TW201840855A - Compositions and methods for template-free enzymatic nucleic acid synthesis - Google Patents

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

Composition and method for templateless enzymatic nucleic acid synthesis

The present disclosure provides compositions and methods for use in templateless enzymatic nucleic acid synthesis using reversible terminating nucleotides.

Over the past decade, the demand for synthetic DNA molecules for a range of molecular biological applications has increased. Advances in DNA sequencing technology have partially contributed to this growth. However, although DNA sequencing technology has developed significantly, DNA synthesis technology has not progressed at the same rate, and the state of the art cannot meet current market demands. The present disclosure provides compositions and methods for template-free enzymatic DNA synthesis that provide a solution to the long-standing unmet needs of the art, which are produced by the compositions and methods of the present disclosure with excellent synthetic precision DNA sequence with length and speed.

The present disclosure provides compositions and methods for the synthesis of non-templated nucleic acid (NA) polymers using reversible terminating nucleotide triphosphate (rtNTP). TdT is used to incorporate individual rtNTPs into the 3' end of a single strand of NA molecule. A unique NA can be synthesized by adding a rtNTP and then removing the blocking group, followed by adding a new rtNTP to the continuous cycle of the reaction.

The present 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 ribozyme having terminal transferase activity. In some embodiments, the composition further comprises (d) a reaction buffer. In some embodiments, the reaction buffer comprises potassium acetate at a final concentration of 50 mM, Tris-Acetate at 20 mM, and magnesium acetate at 10 mM. In some embodiments, a source of divalent cations is provided, the divalent cations including, but not limited to, Zn ²⁺ , Co ²⁺ , Mn ²⁺ . In some embodiments, the composition further comprises one or more divalent cations. In some embodiments of the composition, the divalent cation is Zn ²⁺ , Co ²⁺ or Mn ²⁺ . In some embodiments, the reaction buffer further comprises one or more divalent cations. In some embodiments of the reaction buffer, the divalent cation is Zn ²⁺ , Co ²⁺ or Mn ²⁺ .

In some embodiments of the compositions of the present disclosure, the at least one free reversible terminating nucleotide (rtNTP) comprises a chemically reversible blocking group. In some embodiments, the composition further includes an agent that chemically reverses the blocking group. In some embodiments, the reagent is Lewis acid. In some embodiments, the Lewis acid comprises CoCl ₂ . In some embodiments, the chemically reversible barrier group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group, or an allyl group. In some embodiments, the chemically reversible barrier group comprises a 2-nitrobenzyl group.

In some embodiments of the compositions of the present disclosure, the at least one free reversible termination nucleotide (rtNTP) comprises a photoreversible barrier group. In some embodiments, the photoreversible barrier group comprises a 2-nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group, or a 7-methoxy group. 4-methyoxy-4-methylcoumarin group. In some embodiments, the photoreversible barrier group comprises a 2-nitrobenzyl group.

In some embodiments of the compositions of the present disclosure, the primers comprise a 5' modification. In some embodiments, the 5' modification comprises a selectable tag. A selectable label can be used to isolate or purify the resulting synthetic DNA polymer from the reaction. In some embodiments, the selectable marker is a linkage or hybridization to a primer. In some embodiments, the selectable label is a bond to a substrate or hybridization to an NA chain bonded to a substrate. In some embodiments, the substrate comprises a flat surface or beads. In some embodiments, the substrate comprises a glass, a polymer or a matrix. In some embodiments, the substrate comprises a hole or channel. In some embodiments, the substrate comprises a polymer, wherein the polymer comprises a polypropylene amide gel. In some embodiments, the substrate comprises an anchor.

In some embodiments of the compositions of the present disclosure, the primers comprise a 5' modification. In some embodiments, the 5' modification comprises biotin. In some embodiments, the matrix comprises beads, wherein the beads comprise a polypropylene amide gel, wherein the polypropylene guanamine gel comprises a fixture and wherein the fixture comprises avidin or streptavidin ).

In some embodiments of the compositions of the present disclosure, the primers include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), amino acids, or any combination thereof. In some embodiments, the primer comprises at least one non-naturally occurring base or at least one non-naturally occurring backbone. In some embodiments, the primer comprises at least one non-naturally occurring base and at least one non-naturally occurring backbone. In some embodiments, the at least one non-naturally occurring base comprises dBTP, dKTP, dPTP, dXTP, dZTP, dInDTP, 5-fluoroindolyl-2'-deoxyribonucleoside triphosphate (d5FITP), 5 -Amino-mercapto-2'-deoxyribonucleoside triphosphate (dAITP), 5-nitro-indolyl-2'-deoxyribonucleoside triphosphate (dNITP), 5-cyclohexyl- Mercapto-2'-deoxyribonucleoside triphosphate (dCHITP), dCEITP, 5-phenylmercapto-2'-deoxyribonucleoside triphosphate (d5PhITP), 5-naphthylfluorenyl- 2'-deoxyribonucleoside triphosphate (d5NapITP), or d5AnITP. In some embodiments, the at least one non-naturally occurring backbone comprises cyclohexenyl nucleic acid (CeNA), arabinucleic acid (ANA), 2'-fluoro-arabino nucleic acid (FANA), alpha- L-threofuranosyl nucleic acid (TNA), or locked nucleic acid (LNA). In some embodiments, the LNA comprises 2'-O, 4'-C-methylene-[beta]-D-ribonucleic acid.

In some embodiments of the compositions of the present disclosure, the primer comprises a reversible terminating nucleotide (rtNTP) and the rtNTP comprises a chemically reversible blocking group. In some embodiments, the chemically reversible barrier group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group, or a propylene group. In some embodiments, the chemically reversible barrier group comprises a 2-nitrobenzyl group.

In some embodiments of the compositions of the present disclosure, the primer comprises a reversible terminating nucleotide (rtNTP) and the rtNTP comprises a photoreversible blocking group. In some embodiments, the photoreversible barrier group comprises a 2-nitrobenzyl group, a dansyl group, a p-hydroxyphenacyl group, or a 7-methoxy group. 4-methyoxy-4-methylcoumarin group. In some embodiments, the photoreversible barrier group comprises a 2-nitrobenzyl group.

In some embodiments of the compositions of the present disclosure, the primers are in solution.

In some embodiments of the compositions of the present disclosure, the primers are linked to the substrate.

In some embodiments of the compositions of the present disclosure, the primer is attached to the substrate. In some embodiments, the primer is attached directly to the substrate and the primer contacts the substrate. In some embodiments, the primer is indirectly attached to the substrate and the primer contacts a linker that is in contact with the substrate.

