WO2018152323A1

WO2018152323A1 - Compositions and methods for template-free enzymatic nucleic acid synthesis

Info

Publication number: WO2018152323A1
Application number: PCT/US2018/018365
Authority: WO
Inventors: Derek L. Stemple; Sylwia A. MANKOWSKA; Steven A. HARVEY
Original assignee: Camena Bioscience Limited
Priority date: 2017-02-17
Filing date: 2018-02-15
Publication date: 2018-08-23
Also published as: TW201840855A

Abstract

Disclosed are compositions and methods for template-free nucleic acid synthesis. Exemplary methods comprise deprotecting a primer comprising at least three nucleotides, wherein the primer comprises a 3' reversible terminating nucleotide (rtNTP), incorporating at least one free rtNTP by an enzyme or a ribozyme having a terminal transferase activity and repeating these steps until the desired synthetic nucleic acid is generated. Methods of the disclosure may be performed using primers in solution as well as primers linked to a substrate (e.g. including an array).

Description

COMPOSITIONS AND METHODS FOR TEMPLATE-FREE ENZYMATIC

NUCLEIC ACID SYNTHESIS

RELATED APPLICATIONS

[01] This application claims the benefit of provisional application USSN 62/460,429, filed February 17, 2017, the contents of which are herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

[02] The contents of the text file named "DNWR OOl/OOlWO SeqList.txt," which was created on February 15, 2018 and is 1 KB in size, are hereby incorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

[03] The disclosure provides compositions and methods for template-free enzymatic nucleic acid synthesis using reversibly terminating nucleotides.

BACKGROUND

[04] Over the last decade there has been an increase in demand for synthetic DNA molecules, which are used in a range of molecular biology applications. This increase has, in part, been driven by advances in DNA sequencing technology. However, while there have been significant developments in DNA sequencing technology, DNA synthesis technology has not progressed at a comparable pace and consequently the state of the art technology does not satisfy the current market needs. The disclosure provides compositions and methods for template-free enzymatic DNA synthesis that provide a solution to the long-felt yet until, now, unmet need in the art for the efficacious production of long DNA sequences having the superior accuracy and speed of synthesis demonstrated by the compositions and methods of the disclosure.

SUMMARY

[05] The disclosure provides compositions and methods for the enzymatic, template- independent, synthesis of nucleic acid (NA) polymers using reversible terminating nucleotide triphosphates (rtNTPs). TdT is used to incorporate individual rtNTPs to the 3' end of single stranded NA molecules. Unique NA sequences can be synthesized by successive cycles of addition of one rtNTP, then removal of the blocking groups followed by the addition of a new rtNTP to the reaction.

[06] The disclosure provides a composition comprising: (a) a primer comprising at least three nucleotides, wherein the primer comprises a 3' reversible terminating nucleotide (rtNTP); (b) at least one free rtNTP; and (c) an enzyme or a ribozyme having a terminal transferase activity. In certain embodiments, the composition further comprises (d) a reaction buffer. In certain embodiments, the reaction buffer comprises a final concentration of 50 mM Potassium Acetate, 20 mM Tris-Acetate and 10 mM Magnesium Acetate. In certain embodiments, sources of the divalent cations, including, but not limited to, Zn²⁺, Co²⁺, Mn²⁺, are provided. In certain embodiments, the composition further comprises one or more divalent cations. In certain embodiments of the composition, the divalent cations are Zn²⁺, Co²⁺ or Mn²⁺. In certain embodiments, the reaction buffer further comprises one or more divalent cations. In certain embodiments of the reaction buffer, the divalent cations are Zn²⁺, Co²⁺ or Mn²⁺.

[07] In certain embodiments of the compositions of the disclosure, the at least one free reversible terminating nucleotide (rtNTP) comprises a chemically-reversible blocking group. In certain embodiments, the composition further comprises an agent to chemically-reverse the blocking group. In certain embodiments, the agent is a Lewis acid. In certain

embodiments, the Lewis acid comprises CoCh. In certain embodiments, the chemically- reversible blocking group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group or an allyl group. In certain embodiments, the chemically-reversible blocking group comprises a 2-nitrobenzyl group.

[08] In certain embodiments of the compositions of the disclosure, the at least one free reversible terminating nucleotide (rtNTP) comprises a photo-reversible blocking group. In certain embodiments, the photo-reversible blocking group comprises a 2-nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group or a 7-methyoxy-4-methylcoumarin group. In certain embodiments, the photo-reversible blocking group comprises a 2-nitrobenzyl group.

[09] In certain embodiments of the compositions of the disclosure, the primer comprises a 5' modification. In certain embodiments, the 5' modification comprises a selectable tag. The selectable tag may be used to isolate or purify the resultant synthetic DNA polymer from the reaction. In certain embodiments, the selectable tag is bound or hybridized to the primer. In certain embodiments, the selectable tag is bound to a substrate or hybridized to a substrate- bound NA chain. In certain embodiments, the substrate comprises a flat surface or a bead. In certain embodiments, the substrate comprises a glass, a polymer or a matrix. In certain embodiments, the substrate comprises pores or channels. In certain embodiments, the substrate comprises a polymer and wherein the polymer comprises a polyacrylamide gel. In certain embodiments, the substrate comprises an anchor.

[010] In certain embodiments of the compositions of the disclosure, the primer comprises a 5' modification. In certain embodiments, the 5' modification comprises biotin. In certain embodiments, the substrate comprises a bead, wherein the bead comprises a polyacrylamide gel, wherein the polyacrylamide gel comprises an anchor and wherein the anchor comprises avidin or streptavidin.

[Oil] In certain embodiments of the compositions of the disclosure, the primer comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an amino acid or any combination thereof. In certain embodiments, the primer comprises at least one non-naturally occurring base or at least one non-naturally occurring backbone. In certain embodiments, the primer comprises at least one non-naturally occurring base and at least one non-naturally occurring backbone. In certain embodiments, the at least one non-naturally occurring base comprises a a dBTP, a dKTP, a dPTP, a dXTP a dZTP, a dlnDTP, a 5fluoroindolyl-2'-deoxyriboside triphosphate (d5FITP), a 5-amino-indolyl-2'-deoxyriboside triphosphate (dAITP), a 5-nitro- indoly 1-2 '-deoxy riboside triphosphate (dNITP), a 5-cyclohexyl-indolyl-2'deoxyriboside triphosphate (dCHITP), a dCEITP, a 5-phenylindolyl-2'-deoxyriboside triphosphate (d5PhITP), a 5-napthylindolyl-2'-deoxyriboside triphosphate (d5NapITP) or a d5AnITP. In certain embodiments, the at least one non-naturally occurring backbone comprises a cyclohexenyl nucleic acid (CeNA), an arabinonucleic acid (ANA), a 2'-fluoro- arabinonucleic acid (FANA), a a-L-threofuranosyl nucleic acid (TNA) or a locked nucleic acid (LNA). In certain embodiments, the LNA comprises a 2'-0,4'-C-methylene- -D- ribonucleic acid.

