACCGTTGGTAGCG GTGGTTTTTCTTTATTTATGAGATGATGAATCAATCGGTCTATCAAGTCAACGAAC AGCTATTCCgggccggcCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCC TGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCA GCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATG CGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCT CAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCT ACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCT GACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGG AGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCAGAT CAATTCGCGCGCGAAGGCGAAGCGGCATGCATAATGTGCCTGTCAAATGGACGAAG

45292464vl 131 CAGGGATTCTGCAAACCCTATGCTACTCCGTCAAGCCGTCAATTGTCTGATTCGTT ACCAATTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACG GAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTT GATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTC AAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAAT CCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCT GTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGA CAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTC C AT G C G C C G C AG T AAC AAT T G C T C AAG C AG AT T T AT C G C C AG C AG C T C C G AAT AG C GCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGC TGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTT AAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATT CGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAA ATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACC GCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAA AATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTA AACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACT C C C G C C AT T C AG AG AAG AAAC C AAT T G T C C AT AT T G C AT C AG AC AT T G C C G T C AC T GCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGC ATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGT (SEQ

ID NO:92) pMWcat_Se

FEATURES Location/Qualifiers

misc_feature 1..392

/label=partition (par) locus

misc_feature 482..492

/label=dnaA binding

misc_feature 547..558

/label=IHF binding

misc_feature 577..601

/label=RepA binding

misc_feature 692..697

/label=-35

45292464vl 132 misc_feature 714..719

/label=-10

misc_feature 750..1700

/label=repA

misc_feature 1614..1614

/label=T

misc_feature complement(2020..2906)

/label=cat

misc_feature complement(2907..4654)

/label=Ec trxA(Amb)-BstFDH

misc_feature complement(4655..4912)

/label=Ec ytiA

misc_feature complement(4926..6519)

/label=Ec yedEF

misc_feature complement(6520..6553)

/label=Trho

misc_feature complement(6561..7727)

/label=Td SCL

misc_feature complement(7740..7789)

/label=PcsdA

misc_feature complement(7790..8050)

/label=Td DUF3343

misc_feature complement(8066..9283)

/label=Ec trxB

ORIGIN

GACAGTAAGACGGGTAAGCCTGTTGATGATACCGCTGCCTTACTGGGTGCATTAGC CAGTCTGAATGACCTGTCACGGGATAATCCGAAGTGGTCAGACTGGAAAATCAGAG GGCAGGAACTGCTGAACAGCAAAAAGTCAGATAGCACCACATAGCAGACCCGCCAT AAAACGCCCTGAGAAGCCCGTGACGGGCTTTTCTTGTATTATGGGTAGTTTCCTTG CATGAATCCATAAAAGGCGCCTGTAGTGCCATTTACCCCCATTCACTGCCAGAGCC GTGAGCGCAGCGAACTGAATGTCACGAAAAAGACAGCGACTCAGGTGCCTGATGGT CGGAGACAAAAGGAATATTCAGCGATTTGCCCGAGCTTGCGAGGGTGCTACTTAAG CCTTTAGGGTTTTAAGGTCTGTTTTGTAGAGGAGCAAACAGCGTTTGCGACATCCT

45292464vl 133 T T T G T AAT AC T G C G G AAC T GAC T AAAG T AG T G AG T T AT AC AC AG G G C T GG G AT C T A TTCTTTTTATCTTTTTTTATTCTTTCTTTATTCTATAAATTATAACCACTTGAATA TAAACAAAAAAAACACACAAAGGTCTAGCGGAATTTACAGAGGGTCTAGCAGAATT TACAAGTTTTCCAGCAAAGGTCTAGCAGAATTTACAGATACCCACAACTCAAAGGA AAAGGACTAGTAATTATCATTGACTAGCCCATCTCAATTGGTATAGTGATT AAAAT CACCTAGACCAATTGAGATGTATGTCTGAATTAGTTGTTTTCAAAGCAAATGAACT AGCGATTAGTCGCTATGACTTAACGGAGCATGAAACCAAGCTAATTTTATGCTGTG TGGCACTACTCAACCCCACGATTGAAAACCCTACAAGGAAAGAACGGACGGTATCG TTCACTTATAACCAATACGCTCAGATGATGAACATCAGTAGGGAAAATGCTTATGG TGTATTAGCTAAAGCAACCAGAGAGCTGATGACGAGAACTGTGGAAATCAGGAATC CTTTGGTTAAAGGCTTTGAGATTTTCCAGTGGACAAACTATGCCAAGTTCTCAAGC GAAAAATTAGAATTAGTTTTTAGTGAAGAGATATTGCCTTATCTTTTCCAGTTAAA AAAATTCATAAAATATAATCTGGAACATGTTAAGTCTTTTGAAAACAAATACTCTA TGAGGATTTATGAGTGGTTATTAAAAGAACTAACACAAAAGAAAACTCACAAGGCA AAT AT AG AG AT T AG C C T T GAT G AAT T T AAG TTCATGTTAATGCTT G AAAAT AAC T A CCATGAGTTTAAAAGGCTTAACCAATGGGTTTTGAAACCAATAAGTAAAGATTTAA ACACTTACAGCAATATGAAATTGGTGGTTGATAAGCGAGGCCGCCCGACTGATACG TTGATTTTCCAAGTTGAACTAGATAGACAAATGGATCTCGTAACCGAACTTGAGAA C AAC C AG AT AAAAAT G AAT GG T GAC AAAAT AC CAACAACCATTACAT C AG AT T C C T ACCTACATAACGGACTAAGAAAAACACTACACGATGCTTTAACTGCAAAAATTCAG CTCACCAGTTTTGAGGCAAAATTTTTGAGTGACATGCAAAGTAAGCATGATCTCAA TGGTTCGTTCTCATGGCTCACGCAAAAACAACGAACCACACTAGAGAACATACTGG CTAAATACGGAAGGATCTGAGGTTCTTATGGCTCTTGTATCTATCAGTGAAGCATC AAG AC T AAC AAAC AAAAG T AG AAC AAC TGTTCACCGT T AC AT AT C AAAGG G AAAAC TGTCCATATGCACAGATGAAAACGGTGTAAAAAAGATAGATACATCAGAGCTTTTA CGAGTTTTTGGTGCATTCAAAGCTGTTCACCATGAACAGATCGACAATGTAACAGA TGAACAGCATGTAACACCTAATAGAACAGGTGAAACCAGTAAAACAAAGCAACTAG AACATGAAATTGAACACCTGAGACAACTTGTTACAGCTCAACAGTCACACATAGAC AGCATTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATT C T G C C G AC AT G G AAG C C AT C AC AG AC G G C AT G AT G AAC C T G AAT C G C C AG C GG C AT CAGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGA AGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCACCCAGGGATTG GCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAAATAGGCCAGGTTTTC ACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGT GGTATTCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAA CAAGGGTGAACACTATCCCATATCACCAGCTCACCGTCTTTCATTGCCATACGGAA

45292464vl 134 TTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACT TGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTC TGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATG CCATTGGGATATATCAACGGTGGTATATCCAGTGATTTTTTTCTCCATTTTAGCTT CCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGATCTTATT TCATTATGGTGAAAGTTGGAACCTCTTACGTGCCGATCAACGTCTCATTTTCGCCA AAAGTTGGCCCAGGGCTTCCCGGTATCAACAGGGACACCAGGATTTATTTATTCTG CGAAGTGATCTTCCGTCACAGGTATTTATTCGGCGCAAAGTGCGTCGGGTTTAGGT CAGACGATAGCTCTGTGCACCTGTACCTGCCAGTGTACCACCATCCACAATCAGAT ATTCGTTACGAATCGGTTTACCATCAAACCAACACTGCAGAATTTCCAGGGTGCCT GCTGCATAACGTGCCTGTGCGCTCAGGCTGGTGCCGCTAATATGCGGTGTCATACC ATTAAACGGCATGGTACGCCACGGATGATCTGCCGGTGCAGGCTGCGGAAACCAAA CATCACCACCATAACCTGCCAGATGACCGCTGGTCACTGCACGAACAACGGCATCA CGATCAACCAGTTTTGCACGTGCGGTATTAACCAGATAGGCACCACGTTTCATACG TGCAATCATGGCTGCATCAAACAGGTGTTCGGTGCTCGGATACAGCGGAATCTGCA GGTTAACAATATCAACTGCGCCTGCCAGGCTTGCTGCATCGGCATGATAGGTCAGG GCCAGTTCTTGTTCAATTGCGGCATCCAGACGATGACGCTGGGTATAATGCAGATG C AG AC C AAAC G G T T T C AG AC G AC G C AG AAC T G C C AG AC C AAT AC G AC C T G C AC C AA CGGTGCCAAAATGCATACCTTCAACATCATAGCTACGGCTAACACAATCTGCAATA TTCCAACCACCTTGCTGTGCAATTTCATGGCTCGGCAGATAATTACGAACCAGTGC CAGGGTTGTCATCACAACATGTTCGGCAACGCTAATGCTATTGCTACCGGTAACTT CTGCAACGGTAATATGTGCACGTGCTGCTGCATCCAGATCAACATGATCGCTACCA ATACCTGCTGTCAGTGCCAGTTTCAGTTTCGGTGCACGGGCAATACGTTCTGCGGT C AG AT AT G C AG G C C AAAAC GG C T G G C T AAT AAC AAC AT C T G C T T C C G G C AG AC G AC GTTCAAATTCGCTATCCGGACCATCTTTATCGCTGGTAACAATCAGGGTATGACCA TGTGCTTCCAGATAACCACGCAGACCCAGTGCACCGCTAACGCTACCAACCAGTTC ACCCGGACGAAAACCCAGCGGACCTGCCGGTGTCGGTGCGGTCTGACCATCTGCAT ACTGGGTAATAACCGGAATTGCATCACGAACATAACGAGGCGGATAACCATCAACA GGATCCGGATACAGAACACACAGAACGGTTGCAGAGCCAGAACCAGACGCCAGGTT AGCGTCGAGGAACTCTTTCAACTGACCTTTAGACAGTGCACCCACTTTGGTTGCCG CCACTTCACCGTTTTTGAACAGCAGCAGAGTCGGGATACCACGGATGCCATATTTC GGCGCAGTGCCAGGGTTTTGATCGATGTTCAGTTTTGCAACGGTCAGTTTGCCCTG ATATTCGTCAGCGATTTCATCCAGAATCGGGGCGATCATTTTGCACGGACCCTACC ACTCTGCCCAGAAATCGACGAGGATCGCCCCGTCCGCTTTGAGTACATCCGTGTCA AAACTGTCGTCAGTCAGGTGAATAATTTTATCGCTCATATATAACTCCACAGGAAT AAGCCTGGCGTGTTGGTTTCGTTGTTGGTGTAACATTAACCAACTAAAGGTTGACT

45292464vl 135 T T AT T T C AC C G G AT AC G C T T T C G T AAAG C AAT AG T AAG C T G AT AT T C T AC C AC AC T ATGAGCAAAACACATTTAACAGAACAGAAGTTTTCCGACTTCGCCCTGCATCCGAA GGTTGTAGAAGCCCTTGAAAAAAAAGGGTTTCATAACTGTACGCCCATTCAGGCAC TGGCCCTTAACGCGGAGAAACCCCGTAATGTGCAATGCAGCGCCAGACACCATCCT GCTGGCAATAAATTGACTCAGTCAGCCCAGGGATCACCCATTGTATTTCCTCGCCG GGGGGACAGGTCAGCCAAATGCATAACCCGCAGCCGCCGCGTAAATCACGGGGAAT ATCGCTGACGCGAAAAGTCATGCCCGCTGCCTGCAACGCTTTGCGGGTTTGTATGA CGCCGACCGTGGAGTGAAATAAAAATAAAAACTCTTTCATCGGCGTGTCCTCATTA CTTCTGAATTAAATAACGAATGGTCGGCCCGTCTTGCTGAATATCCAGCACCGTAT AGCCGTGATTACGCGCATCCAGTGGAATATTATTGATCGACTGCGGACAGTCGCTC ACCACTTCCAGGATTTCCCCTTTTTTTAACTGCGGCATCGCCTCAAGGGTTGCGAC TGCCGGATAAGGGCAGGGTTCACCCACCATATCAAGGCGGTAATCAGGAACGATAT TTTTCATGCGATCTCCTTAGCAGTCTGCGGTGCCGCACGGCGGAAGAAGCGTTTTT CCCAGCCGATAATCAACATCAGCGCAGCAAACAACAGCAAATATGTCACCAGCAGG CCACCCATCGGACCAAAGGTTTTCAGCAGGTTGATTTTGTCCCAGTCGGTGGCCAG CGCCGGAGCGAAATCATCCCAGTAATACGCCAGAATCGTTGAGCCGATCACATTGC CCAGACCGACCCACCAGTAGTGCACCTGGCCTTCTACCGCGCGGTACATCCAGCCG GTTTCGCAGCCGCCAGCCAGCACGATGCCAAAACCAAACAGTAAACCACCAATTAC CGCGTTTGGTCCCGCCCACATGATTTTGGGTTCAACGCCTAACTGTACGTAACTGA AGATCCCGATGGCACTCACCGCCATACCGATAATGATTGCTTTCGCCATATGGGTA CGTCCGGTGATCCACATATCGCGGAACGCTGAAGTAAAGCAGATTTGCGCACGTTC AATCAGTAAACCAAAGCCGACGCCAAACAGCATTGCCAGCCCCAGTTTGGGTTGGT TCATCGCTGTGAGCAGCGCCCAGCCCAGCATGCCGAAAAAGACCAGCATCCCGAGA CGAAAACGACGCCGCGCCTGATCCGGTTTTTGCGTCAGCGGTGAGGCGGCAGAAAC TTTCTGCATTTTCACGGGAATACGGAAGATGGGCAGCAGGGTAAAGCGCGCGCCAA AC C AT G AAC C AAT G G C AG T GG C G AT G G C AAAG AAC C AG GC AT G C AG C G AG AAC T G A GGAATACCGGTAAAGAACGCCGCCAGGTTACAGCCCATTGCCAGACGCGCGCCAAA ACCGGCGATAATGCCGCCAATGATGGCCTGCATAATGCGGATACGGCTGCGCGGCA TTCGCAGTTTGACATTGTTGGCCCACAGCGCTGCGGCAAAGCAGCCGCCAAACATA CCGAGGATCATCATCCCGTCGATGCGGGTTAATGGCGATCCTTCCAGATGGATAAT TTTAAAGTAACCCCACTCTTCAGCATGGACGCCGAACAGTTGCAGGAGCTGGCCGC CCCAACGGGTAAATTCACCCGTGACAGCCCAAAAGGTGCCAGTAATGCCAAAATAG T AAG T AG AG AG AAT AC C C G C C G C G AT G AC C G C AG G G AT GG G G G C C C AG AAT T T AAT CAACCAGGCGTGTTTGAATTGCTGCCATGACATGAATGTAGCCTCTGAACTCAGAA GGTTTGAAACAAGAGTCGCGAATCATACACCATGCAAATTTATTTTCGGGGTAATA AT T C AC AAAAAT AAT AG T T AG AT C AAAT T T C T C T C T G AAC C T T G AAAG GC T GG C G A