In some embodiments of the compositions of the present disclosure, the primer is attached to the substrate. In some embodiments, the primer is attached directly to the substrate and the primer contacts the substrate. In some embodiments, the primer is indirectly attached to the substrate and the primer contacts the linker that is in contact with the substrate. In some embodiments, the substrate is a bead. In some embodiments, the beads comprise a glass, a polymer, or a matrix. In some embodiments, the beads are porous. In some embodiments, the beads comprise a polypropylene amide gel.

In some embodiments of the compositions of the present disclosure, the primer is attached to the substrate. In some embodiments, the primer is attached directly to the substrate and the primer contacts the substrate. In some embodiments, the primer is indirectly attached to the substrate and the primer contacts the linker that is in contact with the substrate. In some embodiments, the substrate is a substantially planar surface. In some embodiments, the substrate is a flat surface. In some embodiments, the substrate comprises a glass, a polymer, or a substrate.

In some embodiments of the compositions of the present disclosure, the primer is attached to the substrate. In some embodiments, the primer is attached directly to the substrate and the primer contacts the substrate. In some embodiments, the primer is indirectly attached to the substrate and the primer contacts the linker that is in contact with the substrate. In some embodiments, the substrate is a substantially planar surface. In some embodiments, the substrate is a flat surface. In some embodiments, the substrate comprises a glass, a polymer, or a substrate. In some embodiments, the primer is a plurality of primers, and wherein each of the plurality of primers is attached to the substrate in an array. In some embodiments, the plurality of primers comprise a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, the plurality of primers includes at least one duplicate of the at least first primer and at least one repetition of the second primer.

In some embodiments of the compositions of the present disclosure, the primer is attached to the substrate. In some embodiments, the primer is attached directly to the substrate and the primer contacts the substrate. In some embodiments, the primer is indirectly attached to the substrate and the primer contacts the linker that is in contact with the substrate. In some embodiments, the substrate is a substantially planar surface. In some embodiments, the substrate is a flat surface. In some embodiments, the substrate comprises a glass, a polymer, or a substrate. In some embodiments, the primer is a plurality of primers, and wherein each of the plurality of primers is attached to the substrate in an array. In some embodiments, the plurality of primers comprise a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, each of the plurality of primers comprises a unique sequence.

In some embodiments of the compositions of the present disclosure, the enzyme is a terminal deoxynucleotidyl transferase (TdT).

In some embodiments of the compositions of the present disclosure, the enzyme is pol θ, pol λ, pol μ, Dpo 1, or primase.

In some embodiments of the compositions of the present disclosure, the ribozyme is an RNA dependent RNA polymerase.

The present disclosure provides a method for synthesizing a template-free nucleic acid comprising (a) obtaining a composition of the present disclosure; (b) deprotecting a 3' rtNTP of a primer of the composition; and (c) enzymatic or ribozyme by a composition At least one free rtNTP of the composition is incorporated into the primer of the composition to synthesize the nucleic acid. In some embodiments, the at least one free rtNTP of the composition is a plurality of free rtNTPs. In some embodiments, steps (b) and (c) are completed in less than one minute. In some embodiments, the method further comprises a first rinse after the deprotection step (b) and a second rinse after the incorporation step (c). In some embodiments, including the method further comprises a first rinse after the deprotection step (b) and a second rinse after the incorporation step (c), the steps (b) and (c) being less than Complete in 1 minute. In some embodiments, including those wherein the method further includes a first rinse after the deprotection step (b) and a second rinse after the step (c), the method further comprises the step (d) Repeat steps (b) and (c). In some embodiments, including those wherein the method further includes a first rinse after the deprotection step (b) and a second rinse after the step (c), the method further comprises the step (e) Unincorporated rtNTPs were removed prior to performing step (d).

In some embodiments of the method of templateless nucleic acid synthesis of the present disclosure, the 3' rtNTP of the primer comprises a photoreversible barrier group, and deprotecting comprises exposing the photoreversible barrier group to light illumination. In some embodiments, the light illumination comprises UV illumination.

In some embodiments of the method of templateless nucleic acid synthesis of the present disclosure, the 3' rtNTP of the primer comprises a chemically reversible blocking group, and the deprotecting comprises exposing the chemically reversible blocking group to Lewis acid. In some embodiments, the Lewis acid comprises CoCl ₂ .

In some embodiments of the disclosed methods of templateless nucleic acid synthesis, the primers are in solution.

In some embodiments of the methods of the present disclosure for templateless nucleic acid synthesis, the primers are attached to a substrate.

In some embodiments of the methods of the present disclosure for templateless nucleic acid synthesis, the primers are attached to a substrate. In some embodiments, the primer is a plurality of primers, and wherein each of the plurality of primers is attached to the substrate in the array. In some embodiments, the plurality of primers comprise a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, the plurality of primers includes at least one repetition of the first primer and at least one repetition of the second primer.

In some embodiments of the methods of the present disclosure for templateless nucleic acid synthesis, the primers are attached to a substrate. In some embodiments, the primer is a plurality of primers, and wherein each of the plurality of primers is attached to the substrate in the array. In some embodiments, the plurality of primers comprise a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, each of the plurality of primers comprises a unique sequence.

In some embodiments of the method of templateless nucleic acid synthesis of the present disclosure, the method further comprises the steps of: contacting the synthetic nucleic acid with a random primer, a non-specific primer, a set of random short-terminated nucleic acid sequences, One or more of a catalytic single-stranded binding protein and a non-catalytic single-stranded binding compound to maintain substantially linear conformation, inhibition during deprotection and incorporation of one or more rtNTPs The secondary and/or tertiary structure forms, or untangles, the inhibitory conformation of the synthetic nucleic acid. In some embodiments, the non-catalytic single-stranded binding protein or non-catalytic single-stranded binding compound comprises a polyamine. In some embodiments, the polyamine comprises spermamine, penta-L-lysine, polydisperse poly-L-lysine, or Spermidine.