[012] In certain embodiments of the compositions of the disclosure, the primer comprises a reversible terminating nucleotide (rtNTP) and the rtNTP comprises a chemically-reversible blocking group. In certain embodiments, the chemically-reversible blocking group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group or an allyl group. In certain embodiments, the chemically-reversible blocking group comprises a 2-nitrobenzyl group.

[013] In certain embodiments of the compositions of the disclosure, the primer comprises a reversible terminating nucleotide (rtNTP) and the rtNTP comprises a photo-reversible blocking group. In certain embodiments, the photo-reversible blocking group comprises a 2- nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group or a 7-methyoxy-4- methylcoumarin group. In certain embodiments, the photo-reversible blocking group comprises a 2-nitrobenzyl group.

[014] In certain embodiments of the compositions of the disclosure, the primer is in solution.

[015] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate.

[016] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is directly linked to the substrate and the primer contacts the substrate. In certain embodiments, the primer is indirectly linked to the substrate and the primer contacts a linker that contacts the substrate.

[017] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is directly linked to the substrate and the primer contacts the substrate. In certain embodiments, the primer is indirectly linked to the substrate and the primer contacts a linker that contacts the substrate. In certain embodiments, the substrate is a bead. In certain embodiments, the bead comprises a glass, a polymer, or a matrix. In certain embodiments, the bead is porous. In certain embodiments, the bead comprises a polyacrylamide gel.

[018] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is directly linked to the substrate and the primer contacts the substrate. In certain embodiments, the primer is indirectly linked to the substrate and the primer contacts a linker that contacts the substrate. In certain embodiments, the substrate is a substantially flat surface. In certain embodiments, the substrate is a flat surface. In certain embodiments, the substrate comprises a glass, a polymer, or a matrix.

[019] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is directly linked to the substrate and the primer contacts the substrate. In certain embodiments, the primer is indirectly linked to the substrate and the primer contacts a linker that contacts the substrate. In certain embodiments, the substrate is a substantially flat surface. In certain embodiments, the substrate is a flat surface. In certain embodiments, the substrate comprises a glass, a polymer, or a matrix. In certain embodiments, the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array. In certain embodiments, the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, the plurality of primers comprises at least one duplicate of the first primer and at least one duplicate of the second primer.

[020] In certain embodiments of the compositions of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is directly linked to the substrate and the primer contacts the substrate. In certain embodiments, the primer is indirectly linked to the substrate and the primer contacts a linker that contacts the substrate. In certain embodiments, the substrate is a substantially flat surface. In certain embodiments, the substrate is a flat surface. In certain embodiments, the substrate comprises a glass, a polymer, or a matrix. In certain embodiments, the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array. In certain embodiments, the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, each primer of the plurality of primers comprises a unique sequence.

[021] In certain embodiments of the compositions of the disclosure, the enzyme is a terminal deoxynucleotidyl transferase (TdT).

[022] In certain embodiments of the compositions of the disclosure, the enzyme is a pol theta, a pol lambda, a pol mu, a Dpo 1, or a primase.

[023] In certain embodiments of the compositions of the disclosure, the ribozyme is a RNA- dependent RNA polymerase.

[024] The disclosure provides a method of template-free nucleic acid synthesis comprising: (a) obtaining a composition of the disclosure; (b) deprotecting the 3' rtNTP of the primer of the composition; and (c) incorporating the at least one free rtNTP of the composition by the enzyme or ribozyme of the composition into the primer of the composition, thereby synthesizing a nucleic acid. In certain embodiments, the at least one free rtNTP of the composition is a plurality of free rtNTPs. In certain embodiments, steps (b) and (c) are completed in less than 1 minute. In certain embodiments, the method further comprises a first rinse after the deprotecting step (b) and a second rinse after the incorporating step (c). In certain embodiments, including those where the method further comprises a first rinse after the deprotecting step (b) and a second rinse after the incorporating step (c), steps (b) and (c) are completed in less than 1 minute. In certain embodiments, including those where the method further comprises a first rinse after the deprotecting step (b) and a second rinse after the incorporating step (c), the method further comprises the steps of: (d) repeating steps (b) and (c). In certain embodiments, including those where the method further comprises a first rinse after the deprotecting step (b) and a second rinse after the incorporating step (c), the method further comprises the step of: (e) removing unincorporated rtNTPs prior to performing step (d).

[025] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the 3' rtNTP of the primer comprises a photo-reversible blocking group and the deprotecting comprises exposing the photo-reversible blocking group to light radiation. In certain embodiments, the light radiation comprises UV radiation.

[026] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the 3' rtNTP of the primer comprises a chemically reversible blocking group and the deprotecting comprises exposing the chemically reversible blocking group to a Lewis acid. In certain embodiments, the Lewis acid comprises CoCh.

[027] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the primer is in solution.

[028] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the primer is linked to a substrate.

[029] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array. In certain embodiments, the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, the plurality of primers comprises at least one duplicate of the first primer and at least one duplicate of the second primer.

[030] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the primer is linked to a substrate. In certain embodiments, the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array. In certain embodiments, the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, each primer of the plurality of primers comprises a unique sequence.

[031] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, the method further comprises the step of: contacting the synthetic nucleic acid and one or more of a random primer, a non-specific primer, a set of random short terminated nucleic acid sequences, a non-catalytic single-stranded binding protein and a non-catalytic single-stranded binding compound during synthesis to maintain a substantially linear conformation, to inhibit formation of a secondary and/or a tertiary structure, or to untangle an inhibitory conformation of the synthetic nucleic acid during one or more rounds of deprotection and incorporation of a rtNTP. In certain embodiments, the non-catalytic single- stranded binding protein or the non-catalytic single-stranded binding compound comprises a polyamine. In certain embodiments, the polyamine comprises spermine, penta-L-lysine, poly disperse poly-L-lysine or spermidine.