45292464vl 136 CATCAGGGCTTTAATGCCACAATATAAAAGCAAAACGCCACGTAAACACGTGGCGT TCTCGAGATTATGCACGCGGACACAGTTCTTTAATGGCACGGATTGCGGTTTCGAT TTCTTCTTCAATGGTAAACGGACCGGTGCTAAAACGAACTGTACCCTGCGGAAATG TACCAATGCTTTTATGTGCGCTCGGGCTACAATGCAGACCACAACGGGTCAGAATG CCATATTTCTCTTCCAGGCTTGCACCGGCTTCGGCATTGTCCATAAATTCGCTGAA ATCAATGCTCACAATACCAACACGATTTTCTGCGCTTTCACCACCGGCAATTTTGA TCGGCAGGCCTTTAATACCATCCAGAAACAGTTTCAGCAGTTTCTGTTCGCGTTTA TGGATATTCTGAATGCCAAATGCATCCAGCCATTTCAGGCTATGATGCAGACCTGC AATACCAATAATGTTCTGGGTGCCTGCTTCAAATTTATCCGGCATAAAGCTCGGGG TTTCTTCGCTATCGCTTGCGCTGCCGGTGCCACCGGTAATCAGCGGTTCGACTTCT TTGGCAAAATCTTTATCAAACAGAATACCACCGGTGCCTTGCGGACCCAGCAGACC TTTATGACCGGTAAAACAAAATGCTGCCGGATTCAGCTGGGTCAGATCAACCGGAA TATGACCTGCGCTCTGTGCACCATCAATCACCAGCGGAATATTATGTTTCTTCAGG ATGCTGGCAATTTCTTCAATCGGCTGAATAAAACCGGTCACATTGCTTGCATGGCT GAATACGGCCAGACGTGTTTCCGGACGAATCATGCTTTCAATCAGGCTGGTATCAA CAAAGGTGCCACCATTCTTCAGAATTGCCGGAACGCGATCAATTTTAACACCAATT TTCTCCATCTGAACCAGCGGACGCATAACTGCATTATGTTCAAAGCTGCTGGTCAG AACACGATCACCGCTTTTCAGAAAGCCTTTAATGATATAGTTCAGGCTTGCGGTAA CACCGCTGGTGAAGATCACATGCGTTGCAGGCTGAAAGTTAAACAGTTTACACAGC AGTTCACGGGTTTCAATAACTGCCAGACCTGCTTCTTCGGTTTCGGTATAGGTGCT ACGATTAATGTTACCGGTGCCCATATTCAGTGCCATTGCCAGTGCCTGATCCAGAC CAGGTGTTTTCGGAAATGCACCCGCTGCATTATCCAGAAAGATGCGTTTCTTCATG GAAGAATTCCTTTAGGCGTTAATTTTAGCCTGTTAATCAATACTATTAAGATTTAT T AAT C T T AAAC C T C G AC AT AT T T AC C AT C C T C G C T C AG AT AAT AG AAG GC G T C G T A ATCAAACTGGCTGAAATAATCTTTGTCGAAGGCCAGACCTTCTTTGAAAATCAGTT TCAGTGCGGTGCCACAGCTGCTGCTAACGCTACGCGGAACCGGAACCAGTTTTGCG GTCAGTTCACCGGTCTTAGCATTATCGGTTTTATTCACTGCACGCATACAAACCAG GCTATCATAATGGGTGTGAAAGGTGATCAGGTACTCTTTCATGGTCTGTTTCCTAA ATTATTTTGCGTCAGCTAAACCATCGAGGTAGCGTTCCGCATCAAGTGCTGCCATG CAGCCTGTACCGGCCGAAGTAATGGCCTGGCGATAAATGTGATCCATCACGTCGCC TGCGGCAAAGACGCCAGGAATGCTGGTCTGGGTGGCATTACCATGAATACCCGACT GTACTTTGATGTAGCCGTTTTCCAGTTCCAGCTGCCCTTCGAAAATCGCAGTATTC GG G C T G T G AC C G AT AG C AAC AAAC AG AC C G G C AAC G T C GAG T G AC T C G AT G T T AT C GCTGTTTTGCGTATCGCGCAGACGAACGCCAGTGACACCCATTTGATCGCCGGTCA CTTCTTCCAGCGTACGGTTGGTGTGCAGAATGATGTTGCCGTTCTCCACTTTATCC ATCAGGCGCTTAATGAGGATTTTTTCCGCGCGGAAACCGTCACGGCGGTGAATCAG

45292464vl 137 ATGCACTTCCGAAGCGATGTTAGACAGATACAGCGCCTCTTCAACCGCGGTATTGC CGCCGCCGATGACCGCAACTTTCTGGTTGCGATAGAAGAAACCGTCGCAGGTTGCA CAAGCAGAAACCCCACGGCCTTTAAAGGCTTCTTCAGAGGGCAGGCCGAGATAGCG TGCAGAAGCTCCGGTGGCAATAATCAGCGCGTCGCAAGTGTATTCGCCGTTATCGC C AT T C AG AC GG AAC G G AC G GT T T T G C AG AT C C AC C T T G T T G AT AT G AT C AAAAAT G ATCTCAGTTTCAAACTTGGTGGCATGTTCGTGCATGCGCTCCATTAATAACGGACC GGTCAGATCGTTTGGATCGCCAGGCCAGTTTTCCACTTCCGTGGTGGTGGTCAGTT GGCCGCCTTTTTCCATGCCGGTAATCAGCACAGGTTGCAGGTTGGCGCGCGCCGCG TAGACAGCAGCGGTGTATCCCGCCGGGCCTGAACCCAGGATAAGCAGTTTACTGTG TTTGGTCGTGCCCATGAGATCCCCATAGTTGTTGGCAGACAATGGGCAGGATTGTA GGGAATTTACAGACGTAAAAAAAGAGTATGACGATTTTGTTAACAATTTGTGCAAT CGGCAGCATCGATAAGCAGGTCAAATTCTCCCGTCATTATCACCTCTGCTACTTAA ATTTCCCGCTTTATAAGCCGATTAAATGATGAATAAACGCCCCTGTTAATGAATAT CTGGCATGTTGTACTAAAAATCGATGTTTTGCTTTGACAATCCAGGCCCTTTCGTC

TTCAAGAATT (SEQ ID NO: 115)

>Homo sapiens GPxl(49UAG)-6His

ATGGGCATGTGTGCTGCTCGGCTAGCGGCGGCGGCGGCGGCGGCCCAGTCGGTGTA TGCCTTCTCGGCGCGCCCGCTGGCCGGCGGGGAGCCTGTGAGCCTGGGCTCCCTGC GGGGCAAGGTACTACTTATCGAGAATGTGGCGTCCCTCTAGGGCACCACGGTCCGG GACTACACCCAGATGAACGAGCTGCAGCGGCGCCTCGGACCCCGGGGCCTGGTGGT GCTCGGCTTCCCGTGCAACCAGTTTGGGCATCAGGAGAACGCCAAGAACGAAGAGA TTCTGAATTCCCTCAAGTACGTCCGGCCTGGTGGTGGGTTCGAGCCCAACTTCATG CTCTTCGAGAAGTGCGAGGTGAACGGTGCGGGGGCGCACCCTCTCTTCGCCTTCCT GCGGGAGGCCCTGCCAGCTCCCAGCGACGACGCCACCGCGCTTATGACCGACCCCA AGCTCATCACCTGGTCTCCGGTGTGTCGCAACGATGTTGCCTGGAACTTTGAGAAG TTCCTGGTGGGCCCTGACGGTGTGCCCCTACGCAGGTACAGCCGCCGCTTCCAGAC CATTGACATCGAGCCTGACATCGAAGCCCTGCTGTCTCAAGGGCCCAGCTGTGCCC TCGAGCACCACCACCACCACCACTAA (SEQ ID NO:93)

>Parcubacteria DG 74 2 bin SepCysS

AT GAT C T AC AAAC G C C AG AAC AAAAAC AAAAT T AAC AT C AAC CCGATTCAGGCAGG CGGTATTCTGACCAAAGATGCACGTAAAACCCTGATTGAATGGGGTGATGGTTATA GCGTTTGCGATATTTGGTATAGCGGCAAAATCGATAAAATCGAAAATCCGCAGATC CGCAAATTCATCAATGAAGATCTGCCGAAATTTCTGGGTAGCGATATTGCACGTAT TATTGGTGGTGCACGTGAAGGTATTTGTGCAATTATGCATGCAGTTGCAAAACCGG GT G AT AT T AT T C T G G T G G AT G AG AAC AAAC AC T AT AC C AC C AT T C T G G C AG C AG AA

45292464vl 138 AAAAAT G G T C T G AAAG T T G T T G AAG T T C C G AAT AG C G G T C AT C C G G AAT AC AAAAT TGATGTGCGCGATTATGAAAAACTGATCAAAAAACATAAACCGGCACTGATCCTGC TGACCTATCCGGATGGTAATTATGGTAATATGCCGGATGCAAAAAAACTGGGCGAA ATCGTGATCAAATATAACATTCCGTATCTGCTGAATGCAGCATATAGCGCAGGTCG TCTGCCGGTTGATCTGATTGCAATTAATGGTGATTTTATTGTGGCCAGCGGTCATA AAAGCATGGCAGCAAGCGAACCGATTGGTGTTCTGGGTTTTCGTAAAAAATGGAAA GACACCCTGTTCAAAAAAAGCTTCTTCTATCCGGACAAAGAGATTGAATTCCTGGG CCATTATCAGAAAGGTGCACCGATGATGACCCTGATGGCAAGCTTTCCGTATGTGA AAAAAC G T G T T G AAG AG T G GG AAAAAC AAAT C G AG AAAGC AC G T T G G T T T AGC G C A GAAAT G G AAAAAC T G G G T T T T AAAC AG C T GG G T G AAAAAC C G C AT AAT C AC GAT C T GCTGTTTTTCGAATCACCGCAGCTGTACAAAATTAGCCAGAAACATAAAGAGGGTC GG T T T T T C C T G T AC AAAG AAC T G AAAAAAAAAG G C AT C T AC G G C AT T AAAC C G G G T CTGACGAAACATTTTAAACTGAGCACCTTTGCAGCCAGCAAAGAGGAACTGAAAAA ACTGCTGGAAGTGTTCAAAGAGATCCTGATTAAATAA (SEQ ID NO:94)

GPxl expression and purification

E. coli ME6 cells transformed with pSecUAG-AD and pET- GPxl(49UAG) were grown in LB media containing 50 μg/ml ampicillin, 25 μ^ητΐ kanamycin, 10 μΜ or 100 μΜ Na₂Se0₃ at 37°C until the A600 reached 0.8. In order to raise the Na2Se03 concentration to 100 μΜ, a solution of 50 mg/ml L-cystine in IN HC1 was made and added into the growth medium at a final concentration of 100 μg/ml together with 1/5 volume of 5N NaOH for the neutralization of the medium (31). The culture was then induced by the addition of 1 mM IPTG and 0.1% L-arabinose, and then shifted to 25°C for approximately 16 h before harvesting. The cells were harvested and resuspended in buffer A [50 mM Tris-HCl (pH 8.5), 300 mM NaCl, 5 mM MgCl₂, 10% glycerol, 2 mM 2-mercaptoethanol (or ImM DTT), 0.5 mg/ml lysozyme, 0.1 mg/ml DNase]. After the cell disruption by BugBuster (Millipore), the His6-tagged protein was purified by immobilized metal-ion affinity chromatography using a Ni-NTA (Qiagen). The protein bound to the column was washed with buffer B [20 mM Tris-HCl (pH 8.5), 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol with 5 mM MgCh (or ImM DTT without MgCl₂), 15 mM imidazole] and eluted by buffer B containing 250mM imidazole. Eluted proteins were buffer exchanged into water using 10 kDa molecular weight cut off filters (Millipore).