In some embodiments of the method of the present invention for template-free nucleic acid synthesis, the method further comprises: in the synthesis process, contacting the synthetic nucleic acid with a random primer, a non-specific primer, a random short-terminated nucleic acid sequence, and non-catalytic Steps of one or more of a single-stranded binding protein and a non-catalytic single-stranded binding compound. Those embodiments, 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, The contacting step is performed at 850, 900, 950, 1000 nucleotides or any number of nucleotides in between. Alternatively, or in addition, in some embodiments, the contacting step is performed prior to generating a sequence in the synthetic nucleic acid that can form a secondary or tertiary structure. Alternatively, or in addition, in some embodiments, the contacting step is performed prior to producing a sequence of sufficient length such that the synthetic nucleic acid can be folded into a substantially non-linear configuration. In some embodiments, the method further comprises the step of (f): removing random primers, non-specific primers, a set of random short-terminated nucleic acid sequences, non-catalytic single-stranded binding proteins from the synthesis of the nucleic acid after completion of the synthesis One or more of the compounds are combined with a non-catalytic single-strand.

Compositions and methods for enzymatic, template-independent nucleic acid (NA) polymer synthesis using reversible termination nucleotide triphosphates (rtNTPs) are disclosed. According to the method of the present disclosure, a terminal deoxynucleotidyl transferase (TdT), which is also known as a DNA nucleotidylexotransferase (DNTT) or a terminal transferase, will be individually rtNTPs are incorporated into the 3' end of a single strand of NA molecule. A unique NA sequence can be synthesized by adding an rtNTP followed by removal of the blocking group followed by the addition of a new rtNTP to the continuous cycle of the reaction. Increase the speed and length of DNA synthesis and reduce costs

The compositions and methods of the present disclosure produce longer synthetic DNA molecules and reduce the cost of NA synthesis compared to the prior art.

Phosphoramidite DNA synthesis is an organic chemical method that has been used for many years to synthesize short nucleic acid (NA) molecules, called oligonucleotides. This method has been automated since the 1980s and is commonly used to produce oligonucleotides of 15 to 25 nucleotides in length. However, due to incomplete reactions and side reactions, the length of DNA molecules that can be synthesized by this method is limited. In fact, this limit is 100 nucleotides. This presents a problem because the coding sequence of the average human gene is 1000 nucleotides. Therefore, in order to synthesize an entire gene by the prior art, it is necessary to splicing shorter DNA fragments together, which increases the cost of the commodity and limits many applications in synthetic biology. In addition, the phosphonium amide method typically requires at least 5 minutes of cycle time for each additional nucleotide to increase in the growing NA chain, making the production of long NA chains prohibitive. Therefore, new NA synthesis techniques (i.e., the compositions and methods of the present disclosure) are required to produce longer NA molecules, increase the speed of synthesis, and reduce the cost of NA synthesis.

The compositions and methods of the present disclosure increase the speed of synthesis and reduce the cost of NA synthesis compared to prior methods. For example, the deprotection of the 3' rtNTP of the primer or the growing NA and each cycle incorporating the new rtNTP can be completed in less than one minute. This cycle can be completed in less than one minute even after the rinsing step is inserted after the deprotection step and the incorporation step. Increased diversity of nucleic acids

The compositions and methods of the present disclosure increase the diversity of available NA for use in other applications, such as the detection of small molecules (e.g., metabolites).

There is an additional need in the art to synthesize NA with unnatural bases or backbones. This need comes from the utility of aptamers, which are NAs that specifically bind small molecules (eg, various metabolites and short peptides). Although traditionally single-stranded RNA has been used as an aptamer, various non-natural bases and backbones are currently used to increase the diversity of small molecule binding and enzymatic possibilities. The compositions and methods of the present disclosure can include surrogate nucleotides having non-naturally occurring bases and/or non-naturally occurring backbones to produce non-naturally occurring NA. Non-naturally occurring NA can be used to increase the specificity and diversity of small molecules that bind to the NA disclosed herein. Nucleotidyltransferase deoxy terminal (Terminal Deoxynucleotidyl Transferases)

The present disclosure demonstrates that the TdT of bovine incorporates 3'-O-(2-nitrobenzyl)-2'-dNTP into the 3' end of the single stranded DNA molecule to be synthesized. Light is provided to remove the 2-nitrobenzyl blocking group and another 3'-O-(2-nitrobenzyl)-2'-dNTP is allowed to be added. Other enzymes with appropriate terminal transferase activity can be used. In addition, various evolutionarily different terminal transferases have been described and these can also be used. In general, the rate of incorporation using efficient enzymes is very fast, usually less than 1 minute.

The terminal deoxynucleotidyl transferase (TdT) is a polymerase that can add deoxynucleotides or ribonucleotides to the 3' end of a single strand of NA molecule in a template-independent manner. TdT can synthesize DNA molecules over 1500 nucleotides in length. Many examples have shown that DNA polymerases can incorporate dNTPs with removable barrier groups at their 3' ends into template-dependent synthesis. Using a loop, this method allows for the incorporation of individual nucleotides. TdT can also incorporate modified dNTPs into a single strand DNA synthesis reaction, including dNTPs having an azide group at the 3' or 2' position. In some embodiments, the compositions and methods of the present disclosure comprise a dNTP having a modification at the 3' position comprising 3'-O-azidomethyl dATP and 3'-O-azidomethyl dCTP.

Although mammalian TdT is the most commonly used and most well known terminal transferase, other different enzymes with terminal transferase activity, i.e., the ability to extend the nucleic acid strand in a non-templated manner. These activities are usually found under other biochemical conditions, such as in an environment where divalent cations alternate. Alternative enzymes and ribozymes having terminal transferase activity in the compositions and methods of the present disclosure may be used, but are not limited to, Pol θ, Pol λ, Pol μ, Dpo 1, Primase, RdRPs, and ribozymes ( Ribozyme).

Pol θ can be switched between templated and non-templated terminal transferase activities by changing divalent cations from Mg ²⁺ to Mn ²⁺ .

Pol μ (another member of the PolX family, which contains TdT) can switch between templated and non-templated activities after mutation in the Loop 1 region (see DNA Repair (Amst) by AF Moon et al. 6(12) (2007) 1709-25; Proc Natl Acad Sci USA 106(38) (2009) 16203-8 by P. Andrade; and Biochim Biophys Acta 1804(5) by J. Yamtich and JB Sweasy (2010) 1136-50; the respective contents are incorporated herein by reference in its entirety. Related to this is that TdT can be switched from non-templated to templated with mutations in the Loop 1 region (see Nucleic Acids Res 37(14) (2009) 4642-56 by F. Romain et al; The content is incorporated into the present application by reference to the entire text.

Pol λ (another member of the PolX family) has intrinsic terminal transferase activity and its DNA polymerase activity (see K. Ramadan et al., J Mol Biol 328(1) (2003) 63-72; This article is incorporated by reference in its entirety.