[032] In certain embodiments of the method of template-free nucleic acid synthesis of the disclosure, including those embodiments wherein the method further comprises the step of contacting the synthetic nucleic acid and one or more of a random primer, a non-specific primer, a set of random short terminated nucleic acid sequences, a non-catalytic single- stranded binding protein and a non-catalytic single-stranded binding compound during synthesis, the contacting step is performed once the synthetic nucleic acid comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 nucleotides, or any number of nucleotides in between. Alternatively, or in addition, in certain

embodiments, the contacting step is performed prior to the generation of a sequence within the synthetic nucleic acid that may form a secondary or tertiary structure. Alternatively, or in addition, in certain embodiments, the contacting step is performed prior to the generation of a sequence having sufficient length that the synthetic nucleic acid can fold into a substantially non-linear conformation. In certain embodiments, the method further comprises the step of (f) removing the one or more of a random primer, a non-specific primer, a set of random short terminated nucleic acid sequences, a non-catalytic single-stranded binding protein and a non-catalytic single-stranded binding compound from the synthetic nucleic acid after synthesis is complete.

BRIEF DESCRIPTION OF THE DRAWINGS

[033] Figure 1 is a diagram depicting the structure of 3'-0-(2-nitrobenzyl)-2'-dATP.

[034] Figure 2A is a sequence of the single stranded oligonucleotide (SEQ ID NO: 1) that was used as a starting point for the DNA synthesis shown in Figure 2B. The star at the 5' end represents an alexa-488 label.

[035] Figure 2B is a photograph showing a 20% polyacrylamide gel (8M urea) run for 35 mins at 100V. The gel was then stained by fixation with 10% ethanol for 5 mins, followed by washing with lxTBE containing SybrGold for 3 mins. Lane 1 = a ladder, lane 2 = the oligo shown in Fig. 2A, lane 3 = oligo plus TdT, lane 4 = oligo incubated with TdT and 3'-0-(2- nitrobenzyl)-2'-dATP, lane 5 = the sample shown in lane 4 but also UV irradiated, followed by clean-up and a further incubation with TdT and 3'-0-(2-nitrobenzyl)-2'-dATP, lane 6 = the sample shown in lane 4, not UV irradiated and then further incubated with unblocked dNTPs, lane 7 = oligo incubated with free dNTPs.

[036] Figure 3 is a schematic diagram depicting Watson-Crick pairing rules of size complementarity and hydrogen bonding complementarity for dBTP, dKTP, dPTP, dXTP and dZTP nucleotides. Reproduced from K. Sefah et al. PNAS 2014; 111 : 1449-1454.

[037] Figure 4 is a schematic diagram depicting structures of exemplary non-naturally occurring nucleic acid bases (2'-deoxynucleoside triphosphates) of the disclosure. "dR" is used to represent the deoxyribose triphosphate portion of the nucleotides. Reproduced in part from A.J. Berdis and D. McCutcheon. Chembiochem 8(12) (2007) 1399-408.

DETAILED DESCRIPTION

[038] Disclosed are compositions and methods for the enzymatic, template-independent, synthesis of nucleic acid (NA) polymers using reversible terminating nucleotide

triphosphates (rtNTPs). According to the methods of the disclosure, terminal

deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, incorporates individual rtNTPs to the 3' end of single stranded NA molecules. Unique NA sequences can be synthesized by successive cycles of addition of one rtNTP, then removal of the blocking groups followed by the addition of a new rtNTP to the reaction.

Increased Speed and Length as well as Decreased Cost of DNA Synthesis

[039] The compositions and methods of the disclosure produce longer synthetic DNA molecules and, moreover, reduce the cost of NA synthesis when compared to current technologies.

[040] Phosphoramidite DNA synthesis is an organic chemical method that has been used for many years to synthesize short nucleic acid (NA) molecules, referred to as oligonucleotides. This method has been automated since the 1980s and is commonly used to produce oligonucleotides of 15-25 nucleotides long. However, due to incomplete reactions and side reactions, there is a limit to the length of DNA molecules that can be synthesized by this method. Practically, this limit is 100 nucleotides. This presents a problem, as the coding sequence of an average human gene is 1000 nucleotides. Therefore, to synthesize a whole gene with current technology requires the stitching together of shorter DNA fragments, which increases the cost of goods to a point that limits many applications in synthetic biology. Additionally, phosphoramidite methods typically require a cycle time of at least 5 minutes for each added nucleotide in a growing NA chain, making the generation of long NA chains prohibitive. There is therefore a need for a new NA synthesis technology (i.e. , the compositions and methods of the disclosure) that produces longer NA molecules increases the speed of synthesis and reduces the cost of NA synthesis.

[041] The compositions and methods of the disclosure increase the speed of synthesis and reduce the cost of NA synthesis when compared to existing methods. For example, each cycle of deprotection of a 3' rtNTP of a primer or a growing NA and incorporation of a new rtNTP can be completed in less than one minute. Even with a rinsing step inserted after the deprotecting step and the incorporation step, this cycle can be completed in less than one minute.

Increased Diversity of Nucleic acids

[042] The compositions and methods of the disclosure increase the diversity of available NAs for other application such as the detection of small molecules such as metabolites.

[043] There is an additional need in the field to synthesize NAs with non-natural bases or backbones. This need arises from the increasing utility of aptamers, NAs that specifically bind small molecules, such as a variety of metabolites and short peptides. While single- stranded RNA traditionally has been used as aptamers, a variety of non-natural bases and backbones are now used to increase the diversity of small molecule binding and enzymatic possibilities. The compositions and methods of the disclosure can comprise alternative nucleotides possessing non-naturally occurring bases and/or non-naturally occurring backbones to generate non-naturally occurring NAs. Non-naturally occurring NAs may be used to increase the specificity and diversity of small molecules that bind NAs of the disclosure.

Terminal Deoxynucleotidyl Transferases

[044] The disclosure demonstrates that bovine TdT incorporates 3'-0-(2-nitrobenzyl)-2'- dNTPs onto the 3' end of a single stranded DNA molecule that is being synthesized.

Providing light removes the 2-nitrobenzyl blocking group and permits the addition of another 3'-0-(2-nitrobenzyl)-2'-dNTP. Other enzymes, possessing appropriate terminal transferase activity may be used. Additionally, a variety of evolutionarily distinct terminal transferases have been described and these may also be used. In general the speed of incorporation using efficient enzymes is very quick, generally less than 1 minute. [045] Terminal deoxynucleotidyl transferase (TdT) is a polymerase that can add

deoxynucleotides or ribonucleotides to the 3 ' end of a single stranded NA molecule in a template-independent manner. TdT is capable of synthesizing DNA molecules over 1500 nucleotides long. Numerous examples have shown the DNA polymerases can incorporate into template-dependent synthesis dNTPs that have removable blocking groups at their 3 ' position. Using cycles, this approach allows the incorporation of individual nucleotides. TdT can also incorporate modified dNTPs into single stranded DNA synthesis reactions, including dNTPs with an azide group at either the 3 ' or 2' position. In certain embodiments, the compositions and methods of the disclosure comprise dNTPs that have modifications at the 3' position, including 3'-0-azidomethyl dATP and 3 '-0-azidomethyl dCTP.