45292464vl 139 E. coli ME6 cells transformed with pMWcat-Se, pSecUAG-ADT and pET-GPxl(49UAG) were cultured with additional 34

chloramphenicol. For activating some proteins encoded in pMWcat-Se, the growth media were supplemented with arabinose, sodium selenite, and sodium formate at final concentrations of 0.1%, 10 μΜ, and 5 mM, respectively, prior to IPTG induction. E. coli ME68z cells transformed with pSecUAG-Evoll/2 and pET-GP l(49UAG) were cultured without zeocin. Their growth media were supplemented with arabinose and sodium selenite at final concentrations of 0.01% or 0.001% and 10 μΜ, respectively, prior to IPTG induction. Glutathione peroxidase activities were measured with Nanodrop 2000c by using the Glutathione Peroxidase Cellular Activity Assay Kit (Sigma- Aldrich) (Aldag, et al., Angew Chem Int Ed Engl 2013, 52, 1441.)

Mass spectrometry

Electrospray mass spectrometry analyses were done at the W.M.

Keck Biotechnology Resource Laboratory at Yale. The procedure of Keck: Intact proteins samples were diluted in 50% acetonitrile containing 0.1% formic acid and loaded into glass nanospray emitters. The samples were analyzed by direct infusion on an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific, San Jose, CA). The mass spectrometer was operated in Intact Protein mode with an ion routing multipole pressure of 3mTorr or 8mTorr. Spectra were acquired at 120K resolution. The isotopically resolved data were processed using Protein Deconvolution 4.0 software (ThermoFisher Scientific).

Results

In order to improve the conversion rate of Ser-allo-tRNA^UTu to Sec- allo-tRNA^UTu, five modifications to the original pSecUAG-A system were made (Fig. 9A). Open reading frames for As SelA and phosphoseryl-tRNA kinase (PTSK) of Trypanosoma brucei (Aeby, et al., Proc Natl Acad Sci U S A, 106:5088-5092 (2009)) or Homo sapiens (Carlson, et al., Proc Natl Acad

Sci U S A, 101: 12848-12853 (2004)) were cloned under the trc promoter in the pTrc99A plasmid vector. The selD gene of As 34mel was cloned into pSecUAG-A and the start codon was changed from AUG to GUG to reduce

45292464vl 140 the expression level of As SelD to produce pSecUAG-AD (Fig. 9E). In addition, an ORF for Treponema denticola Trxl (Sec-containing

thioredoxin) (Kim, et al., Biochem Biophys Res Commun, 461 :648-652 (2015)) was cloned after the As SelD ORF of pSecUAG-AD with a Shine- Dalgarno (SD) sequence. The original UGA Sec codon of Td Trxl ORF was mutated to UAG for its allo-tRNA^UTu-mediated expression. Previous studies indicate that thioredoxin may be involved in Se-transfer in the cytoplasm of bacteria (Tamura, et al., Biosci Biotechnol Biochem, 75: 1184-1187 (2011); Kumar, et al., Eur J Biochem, 207:435-439 (1992)) and that recombinant expression of Td Trxl in E. coli may modulate Sec incorporation in the E. coli proteome (Kim, et al., Biochem Biophys Res Commun, 461:648-652 (2015)).

It was revealed that As SelA should be expressed at a proper level, because excess As SelA molecules sequestered allo-tRNA^UTu molecules in a dose-dependent manner (Fig. 12A-12B). PSTK might prevent Ser-allo- tRNA^UTu translating UAG codons (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013)), because PSTK can convert Ser-tRNA to

phosphoseryl-tRNA which is a poor substrate of EF-Tu but is a good substrate of SelA (Aldag, et al., Angew Chem Int Ed Engl, 52: 1441-1445 (2013); Xu, et al., PLoS Biol, 5, e4 (2007)). Both PSTK species recognized Ser-allo-tRNA^UTu according to the observation that their co-expression with a Sep-tRNA:Cys-tRNA synthase (SepCysS) (Yuan, et al., FEBS Lett, 584:2857-2861 (2010); Mukai, et al., MBio, 8, e00561-00517 (2017)) resulted in the formation of Cys-allo-tRNA^UTu which inserted Cys into the UAG 140 position of FDH_H (Fig. 12C). The FDH_H (Cys 140) variant retains the activity (Yuan, et al., FEBS Lett, 584:2857-2861 (2010); Axley, et al., Proc Natl Acad Sci U S A, 88:8450-8454 (1991)). T brucei PSTK molecules sequestered allo-tRNA^UTu molecules in a dose-dependent manner, whereas H. sapiens PSTK did not affect the pSecUAG-A system (Fig. 12A-12B). Therefore, application of PSTK does not help.

Experiments were also designed to determine whether As SelD improved the expression levels of the FDHH variants carrying four or five

Sec residues. Note that the E. coli strain tested has its own selD gene in the

141

45292464vl chromosome. The new pSecUAG-AD system (Fig. 9E) drastically improved the yield of the FDHH variant carrying five Sec residues and also improved the yield of the four Sec variants. The FDHH activities of cell spots carrying pSecUAG-A +fdhF(4 UAG codons) and pSecUAG-AD +fdhF(5 UAG codons) were comparable. The cells carrying pSecUAG-AD +fdhF(5 UAG codons) became darkened within a few minutes, whereas cells carrying pSecUAG-A +fdhF(5 UAG codons) were colorless within a short incubation time. Thus, supply of selenophosphate from SelD to SelA was revealed as a limiting step. While the wildtype As selD gene carrying the AUG start codon gave similar results (Fig. 13A), its effect was not robust (Fig. 13B), possibly due to the SelD overexpression. Since three Sec residues plus one Ser residue failed to properly accommodate the iron-sulfur cluster in FDHH variants (Fig. 13C), the pSecUAG-AD system actually produced FDHH with five Sec residues.

Human selenoprotein glutathione peroxidase 1, or GPxl(Sec49), was expressed and analyzed by mass analysis. The pSecUAG-AD system fully supported the overexpression of GPxl proteins by the standard pET expression system. Intact mass spectrometry confirmed Sec incorporation (Figs. 9F and 14A-14F). However, Ser was also incorporated at a significant rate because the peak for GPxl(Ser49) is higher than that for GPxl(Sec49) (Figs. 9F and S4A). Increasing the concentration of sodium selenite

(selenium source) in the growth medium from 10 μΜ to 100 μΜ did not improve the Sec incorporation ratio (Fig. 14B). In order to facilitate continuous selenium supply to SelD, pSecUAG-AD was modified to additionally encode the Sec-containing thioredoxin of T. denticola (Kim, et al., Biochem Biophys Res Commun, 461 :648-652 (2015)). The developed pSecUAG-ADT system produced mixtures of GPxl(Sec49) and

GPxl(Ser49) in which the former is the major product (Figs. 9G and 14C, 14D).

Since this pSecUAG-ADT system (Fig. 9H and 9E) produced red- colored elemental selenium from 10 μΜ selenite in the growth media, the cells may have reduced enough amount of selenite. Actually, addition of other proteins and enzymes (e.g., thioredoxin reductase, selenocysteine lyase,

142

45292464vl putative S-transporters possibly involved in Se-transfer) made no improvement (see, e.g., Figs. 14C).

Thus, selenium supply to SelD was revealed as a limiting step. Example 10: Variants of allo-tRNA^UTu are also functional

Materials and Methods

Sequences

>allo-tRNA^UTu (Ac-3U variant)

GGAGGTTGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCT CGCCGCATTGGGTTCGATTCCCTTCTCCTCCGCCA (SEQ ID NO:32, DNA) GGAGGUUGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCU CGCCGCAUUGGGUUCGAUUCCCUUCUCCUCCGCCA (SEQ ID NO: 137, RNA) >allo-tRNA^UTu (Ac-bU variant)

GGAGGTGGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCT CGCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:33, DNA) GGAGGUGGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCU CGCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO: 138, RNA) >allo-tRNA^UTu (D-3b variant) (also referred to as allo-tRNA^UTulD)

GGAGGGGAACTTCTGTCTGGTGGCAGACGGGAACTCTAAATTCCTTGAAATGCCTC GCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:34, DNA) GGAGGGGAACUUCUGUCUGGUGGCAGACGGGAACUCUAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO: 139, RNA) >allo-tRNA^UTu2

GGACGGGGGTTCCGTCTGGTGACGGTCGCGGGCTCTAAACCCGTCAGGACGCTGTG CAGGCGTTAGGTTCGATTCCTCCCCCGTCCGCCA (SEQ ID NO:35, DNA) GGACGGGGGUUCCGUCUGGUGACGGUCGCGGGCUCUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCGCCA (SEQ ID NO:58, RNA) >allo-tRNA^UTu2 (G21 variant) (also referred to as allo-tRNA^UTu2D)

GGACGGGGGTTCCGTCTGGTGGCGGTCGCGGGCTCTAAACCCGTCAGGACGCTGTG CAGGCGTTAGGTTCGATTCCTCCCCCGTCCGCCA (SEQ ID NO:36, DNA) GGACGGGGGUUCCGUCUGGUGGCGGUCGCGGGCUCUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCGCCA (SEQ ID NO: 140, RNA)

45292464vl 143 >2225 (also referred to as allo-tRNA^UTu) (also referred to as allo-tRNA^UTul)

GGAGGGGAACTTCTATCTGGTGATAGACGGGAACTCTAAATTCCTTGAAATGCCTC GCCGCATTGGGTTCGATTCCCTTCCCCTCCGCCA (SEQ ID NO:31, DNA) GGAGGGGAACUUCUAUCUGGUGAUAGACGGGAACUCUAAAUUCCUUGAAAUGCCUC GCCGCAUUGGGUUCGAUUCCCUUCCCCUCCGCCA (SEQ ID NO:57, RNA)

>2459

GGAGTGGGGTTCCGGCTGGTGCCGGTCGCGGGCTCTAAACCCGTCAGGACGCTGCG ACGCGTAAGGTTCGATTCCTCCCCACTCCGCCA (SEQ ID NO:37, DNA) GGAGUGGGGUUCCGGCUGGUGCCGGUCGCGGGCUCUAAACCCGUCAGGACGCUGCG ACGCGUAAGGUUCGAUUCCUCCCCACUCCGCCA (SEQ ID NO:141, RNA) >S15 UU variant

GGAGGGCATTTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:38, DNA) GGAGGGCAUUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 142, RNA) >S15 CU variant

GGAGGGCACTTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:39, DNA) GGAGGGCACUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 143, RNA) >S15 UC variant

GGAGGGCATCTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:40, DNA) GGAGGGCAUCUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 144, RNA) >S15 AA variant

GGAGGGCAAATTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:41, DNA) GGAGGGCAAAUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 145, RNA) >S15 AU variant

GGAGGGCAATTTCAGTCGGTACTGGACGCCGTCTCTAAAACGGTTGCAGGGTCTTA GTCAGCTCTGGGAGTTCGACTCTCCTGCCCTCCGCCA (SEQ ID NO:42, DNA)

45292464vl 144 GGAGGGCAAUUUCAGUCGGUACUGGACGCCGUCUCUAAAACGGUUGCAGGGUCUUA GUCAGCUCUGGGAGUUCGACUCUCCUGCCCUCCGCCA (SEQ ID NO: 146, RNA)

The nucleotide sequences of Rx and Sh SelA were optimized for use in E. coli:

>Rubrobacter xylanophilus SelA

ATGCTGGATGCAGAACGTCAGAGCCGTCTGCGTAGCCTGCCTGCAGTTGATGCAGT TCTGCGTGGTCCGGCAGCAGGTCTGGCAGCACGTCATGGTCGTGCAGCAGTTGCAG CAGCAGTTCGTGAAGTTCTGGAAGGTCTGCGTCGTGAAATTGCAGCCGGTGGTAGT CCGGATGTTAGCGGTCGTGCCGTTGCAGAAGGTGCAGCCCGTCTGCTGAGTGGTCG TGGCCTGCGTCGCGTTGTTAATGCAACCGGTGTTGTTCTGCATACCAATCTGGGTC GTGCGGTTCTGAGCGAACGTGCAGCCGCAGCAGCGGCACGTGCAGGCACCAGCTAT AGCAATCTGGAATATGATCTGAGCCGTGGTCGTCGTGGTAGCCGTTATGATCATGC AGTTCCTCTGCTGCGTGAACTGACCGGTGCAGAAGATGCACTGGTTGTTAATAACT GTGCCGGTGCAACCCTGCTGGCACTGAGCGCACTGGCAGGCGAAGAAGGTGAAGGT CCGCCTGAAGTTGTTGTTAGTCGTGGTCAGCTGATTGAAATTGGTGGTGGTTTTCG TATTCCGGAAGTGCTGGAACTGAGTGGTGCCGTTCTGCGCGAAGTTGGTACAACCA ATCGTACCCGTCTGAGCGATTATGAACGTGCACTGAGTGAACGTACCCGTGCAATT CTGTGGGTTCATCCGAGCAATTTTGAAATTCGCGGTTTTACCGAAAGCGCAGGTAT TGCAGAACTGGCTGGTCTGGGTCCTCCGGTTGTTGCAGATCTGGGTAGCGGTGCAC TGCTGCCGCTGGGTGGTGAACCGCTGGTTCAGGCAGCACTGCGTGATGGTGCCGAA CTGGCACTGTTTAGCGGTGATAAACTGCTGGGTGGACCGCAGGCTGGTATTGCCGC AGGTAGCAGCCGTCTGGTTCGTCGTATGCGTCGTCATCCGCTGGTGCGTGCCCTGC GTGCAGATAAACTGTGCCTGGCAGCCCTGGAAGCAACACTGCGTGCATATCTGGAA GGCCGTGCCGAAGAAGAAGTTCCGGCACAGCGTATGCTGCGCGAACCACTGGAAGG TGTTGAAGCACGTGCCCGTCGTCTGGCAAGCGCACTGAGTCGTGAAGTGCCTGGTC TGGAAGTTGGTGTTGTGCCGAGCGTTGCACGTAGCGGTGGTGGCACCCTGCCTGGT TATGAAATTCCGAGCTTTGCAGCACGTGTTCTGGGTGCAGATGCAGAAGCCCTGGC AGCGCGTCTGCGTGCCGCAGAACCGCCTGTTGTGGGTCGTGTTCATGAAGGTGCCC TGCTGCTGGATGCCCGTACCCTGCTGCCAGGTGATGAAGAAGCAGTTGTTGAAGCG CTGCGTGAGGCAGCCCGTGGTTAA (SEQ ID NO:83)