Primers, which are involved in the replication of DNA, typically produce short RNA primers to initiate DNA synthesis. Very different versions of these primer enzymes from archaeal organisms have non-templated terminal transferase activity (see Nucleic Acids Res 32 (17) (2004) 5223-30 by M. De Falco et al; SH Lao- Posted by Sirieix et al., Trends Genet 21(10) (2005) 568-72; S. Gill et al., Nucleic Acids Res 42(6) (2014) 3707-19; and SH Lao-Sirieix and SD Bell. J Mol Biol 344(5) (2004) 1251-63; the respective contents of which are incorporated herein by reference.

The archaeal DNA synthetase I (Dpo1 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 in its entirety. In this case).

RNA-dependent RNA polymerase (RdRP) of some viruses has demonstrated terminal transferase activity, and all RdRPs have terminal transferase activity (see J Virol 75(18) (2001) 8615 by CT Ranjith-Kumar et al. -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, published by Man, the contents of which are incorporated herein by reference.

The ribozyme version of RdRP that has recently been produced may also have terminal transferase activity (see DP Horning and GF Joyce et al., Proc Natl Acad Sci USA 113(35) (2016) 9786-91; And incorporated into the case). Reversible terminator nucleotide (Reversible-Terminating Nucleotide)

With respect to controlled NA chain synthesis, the disclosed method adds only one base at a time. If TdT is present with native NTP, TdT will incorporate NTP over time to form a long random sequence. If only one nucleotide is present, TdT will produce a long homopolymeric tract of the nucleotide. Thus, the compositions of the present disclosure are controlled in response to methods such that only one nucleotide is incorporated per cycle, and then any but specific NA sequence can be generated.

In order to obtain one and only one nucleotide of the polymerase, Sanger and Coulson designed the dideoxy nucleotide terminator method, which allows only the 3' end of each chain. A single nucleotide is introduced (see F. Sanger and AR Coulson, J Mol Biol 94(3) (1975) 441-8; the contents of which are incorporated herein by reference in its entirety. However, the di-deoxynucleotides cannot be prolonged. Thus, the compositions and methods use various reversible terminating nucleotides that allow for the re-production of 3'-OH groups after controlled removal of the blocking group. This disclosure refers to this modified nucleotide as a reversible terminating nucleotide triphosphate (rtNTP).

In accordance with the compositions and methods of the present disclosure, one or more photoremovable protecting groups that can be used to block the 3'-OH of a given nucleotide include, but are not limited to, 2-nitrobenzyl, dansulfonyl ( Dansyl group), p-hydroxyphenacyl group, and 7-methyoxy-4-methylcoumarin group. Alternatively, or in addition, according to the compositions and methods of the present disclosure, one or more chemically resectable protecting groups that can block the 3'-OH of a given nucleotide include, but are not limited to, an amine group, an azide group. Azidomethyl group or allyl group. In accordance with the compositions and methods of the present disclosure, a removable protecting group (both light and chemical can be cleaved) can be used to block the 3'-OH of a given nucleotide. For example, 2-nitrobenzyl group is photoswitchable and chemically resectable by a Lewis acid/amine combination treatment.

Modification of other portions of the nucleotide provides reversible termination activity. For example, a 2' modification of a nucleotide can reversibly terminate the growing RNA strand that is mediated by T7 RNA polymerase. Thus, in some embodiments of the compositions and methods of the present disclosure, a ribonucleotide can include one or more 2' modifications that reversibly terminate the RNA polymer in the synthesis by T7 RNA polymerase. Alternatively, or in addition, one or more 2' modified ribonucleotides (which reversibly terminate the RNA polymer during synthesis by T7 RNA polymerase) may include one or more removable blocking groups (eg, a photoreversible or chemically reversible barrier group).

Although various RNA and DNA polymerases have been shown to incorporate different rtNTPs, such activities have not been demonstrated for TdT or other enzymes with terminal transferase activity. 5' nucleic acid modification

According to the compositions and methods of the present disclosure, the non-templated synthesis reaction of TdT catalyzes the addition of nucleotides to the growing NA chain, which requires at least three nucleotides in length. Thus, the methods of the present disclosure use a NA primer having at least three 3' available nucleotides to initiate synthesis using TdT.

Regarding the synthesis in a solution using TdT and rtNTP, it is important to be able to separate the growing NA chain in the case of one base at a time. In some embodiments of the compositions and methods of the present disclosure, the growing NA chain can include a 5' modification for isolating the resulting synthetic DNA polymer. In some embodiments, the 5' modification can comprise a label or oligonucleotide that binds or hybridizes to the growing NA chain or resulting synthetic DNA polymer, and can optionally be further attached to the substrate.

In some embodiments, the 5' modification can comprise a biotin label that can be bonded to an avidin coated substrate or a streptavidin coated substrate. In some embodiments, the substrate can be a solid or semi-solid substrate. The semi-solid substrate of the present disclosure may comprise glass. Alternatively, or in addition, the semi-solid substrate of the present disclosure may include one or more holes or channels. In some embodiments, the substrate form can be a substantially flat surface or bead. Enzymatic synthesis linked to a substrate

Typically, phosphoramidite synthesis uses controlled porous glass or polystyrene beads for the mass production of a single NA sequence. Recently, arrays have been used for the synthesis of multiple NA sequences. These array reactions are performed in a variety of ways, including the use of a light mask to initiate a particular region of light such that only a limited number of growing links in each cycle are subjected to the next nucleotide; using a micromirror-based scanning system (micromirror- Based scanning system) using a specific light to initiate the incorporation of the next nucleotide; using an inkjet printer technology to produce an array of synthetic NA sequences; and using surface derivatization controlled by microheating, However, the length of the DNA polymer synthesized in each of these systems is short compared to the synthetic DNA polymer produced by the compositions and methods of the present disclosure. Although these four microarray generation methods have proposed or used phosphoramidite synthesis, when combined with the disclosed compositions and enzymatic synthesis methods (eg, by TdT and light reversible dNTPs on dense aperture arrays, and can be separate These existing methods for array generation can be modified to provide many long NA sequences in a very short time. Microfluidic system

A large amount of synthesis in solution or on beads produces a sufficient amount of small amounts of NA sequences for many molecular biology experiments. At the other extreme, arrays of many different NA sequences can be used for whole genome and aptamer experiments, however, the amount of material available for each NA sequence may be limited. As a medium scale synthesis, the NA sequence can be synthesized in a microfluidic droplet system using a composition comprising TdT and rtNTP in accordance with the methods of the present disclosure. Microfluidic systems can be adapted to use chemically or photoreversible rtNTPs and are suitable for the synthesis of many different NA sequences, each in an amount that can be used in many applications. Non-catalytic single-stranded binding protein

When single-stranded NAs are synthesized, they begin to form secondary and tertiary structural configurations that may inhibit the incorporation of other nucleotides. For example, a short region that is self-complementary may mask 3'-terminal three bases in such a way that TdT cannot extend the NA chain. There are ways to overcome this suppression.