[046] Although mammalian TdTs are the most frequently used and best understood terminal transferases, there other diverse enzymes that possess terminal transferase activity, that is the ability to extend a nucleic acid chain in a non-templated fashion. These activities are often revealed under alternate biochemical conditions, such as in a milieu of alternate divalent cations. Alternative enzymes and ribozymes having terminal transferase activity that may be used in the compositions and methods of the disclosure include, but are not limited to, Pol Theta, Pol Lambda, Pol Mu, Dpo 1 , Primase, RdRPs and Ribozyme.

[047] Pol theta can switch between templated and non-templated terminal transferase activity by changing the divalent cation from Mg²⁺ to Mn²⁺.

[048] Pol mu, another member of the PolX family, which includes TdT, can switch between templated and non-templated activity after mutation in the Loop 1 domain (see, A.F. Moon, et al. DNA Repair (Amst) 6(12) (2007) 1709-25; P. Andrade, et al. Proc Natl Acad Sci U S A 106(38) (2009) 16203-8; and J. Yamtich and J.B. Sweasy. Biochim Biophys Acta 1804(5) (2010) 1136-50; the contents of each of which are incorporated by reference herein in their entirety). Related to this, TdT can be switched from non-templated to templated with a mutation in its Loop 1 domain (see, F. Romain, et al. Nucleic Acids Res 37(14) (2009) 4642- 56; the contents of which are incorporated by reference herein in their entirety).

[049] Pol lambda, a further member of the PolX family, possesses an intrinsic terminal transferase activity as well as its DNA polymerase activity (see, K. Ramadan, et al. J Mol Biol 328(1) (2003) 63-72; the contents of which are incorporated by reference herein in their entirety).

[050] Primases, which are involved in replicative DNA synthesis, normally generate short RNA primers to initiate DNA synthesis. Very divergent versions of these primases from archaeal organisms possess non-templated terminal transferase activity (see, M. De Falco, et al. Nucleic Acids Res 32(17) (2004) 5223-30; S.H. Lao-Sirieix et al. Trends Genet 21(10) (2005) 568-72; S. Gill, et al. Nucleic Acids Res 42(6) (2014) 3707-19; and S.H. Lao-Sirieix and S.D. Bell. J Mol Biol 344(5) (2004) 1251-63; the contents of each of which are incorporated by reference herein in their entirety).

[051] An archaeal DNA polymerase I, Dpol from Sulfolobus solfataricus has terminal transferase activity (see, Z. Zuo, et al. Biochemistry 50(23) (2011) 5379-90 the contents of which are incorporated by reference herein in their entirety).

[052] Several viral RNA-dependent RNA polymerases (RdRPs) have demonstrated terminal transferase activity and all RdRPs may possess terminal transferase activity (see, C.T.

Ranjith-Kumar, et al. J Virol 75(18) (2001) 8615-23; Z. Wang, et al. J Biol Chem 288(43) (2013) 30785-801; T. Yamashita, et al. J Biol Chem 273(25) (1998) 15479-86; and W. Wu, et al. PLoS One 9(1) (2014) e86876; the contents of each of which are incorporated by reference herein in their entirety).

[053] Ribozyme versions of RdRPs, which have been recently created, may also possess terminal transferase activity (see, D.P. Horning and G.F. Joyce. Proc Natl Acad Sci U S A 113(35) (2016) 9786-91; the contents of which are incorporated by reference herein in their entirety).

Reversible-Terminating Nucleotides

[054] For a controlled synthesis of a NA chain, the methods of the disclosure add only one base at a time. If TdT is presented with natural NTPs it will incorporate them over time to form a long but random sequence. If only one species of nucleotide is presented, TdT will generate long homopolymeric tracts of that nucleotide. Therefore, the compositions and methods of the disclosure control the reactions so that one and only one nucleotide is incorporated at each cycle, then an arbitrary but specific NA sequence can be generated.

[055] To obtain a one and only one nucleotide incorporation of a polymerase, Sanger and Coulson devised the dideoxy nucleotide terminator method, which allows the incorporation of only a single nucleotide at the 3' end of each chain (see, F. Sanger and A.R. Coulson. J Mol Biol 94(3) (1975) 441-8; the contents of which are incorporated by reference herein in their entirety). However, dideoxynucleotides are unable to be extended. Thus, the compositions and methods use a variety of reversible-terminating nucleotides, which allow the re-generation of the 3'-OH group after a controlled removal of a blocking group. The disclosure refers to such modified nucleotides as reversible, terminating nucleotide triphosphates (rtNTP). [056] According to the compositions and methods of the disclosure, one or more photoremovable protecting groups that may be used to block the 3' -OH of a given nucleotide include, but are not limited to, a 2-nitrobenzyl group, a dansyl group, a p-hydrozyphenacyl group and a 7-methoxy-4-methylcoumarin group. Alternatively, or in addition, according to the compositions and methods of the disclosure, one or more chemically-cleavable protecting groups that may be used to block the 3'-OH of a given nucleotide include, but are not limited to, an amine group, an axidomethyl group and an allyl group. According to the compositions and methods of the disclosure, a removable protecting group that is both photo- and chemically-cleavable may be used to block the 3' -OH of a given nucleotide. For example, a 2-nitrobenzyl group is photoconvertible and chemically cleavable by treatment with Lewis acid/amine combinations.

[057] Modifications of other parts of a nucleotide provide a reversibly -terminating activity. For example, 2' modifications of ribonucleotides can reversibly terminate a growing RNA chain mediated by T7 RNA polymerase. Thus, in certain embodiments of the compositions and methods of the disclosure, ribonucleotides may comprise one or more 2' modifications that reversibly terminate an RNA polymer during synthesis by a T7 RNA polymerase.

Alternatively, or in addition, ribonucleotides comprising one or more 2' modifications that reversibly terminate an RNA polymer during synthesis by a T7 RNA polymerase may comprise one or more removable blocking groups (e.g. photo-reversible or chemically- reversible blocking groups).

[058] While a variety of different RNA and DNA polymerases have been shown to incorporate different rtNTPs, such activity has not yet been demonstrated for TdT or other enzymes with terminal transferase activity.

5' Nucleic Acid Modifications

[059] According to the compositions and methods of the disclosure, a non-templated synthesis reaction for TdT to catalyze the addition of nucleotides to a growing NA chain, the chain needs to be at least three nucleotides long. Thus, the methods of the disclosure use a NA primer that has at least three available 3' nucleotides to initiate synthesis using TdT.