>Sulfurimonas honglongensis SelA

ATGTTCCTGCTGAAAAGCATTCCGAAAGTGGATAAGTTTATCGCCAAGAAAGAGTT TAAAACCCTGGGTAGCGCACTGGTTATGAGCCTGACCAAAGAACTGCTGAGCGAAC

45292464vl 145 TGCGTGAAAACATTCTGAATGGTCGTGTTACCACCTTTAGCGAAGATGAACTGGTT AAAGAGCTGCTGCAGCGTTATACCGAACTGACCAAACCGAGCCTGCAGACCCTGAT TAATGCAACCGGTATTATTGTTCATACCAATCTGGGTCGTAGCCTGATTGATGCAG ATGCATTTGATCGTGTTAAAGAACTGATGACCAACTATAACAACCTGGAATTTAAT CTGGAAAGCGGTAAACGTGGTGAACGCTATAGTCTGATTAGCAAAAGCGTTTGTAG CCTGCTGGGTTGTGAAGATGTTCTGATTGTGAATAATAACGCCAGCGCAGTTTTTC TGATTCTGAACACCTTTGCGCGTAAAAAAGAAGTTGTTGTTAGTCGCGGTGAACTG GTGGAAATTGGTGGTAGCTTTCGTGTTCCGGATGTTATGAAACAGAGCGGTGCAAA AC T G G T T G AAG T T G G C AC C AC C AAT AAAAC C C AT C T G T AT G AT T AT G AAG AT G C C A TCGGTAAAAAAACGAGCATGCTGATGAAAGTGCACAAAAGCAACTATAGCATTGAA GGTTTTAGCAGCGACGTGGAATTTGGCGAAATTGTTAAACTGGCATGTGAAAAAGG CCTGATCGATTATTATGATATGGGTAGCGGTCACCTGTTTGATCTGCCGTATGGTC TGGATGAACCGAGCGTTCTGGACTTTATGAAACTGAATCCGAGTCTGCTGAGCTTT AGCGGTGATAAACTGCTGGGTAGTGTTCAGGCAGGCATTATTGTTGGCAAAAAAAA GTATATCGACATGCTGAAGAAAAACCAGCTGCTGCGTATGCTGCGTGTGGATAAAC TGACCCTGGCACTGCTGGAAGAAAGTTTTAAAGCAATTCTGCTGGGCAACAAAGAG CAGATTCCGACCGCACGTATGCTGTTTCGTAGCACCGATGAACTGCGCGAAGATGC AATGCAGGTTCAGCAGAAACTGAAAAAAAACATCAAGACCAACATCGTGGATACCA AAACACTGATTGGTGGCGGTACAACCCCGAATAAAACCATTCCGAGCGTTGCCCTG GT T AT T G AAAG C AAAAAC AT T AAG G T G AAAAAAC T G C AGAAG C T G T T T C G C C AG AA AAGTATTATTGGTCGCATCGAGGATGATGAATTTCTGCTGGATTTTCGTACGATTC AGAAAACCCAACTGCAGCAGGTTGTTGATGCAATTGATGAAATTACCGACGTGTAA

(SEQ ID NO:85)

Figure 15 A

Figure 15B

GGACGGGGGUUCCGUCUGGUGACGGUCGCGGGCUCUAAACCCGUCAGGACGCUGUG CAGGCGUUAGGUUCGAUUCCUCCCCCGUCCGCCA (SEQ ID NO:58)

Results

Variants of the nucleotide sequence of allo-tRNA^UTu and the amino acid sequence of As SelA were engineered. Since allo-tRNA^UTu is not the original substrate of As SelA, there maybe room for improvement (Miller, et al., FEBS Lett, 589:2194-2199 (2015)). Some characteristic features of Aeromonas tRNA^Sec were each transplanted to allo-tRNA^UTu (Fig. 15A-15B).

45292464vl 146 The tRNA^Sec species of Aeromonas and some other bacteria have a bulged pyrimidine at position 5 or 5a in the 7-bp acceptor stem (Mukai, et al., Angew Chem Int Ed Engl, 55, 5337-5341 (2016); Santesmasses, et al., PLoS Comput Biol, 13, el005383 (2017)). Two allo-tRNA^UTu variants having a bulged 5aU were made (Fig. 15A-15B). The U14:G21 wobble base pair in the D-stem of As tRNA^Sec was transplanted to make variant D-3b (Figs. 10A and 15A-15B).

Another type of UTu tRNA from an alanine-accepting allo-tRNA species (named 9/3-3, see also Examples above) (Mukai, et al., Nucleic Acids Res, 45:2776-2785 (2017)) to make allo-tRNA^UTu2 (Fig. 15A-15B). Effects of allo-tRNA engineering were assessed by observing the yields of the FDHH variant carrying five Sec residues (Fig. 15C).

To facilitate experiments, the expression level of As SelA was significantly reduced. First, the As SelA expression cassette of pSecUAG- AD (100 copies per cell) was removed and transferred to a low-copy-number plasmid (5-8 copies per cell). Then the AUG start codon for As SelA was mutated to GUG to further decrease its translation level. With these limited amounts of As SelA molecules, the D-3b variant produced the largest amount of the FDHH variant carrying five Sec residues (Fig. 15C). On the other hand, allo-tRNA^UTu2 and its variant carrying U14:G21 were less active than the original allo-tRNA^UTu (Fig. 15D), probably because they are not an inherent substrate of SerRS. Intact mass analysis was performed using the same GPxl reporter. A modified pSecUAG-ADT system (Fig. 10B) (named pSecUAG-AD3T) expressing the D-3b variant instead of allo-tRNA^UTu gave similar results as those obtained with the original pSecUAG-ADT system (Figs. IOC and 14C-14F). A small improvement was observed (Figs. IOC and 14E).

Two other SelA species from Rubrobacter xylanophilus and

Sulfurimonas honglongensis and six other allo-tRNA variants were also tested. R. xylanophilus and S. honglongensis have a small tRNA^Sec like A. salmonicida.

The results are shown in Figures 16A-16B. Sh, As, Rx denote

Sulfurimonas honglongensis, Aeromonas salmonicida, Rubrobacter

147

45292464vl xylanophilus , respectively. 2225, 2459, S15 were derived from 9/3-1, 9/3-2, 8/4-1, respectively (Mukai, et al., Nucleic Acids Res, 45:2776-2785 (2017)). Figure 16A shows that all of the combinations of allo-tRNA and SelA inserted Sec. The/<i/2 (140Amb) gene variant was used as reporter. Figure 16B shows that the two allo-tRNA^UTu species derived from (9/3-1 and 9/3-2) were more active than the five allo-tRNA^UTu species derived from (8/4-1). As SelA was used. The/<i/2 (3 UAG codons) gene variant was used as reporter.

The results indicated that diverse SelA species can be used for selenocysteinylation of allo-tRNA (Fig. 16A) and that allo-tRNA with a 9/3 structure is more active than allo-tRNA with an 8/4 structure (Fig. 16B). Example 11: Engineered SelA further improves the translation system Materials and Methods

As SelA library construction and screening

To select As SelA variants with improved selenation capacity a plasmid pNMC-A(2xAm) was constructed which carries an NMC-A variant gene (with two UAG codons at positions 70 and 240) in place of the As SelA gene from pSecUAG-AD-allo-tRNAUTu2D (see above). This NMC-A gene product is only functional if both positions are translated as either Cys or Sec, but not Ser (Thyer, et al., J Am Chem Soc 2015, 137, 46) Thus, higher conversion levels of Ser-allo-tRNAUTulD to Sec-allo-tRNAUTulD are expected to result in resistance to higher ampicillin levels in the strain carrying the NMC-A(2xAm) gene. Two libraries of As selA variants (NTT and GST libraries) were generated by using pMWcat-AsSelA(GUG) as PCR template. The codon for Cys 173 was randomized as NNK. The codons for Pro68, Leu69, and Gln72 were changed to SST, NTT or GST, and NMK, respectively. An "Amber- less" ARF-l E. coli strain (Kuznetzov, et al., Genome Biol 2017, 18, 100) was used because non-Amber-less cells overexpressing allo-tRNAUTu ID were unable to resume growth upon subculturing. To carry out selection, 105 electrocompetent cells of E. coli

C321.AA.opt AselAB strain carrying a selection vector were transformed with mixture of the NTT and GST libraries (500 ng each). Cells were recovered is 50 ml of SOC media for 1 hour prior to addition of antibiotics,

45292464vl 148 and a small aliquot of culture was harvested for colony count. After overnight growth at 30°C, 200 μΐ aliquots of cell culture were plates on selection plates (0150 mm) containing 17 μg/ml chloramphenicol, 50 μg/ml kanamycin, 5 μΜ sodium selenite, 0.1% arabinose and increasing concentration of ampicillin. After 30 hours of growth at 30°C, 20 colonies from the plate containing 125 μg/ml ampicillin were subcultured and analyzed individually, and colonies from plates containing 100 and 80 μg/ml ampicillin were pooled together. After plasmid DNA was isolated from each sample, the region of selA with promoter was amplified with oligos err_AsA_RI (5' - CATACGGAATTCCGGATGAGC-3' (SEQ ID NO: 116)) and err_AsA_BH (5'-aaaaaaggatcCACGTGTTGACAATTAATCATCG-3' (SEQ ID NO: 117)) using Herculase II Fusion polymerase (Aglient) for individual clones or GeneMorph II Random Mutagenesis kit (Aglient) for combined clones according to manufacturer's instructions. The original pMWcat- AsSelA(GUG) vector was linearized by PCR with oligos pMW_F_BH (5'-aaaaaaggatccAGGCCCTTTCGTCTTCAAG-3' (SEQ ID NO: 118)) and pMW_R_RI (5'-CATCCGGAATTCCGTATGGC-3' (SEQ ID NO: 119)) using Herculase II Fusion polymerase. PCR products were purified by a column- based kit, treated with EcoRI, BamHI and Dpnl restriction enzymes and purified again. selA fragments from separate clones were ligated with vector individually according to manufacturer instructions, transformed into chemically competent DH5a cells and plated onto LB-agar plates containing chloramphenicol. Recloned selA variants from the 1^st round of selection were transformed into of E. coli C321.AA.opt AselAB strain carrying pNMC- A(2xAm), and compared to the original pMWcat-AsSelA(GUG) in ampicillin-resistance assay. Ligation was performed using 10 μg of each vector and insert amplified from combined pDNA in 500 μΐ reaction volume at 16°C for 18 hours. DNA was purified and concentrated by column, transformed into ElectroMax DH10B competent cells (ThermoFisher) and recovered in 10 ml in SOC. After 1 hour cells a small aliquot of culture was harvested for colony count, while the rest was transferred into 200 ml of LB containing chloramphenicol and allowed to grow overnight prior to isolation of pDNA. For the 2nd round of selection, 1 μg of pDNA was transformed

45292464vl 149 into of E. coli C321.AA.opt AselAB strain carrying pNMC-A(2xAm) and processed as described above, except concentration of ampicillin on the selection plates was higher.

Individual colonies from plates containing 125, 145 and 210 μg/ml ampicillin were subcultured individually for pDNA isolation. It was revealed that all obtained selA variants lack the BamHI site in the plasmid vector region, indicating that they derive from undigested template plasmids for the error-prone PCR, since the BamHI site was added in this PCR step with the oligo pMW_F_BH. Therefore, our random mutagenesis was not productive. selA ORF with the EM7 promoter but without the trp terminator was amplified with oligos PP464 (5'- aagacgaaagggcctCACGTGTTGACAATTAATCATCG-3' (SEQ ID NO: 120)) and PP313 (5' -ctggcggctgtgggaTCAGGGCTCCTCGGTCGCAG-3' (SEQ ID NO: 122)) and cloned into pMWcat vector linearized by PCR oligos PP414 (5' -AGGCCCTTTCGTCTTCAAG-3' (SEQ ID NO: 123)) and PP485 (5'-

TCCCACAGCCGCCAGTTCCGCTGGCGGCATTTTACCCGACGCACTTTGCGCCG-3' (SEQ ID NO: 124)) by Infusion. Recloned selA variants from the 2nd round of selection were transformed into of E. coli C321.AA.opt AselAB strain carrying pNMC-A(2 Am), and compared to the original pMWcat- AsSelA(GUG) in ampicillin-resistance assay. Also, selA variants listed in Table 5 and recloned in the pMWcat vector were compared to the original pMWcat-AsSelA(GUG) and examined in FDHH assay.