In some embodiments of the compositions and methods of the present disclosure, after some incorporation cycles, specific or random primers are added and a second synthesis reaction is performed to unwind the inhibition configuration. To maintain a substantially linear configuration of the double-stranded NA, the 3' end of the original growing NA can be freely incorporated into other nucleotides by the TdT vector.

In some embodiments of the compositions and methods of the present disclosure, after some incorporation cycles, a set of random short-terminating NA sequences (without 5'-) is provided under conditions sufficient to effect hybridization to unwind the inhibitory conformation. PO ₄ , 3'-dideoxy).

In some embodiments of the compositions and methods of the present disclosure, a non-catalytic single-stranded binding protein is added to free the 3' end of the original growing NA chain. In some embodiments of the compositions and methods of the present disclosure, a non-catalytic single-stranded binding compound is added to free the 3' end of the original growing NA chain. For example, polyamines can be used: spermine, penta-L-lysine, polydisperse poly-L-lysine, and polydisperse poly-L-lysine. One or more of spermidines to untie the knotted NA chain by interacting with a negatively charged NA backbone. Non-naturally occurring DNA nucleotides

Various non-naturally occurring nucleotides having non-natural bases and/or backbones can be used in the compositions and methods of the present disclosure.

Exemplary non-naturally occurring nucleotides including non-natural bases include, but are not limited to, dBTP, dKTP, dPTP, dXTP, and dZTP (see Figure 3 and K. Sefah et al., Proc Natl Acad Sci USA 111 (4) (2014) 1449-54; the contents of which are incorporated herein by reference. Non-naturally occurring nucleotides including non-natural bases include, but are not limited to, dInDTP, d5FITP, dAITP, dNITP, dCHITP, dCEITP, d5PhITP, d5NapITP, and d5AnITP (see Chembiochem 8 by AJ Berdis and D. McCutcheon) 12) (2007) 1399-408; the contents of which are incorporated herein by reference.

Illustrative non-naturally occurring nucleotides comprising a non-natural backbone include, but are not limited to, CeNA (cyclohexenyl nucleic acid), ANA (arabinonucleotide), FANA (2'-fluoro-arabinonucleotide), TNA (alpha-L) - threofuranosyl nucleic acid) and LNA (2'-O, 4'-C-methylene-β-D-ribonucleic acid; locked nucleic acid) (see Science 336 (6079) by VB Pinheiro et al. 2012) 341-4; the contents of which are incorporated herein by reference. DNA synthesis method

In order to use the DNA synthesis technique of TdT independent of template synthesis properties and to develop cores, the disclosed compositions and methods use TdT to incorporate rtNTPs into the synthesis reaction at the 3' end of the growing NA chain. Incorporation of rtNTPs at the 3' end of the growing NA chain blocks further synthesis, thus resulting in the addition of only one nucleotide during the NA synthesis process. Removal of the terminator modification allows for the addition of another modified nucleotide and the use of this stepwise approach results in the production of synthetic DNA molecules. DNA synthesis array

The composition of the present disclosure may comprise (a) a plurality of primers, wherein each primer comprises at least three nucleotides, wherein each primer comprises a 3' reversible termination nucleotide (rtNTP), and wherein each primer is linked to the substantial An array of upper flat substrates; (b) a plurality of free rtNTPs; and (c) an enzyme or ribozyme having terminal transferase activity.

In some embodiments of the array of the present disclosure, the primer can be attached directly to the substrate such that the primer contacts the substrate. Alternatively, in some embodiments, the primer can be indirectly attached to the substrate such that the primer contacts and thereby directly contacts the linker of the substrate. The linkers disclosed herein can be of any length. Exemplary linkers of the present disclosure can include any material including, but not limited to, organic or inorganic molecules or polymers. In some embodiments, the organic polymer comprises one or more of DNA, RNA or amino acid monomers.

The plurality of primers attached to the substrate can include a first primer having a first sequence and a second primer having a second sequence, wherein the first sequence is different from the second sequence. In some embodiments, the plurality of primers attached to the substrate can include 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 A sequence, a second sequence, and a third or subsequent sequence are different. In some embodiments, the plurality of primers includes at least one duplicate of each of the first primer, the second primer, and the third or subsequent different primers. In some embodiments, the plurality of primers includes two or more repetitions of each of the different primers. In some embodiments, the plurality of primers comprises 5, 10, 50, 100, 200, 500, 100 repeats of each of the different primers or any number of repetitions therebetween.

In some embodiments, each of the plurality of primers attached to the substrate can comprise a unique sequence of each of the other primers different from the plurality of primers (ie, each of the plurality of primers is a different primer) ).

In some embodiments of the array composition of the present disclosure, the synthetic nucleic acid produced by the array can include a first synthetic nucleic acid having a first sequence and a second synthetic nucleic acid having a second sequence, wherein the first sequence is different from the second sequence . In some embodiments, a plurality of synthetic nucleic acids linked to a substrate can include a first synthetic nucleic acid having a first sequence, a second synthetic nucleic acid having a second sequence, and a third or subsequent synthesis having a third or subsequent sequence A nucleic acid in which the first, second and third or subsequent sequences are different. In some embodiments, the plurality of synthetic nucleic acids comprises at least one repeat of each of the first synthetic nucleic acid, the second synthetic nucleic acid, and the third or subsequent different synthetic nucleic acids. In some embodiments, the plurality of synthetic nucleic acids comprises two or more repeats of each of the different synthetic nucleic acids. In some embodiments, the plurality of synthetic nucleic acids comprises 5, 10, 50, 100, 200, 500, 100 repeats of each of the different synthetic nucleic acids or any number of repeats therebetween.

In some embodiments of the array of the present disclosure, each of the plurality of synthetic nucleic acids attached to the substrate can comprise a unique sequence of each of the other synthetic nucleic acids different from the plurality of synthetic nucleic acids (ie, the plurality of synthetics) Each synthetic nucleic acid of a nucleic acid is a different synthetic nucleic acid).