[060] For synthesis in a solution using TdT and rtNTPs in a one-base-at-a-time context, it may be important to be able to isolate the growing NA chains. In certain embodiments of the compositions and methods of the disclosure, the growing NA chain may comprise a 5' modification for isolation of the resultant synthetic DNA polymer. In certain embodiments, the 5' modification may include a tag or an oligonucleotide that is bound or hybridized to the growing NA chain or resultant synthetic DNA polymer and, optionally, may be further attached to a substrate.

[061] In certain embodiments, the 5' modification may include a biotin tag that may be bound to an avidin-coated or streptavidin-coated substrate. In certain embodiments, the substrate may be a solid or semi-solid substrate. Semi-solid substrates of the disclosure may be comprised of a glass. Alternatively, or in addition, semi-solid substrates of the disclosure may comprise one or more pores or channels. In certain embodiments, the substrate may take the form of a substantially flat surface or a bead.

Enzymatic Synthesis Linked to a Substrate

[062] Typically, phosphoramidite synthesis employs either controlled porous glass or polystyrene beads for bulk production of single NA sequences. More recently, for the synthesis of a multiplicity of NA sequences arrays have been used. These array reactions are carried out in a variety of ways, including the use of photo masks to photo-activate specific regions allowing only a limited number of growing chains to accept the next nucleotide in each cycle, micromirror-based scanning systems to specifically photo-activate sites for incorporation of the next nucleotide, inkjet printer technology to generate arrays of synthesized NA sequences and surface derivatization controlled by micro heating, however, in each of these systems the length of synthesized DNA polymers is short when compared to the synthetic DNA polymers produced by the compositions and methods of the disclosure. Though these four micro array generation methods have all proposed or used

phosphoramidite synthesis, when combined with the compositions and enzymatic synthesis methods of the disclosure (e.g. by TdT along with photoreversible dNTPs on a dense aperture array, with individually addressable regions), these existing micro array generation methods could be improved to provide many long NA sequences in very little time.

Microfluidic Systems

[063] Bulk synthesis either in solution or on beads produces a small number of NA sequences at a sufficient quantity for many molecular biology experiments. At the other extreme, arrays of many different NA sequences are useful for genome wide and aptamer experiments, however, the amount of material available for each NA sequence could be limited. As an intermediate scale of synthesis, compositions comprising TdT and rtNTPs may be used to synthesize NA sequences in a microfluidic droplet system according to the methods of the disclosure. A microfluidic system may be adapted to employ either chemical- or photo-reversible rtNTPs and to synthesize a large number of different NA sequences, each produced in a quantity useful for many applications. Non-Catalytic Single-Stranded Binding Proteins

[064] As single stranded NAs are synthesized they will begin to take on secondary and tertiary structural conformations that may inhibit incorporation of additional nucleotides. A short region of self-complementarity, for example, may mask the 3 '-terminal three bases in such a way that TdT cannot extend the NA chain. There are several ways this inhibition may be overcome.

[065] In certain embodiments of the compositions and methods of the disclosure, after some cycles of incorporation, specific or random primers are added and a second strand synthesis reaction is carried out to untangle the inhibitory conformation. With double-stranded NAs, which maintain a substantially linear conformation, the 3 ' end of the original growing NA chain would be free to incorporate additional nucleotides mediated by TdT.

[066] In certain embodiments of the compositions and methods of the disclosure, after some cycles of incorporation, a set of random short terminated NA sequences (5'-P04 free, 3'- dideoxy) are provided under conditions sufficient for hybridization to untangle the inhibitory conformation.

[067] In certain embodiments of the compositions and methods of the disclosure, non- catalytic single-stranded binding proteins are added to free the 3 ' end of the original growing NA chain. In certain embodiments of the compositions and methods of the disclosure, non- catalytic single-stranded binding compounds are added to free the 3 ' end of the original growing NA chain. For example, one or more of the poly amines: spermine, penta-L-lysine, penta-L-arginine, poly disperse poly-L-lysine and spermidine may be used to untangle knotted NA chains by interaction with the negatively charged NA backbone.

Non-Naturally Occurring DNA Nucleotides

[068] A variety of non-naturally-occurring nucleotides, possessing non-natural bases and/or backbones may be used in the compositions and methods of the disclosure.

[069] Exemplary non-naturally-occurring nucleotides comprising a non-natural base include, but are not limited to, dBTP, dKTP, dPTP, dXTP and dZTP (see, Fig. 3 and K. Sefah, et al. Proc Natl Acad Sci U S A 1 11 (4) (2014) 1449-54; the contents of which are incorporated by reference herein in their entirety). Exemplary non-naturally-occurring nucleotides comprising a non-natural base, but are not limited to, dlnDTP, d5FITP, dAITP, dNITP, dCHITP, dCEITP, d5PhITP, d5NapITP and d5AnITP (see, A.J. Berdis and D.

McCutcheon. Chembiochem 8(12) (2007) 1399-408; the contents of which are incorporated by reference herein in their entirety). [070] Exemplary non-naturally-occurring nucleotides comprising a non-natural backbone, but are not limited to, CeNA (cyclohexenyl nucleic acids), ANA (arabinonucleic acids), FANA (2'-fluoro-arabinonucleic acid), TNA (a-L-threofuranosyl nucleic acids) and LNA (2'- 0,4'-C-methylene- -D-ribonucleic acids; locked nucleic acids) (see, V.B. Pinheiro, et al. Science 336(6079) (2012) 341-4; the contents of which are incorporated by reference herein in their entirety).

DNA Synthesis Methods

[071] To utilize the template-independent synthesis properties of TdT and develop a new DNA synthesis technology, the compositions and methods of the disclosure utilize TdT to incorporate rtNTPs into synthesis reactions at the 3' end of growing NA chains.

Incorporation of an rtNTP at the 3' end of the growing NA chain blocks further synthesis and therefore results in the addition of just one nucleotide during NA synthesis. Removing the terminator modification permits the addition of another modified nucleotide and, using this step-wise method, leads to the production of synthetic DNA molecules.

DNA Synthesis Arrays

[072] Compositions of the disclosure may comprise (a) a plurality of primers, wherein each primer comprises at least three nucleotides, wherein each primer comprises a 3' reversible terminating nucleotide (rtNTP), and wherein each primer is linked to a substantially flat substrate in an array; (b) a plurality of free rtNTP; and (c) an enzyme or a ribozyme having a terminal transferase activity.

[073] In certain embodiments of the arrays of the disclosure, the primer may be linked to the substrate directly such that the primer contacts the substrate. Alternatively, in certain embodiments, the primer may be linked to the substrate indirectly such that the primer contacts a linker that, in turn, contacts the substrate directly. Linkers of the disclosure may be of any length. Exemplary linkers of the disclosure may be comprised of any material, including, but not limited to, an organic or inorganic molecule or polymer. In certain embodiments, the organic polymer comprises one or more of a DNA, an RNA or an amino acid monomer.