>Treponema denticola selenocysteine lyase (SCL)

ATGAAGAAACGCATCTTTCTGGATAATGCAGCGGGTGCATTTCCGAAAACACCTGG TCTGGATCAGGCACTGGCAATGGCACTGAATATGGGCACCGGTAACATTAATCGTA GCACCTATACCGAAACCGAAGAAGCAGGTCTGGCAGTTATTGAAACCCGTGAACTG CTGTGTAAACTGTTTAACTTTCAGCCTGCAACGCATGTGATCTTCACCAGCGGTGT TACCGCAAGCCTGAACTATATCATTAAAGGCTTTCTGAAAAGCGGTGATCGTGTTC TGACCAGCAGCTTTGAACATAATGCAGTTATGCGTCCGCTGGTTCAGATGGAGAAA ATTGGTGTTAAAATTGATCGCGTTCCGGCAATTCTGAAGAATGGTGGCACCTTTGT TGATACCAGCCTGATTGAAAGCATGATTCGTCCGGAAACACGTCTGGCCGTATTCA GCCATGCAAGCAATGTGACCGGTTTTATTCAGCCGATTGAAGAAATTGCCAGCATC

45292464vl 150 C T G AAG AAAC AT AAT AT T C C G C T G G T G AT T G AT G G T G C AC AG AG C G C AGG T C AT AT TCCGGTTGATCTGACCCAGCTGAATCCGGCAGCATTTTGTTTTACCGGTCATAAAG GTCTGCTGGGTCCGCAAGGCACCGGTGGTATTCTGTTTGATAAAGATTTTGCCAAA GAAGTCGAACCGCTGATTACCGGTGGCACCGGCAGCGCAAGCGATAGCGAAGAAAC C C C G AG C T T T AT G C C G G AT AAAT T T G AAGC AG G C AC C C AG AAC AT T AT T G G T AT T G CAGGTCTGCATCATAGCCTGAAATGGCTGGATGCATTTGGCATTCAGAATATCCAT AAACGCGAACAGAAACTGCTGAAACTGTTTCTGGATGGTATTAAAGGCCTGCCGAT CAAAATTGCCGGTGGTGAAAGCGCAGAAAATCGTGTTGGTATTGTGAGCATTGATT TCAGCGAATTTATGGACAATGCCGAAGCCGGTGCAAGCCTGGAAGAGAAATATGGC ATTCTGACCCGTTGTGGTCTGCATTGTAGCCCGAGCGCACATAAAAGCATTGGTAC ATTTCCGCAGGGTACAGTTCGTTTTAGCACCGGTCCGTTTACCATTGAAGAAGAAA TCGAAACCGCAATCCGTGCCATTAAAGAACTGTGTCCGCGTGCATAA (SEQ ID

NO:125)

>Treponema denticola DUF3343 protein

ATGAAAGAGTACCTGATCACCTTTCACACCCATTATGATAGCCTGGTTTGTATGCG TGCAGTGAATAAAACCGATAATGCTAAGACCGGTGAACTGACCGCAAAACTGGTTC CGGTTCCGCGTAGCGTTAGCAGCAGCTGTGGCACCGCACTGAAACTGATTTTCAAA GAAGGTCTGGCCTTCGACAAAGATTATTTCAGCCAGTTTGATTACGACGCCTTCTA TTATCTGAGCGAGGATGGTAAATATGTCGAGGTTTAA (SEQ ID NO: 126) >Burkholderia stabilis FDH

ATGGCAACCGTTCTGTGTGTTCTGTATCCGGATCCTGTTGATGGTTATCCGCCTCG TTATGTTCGTGATGCAATTCCGGTTATTACCCAGTATGCAGATGGTCAGACCGCAC CGACACCGGCAGGTCCGCTGGGTTTTCGTCCGGGTGAACTGGTTGGTAGCGTTAGC GGTGCACTGGGTCTGCGTGGTTATCTGGAAGCACATGGTCATACCCTGATTGTTAC CAGCGATAAAGATGGTCCGGATAGCGAATTTGAACGTCGTCTGCCGGAAGCAGATG TTGTTATTAGCCAGCCGTTTTGGCCTGCATATCTGACCGCAGAACGTATTGCCCGT GC AC C G AAAC T G AAAC T G G C AC T G AC AG C AG G T AT T G G T AG C G AT C AT GT T GAT C T GGATGCAGCAGCACGTGCACATATTACCGTTGCAGAAGTTACCGGTAGCAATAGCA TTAGCGTTGCCGAACATGTTGTGATGACAACCCTGGCACTGGTTCGTAATTATCTG CCGAGCCATGAAATTGCACAGCAAGGTGGTTGGAATATTGCAGATTGTGTTAGCCG TAGCTATGATGTTGAAGGTATGCATTTTGGCACCGTTGGTGCAGGTCGTATTGGTC TGGCAGTTCTGCGTCGTCTGAAACCGTTTGGTCTGCATCTGCATTATACCCAGCGT CATCGTCTGGATGCCGCAATTGAACAAGAACTGGCCCTGACCTATCATGCCGATGC AGCAAGCCTGGCAGGCGCAGTTGATATTGTTAACCTGCAGATTCCGCTGTATCCGA GCACCGAACACCTGTTTGATGCAGCCATGATTGCACGTATGAAACGTGGTGCCTAT CTGGTTAATACCGCACGTGCAAAACTGGTTGATCGTGATGCCGTTGTTCGTGCAGT

45292464vl 151 GACCAGCGGTCATCTGGCAGGTTATGGTGGTGATGTTTGGTTTCCGCAGCCTGCAC

CGGCAGATCATCCGTGGCGTACCATGCCGTTTAATGGTATGACACCGCATATTAGC

GGCACCAGCCTGAGCGCACAGGCACGTTATGCAGCAGGCACCCTGGAAATTCTGCA

GTGTTGGTTTGATGGTAAACCGATTCGTAACGAATATCTGATTGTGGATGGTGGTA

CACTGGCAGGTACAGGTGCACAGAGCTATCGTCTGACCTAA (SEQ ID NO: 127)

> Grxl(l lUAG/14Ser)

ATGGTGCAAACCGTTATTTTTGGTCGTTCGGGTtagCCTTACtcgGTGCGTGCAAA

AGATCTGGCTGAGAAATTGAGCAATGAACGCGATGATTTTCAGTATCAGTATGTAG

ATATTCGTGCGGAAGGGATCACTAAAGAAGATCTACAACAAAAGGCAGGTAAACCC

GTAGAAACCGTGCCGCAGATTTTTGTCGATCAGCAACATATCGGCGGCTATACCGA

TTTTGCTGCATGGGTGAAAGAAAATCTGGACGCCGCAGCTGCGCATCATCACCACC

ATCACTAA (SEQ ID NO: 128)

>NMC-A(2xAm)

ATGAGCCTGAATGTTAAACAGAGCCGTATTGCAATTCTGTTTAGCAGCTGTCTGAT TTCGATCAGCTTCTTCAGCCAGGCAAATACCAAAGGCATCGACGAAATCAAGAATC TGGAAACCGATTTTAATGGTCGCATTGGTGTTTATGCACTGGATACCGGTAGCGGT AAAAGCTTTAGCTATCGTGCAAATGAACGTTTTCCGCTGtagTCTAGCTTTAAAGG TTTTCTGGCAGCAGCAGTTCTGAAAGGTAGCCAGGATAATCGTCTGAATCTGAACC AGATTGTGAATTATAACACCCGCAGCCTGGAATTTCATAGCCCGATTACCACCAAA TACAAAGATAATGGTATGAGCCTGGGTGATATGGCAGCCGCAGCACTGCAGTATAG CGATAATGGTGCAACCAACATTATTCTGGAACGCTATATTGGTGGTCCGGAAGGTA TGACCAAATTTATGCGTAGCATTGGCGACGAAGATTTTCGTCTGGATCGTTGGGAA TTAGATCTGAATACCGCAATTCCGGGTGATGAACGTGATACCAGCACACCGGCAGC AGTTGCTAAGAGCCTGAAAACCCTGGCACTGGGTAATATTCTGAGCGAACATGAGA AAGAGACCTATCAGACCTGGCTGAAAGGCAATACCACCGGTGCAGCACGTATTCGT GCAAGCGTTCCGAGCGATTGGGTTGTTGGTGATAAAACCGGTAGCtagGGTGCCTA TGGCACCGCAAATGATTATGCAGTTGTTTGGCCGAAGAATCGTGCACCGCTGATTA TTAGCGTTTACACCACCAAGAACGAGAAAGAAGCAAAACACGAGGATAAAGTTATT GCAGAAGCAAGCCGCATTGCCATTGATAATCTGAAATAA (SEQ ID NO: 129)

Grxl expression and purification

Grxl was expressed and purified as previously described (Aldag, et al., Angew Chem Int Ed Engl 2013, 52, 1441). In brief, E. coli ME68z cells transformed with pSecUAG-Evol2 and pET-Grxl(HUAG/14Ser) were grown in 2x1 liter LB media containing 100 μg/ml ampicillin, 30 μg/ml kanamycin, 10 μΜ Na2Se03, and 0.001% L-arabinose 37°C until the A600

152

45292464vl reached 1.2. The culture was then shifted down to 20°C and protein expression was then induced at an A600 of 1.5 with 1 mM IPTG and continued for approximately 18 h at 20°C. Starting with cell harvest all procedures were carried under anaerobic conditions (90% N2, 5% H2, 5% C02) in an anaerobic tent (Coy Laboratories). The cells were harvested and washed in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 30 mM imidazole) with 2 mM 2-mercaptoethanol (buffers were kept in the anaerobic tent for 4-5 days). Later, the cells were supplemented with 1 mg/ml EDTA-free protease inhibitor cocktail (Roche) and lysozyme (0.1 mg/mL) and subsequently disrupted by BugBuster (Millipore) in the presence of 10 U/ml DNasel (ThermoFisher). The resulting cell lysate was clarified by centrifugation at 18,000 g for 20 min and filtration (0.22 μιη) prior to application to Ni-NTA resin (1.5 ml bed volume, Qiagen) in a gravity flow column equilibrated with 100 mL lysis buffer. Unbound proteins were removed by washing the column with 150 ml lysis buffer. Proteins bound to the column were eluted by 3 ml elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 230 mM imidazole). Up to 1.2 mg Grxl were purified from 2 L of culture. The Grxl(CllU/C14S) was further separated from the Grxl(Cl lS/C14S) species by a second-step affinity purification step using thiol-selenol coupling on Activated Thiol Sepharose 4B (GE healthcare). Proteins were dialyzed in 4 liter Thio- purification buffer (100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM EDTA) using Slide- A-Lyze dialysis cassette (3.5 kDa

molecular weight cut-off, 3 ml capacity, Thermo Fisher Scientific). Total 1.0 mg of the Grxl mixture was loaded onto 2 mL of Activated Thiol

SepharoseTM 4B resin in a gravity flow column equilibrated with 100 mL Thio-purification buffer. The Grxl mixture was then incubated with the Activated Thiol SepharoseTM 4B for 40 min at room temperature to couple the Grxl(CllU/C14S) to the sepharose. After washing with 150 mL Thio- purification buffer to remove Grxl(Cl 1S/C14S), Grxl(Cl 1U/C14S) was eluted from the resin with 3 mL Thio-elution buffer (100 mM Tris-HCl, pH

8.0, 300 mM NaCl, 10% glycerol, 1 mM EDTA, 50 mM reduced glutathione). 0.5 mg (50%) of the Grxl mixture was recovered using the

45292464vl 153 Activated Thiol Sepharose. Both flow-through and eluted proteins were buffer exchanged into thiol sephorase buffer using 3 kDa molecular weight cut off filters (Millipore). After buffer exchange, protein concentration was measured using NanoDrop spectrophotometer at 280 nm. After TCA precipitation, proteins were mixed with SDS reducing buffer followed by SDS-PAGE and stained by Coomassie Brilliant Blue for visualization. For mass spectrometry, protein samples were newly produced and purified. The modifications in the protein purification protocol are: 1) after harvest, cells resuspended in lysis buffer supplemented with EDTA-free protease inhibitors were subjected to freeze-thaw cycle to facilitate the lysis; 2) MgC12 was added to the lysis buffer at 2 mM level to enhance genomic DNA cleavage by DNase I; 3) to remove the excess of glutathione and 2-thiopyridone, the elution and wash fractions after the thiol- sepharose chromatography were buffer exchanged into the buffer containing 50 mM Tris HC1, pH 8.0, 250 mM NaCl, 0.5 mM EDTA and 5% glycerol using Slide-A-Lyze dialysis cassette (3.5 kDa MWCO); 3) the TCA precipitation was omitted. Together, these modification improved the total Grxl yeild to 3 mg, and pure Grxl(Cl 1U/C14S) to 0.9 mg per 2 liter of cell culture (note that this yield is underestimated because about half of the lysate was lost during purification).