In some embodiments of the arrays disclosed herein, the plurality of synthetic nucleic acids attached to the substrate can include at least one synthetic DNA. Alternatively, or in addition, in some embodiments of the array of the present disclosure, the plurality of synthetic nucleic acids attached to the substrate can include at least one synthetic RNA. In some embodiments, those embodiments in which a plurality of synthetic nucleic acids linked to a substrate can include at least one synthetic RNA, the compositions and/or methods of the present disclosure can additionally include the use of an RNAse inhibitor.

In some embodiments of the arrays disclosed herein, each of the plurality of synthetic nucleic acids attached to the substrate comprises synthetic DNA.

In some embodiments of the arrays disclosed herein, each of the plurality of synthetic nucleic acids attached to the substrate comprises synthetic RNA. In some embodiments, the compositions and/or methods of the present disclosure may additionally comprise the use of an RNAse inhibitor.

In some embodiments of the arrays disclosed herein, a plurality of primers are disposed on the substrate in a repeatable or non-repeating pattern from a plurality of synthetic nucleic acids produced therefrom. In some embodiments, the repeating pattern can be used to include repeated primers and/or repeated synthetic nucleic acids. The inclusion of repeated primers and/or repeated synthetic nucleic acids can increase the statistical power of the assay using the array in which an analyte is detected or hybridized to one or more of the synthetic nucleic acids. In some embodiments, non-repetitive patterns can be used to increase the diversity of primers and/or synthetic nucleic acids present on the array. This increase in diversity can be particularly useful for initial screening of analytes to identify targets for further analysis or to identify rare analytes in a large number of analytes.

In some embodiments of the arrays disclosed herein, the array can be used in a device for detecting analytes that bind or hybridize to the synthetic nucleic acids of the array. An exemplary detection device is described in U.S. Patent No. 9,410,887, the disclosure of which is incorporated herein in its entirety. EXAMPLES Example 1: 3'-O-(2-nitrobenzyl)-2'-dNTP composition was combined with T7 RNA polymerase and UV radiation to remove the 2-nitrobenzyl moiety.

In some embodiments of the compositions and methods of the present disclosure, the modified dNTP (having a 2-nitrobenzyl group at the 2' position) is incorporated into a template-dependent reaction by T7 RNA polymerase. When only one modified dNTP was added, 2-nitrobenzyl was used as the reversible terminator, and UV radiation removed the 2-nitrobenzyl moiety, leaving the OH group as the site for subsequent synthesis. Although the TdT of the calf can incorporate a modified dNTP at the 3' position, prior to the disclosure, it was not shown that TdT can be incorporated into 3'-O-(2-nitrobenzyl)-2'-dNTP or this can be used NA synthesis.

To test this method, 3'-O-(2-nitrobenzyl)-2'-dATP (taken from Trilink, see Figure 1) and recombinant calf TdT (taken from NEB) were used. To initiate template-dependent synthesis, TdT requires a single strand of DNA molecules of at least three nucleotides in length. A 24 nucleotide oligonucleotide was used which has an alexa-488 label at the 5' end (Fig. 2A).

If TdT successfully incorporates 3'-O-(2-nitrobenzyl)-2'-dATP at the 3' end of the oligonucleotide, there will be reduced mobility on the denaturing polypropylene guanamine gel. The mobility is equal to the addition of one nucleotide. When analyzed on a denaturing polyacrylamide gel, the presence of 5' reduces the mobility of this oligonucleotide, thus compared to the ladder (Figure 2B, lines 1 and 2), Analyzed to 27 nucleotides. When the oligonucleotide was incubated with TdT but no nucleotide, there was no non-specific change in the resolution of the oligonucleotide on the gel (Fig. 2B, line 3). Oligonucleotide incubation with TdT and unmodified dNTPs resulted in polymerization of long DNA molecules (Fig. 2B, line 7). However, oligos with TdT and 3'-O-(2-nitrobenzyl)-2'-dATP culture caused a shift in the analytical results on the gel, which is equivalent to adding only one nucleotide (Figure 2B). , line 4). After culturing the oligonucleotide with TdT and 3'-O-(2-nitrobenzyl)-2'-dATP, the sample is irradiated with UV, and then the sample is cleaned by rotating the column (Zymo, oligonucleotide cleaning and concentration) Column) and further cultured with 3'-O-(2-nitrobenzyl)-2'-dATP and TdT. This caused the resolution of the gel to shift, which was equivalent to the addition of another blocked nucleotide (Fig. 2B, line 5), confirming the gradual addition of a single nucleotide at the 3' end of the oligonucleotide.

After the oligonucleotides were incubated with TdT and 3'-O-(2-nitrobenzyl)-2'-dATP, the samples were further incubated with unmodified dNTPs (Fig. 2B, line 6). Gel analysis of this sample showed a weak band comparable to that observed after culturing the oligonucleotide with TdT and 3'-O-(2-nitrobenzyl)-2'-dATP. However, longer DNA molecules were also observed (Fig. 2B, line 6).

Overall, the results confirmed that although TdT can add 3'-O-(2-nitrobenzyl)-2'-dATP at the 3' end of the oligonucleotide, some oligonucleotides have not yet received 3'- O-(2-nitrobenzyl)-2'-dATP and still remain free for further synthesis. Therefore, the addition of unmodified dNTPs results in the synthesis of long DNA molecules on these unblocked oligonucleotides. However, this longer DNA molecule is still shorter than the length of the DNA molecule observed after culturing the oligonucleotide with TdT and unmodified dNTP (Fig. 2B, comparing lines 6 and 7). This is the result of retaining free 3'-O-(2-nitrobenzyl)-2'-dATP in the reaction. When TdT is synthesized as unmodified dNTPs, it also incorporates 3'-O-(2-nitrobenzyl)-2'-dATP, which blocks the addition of other dNTPs. This is comparable to the Sanger DNA sequencing ladder caused by the use of dideoxynucleotides which lack 3' OH groups and block further DNA polymerization. This observation further supports the scope of the patent application in which TdT can be incorporated into 3'-O-(2-nitrobenzyl)-2'-dATP and this blocks further DNA synthesis. Example 2: DNA synthesis reaction conditions