[074] The plurality of primers linked to the substrate may comprise a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, the plurality of primers linked to the substrate may comprise a first primer having a first sequence, a second primer having a second sequence and a third or subsequent primer having a third or subsequent sequence wherein the first sequence, the second sequence and the third or subsequent sequence are not identical. In certain embodiments, the plurality of primers comprises at least one duplicate of each of the first primer, the second primer and the third or subsequent distinct primer. In certain embodiments, the plurality of primers comprises 2 or more duplicates of each distinct primer. In certain embodiments, the plurality of primers comprises 5, 10, 50, 100, 200, 500, 100 duplicates or any number of duplicates in between of each distinct primer.

[075] In certain embodiments, each of the plurality of primers linked to the substrate may comprise a unique sequence from every other primer of the plurality of primers (i.e. each primer of the plurality of primers is a distinct primer).

[076] In certain embodiments of the array compositions of the disclosure, the synthetic nucleic acids generated by the array may comprise a first synthetic nucleic acid having a first sequence and a second synthetic nucleic acid having a second sequence, wherein the first sequence and the second sequence are not identical. In certain embodiments, the plurality of synthetic nucleic acids linked to the substrate may comprise a first synthetic nucleic acid having a first sequence, a second synthetic nucleic acid having a second sequence and a third or subsequent synthetic nucleic acid having a third or subsequent sequence wherein the first sequence, the second sequence and the third or subsequent sequence are not identical. In certain embodiments, the plurality of synthetic nucleic acids comprises at least one duplicate of each of the first synthetic nucleic acid, the second synthetic nucleic acid and the third or subsequent distinct synthetic nucleic acid. In certain embodiments, the plurality of synthetic nucleic acids comprises 2 or more duplicates of each distinct synthetic nucleic acid. In certain embodiments, the plurality of synthetic nucleic acids comprises 5, 10, 50, 100, 200, 500, 100 duplicates or any number of duplicates in between of each distinct synthetic nucleic acid.

[077] In certain embodiments of the arrays of the disclosure, each of the plurality of synthetic nucleic acids linked to the substrate may comprise a unique sequence from every other synthetic nucleic acid of the plurality of synthetic nucleic acid (i.e. each synthetic nucleic acid of the plurality of synthetic nucleic acids is a distinct synthetic nucleic acid).

[078] In certain embodiments of the arrays of the disclosure, the plurality of synthetic nucleic acids linked to the substrate may comprise at least one synthetic DNA. Alternatively or in addition, in certain embodiments of the arrays of the disclosure, the plurality of synthetic nucleic acids linked to the substrate may comprise at least one synthetic RNA. In certain embodiments, including those wherein the plurality of synthetic nucleic acids linked to the substrate may comprise at least one synthetic RNA, the compositions and/or methods of the disclosure may further comprise the use of an RNAse inhibitor. [079] In certain embodiments of the arrays of the disclosure, each of the plurality of synthetic nucleic acids linked to the substrate comprises a synthetic DNA.

[080] In certain embodiments of the arrays of the disclosure, each of the plurality of synthetic nucleic acids linked to the substrate comprises a synthetic RNA. In certain embodiments, the compositions and/or methods of the disclosure may further comprise the use of an RNAse inhibitor.

[081] In certain embodiments of the arrays of the disclosure, the plurality of primers, and, consequently, the plurality of synthetic nucleic acids generated therefrom, may be arranged on the substrate in a repeating or non-repeating pattern. In certain embodiments, a repeating partem may be used to include duplicate primers and/or duplicate synthetic nucleic acids. The inclusion of duplicate primers and/or duplicate synthetic nucleic acids may increase the statistical power of an analysis using the array wherein an analyte that binds or hybridizes to one or more of the synthetic nucleic acids of the array is detected. In certain embodiments, a non-repeating partem may be used to increase the diversity of primers and/or synthetic nucleic acids present on the array. This increase of diversity may be particularly useful for initial screenings of analytes to identify targets for further analysis or to identify rare analytes in a large population of analytes.

[082] In certain embodiments of the arrays of the disclosure, the array may be used in a device for detection of an analyte that binds to or hybridizes to a synthetic nucleic acid of the array. Exemplary detection devices are described for example, in U.S. Patent 9,410,887 (the contents of which are herein incorporated by reference in their entirety).

EXAMPLES

Example 1 : 3'-Q-(2-nitrobenzyl)-2'-dNTP compositions for use with T7 RNA polymerase and UV irradiation to remove the 2-nitrobenzyl moiety

[083] In certain embodiments of the compositions and methods of the disclosure, modified dNTPs, with a 2-nitrobenzyl at the 2' position, are incorporated into template-dependent reactions by T7 RNA polymerase. The 2-nitrobenzyl acts as a reversible terminator as only one modified dNTP is added and UV irradiation removes the 2-nitrobenzyl moiety, leaving an OH group that acts as the site for subsequent synthesis. While calf TdT can incorporate dNTPs with modifications at the 3' position, prior to this disclosure, it had not been shown that TdT can incorporate 3'-0-(2-nitrobenzyl)-2'-dNTPs or that this could be used for NA synthesis. [084] To test this method, a composition comprising 3 '-0-(2-nitrobenzyl)-2'-dATP (from Trilink, see Fig. 1) and recombinant calf TdT (from NEB) was used. To initiate template- independent synthesis, TdT requires a single stranded DNA molecule of at least three nucleotides long. A 24-nucleotide oligo was used, which had an alexa-488 label at the 5 ' end (Fig. 2A).

[085] If TdT successfully incorporated the 3'-0-(2-nitrobenzyl)-2'-dATP onto the 3' end of the oligo there will be a reduced mobility on a denaturing polyacrylamide gel, equaling the addition of one nucleotide. When analyzed on a denaturing polyacrylamide gel, the presence of the 5 ' label reduced the mobility of this oligo, such that it resolved at 27 nucleotides, when compared to a ladder (Fig. 2B, lanes 1 and 2). When the oligo was incubated with TdT and no nucleotides there was no non-specific change in the resolution of the oligo on the gel (Fig. 2B, lane 3). Incubation of the oligo with TdT and unmodified dNTPs resulted in

polymerization of long DNA molecules (Fig. 2B, lane 7). However, incubation of the oligo with TdT and 3 '-0-(2-nitrobenzyl)-2'-dATP resulted in a shift in resolution on the gel that corresponded to the addition of just one nucleotide (Fig. 2B, lane 4). Following incubation of the oligo with TdT and 3'-0-(2-nitrobenzyl)-2'-dATP the sample was UV irradiated followed by spin column clean-up of the sample (Zymo, oligo clean and concentrator columns) and further incubated with 3 '-0-(2-nitrobenzyl)-2'-dATP and TdT. This resulted in a shift of resolution on the gel that corresponded the addition of another blocked nucleotide (Fig. 2B, lane 5) demonstrating a step-wise addition of individual nucleotides the 3 ' end of the oligo.