MXB expression, purification, and assay

E. coli ME68z cells transformed with a pSecUAG-Evol2 derivative and pET-MXB(384UAG) were grown in LB media containing 50 μg/ml ampicillin, 25 μg/ml kanamycin, 10 μΜ Na2Se03 and 0.001% L-arabinose at 37°C until the A600 reached 0.8. The culture was then induced by the addition of 1 mM IPTG, and then shifted to 25°C for approximately 16 h before harvesting. The cells were harvested and disrupted with a lysis buffer [BugBuster, 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% glycerol, lysozyme, benzonase, and cOmplete protease inhibitor]. The

His6-tagged protein was purified by Ni-NTA (Qiagen). The Ni-NTA elution fractions were buffer exchanged into an intein reaction buffer [20 mM

HEPES, pH 8.5, 200 mM NaCl, and 0.1 mM EDTA] (Haruna, et al., Nucleic

Acids Res 2014, 42, 9976) by using amicon ultra (30k). About 7 μg of

154

45292464vl proteins were incubated with DTT (100 mM final) at room temperature for 16 h. After the intein cleavage reaction, proteins were mixed with SDS- PAGE buffer with 100 mM DTT and boiled, followed by SDS-PAGE and CBB staining. The intein cleavage efficiencies were calculated by comparing the protein band intensities by using NIH ImageJ.

Results

Next, the As SelA structure was engineered for a better positioning of allo-tRNA (Figure 17A). The SelA N-terminal domain binds the elbow region of Ser-tRNA^Sec to bring the Ser moiety into the catalytic site of the SelA core domain, and is fixed to the core domain by hydrophobic interactions with a few residues (Figure 17A) (Itoh, et al.,

Science. 2013;340:75). Engineering of this inter-domain interaction may fine-tune the orientation of the N-terminal domain and consequently improve Ser-to-Sec conversion on engineered allo-tRNA. Based on structural information from a SelA-tRNA^Sec complex, residues Pro68, Leu69, Gln72, and Cysl73 of As SelA, which corresponds to Ile25, Tyr26, Lys29, and Glul29 of Aquifex aeolicus SelA were modified (Figure 17A). For screening a mutant library of As SelA, a NMC-A β-lactamase variant was utilized in which the important disulfide bond must be replaced by a diselenide bond in E. co/zC321.AA.opt AselAB (Figure 17B). Several good variants were obtained (Table 5 Figure 17B). Variants #1.9 and #2.1 carry multiple mutations (A68-I69-E72-R173 and P68-F69-S72-V173), while variant #1.9 also gained a spontaneous Pro2-to-Thr2 mutation. These As SelA variants drastically enhanced the expression of FDHH (with 5 Sec residues) at 30 °C (Figure 17C ) and 37°C. Furthermore, they remained orthogonal to E.

coli tRNA^Ser. Finally, #2.1 was combined with Thr2 to develop SelA^Evo1.

SelA^Evo1 with allo-tRNA^UTu2D were subcloned to develop a series of pSecUAG-Evol (No. 1-4) variants, since the relative expression levels of tRNA and SelA appear to be important. The SelA^Evo1 expression level is controlled with four different transcription/translation initiation signals (No.

1 > 2 >3 >4). In this way, the allo-tRNA expression level can be regulated by arabinose concentration. Since unwanted suppression of amber codons at the ends of essential genes could impose burdens on the cells, the E. coli ME6

45292464vl 155 strain was transformed with a plasmid encoding essential genes with a UAG- to-UAA replacement, (Mukai T, et al. Biochem Biophys Res

Commun. 2011;411 :757) to establish ME68z (see Materials and Methods). For FDHH yields at 37 °C, pSecUAG-Evol3 with 0.01% arabinose was the best (Figure 17D). By using pSecUAG-Evol2 with 0.001% arabinose at 20°C, selenoglutaredoxin (Aldag, et al., Angew Chem Int Ed. 2013;52: 1441, Miller, et al., FEBS Lett. 2015;589:2194, 20) an E. coli glutaredoxin

Grxl(Cysl lSec/Cysl4Ser) variant, was produced in a yield of 0.9 mg from 2 L cell culture in a pure form using thiolsepharose chromatography (Aldag, et al., Angew Chem Int Ed. 2013;52: 1441).

GPxl was produced using pSecUAG-Evol2 with 0.001% arabinose at 25 °C. The protein yield and suppression efficiency were about 2-3 mgL^"1 and 70%, respectively. An intact mass analysis indicated predominant (>80%) Sec incorporation (Figure 18 A). The increase in the mass of GPxl(49Sec) by 44-46 Da (Figure 18A) may correspond to a Cys-to-Sec substitution at any of the five Cys positions (Guo, et al., IUBMB Life. 2014 doi: 10.1002/iub.l255). High levels of SelA^Evo1 expression may lead to Sec- allo-tRNA levels in excess of the amount needed for UAG translation;

hydrolysis of Sec-allo-tRNA may generate free Sec, which would produce cysteinyl tRNA synthetase mediated Sec misincorporation at Cys codons. Therefore, expression levels of SelA^Evo1 and allo-tRNA^UTu2D should be kept moderate. Curiously, in SDS-PAGE analysis, the GPxl band disappeared, while another main band emerged around 55 kDa when the highest

SelA^Evolexpression level (pSecUAG-Evoll) was employed. Excess

SelA^Evo1 may have sequestered allo-tRNA^UTu2D. Furthermore, SelA^Evo1 was revealed to have affinity to nickel resin through the N-terminal His-rich region. Therefore, its AHSHS (SEQ ID NO: 130) sequence was changed to PYR (from another AeromonasSelA) or ASSAS (SEQ ID NO: 110). GPxl proteins produced with SelA^Evo1 and the ASSAS (SEQ ID NO: 110) variant exhibited similar GPx activities, while the PYR variant was slightly less productive (Figure 18B).

Finally, pSecUAG-Evol2 was modified to encode the ASSAS variant and selenocysteine lyase SufS(C364A) (Mihara, et al., /

45292464vl 156 Biochem. 2000; 127:559) to develop pSecUAG2. Predominant Sec incorporation was confirmed by an in vitro intein cleavage assay of an Mxe GyrA intein (MXB) variant with the substitution of a Sec for the catalytic Cys384 (Haruna, et al., Nucleic Acids Res. 2014;42:9976). The yield of the full-length protein (70 kDa) at 25 °C was about 10 mgL^"1. Because less than 10% of the full-length protein remained intact after reaction, and because the Ser384 variant is inactive (Haruna, et al., Nucleic Acids Res. 2014;42:9976), the Sec incorporation rate is estimated to be more than 90% by assuming complete intein reaction.

Compared to earlier tRNA^UTu variants, which carry a 13-bp branch, the allo-tRNA system is intrinsically decoupled from the SelB/SECIS- mediated machinery and fully compatible with the canonical translation apparatus.

Table 5: As SelA variants. Variant #1.9 also has a spontaneous mutation ->Thr2)

Example 12: Design and testing of Saccharomyces cerevisiae tRNA^Ser variants for decoding selenocysteine

Materials and Methods

Strains and Growth Conditions

All yeast strains used in this study were derived from either BY4741 (Winzeler, et al., Curr Opin Genet Dev 1997, 7, 771-776) or MaV203 (Vidal, et al., Proc Natl Acad Sci U S A 1996, 93, 10315-10320; Vidal, et al., P Natl Acad Sci USA 1996, 93, 10321-10326, doi:DOI 10.1073/pnas.93.19.10321)

157

45292464vl (listed in Table 6). Unless otherwise indicated, yeast strains were grown in Yeast Peptone (YP) medium containing 2% glucose, or in minimal medium (MM) supplemented with required amino acids. All cultures were grown at 30°C. KHY1 was created by disrupting the HIS3 allele in MaV203 with a KanMX integration cassette (described in DNA molecules). The KanMX cassette was transformed into MaV203 and plated onto YPD plates containing 200 μg/ml G418 (Sigma). Histidine auxotrophic transformants were validated by patching onto minimal medium plates lacking histidine, and integration of the KanMX cassette was confirmed by PCR.

DNA molecules

Mouse selenocysteine lyase (mSCL), Aeromonas salmonicidia SelA (AsSelA), and A. salmonicidia SelD (AsSelD) were expressed from the ADH1 constitutive promoter (700bp) and terminated by the RPL41B terminator. A construct containing ADH1, mSCL, and the RPL41B terminator (sequence listed below) was synthesized (IDT) and cloned into pRS425 to generate pKH24 (plasmids listed in Table 7). AsSelA and AsSelD were codon optimized (sequences listed below), synthesized (IDT), and cloned into Ncol/Nhel digested KH24 by Infusion, thereby replacing mSCL and creating pKH29 and pKH30, respectively. To generate pKH39, ADH1- SCL-RPL41B was PCR amplified from KH24 template using ASR-F and ASR-R (oligonucleotides listed in Table 8) and cloned by Infusion into Pcil cut pRS424. AsSelA was PCR amplified using primers adhl-F and rpl41b-R and KH29 as template and cloned by Infusion into BamHI/XhoI digested pRS424 and KH39 to create pKH31 and pKH47, respectively.

GAM, from -15 to +2946 of the translational start, was PCR amplified from S. cerevisiae genomic DNA in two fragments using primers gal4a with gal4b and gal4c with gal4d. Full-length ADH1 promoter (1400 base pairs) was amplified using primers fADHl-F and fADHl-R. The three PCR production were cloned by Infusion into BamHI cut pRS424 to produce pKH58. The gal4-CHS, gal4-C21S, gaU-CllAm, and gal4-C21Am alleles were made by Quikchange (Table 8; primer names indicate mutations) using pKH58 as template, generating pKH79, pKH80, pKH81, and pKH82, respectively. Each of the gal4 alleles were moved into pRS423 as a BamHI

45292464vl 158 fragments to create pKH85, pKH99, pKHlOO, and pKHlOl. pKH85 was amplified with primers Cl lAm-F and Cl lAm-R to create pKH95. Similarly, pKH97 was made by amplifying pKH79 template using primers C21S-F and C21S-R, and pKH102 contains the gal4-CHS/C21S allele subcloned from pKH97 into pRS423 as a BamHI fragment.

tS(AGA)D2, including -500bp and +300bp of the tRNA coding sequence, was PCR amplified from S. cerevisiae genomic DNA using primers tSD2-F and tSD2-R, and cloned by Infusion into BamHI digested pRS425 to create pKH66. The anticodon was changed to CUA by two-step PCR using primers tScua-F with tSD2-R and tScua-R with tSD2-F. The two PCR products were mixed at a 1: 1 molar ratio, denatured at 95°C, annealed at 55 °C, and extended for 5 minutes at 72°C to produce full-length tS(CUA)D2. The full-length product was PCR amplified using primers tSD2- F and tSD2-R and cloned into BamHI cut pRS425 by Infusion to generate pKH71. pKH74 was made by inverse PCR using primers tSec-F and tSec-R with KH71 template, followed by Infusion. tU(CUA)l (tRNA sequences listed in Table 4) was subcloned into ΚΉ30 as a BamHI fragment to create pKH77. tU(CUA)2, tU(CUA)3, and tU(CUA)4 were introduced by inverse PCR, amplifying pKH77 template using primers tSec2-F with tSec-R, tSec3- F with tSec-R, and tSec4-F with tSec-R, respectively. Infusion cloning of the PCR products generated pRS425-AsSe\O/tU(CUA)2 (pKH84), pRS425- AsSe\O/tU(CUA)3 (pKH86), and pRS425-AsSelD/i£/fC£/A)4 (pKH103).

To disrupt the HIS3 allele in MaV203, a KanMX disruption cassette was made by three sequential PCRs. KanMX was PCR amplified using primers U2 and D2. This product was used as template for PCR using primers his3-F and his3-R. Primers his3-P and his3-T were used for PCR to extend the HIS3 homologous sequences flanking KanMX to 96 base pairs. Integration of the KanMX marker was validated by PCR using primers kanMX-F and Iys2-R.

Spot plating and growth assays

Suppression of gaU-CllAm (pKHlOl), gal4-C21Am (pKH85), and gal4-CllAm/C21Am (pKH95) was analyzed in yeast strain KHYl containing pKH47, and either pKH77, pKH84, pKH86, pKH103, or pRS425.

Each strain was grown in MM lacking leucine, tryptophan, and histidine to

45292464vl 159 stationary phase, then diluted into YPD medium contain 15 μΜ SeMet and 50 μg/ml sodium selenite and grown for 5 hours. Cells were spotted onto MM plates lacking leucine, tryptophan, histidine, and uracil, but containing 15 μΜ SeMet and 50 μg/ml sodium selenite in 10-fold serial dilutions. A control plate containing uracil was used to spot each strain and show viability and relative cell densities. Each spot plate was grown at 30° for 72 hours. As controls for selenocysteine-specific gal4 suppression, the KHY1 strains containing pKH47, pRS425, and either gal4-CHS (pKH99), gal4- C21S (pKHlOO), or gal4-CHS/C21S (pKH102) were grown and spotted using the same media and procedure described above.

Table 6. Strains used in this Example.

45292464vl 160 Table 7. Plasmids used in this Example.

45292464vl 161 pKH86 pRSA25-AsSelD/tU(CUA )3 This study

pRS423-ga/4- This study

pKH95 CllAm/C21Am

pKH97 pRS424-gal4-CllS/C21S This study

pKH99 pRS423-gal4-CllS This study

pKHlOO pRS423-gal4-C21S This study

pKHlOl pRS423-gal4-CllAm This study

pKH102 pRS423-gal4-CllS/C21S This study

pKH103 pRS425-AsSelD/tU(CUA )4 This study

Table 8. Oligonucleotides used in this Example.