The reaction of Example 1 was carried out in a volume of 5 μl (ml) and incubated at 37 ° C for 1 hour. By culturing 1 microliter of 10 μM oligonucleotide (shown in Figure 2A) with 0.2 μl of TdT (20 units/μl, NEB), 0.5 μl of CoCl ₂ (final concentration 0.25 mM), 0.5 Microliter of reaction buffer (final concentration of 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate) and 1 μl of 10 mM dNTP or dATP or 3'-O-(2-nitrobenzyl) -2'-dATP for the synthesis reaction. This provides an oligonucleotide to nucleotide ester ratio of 1:1000. For control group experiments, the nucleotides were replaced with 1 microliter of water. The incubation was carried out by UV irradiation under UV light for 10 minutes. The reaction was stopped by adding 1 μl of 100 mM EDTA and incubating at 80 ° C for 5 minutes. Sample analysis was performed on a denatured 20% polyacrylamide gel (8 M urea). Incorporated by reference

Each of the documents (including any cross-references or related patents or applications) cited herein is hereby incorporated by reference in its entirety in its entirety in its entirety. The citation of any document is not an admission that it is prior art to any invention disclosed or claimed herein, or it is taught, suggested or disclosed in any combination, either alone or in combination with any other reference. Furthermore, 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 the document incorporated by reference, the meaning or definition given to the term in this document controls. Other embodiments

While the specific embodiments of the present disclosure have been shown and described, various modifications and changes may be made without departing from the spirit and scope of the disclosure. All such variations and modifications are intended to be included within the scope of the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing the structure of 3'-O-(2-nitrobenzyl)-2'-dATP.

Figure 2A is the sequence of a single-stranded oligonucleotide (SEQ ID NO: 1) used as a starting point for DNA synthesis as shown in Figure 2B. The asterisk at the 5' end indicates the alexa-488 marker.

Figure 2B is a photograph illustrating the running of a gel at 100 V on a 20% polyacrylamide gel (8 M urea) for 35 minutes. The gel was then stained by fixing with 10% ethanol for 5 minutes followed by washing with 1 x TBE containing SybrGold for 3 minutes. Line 1 = ladder; row 2 = oligonucleotide shown in Figure 2A; row 3 = oligonucleotide plus TdT; row 4 = TdT and 3'-O- (2-nitrate Benzyl)-2'-dATP incubated oligonucleotide; row 5 = sample shown in row 4, but cleaned by UV irradiation and further with TdT and 3'-O-(2- Nitrobenzyl)-2'-dATP culture; line 6 = sample shown in row 4, but not further irradiated with unblocked dNTPs without UV irradiation; row 7 = oligonucleus cultured with free dNTP Glycosylate.

Figure 3 is a schematic diagram showing Watson-Crick pairing rules in which the size of dBTP, dKTP, dPTP, dXTP and dZTP nucleotides are complementary and hydrogen bonds are complementary. Reproduced from PNAS 2014; 111:1449-1454 published by K. Sefah et al.

Figure 4 is a schematic diagram showing the structure of a non-naturally occurring nucleic acid base (2'-deoxynucleoside triphosphate) exemplified in the present disclosure. "dR" is a portion of a deoxyribose triphosphate used to represent a nucleotide. Partially reproduced from Chembiochem 8(12) (2007) 1399-408 by A.J. Berdis and D. McCutcheon.

Claims (77)