[086] Following incubation of the oligo with TdT and 3 '-0-(2-nitrobenzyl)-2'-dATP the sample was further incubated with unmodified dNTPs (Fig. 2B, lane 6). Gel analysis of this sample showed weak bands at a size comparable to those seen following incubation of the oligo with TdT and 3 '-0-(2-nitrobenzyl)-2'-dATP. However, longer DNA molecules were also observed (Fig. 2B, lane 6).

[087] Collectively the results demonstrate that while TdT can add 3 '-0-(2-nitrobenzyl)-2'- dATP onto the 3' end of the oligo, some oligos have not received 3 '-0-(2-nitrobenzyl)-2'- dATP and remain free for further synthesis reactions. Consequently, the addition of unmodified dNTPs leads the synthesis of long DNA molecules on these unblocked oligos. However, the longer DNA molecules remain shorter than those observed following the incubation of the oligo with TdT and unmodified dNTPs (Fig. 2B, compare lane 6 and 7). This is a consequence of free 3'-0-(2-nitrobenzyl)-2'-dATP remaining within the reaction. As TdT performs synthesis with the unmodified dNTPs it also incorporates 3 '-0-(2- nitrobenzyl)-2'-dATP, which blocks the addition of further dNTPs. This is comparable to a Sanger DNA sequencing ladder that results from the use of dideoxynucleotides, which lack a 3' OH group and block further DNA polymerization. This observation further supports the claim that TdT can incorporate 3'-0-(2-nitrobenzyl)-2'-dATP and that this blocks further DNA synthesis.

Example 2: DNA Synthesis Reaction Conditions

[088] Reactions from Example 1 were performed in 5 μΐ volumes, incubating at 37°C for 1 hour. Synthesis reactions were performed by incubating 1 μΐ of 10 μΜ oligo (shown in Fig 2A) with 0.2 μΐ TdT (20 units/μΐ, NEB), 0.5 μΐ CoCh (final concentration of 0.25mM), 0.5 μΐ reaction buffer (final concentration of 50 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate) and 1 μΐ of lOmM dNTPs or dATP or 3'-0-(2-nitrobenzyl)-2'- dATP. This provided a ratio of 1 : 1000 of oligo compared to nucleotide(s). For control experiments the nucleotides were replaced with Ιμΐ of water. UV irradiation was performed by 10 min incubation on a UV light. Reactions were stopped by adding 1 μΐ of 100 mM EDTA and incubating at 80°C for 5 mins. Samples were analysed by running of a denaturing 20% polyacrylamide gel (8M urea).

INCORPORATION BY REFERENCE

[089] Every document cited herein, including any cross referenced or related patent or application is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

OTHER EMBODIMENTS

[090] While particular embodiments of the disclosure have been illustrated and described, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. The scope of the appended claims includes all such changes and modifications that are within the scope of this disclosure.

Claims

CLAIMS What is claimed is:

1. A composition comprising:

(a) a primer comprising at least three nucleotides, wherein the primer comprises a 3 ' reversible terminating nucleotide (rtNTP);

(b) at least one free rtNTP; and

(c) an enzyme or a ribozyme having a terminal transferase activity.

2. The composition of claim 1, further comprising

(d) a reaction buffer.

3. The composition of claim 2, wherein the reaction buffer comprises a final concentration of 50 mM Potassium Acetate, 20 mM Tris-Acetate and 10 mM Magnesium Acetate.

4. The composition of claim 2 or 3, wherein the reaction buffer comprises Zn²⁺, Co²⁺ or Mn²⁺.

5. The composition of any one of claims 1-4, wherein the at least one free reversible terminating nucleotide (rtNTP) comprises a chemically -reversible blocking group.

6. The composition of claim 4, further comprising an agent to chemically -reverse the blocking group.

7. The composition of claim 6, wherein the agent is a Lewis acid.

8. The composition of claim 7, wherein the Lewis acid comprises CoCh.

9. The composition of any one of claims 1-8, wherein the chemically-reversible blocking group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group or an allyl group.

10. The composition of any one of claims 1-8, wherein the chemically-reversible blocking group comprises a 2-nitrobenzyl group.

1 1. The composition of any one of claims 1-10, wherein the at least one free reversible terminating nucleotide (rtNTP) comprises a photo-reversible blocking group.

12. The composition of any one of claims 1-11 , wherein the photo-reversible blocking group comprises a 2-nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group or a 7- methyoxy-4-methylcoumarin group.

13. The composition of any one of claims 1-11 , wherein the photo-reversible blocking group comprises a 2-nitrobenzyl group.

14. The composition of any one of claims 1-13, wherein the primer comprises a 5' modification.

15. The composition of claim 14, wherein the 5' modification comprises a selectable tag.

16. The composition of claim 15, wherein the selectable tag is bound or hybridized to the primer.

17. The composition of claim 16, wherein the selectable tag is bound or hybridized to a substrate.

18. The composition of claim 17, wherein the substrate comprises a flat surface or a bead.

19. The composition of claim 18, wherein the substrate comprises a glass, a polymer or a matrix.

20. The composition of claim 19, wherein the substrate comprises a polymer and wherein the polymer comprises a polyacrylamide gel.

The composition of any one of claims 17-20, wherein the substrate comprises

22. The composition of any one of claims 14-21, wherein 5' modification comprises biotin.

23. The composition of claim 22, wherein the substrate comprises a bead, wherein the bead comprises a polyacrylamide gel, wherein the polyacrylamide gel comprises an anchor and wherein the anchor comprises avidin or streptavidin.

24. The composition of any one of claims 1-23, wherein the primer comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an amino acid or any combination thereof.

25. The composition of any one of claims 1-24, wherein the primer comprises at least one non-naturally occurring base or at least one non-naturally occurring backbone.

26. The composition of any one of claims 1-24, wherein the primer comprises at least one non-naturally occurring base and at least one non-naturally occurring backbone.

27. The composition of any one of claims 1-26, wherein the at least one non-naturally occurring base comprises a dBTP, a dKTP, a dPTP, a dXTP a dZTP, a dlnDTP, a

5fluoroindolyl-2'-deoxyriboside triphosphate (d5FITP), a 5-amino-indolyl-2'-deoxyriboside triphosphate (dAITP), a 5-nitro-indolyl-2'-deoxyriboside triphosphate (dNITP), a 5- cyclohexyl-indolyl-2'deoxyriboside triphosphate (dCHITP), a dCEITP, a 5-phenylindolyl-2'- deoxyriboside triphosphate (d5PhITP), a 5-napthylindolyl-2'-deoxyriboside triphosphate (d5NapITP) or a d5AnITP.