Name Sequence

ASR-F GCAGGAAAGAACATGGGGTGTACAATATGGACTTCCTCTTTTC (SEQ ID

NO: 159)

ASR-R CCTTTTGCTCACATGTCGGGATGCACCATGAGG (SEQ ID NO: 160) adhl-F CGCGGATCCGGGTGTACAATATGGACTTCCTCTTTTC (SEQ ID NO: 161) rpl41b-R CCGCTCGAGTCGGGATGCACCATGAGG (SEQ ID NO: 162)

gal4a ACAATCAACTGCAAGCCTCCTGAAAGATGA (SEQ ID NO: 163) gal4b TACAAATCATCAAGCATTTTTTTGCACATATAGGA (SEQ ID NO:164) gal4c AAAATGCTTGATGATTTGTAATGAGATTGAGGAGG (SEQ ID NO: 165) gal4d CGCTCTAGAACTAGTGGATCCAACTAATCAATCTCAATTACGTTCG

(SEQ ID NO: 166)

fADHl-F TTCCTGCAGCCCGGGGGATCCGGGATCGAAGAAATGATGGTAAATG

(SEQ ID NO: 167)

fADHl-R GGAGGCTTGCAGTTGATTGTATGCTTGGTATAGC (SEQ ID NO: 168)

C21S-F TTCGGTTTTTCTTTGGAGCTCTTGAGCTTTTTAAGTCGG (SEQ ID NO: 169)

C21S-R CCGACTTAAAAAGCTCAAGAGCTCCAAAGAAAAACCGAA (SEQ ID

NO: 170)

Cl lAm-F GCTTTTTAAGTCGGCAAATATCCTATGCTTGTTCGATAGAAGACAG

(SEQ ID NO: 171)

Cl lAm-R CTGTCTTCTATCGAACAAGCATAGGATATTTGCCGACTTAAAAAGC

(SEQ ID NO: 172)

162

45292464vl C21Am-l CACTTCGGTTTTTCTTTGGACTACTTGAGCTTTTTAAGTCGGCAAATA (SEQ ID NO: 173)

C21Am-2 TATTTGCCGACTTAAAAAGCTCAAGTAGTCCAAAGAAAAACCGAAGTG

(SEQ ID NO: 174)

tSD2-F GCAGCCCGGGGGATCCGGATCTTGATTTGATGGGACTTCCT (SEQ ID

NO: 175)

tSD2-R TAGAACTAGTGGATCCGCGATAAGTCGTGATGCGGT (SEQ ID NO: 176) tScua-F GTTAAGGCGAAAGATTCTAAATCTTTTGGGCT (SEQ ID NO: 177) tScua-R AGCCCAAAAGATTTAGAATCTTTCGCCTTAAC (SEQ ID NO: 178) tSec-F CTAAATCTTTTGGGCTTTGCCCGGGCAGGTTCGATTCCTGCAGTTGTCGT

TTTTACCAAAAATTTTCCTCAATC (SEQ ID NO: 179)

tSec-R GCCCAAAAGATTTAGAATCTTTCGCCGCCACCAGACGGCGAAGTTGCCT

CAATTTACAAGTAATTTGAATATTATTAGATAGT (SEQ ID NO: 180) tSec2-F GCCCAAAAGATTTAGAATCTTTCGCCGCCACCAGGCGGCGTAGTTGCCT

CAATTTACAAGTAATTTGAATATTATTAGATAGT (SEQ ID NO: 181) tSec3-F GCCCAAAAGATTTAGAATCTTTCGCCGCCACCAGGCGGCATAGTTGCCT

CAATTTACAAGTAATTTGAATATTATTAGATAGT (SEQ ID NO: 182) tSec4-F GCCCAAAAGATTTAGAATCTTTCGCCGCCACCAGACGGCATAGTTGCC- TCAATTTACAAGTAATTTGAATATTATTAGATAGT (SEQ ID NO: 183)

U2 CGTACGCTGCAGGTCGAC (SEQ ID NO: 184)

D2 ATCGATGAATTCGAGCTCG (SEQ ID NO: 185)

his3-F ATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAG

ATCGTACGCTGCAGGTCGAC (SEQ ID NO: 186)

his3-R CTACATAAGAACACCTTTGGTGGAGGGAACATCGTTGGTACCATTGGGC

GAATCGATGAATTCGAGCTCG (SEQ ID NO: 187)

his3-P GAAGAATATACTAAAAAATGAGCAGGCAAGATAAACGAAGGCAAAGA

TGACAGAGCAGAAAGCCCTAG (SEQ ID NO: 188)

his3-T ACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCACTAC

ATAAGAACACCTTTGGTGGAG (SEQ ID NO: 189)

kanMX-F GAATCGCAGACCGATACCA (SEQ ID NO: 121)

Lys2-R TTTAAGTGACATCACCCGAAAAG (SEQ ID NO: 131)

163

45292464vl Table 9. tRNA sequences.

164

45292464vl SctRNA^SeccuA-3 tU(CUA)3 GGCAACTATGCCGCCTGGTGGCGGCGAAAG ATTCTAAATCTTTTGGGCTTTGCCCGGGCAG GTTCGATTCCTGCAGTTGTCG (SEQ ID NO: 155, DNA)

GGCAACUAUGCCGCCUGGUGGCGGCGAAAG AUUCUAAAUCUUUUGGGCUUUGCCCGGGCAG GUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 156, RNA, Figure 20E)

SctRNA^SeccuA-4 tU(CUA)4 GGCAACTATGCCGTCTGGTGGCGGCGAAAG

ATTCTAAATCTTTTGGGCTTTGCCCGGGCAG GTTCGATTCCTGCAGTTGTCG (SEQ ID NO: 157, DNA)

GGCAACUAUGCCGUCUGGUGGCGGCGAAAG AUUCUAAAUCUUUUGGGCUUUGCCCGGGCAG GUUCGAUUCCUGCAGUUGUCG (SEQ ID NO: 158, RNA, Figure 20F)

ADH1 -mSCL-RPUlB:

GGGTGTACAATATGGACTTCCTCTTTTCTGGCAACCAAACCCATACATCGGGATTC CTATAATACCTTCGTTGGTCTCCCTAACATGTAGGTGGCGGAGGGGAGATATACAA T AG AAC AG AT AC CAGACAAGACATAATGGGC T AAAC AAGAC T AC AC C AAT T AC AC T GCCTCATTGATGGTGGTACATAACGAACTAATACTGTAGCCCTAGACTTGATAGCC ATCATCATATCGAAGTTTCACTACCCTTTTTCCATTTGCCATCTATTGAAGTAATA ATAGGCGCATGCAACTTCTTTTCTTTTTTTTTCTTTTCTCTCTCCCCCGTTGTTGT C T C AC C AT AT C C G C AAT G AC AAAAAAAT GAT G G AAG AC AC T AAAG G AAAAAAT T AA CGACAAAGACAGCACCAACAGATGTCGTTGTTCCAGAGCTGATGAGGGGTATCTCG AAGCACACGAAACTTTTTCCTTCCTTCATTCACGCACACTACTCTCTAATGAGCAA CGGTATACGGCCTTCCTTCCAGTTACTTGAATTTGAAATAAAAAAAAGTTTGCTGT CTTGCTATCAAGTATAAATAGACCTGCAATTATTAATCTTTTGTTTCCTCGTCATT GTTCTCGTTCCCTTTCTTCCTTGTTTCTTTTTCTGCACAATATTTCAAGCTATACC AAGCATACAATCAACTATCTCATATACACCATGGCTAGCATGGATGCTGCTAGAAA TGGTGCTTTGGGTTCTGTTGAATCTTTGCCAGATAGAAAGGTTTACATGGATTACA AC G C T AC T AC T C C AT T G G AAC C AG AAG T T AT T C AAG C T GT T AC T G AAG C T AT G AAG GAAGCTTGGGGTAATCCATCTTCTTCATATGTTTCTGGTAGAAAGGCCAAGGATAT T AT C AAT G C T G C AAG AG C T T C T T T G G C C AAG AT G AT T G GT G G T AAAC C AC AAG AT A

165

45292464vl TTATCTTCACCTCTGGTGGTACTGAGTCTAACAACTTGGTTATCCATTCTATGGTT AGGTGCTTCCATGAACAACAAACTTTGAAGGGTAACATGGTTGACCAACATTCTCC AG AAG AAG G T AC T AG AC C AC AT T T C AT T AC T T G C AC C G T T G AAC AC G AT T C C AT T A GATTGCCATTAGAACACTTGGTCGAAAACCAGATGGCTGAAGTTACTTTTGTTCCA GTTTCTAAGGTTAACGGTCAAGCCGAAGTTGAAGATATTTTGGCTGCTGTTAGACC AACTACTTGTTTGGTTACTATCATGTTGGCTAACAACGAAACCGGTGTTATTATGC CAGTTTCCGAAATCTCTAGAAGGATCAAGGCTTTGAATCAAATTAGAGCTGCTTCT GGTTTGCCAAGAGTTTTGGTTCATACTGATGCTGCTCAAGCTTTGGGTAAGAGAAG AGTTGATGTTGAAGATTTGGGTGTTGACTTCTTGACTATCGTTGGTCATAAGTTTT ACGGTCCAAGAATTGGTGCCTTGTATGTTAGAGGTGTTGGTAAATTGACTCCACTG TACCCAATGTTGTTTGGTGGTGGTCAAGAAAGAAATTTCAGACCAGGTACTGAAAA CACCCCAATGATTGCTGGTTTGGGTAAAGCTGCTGATTTGGTTTCTGAAAACTGTG AAAC T T AC G AAG C C C AC AT GAG AG AT AT C AG AG AT T AC T T G G AAG AAAGG T T G G AA GCTGAATTCGGTAAAAGAATCCACTTGAACTCTAGATTCCCAGGTGTTGAAAGATT GCCAAACACTTGCAACTTCTCCATTCAAGGTTCTCAATTGCAAGGTTACACTGTTT TGGCTCAATGTAGAACTTTGTTGGCTTCTGTTGGTGCTTCTTGTCATTCTAACCAT GAAGATAGACCATCTCCAGTTTTGTTGTCTTGTGGTATTCCAGTTGATGTCGCAAG AAATGCAGTTAGATTGTCTGTTGGTAGAGGTACTACTAGAGCAGATGTTGATTTGA TCGTTCAAGACTTGAAACAAGCTGTTGCACAATTGGAAGGTCGTTTGTGAATGCAT GCGGATTGAGAGCAAATCGTTAAGTTCAGGTCAAGTAAAAATTGATTTCGAAAACT AATTTCTCTTATACAATCCTTTGATTGGACCGTCATCCTTTCGAATATAAGATTTT GT T AAGAAT AT T T T AGAC AGAGAT C T AC TT T AT AT T T AAT AT C T AGAT AT T AC AT A AT T T C C T C T C T AAT AAAAT AT C AT T AAT AAAAT AAAAAT G AAG C G AT T T G AT T T T G TGTTGTCAACTTAGTTTGCCGCTATGCCTCTTGGGTAATGCTATTATTGAATCGAA GGGCTTTATTATATTACCCTCATGGTGCATCCCGA (SEQ ID NO: 132)

AsSelA:

ATACACCATGGCTAGATGCCAAACTCTTCTCATGCTCCAGCTATTGCTCATTCTCA TTCACAACCAGAATCTTGTCCAACTGCTGATGATTCTTTGCCAGATTCATTACCTG ATTCATTGCCACAACCATCTCAACAACAAGCTAGAAGATTACCACAAGTCGAACAA TTGCTGCAACAACCATTTTTGACCGGTTTCATTGAAGCTTTGTCTAGACCATTGGT T AC C C AAG C T G T T AG AG AT GT T T T G T C C GAAT T G AG AC AAT C C G AAG C T T T T AG AC AACATGGTGTTGCTCCAGAACAAATTGAAGCCTTGATTGCTAAGAGATGCCAACAA CAACTAAGACAAAGACAAACCAGAGTTATTAACGCTACCGGTACTTTGGTTCATAC CAATTTGGGTAGATCTCCATTGTCTAGAGAATTGTGGGATGAAGTTAGAGATTTGA ACACCGGTTACAACAACTTGGAATTGGATTTGGCTACTGGTAAAAGAGGTGGTAGA

45292464vl 166 AAAGGTTTGATTGCTCCTTTGTTAAGATGCTTGACTCAAGCCGAAGATTCCTTGGT TGTAAACAACAATGCTGCCTCTCTGTTCCTTTTGTTGCAAGAAATTGCTAAGGGTA GAGAGGTCATAGTTTCTAGAGGTGAACAAATTCAAATCGGTGGTGGTTTCAGAATC CCAGATATTTTGGCTTTGTCTGGTGCTAAGTTGGTTGAAGTTGGTACTACTAATAT TACCACCGCCAAGGATTACTTGGATGCTATTACTGATCAAACCGCCTTGGTTTTGA TGGTTCATAGATCTAACTTCGCCATTAGAGGTTTCACTGAATCACCTGATATTGGT GAAGTTGCTAGAGCTTTACCAGAACATGTTGTTTTGGCTGTTGATCAAGGTTCTGG T T T G AC T AC T G AAG AAT T T GC T C C T G AC GAAAC T T C T G T T AG AC AG T AT AT T AAG G CTGGTGCTGATTTGGTTTGTTACTCAGGTGATAAGTTGTTAGGTGGTCCACAATCC GG T AT T AT T T C T G G T AG AT C C G AT T T G AT C AAG AG G T T GG AAAAAC AC C C AAT G AT GAGAACTTTCAGACCAAGCAGAATCGTCTACTCTTTGTTGGAAAGACTGTTGATCC ACAAGCTGAACAAATCTCCAATAGGTGAAGGTATTGCTCAGAGAACTTTGTCTAAT CCAGCTGCTATGCAAGCTAGAGCTGACCAATTGATGGCTGCTTTGCCAGGTTGTTT TGTTCCAGTTCCAGCTCAATTGGTTGTTGGTGGTGGTACATTGCCAGATGAATTTT AT C C AG C T C C AG C T T T G G AAT G T AC T G AT C C AAG AC C AGC T C AAC AAT T AT T G G AT GCCTTGAGAAAATTGCCCGTTCCAGTTATTGCTACTGTCAGACAACAAAAAGTCTT GTTGAACATGGCTACTTTGTTGCCAACTGAAATTGCCTTGTTGATCGCTCAATTGA AAGAGTTGTTATTGCCAACTCCAACTACTGCTACAGAAGAACCATAATGCATGCGG