A composition comprising: (a) a primer comprising at least three nucleotides, wherein the primer comprises a 3' reversible termination nucleotide (rtNTP); (b) at least one free rtNTP; and (c An enzyme or ribozyme having terminal transferase activity. For example, the composition of claim 1 of the patent scope further includes: (d) a reaction buffer. The composition of claim 2, wherein the reaction buffer comprises potassium acetate at a final concentration of 50 mM, 20 mM Tris-acetate, and 10 mM magnesium acetate. The composition of claim 2, wherein the reaction buffer comprises Zn ²⁺ , Co ²⁺ or Mn ²⁺ . The composition of any one of claims 1 to 4, wherein the at least one free reversible terminating nucleotide (rtNTP) comprises a chemically reversible blocking group. The composition of claim 4, further comprising a reagent to chemically reverse the barrier group. The composition of claim 6, wherein the reagent is Lewis acid. The composition of claim 7, wherein the Lewis acid comprises CoCl ₂ . The composition of any one of claims 1 to 8, wherein the chemically reversible blocking group comprises a 2-nitrobenzyl group, an amine group, an azidomethyl group (azidomethyl group). ) or allyl. The composition of any one of claims 1 to 8, wherein the chemically reversible barrier group comprises a 2-nitrobenzyl group. The composition of any one of claims 1 to 10, wherein the at least one free reversible terminating nucleotide (rtNTP) comprises a photoreversible blocking group. The composition of any one of claims 1 to 11, wherein the photoreversible blocking group comprises 2-nitrobenzyl, dansyl group, p-hydroxybenzhydrylmethyl ( P-hydroxyphenacyl group), or 7-methyoxy-4-methylcoumarin group. The composition of any one of claims 1 to 11, wherein the photoreversible blocking group comprises 2-nitrobenzyl. The composition of any one of claims 1 to 13, wherein the primer comprises a 5' modification. The composition of claim 14 wherein the 5' modification comprises a selectable tag. The composition of claim 15 wherein the selectable label is a linkage or hybridization to the primer. The composition of claim 16, wherein the selectable label is a bond or hybridization to a substrate. The composition of claim 17, wherein the substrate comprises a flat surface or a bead. The composition of claim 18, wherein the substrate comprises a glass, a polymer, or a substrate. The composition of claim 19, wherein the substrate comprises a polymer, and wherein the polymer comprises a polypropylene amide gel. The composition of any one of claims 17 to 20, wherein the substrate comprises an anchor. The composition of any one of claims 14 to 21, wherein the 5' modification comprises biotin. The composition of claim 22, wherein the substrate comprises a bead, wherein the bead comprises a polyacrylamide gel, wherein the polyamidoamide gel comprises a fixture, and wherein the fixture comprises avidin Avidin or streptavidin. The composition of any one of claims 1 to 23, wherein the primer comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), amino acid, or any combination thereof. The composition of any one of claims 1 to 24, wherein the primer comprises at least one non-naturally occurring base or at least one non-naturally occurring backbone. The composition of any one of claims 1 to 24, wherein the primer comprises at least one non-naturally occurring base and at least one non-naturally occurring backbone. The composition of any one of claims 1 to 26, wherein the at least one non-naturally occurring base comprises dBTP, dKTP, dPTP, dXTP, dZTP, dInDTP, 5-fluoroindolyl-2'- Deoxyribonucleoside triphosphate (d5FITP), 5-amino-mercapto-2'-deoxyribonucleoside triphosphate (dAITP), 5-nitro-mercapto-2'-deoxyribose nucleus Glycoside triphosphate (dNITP), 5-cyclohexyl-mercapto-2'-deoxyribonucleoside triphosphate (dCHITP), dCEITP, 5-phenylmercapto-2'-deoxyribonucleoside triphosphate (d5PhITP), 5-naphthyldecyl-2'-deoxyribonucleoside triphosphate (d5NapITP), or d5AnITP. The composition of any one of claims 1 to 26, wherein the at least one non-naturally occurring backbone comprises cyclohexenyl nucleic acid (CeNA), arabinucleic acid (ANA), 2 '-Fluoro-arabino nucleic acid (FANA), α-L-threofuranosyl nucleic acid (TNA), or locked nucleic acid (LNA). The composition of claim 28, wherein the LNA comprises 2'-O, 4'-C-methylene-β-D-ribonucleic acid. The composition of claim 1, wherein the reversible terminating nucleotide (rtNTP) comprises a chemically reversible blocking group. The composition of claim 30, wherein the chemically reversible barrier group comprises 2-nitrobenzyl, amine, azidomethyl, or allyl. The composition of claim 30, wherein the chemically reversible barrier group comprises 2-nitrobenzyl. The composition of claim 30, wherein the reversible terminating nucleotide (rtNTP) comprises a photoreversible blocking group. The composition of claim 33, wherein the photoreversible blocking group comprises 2-nitrobenzyl, dansyl, hydroxybenzhydrylmethyl, or 7-methoxy-4-methyl Based on coumarin. The composition of claim 33, wherein the photoreversible blocking group comprises 2-nitrobenzyl. The composition of any one of claims 1 to 35, wherein the primer is in solution. The composition of any one of claims 1 to 35, wherein the primer is attached to the substrate. The composition of claim 37, wherein the primer is directly attached to the substrate, and wherein the primer contacts the substrate. The composition of claim 37, wherein the primer is indirectly attached to the substrate, and wherein the primer contacts a linker in contact with the substrate. The composition of any one of claims 37 to 39, wherein the substrate is a bead. The composition of claim 40, wherein the bead comprises a glass, a polymer, or a substrate. The composition of claim 40 or 41, wherein the bead is porous. The composition of any one of claims 40 to 42 wherein the bead comprises a polyacrylamide gel. The composition of any one of claims 37 to 39, wherein the substrate is a substantially flat surface. The composition of claim 44, wherein the substrate is a flat surface. The composition of claim 45, wherein the substrate comprises a glass, a polymer, or a substrate. The composition of any one of claims 45 to 46, wherein the primer is a plurality of primers, and wherein each primer of the plurality of primers is attached to the substrate in the array. 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 is different from the second sequence. The composition of claim 48, wherein the plurality of primers comprises at least one repeat of the first primer and at least one repeat of the second primer. The composition of claim 49, wherein each of the plurality of primers comprises a unique sequence. The composition of any one of claims 1 to 50, wherein the enzyme is a terminal deoxynucleotidyl transferase (TdT). The composition of any one of claims 1 to 50, wherein the enzyme is pol θ, pol λ, pol μ, Dpo 1, or primase. The composition of any one of claims 1 to 50, wherein the ribozyme is an RNA-dependent RNA polymerase. A method for synthesizing a template-free nucleic acid, comprising: (a) obtaining a composition according to any one of claims 1 to 53; (b) removing the 3' rtNTP of the primer of the composition; and (c) The at least one free rtNTP of the composition is incorporated into the primer of the composition by the enzyme or ribozyme of the composition, thereby producing a synthetic nucleic acid. The method of claim 54, wherein the at least one free rtNTP of the composition is a plurality of free rtNTPs. The method of claim 54 or 55, wherein steps (b) and (c) are completed in less than 1 minute. The method of claim 56, further comprising a first rinse after the deprotection step (b) and a second rinse after the incorporation step (c). The method of claim 57, wherein steps (b) and (c) are completed in less than 1 minute. The method of any one of claims 54 to 58 further comprising the steps of: (d) repeating steps (b) and (c). The method of claim 59, further comprising the steps of: (e) removing the unincorporated rtNTP prior to performing step (d). The method of any one of claims 54 to 60, wherein the 3' rtNTP of the primer comprises a photoreversible blocking group, and the deprotecting comprises exposing the photoreversible blocking group to light irradiation. The method of claim 61, wherein the light irradiation comprises UV irradiation. The method of any one of claims 54 to 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 Lewis acid . The method of claim 63, wherein the Lewis acid comprises CoCl ₂ . The method of any one of claims 54 to 64, wherein the primer is in solution. The method of any one of claims 54 to 64, wherein the primer is attached to the substrate. The method of claim 66, wherein the primer is a plurality of primers, and wherein each primer of the plurality of primers is attached to the substrate in the array. 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 is different from the second sequence. The method of claim 68, wherein the plurality of primers comprises at least one repetition of the first primer and at least one repetition of the second primer. The method of claim 68, wherein each of the plurality of primers comprises a unique sequence. The method of any one of claims 54 to 70, further comprising the steps of: contacting the synthetic nucleic acid with a random primer, a non-specific primer, a random short-terminated nucleic acid sequence, and a non-catalytic process during the synthesis process One or more of a single-stranded binding protein and a non-catalytic single-stranded binding compound to maintain a substantially linear conformation and inhibit secondary in one or more rtNTP deprotection and incorporation processes And/or a tertiary structure forms or unfolds the inhibitory conformation of the synthetic nucleic acid. The method of claim 71, wherein the non-catalytic single-stranded binding protein or the non-catalytic single-stranded binding compound comprises a polyamine. The method of claim 72, wherein the polyamine comprises spermine, penta-L-lysine, polydisperse poly-poly-L-lysine (polydisperse poly- L-lysine), or spermidine. The method of any one of claims 70 to 73, wherein 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 The contacting step is carried out with nucleotides or any number of nucleotides in between. The method of any one of claims 70 to 74, wherein the contacting step is performed prior to generating a sequence in the synthetic nucleic acid that may form a secondary or tertiary structure. The method of any one of claims 70 to 74, wherein the contacting step is performed prior to producing a sequence of sufficient length such that the synthetic nucleic acid can be folded into a substantially non-linear configuration. The method of any one of claims 70 to 76, further comprising the steps of: (f) removing the random primer, the non-specific primer, a random short-term termination from the synthetic nucleic acid after the synthesis is completed One or more of a nucleic acid sequence, a non-catalytic single-stranded binding protein, and a non-catalytic single-stranded binding compound.