28. The composition of any one of claims 1-26, wherein the at least one non-naturally occurring backbone comprises a cyclohexenyl nucleic acid (CeNA), an arabinonucleic acid (ANA), a 2'-fluoro-arabinonucleic acid (FANA), a a-L-threofuranosyl nucleic acid (TNA) or a locked nucleic acid (LNA).

29. The composition of claim 28, wherein the LNA comprises a 2'-0,4'-C-methylene- - D-ribonucleic acid.

30. The composition of claim 1, wherein the reversible terminating nucleotide (rtNTP) comprises a chemically-reversible blocking group.

31. The composition of claim 30, wherein the chemically-reversible blocking group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group or an allyl group.

32. The composition of claim 30, wherein the chemically-reversible blocking group comprises a 2-nitrobenzyl group.

33. The composition of claim 30, wherein the reversible terminating nucleotide (rtNTP) comprises a photo-reversible blocking group.

34. The composition of claim 33, wherein the photo-reversible blocking group comprises a 2-nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group or a 7-methyoxy-4- methylcoumarin group.

35. The composition of claim 33, wherein the photo-reversible blocking group comprises a 2-nitrobenzyl group.

36. The composition of any one of claims 1-35, wherein the primer is in solution.

37. The composition of any one of claims 1-35, wherein the primer is linked to a substrate.

38. The composition of claim 37, wherein the primer is directly linked to the substrate and wherein the primer contacts the substrate.

39. The composition of claim 37, wherein the primer is indirectly linked to the substrate and wherein the primer contacts a linker that contacts the substrate.

The composition of any one of claims 37-39, wherein the substrate is a bead.

41. The composition of claim 40, wherein the bead comprises a glass, a polymer, or a matrix.

42. The composition of claim 40 or 41, wherein the bead is porous.

43. The composition of any one of claims 40-42, wherein the bead comprises a polyacrylamide gel.

44. The composition of any one of claims 37-39, wherein the substrate is a substantially flat surface.

45. The composition of claim 44, wherein the substrate is a flat surface.

46. The composition of claim 45, wherein the substrate comprises a glass, a polymer, or a matrix.

47. The composition of any one of claims 45-46, wherein the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array.

48. The composition of claim 47, wherein the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical.

49. The composition of claim 48, wherein the plurality of primers comprises at least one duplicate of the first primer and at least one duplicate of the second primer.

50. The composition of claim 49, wherein each primer of the plurality of primers comprises a unique sequence.

51. The composition of any one of claims 1-50, wherein the enzyme is a terminal deoxynucleotidyl transferase (TdT).

52. The composition of any one of claims 1-50, wherein the enzyme is a pol theta, a pol lambda, a pol mu, a Dpo 1, or a primase.

53. The composition of any one of claims 1-50, wherein the ribozyme is a RNA- dependent RNA polymerase.

54. A method of template-free nucleic acid synthesis comprising:

(a) obtaining the composition of any one of claims 1-53;

(b) deprotecting the 3' rtNTP of the primer of the composition; and

(c) incorporating the at least one free rtNTP of the composition by the enzyme or ribozyme of the composition into the primer of the composition,

thereby generating a synthetic nucleic acid.

55. The method of claim 54, wherein the at least one free rtNTP of the composition is a plurality of free rtNTP s.

56. The method of claim 54 or 55, wherein steps (b) and (c) are completed in less than 1 minute.

57. The method of claim 56, further comprising a first rinse after the deprotecting step (b) and a second rinse after the incorporating step (c).

58. The method of claim 57, wherein steps (b) and (c) are completed in less than 1 minute.

59. The method of any one of claims 54-58, further comprising the steps of:

(d) repeating steps (b) and (c).

60. The method of claim 59, further comprising the step of:

(e) removing unincorporated rtNTPs prior to performing step (d).

61. The method of any one of claims 54-60, wherein the 3' rtNTP of the primer comprises a photo-reversible blocking group and the deprotecting comprises exposing the photo-reversible blocking group to light radiation.

62. The method of claim 61, wherein the light radiation comprises UV radiation.

63. The method of any one of claims 54-60, wherein the 3 ' rtNTP of the primer comprises a chemically reversible blocking group and the deprotecting comprises exposing the chemically reversible blocking group to a Lewis acid.

64. The method of claim 63, wherein the Lewis acid comprises CoCh.

65. The method of any one of claims 54-64, wherein the primer is in solution.

66. The method of any one of claims 54-64, wherein the primer is linked to a substrate.

67. The method of claim 66, wherein the primer is a plurality of primers and wherein each primer of the plurality of primers is linked to the substrate in an array.

68. The method of claim 67, wherein the plurality of primers comprises a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence and the second sequence are not identical.

69. The method of claim 68, wherein the plurality of primers comprises at least one duplicate of the first primer and at least one duplicate of the second primer.

70. The method of claim 68, wherein each primer of the plurality of primers comprises a unique sequence.

71. The method of any one of claims 54-70, further comprising the step of:

contacting the synthetic nucleic acid and one or more of a random primer, a nonspecific primer, a set of random short terminated nucleic acid sequences, a non-catalytic single-stranded binding protein and a non-catalytic single-stranded binding compound during synthesis to maintain a substantially linear conformation, to inhibit formation of a secondary and/or a tertiary structure, or to untangle an inhibitory conformation of the synthetic nucleic acid during one or more rounds of deprotection and incorporation of a rtNTP.

72. The method of claim 71, wherein the non-catalytic single-stranded binding protein or the non-catalytic single-stranded binding compound comprises a polyamine.

73. The method of claim 72, wherein the polyamine comprises spermine, penta-L-lysine, poly disperse poly-L-lysine or spermidine.

74. The method of any one of claims 70-73, wherein the contacting step is performed once the synthetic nucleic acid comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 nucleotides, or any number of nucleotides in between.

75. The method of any one of claims 70-74, wherein the contacting step is performed prior to the generation of a sequence within the synthetic nucleic acid that may form a secondary or tertiary structure.

76. The method of any one of claims 70-74, wherein the contacting step is performed prior to the generation of a sequence having sufficient length that the synthetic nucleic acid can fold into a substantially non-linear conformation.

77. The method of any one of claims 70-76, further comprising the step of:

(f) removing the one or more of a random primer, a non-specific primer, a set of random short terminated nucleic acid sequences, a non-catalytic single-stranded binding protein and a non-catalytic single-stranded binding compound from the synthetic nucleic acid after synthesis is complete.