ATT GAG (SEQ ID NO:133)

AsSelD:

ATACACCATGGCTAGATGTCCTCCATTAGATTGACCCAATATTCTCATGGTGCTGG TTGTGGTTGTAAGATTTCTCCAAAGGTTTTGGACACCATCTTGAAGTCTCAAATTC CAGGTTTTGATGATCCAACCTTGGTTGTTGGTAACTCCTCTAAAGATGATGCTGCC GTTGTTGATATTGGTAATGGTCAAGGTATCGTTTCTACCACCGATTTTTTCATGCC AATTGTCGATGATCCATTCACCTTTGGTAGAATTGCTGCTACCAATGCCATTTCTG ATATCTATGCTATGGGTGGTAAACCTATCGTTGCCATTGCTATTTTAGGTTGGCCA ATCAATACTTTGGCTCCAGAAGTTGCTCAACAAGTTATTGATGGTGGTAGACAAGT TTGTCATGAAGCCGGTATTTCTTTGGCTGGTGGTCATTCTATTGATGCTCCAGAAC CTATTTTTGGTTTGGCTGTTACTGGTATTGTCCCATTGAATGCCATTAAGCAAAAC GATACTGCTCAAGCTGGTGACATCTTGTATTTGACAAAGCCATTAGGTATCGGTAT TTTGACTACCGCTCAAAAGAAGGGTAAATTGAAGCCAGAACATGAACAATTGGCTC CAAATGCTATGTGTACCTTGAACAAGATTGGTCAAAGATTCGCTGAATTGCCAGGT GTTCATGCTATGACTGATGTTACAGGTTTTGGTTTAGCAGGTCACTTGTTGGAAAT GTGTGAAGGTTCTGGTGTTTGTGCTACTTTGGATTTTAAAGCCTTGCCTTTGTTGG ATGAAGTCGACTATTATTTGTCTGAAGGTTGTGTTCCAGGTGGTACTTTGAGAAAT

45292464vl 167 TTTGATTCTTACGGTGCTAAGTTGGGTGCTATGGACGAAAGAACTAGAAACATTAT GTGTGACCCACAAACATCTGGTGGTTTGTTGGTTGCTGTTGGTAAAGAATCTGAAG CTGAGTTGTTGGCTATTGCTACACAAGCTGGTTTGACTTTGTCTCCAATTGGTCAA TTGAAAGCCTACACCGGTAATCAATTCATCGAAGTCATTCAGTAATGCATGCGGAT

TGAG (SEQ ID NO: 134) tS(AGA)D2 (including -500bp and +300bp of the tRNA coding sequence):

GGATCTTGATTTGATGGGACTTCCTTAGAAGTAACCGAAGCAGCGGCGCTACCATG T AG AG AAT G T G G AT T T T G AT G T AAT T G T T G G G AT T C C AT T G T G AT T AAGG C T AT AA T AT T AG G T AT G T AG AT AT AC T AG AAG T T C T C C T C G AG G AT T T AG G AAT C C AT AAAA GG G AAT C T G C AAT T C T AT AC AAT T C T AT AAAT AT TAT TAT CATCATTT TAT AT G T T AATATTCATTGATCCTATTACATTATCAATCCTTGCGTTTCAGCTTCCACTAATTT AGATGACTATTTCTCATCATTTGCGTCATCTTCTAACACCGTATATGATAATATAC T AG T AAC G T AAAT AC T AG T T AG T AG AT G AT AG T T G AT T T T T AT T C C AAC AAGAC G A TAGTGGATTTTTATTCCAACAAGTTGCATGTGTGTTTTATTAACGTTTAGATGTGT GTCATAATACTGCATCATACTATCTAATAATATTCAAATTACTTGTAAATTGAGGC AACTTGGCCGAGTGGTTAAGGCGAAAGATTAGAAATCTTTTGGGCTTTGCCCGCGC AGGTTCGAGTCCTGCAGTTGTCGTTTTTACCAAAAATTTTCCTCAATCTTTTTAAT TTTACTTTAATAGTACCAGAATGACAATGCGATAAAAAAGAATAATTTGAAAGGGC ACAAAAATGGCAAATAGTGCTTGGATCGTGAACTCTAGTCTTTTTTATACCTACGG CTGCCCTTCTGCCTGCAGCATACATTAACTATCAAAGAATTCGGTTGGTCGATGAG CCATAAATCGATGAGAAAAAGTATGCTTTAGTGTTACACGATATTTTACGGACAAA CAGAAGGTTTCGATATCGAGGTAACCGCATCACGACTTATCGC (SEQ ID

NO:135) Results

Transfer RNA^Sec (tRNA^Sec) is naturally a poor substrate for seryl- tRNA synthetase (SerRS) relative to tRNA^Ser, resulting in a low level of tRNA^Sec charged with selenocysteine (Sec) within the cell. This is likely due to the minimal number of Sec incorporations events required for expressing selenoproteins. For recombinant selenoprotein production it is desirable to have high cellular concentrations of Sec-tRNA^Sec, to out-compete release factor for decoding stop codons with Sec. Considering this, and the fact that the anticodon of tRNA^Ser is not an identity element for aminoacylation by

45292464vl 168 SerRS, an efficient nonsense suppressor was designed using Saccharomyces cerevisiae tRNA^Ser (5ctRNA^Ser) for Sec incorporation at amber (UAG) codons. Since eukaryotic SepSecS recognizes a longer tRNA acceptor branch Aeromonas salmonicida SelA (AsSelA) was choosen, which works with shorter acceptor branch tRNA^Sec molecules and results in highly efficient selenoprotein production in bacteria (Mukai, et al., Angew Chem Int Ed Engl 2018, 57, 7215-7219, doi: 10.1002/anie.201713215). Moreover, AsSelD from this system was also used to produce free selenophosphate substrate for AsSelA. Mus musculus selenocysteine lyase (MmSCL) was added to this system to hydrolyze free selenocysteine for the production of selenide used by AsSelD (Figure 19).

To generate a tRNA^Sec species from 5ctRNA^Ser, the anticodon was switched from AGA to CUA and AstRNA^Sec identity elements were introduced. Four tRNA^Sec variants were tested, each having mutations that may influence tRNA stability and AsSelA recognition (Figure 20C-20F; tRNA sequences listed in Table 9). To test suppression efficiency and Sec incorporation of each tRNA^Sec variant, a reporter was designed using the yeast Gal4 transcription factor, which would show a yeast prototrophic phenotype specifically when Sec, but not Ser, is incorporated. In this way, incorporation as a result of Sec-tRNA^Sec biosynthesis, can be distinguished from the Ser-tRNA^Sec intermediate species. A similar Gal4 reporter system has previously been used to sense read-through of stop codons without amino acid specificity (Chin, et al., Chem Biol 2003, 10, 511-519). In this case the important zinc -coordinating residues Cysl l and/or Cys21 were replaced in Gal4 with amber codons so that Sec replacement of Cys would maintain zinc coordination, while Ser insertion or translation termination would result in a non- functional protein (Figure 21A-21B). Functional Gal4 promotes uracil prototrophy through expression of URA3 driven by GALA promoter sequences. Indeed, substitution of Cysl l or Cys21 with Ser did not permit growth on medium lacking uracil (Figure 22). tRNA^Sec variants 1, 3, and 4 resulted in suppression of gal4-CHAm on plates lacking uracil and containing sodium selenite and selenomethionine (Figure 23 A), whereas tRNA^Sec-2 did not. Interestingly, only tRNA^Sec-3 and -4 suppress gal4-

45292464vl 169 C21Am (Figure 24B), and tRNA^Sec-3 was able to suppress gal4- CllAm/C21Am in a selenium dose-dependent fashion (Figure 24C). These results suggest that tRNA^Sec-3 is the most efficient variant tested for incorporation of Sec into Gal4.

To demonstrate the requirement for each system component, each was removed individually and analyzed for gal4 suppression. Yeast strains without either AsSelA, AsSelD, or tRNA^SeccuA do not support growth on medium without uracil (Figure 24D). Removal of only MmSCL did not completely disrupt the system, likely because hydrogen selenide is produced from sodium selenite reduction, which can be used by AsSelD (Falcone, et al., J Bacteriol 1963, 85, 754-762).

45292464vl 170

Claims

We claim:

1. A nucleic acid comprising a nucleic acid sequence encoding a non- naturally occurring tRNA^Sec, wherein the tRNA^Sec comprises the concensus sequence of

i.

optionally, wherein an acceptor stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-80 respectively;

an anti-codon arm can be formed by base pairing between nucleotides

27- 32 with nucleotides 45-40 respectively;

or a combination thereof;

ii.

optionally, an acceptor stem can be formed by base pairing between nucleotides 1-9 with nucleotides 91-83 respectively;

an anti-codon arm can be formed by base pairing between nucleotides

28- 33 with nucleotides 46-41 respectively;

or a combination thereof; or

45292464vl 171 YU 5714 CIP (2)

078245/00390 iii.

GNNNNRYNANNNNNNNNGGYNNNNNNNNNNGNYYNN2VAANCNNNNNNNNN NNNNNNNNNNNNNNNNNGNUCRANNCNNNNNYNNNNCGCCA (SEQ ID NO:21) optionally, an acceptor stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-80 respectively;

or a combination thereof.

2. The nucleic acid of claim 1, wherein the tRNA^Sec comprises the nucleic acid sequence according SEQ ID NO: 19-42 or SEQ ID NO: 137- 146 or the DNA or RNA sequence thereto, or a variant thereof comprising at least 85% sequence identity to any one of SEQ ID NO: 19-42 or 137-146 or the DNA or RNA sequence thereto.

3. The nucleic acid of claim 2, wherein the tRNA^Sec comprises the nucleic acid sequence according SEQ ID NO:57 or a variant thereof comprising at least 85% sequence identity to SEQ ID NO:57, and wherein the anticodon of the tRNA^Sec recognizes a stop codon.

4. The nucleic acid of any one of claims 1-3, wherein the nucleic acid is DNA or RNA.

5. The nucleic acid of anyone of claims 1-3 further comprising a heterologous expression control sequence.

6. An expression vector comprising the isolated nucleic acid

of claim 5.

7. The tRNA^Sec formed by the isolated nucleic acid of any one of claims 1-4.

45292464vl 172 YU 5714 CIP (2)

078245/00390

8. A host cell comprising the isolated nucleic acid of any one of claims 1-4.

9. The host cell of claim 8, wherein the host cell is a prokaryote, archaeon, or eukaryote.

10. The host cell of claim 15, whereint the prokaryote is E. coli.

11. The host cell of claim 8, wherein the nucleic acid is incorporated into the genome of the cell.

12. The host cell of claim 11, wherein the host cell is a genetically recoded organism.

13. A non- naturally occurring tRNA^Sec including sequence elements from a yeast tRNA^Ser in combination with sequence elements from a non-yeast tRNA wherein the non-naturally occurring tRNA^Sec is a substrate for a SerRS and a SelA and binds to eEFla.

14. The non-naturally occurring tRNA^Sec of claim 13, wherein the yeast tRNA^Ser comprises the nucleic acid sequence of SEQ ID NO: 150.

15. The non-naturally occurring tRNA^Sec of claim 14, wherein the non- yeast tRNA is a tRNA^Sec.

16. The non-naturally occurring tRNA^Sec of claim 15, wherein the non- yeast tRNA is an Aeromonas salmonicida tRNA^Sec comprising SEQ ID NO: 148.

17. The non-naturally occurring tRNA^Sec of any one of claims 13-16 comprising at least 85% sequence identity to any one of SEQ ID NO: 152, 154, 156, or 158, or the opal or ochre equivalent thereof.

18. A nucleic acid encoding a non-naturally occurring tRNA^Sec, wherein the non-naturally occurring tRNA^Sec comprises at least 85% sequence identity to any one of SEQ ID NO: 152, 154, 156, or 158, or the opal or ochre equivalent thereof.

19. A method of making a recombinant selenocysteine containing protein comprising expressing a nucleic acid encoding the tRNA^Sec of any one of claims 1-19 in a translation system.

20. A variant of SelA comprising at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any one of SEQ ID NO:79, 80, 82, or 84,

45292464vl 173 YU 5714 CIP (2)

078245/00390 wherein the variant does not have 100% sequence identity to SEQ ID NO:79, 80, 82, or 84.

45292464vl 174 YU 5714 CIP (2)

078245/00390