Compositions and methods relating to enzymatic DNA synthesis

Abstract

The present disclosure provides compositions and methods related to enzymatic nucleic acid synthesis. In particular, the present disclosure provides novel compositions and methods for generating a plurality of modified nucleic acid oligos using a nucleic acid template molecule.

Description

COMPOSITIONS AND METHODS RELATING TOENZYMATIC DNA SYNTHESISCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 580,877 filed September 6, 2023, which is incorporated herein by reference in its entirety for all purposes.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 29,749 Byte XML file named “NCSU_42151_601_SequenceListing” created on September 5, 2024.GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant number ECCS2027655 awarded by the National Science Foundation. The government has certain rights in the invention.FIELD

[0004] The present disclosure provides compositions and methods related to enzymatic nucleic acid synthesis. In particular, the present disclosure provides novel compositions and methods for generating a plurality of modified nucleic acid oligos using a nucleic acid template molecule.BACKGROUND

[0005] The proportion of the economy reliant on biological systems is rapidly growing, and in many cases, it is driven by the ability to synthesize DNA and RNA. For example, DNA is already the information substrate in engineering medical and environmental diagnostics and sensors, cells and organisms for production of biofuels and other commodities, cell-based cancer immunotherapies, vaccines, and even longer-term applications in DNA-based molecular computing and storage. However, it has been known for over half a century that natural DNA and RNA are not just bare As, Cs, Ts, and Gs. Rather, they exist in chemically modified forms including methylated, hydroxy-methylated, and formyl-methylated Cs and As, as just a few predominant examples. The patterns of these modifications are central regulators of geneexpression in cells, stability and function of RNAs, distinguish healthy and cancerous cells, are used by microbes to distinguish their own DNA from foreign invading DNA, and are a nexus in cellular responses to environmental toxins and drugs of abuse. In short, the nucleic acids that can currently be synthesized are unmodified; yet, natural nucleic acids with biomedical, agricultural, and industrial applications are richly modified with diverse chemical groups. Completely artificial nucleic acid modifications would also provide additional advantages, including increasing nucleic acid stability and providing chemical handles to link nucleic acids to other biomolecules. In other words, the market problem is that the current nucleic acid-based economy is artificially restricted to a narrow range of applications that exclude the central importance of ‘epi ’-genetics and epitranscriptomics in bioengineering, the layer of modifications ‘on’ DNA and RNA.SUMMARY

[0006] Embodiments of the present disclosure include a nucleic acid template molecule for generating a plurality of modified nucleic acid oligos comprising a hairpin region chemically configured for attachment to a substrate, a double-stranded region comprising at least one nucleic acid processing site, and a single- or double-stranded region comprising a nucleic acid template sequence.

[0007] In some embodiments, embodiments of the present disclosure include methods for RNA production through in vitro transcription. In some embodiment, an RNA polymerase promoter sequence is positioned between the hairpin region and the at least one nucleic acid processing site, or after the at least one nucleic acid processing site. In some embodiments, the promoter sequence can comprise a T7, SP6, or T3 promoter.

[0008] In some embodiments, the chemically configured region of the hairpin comprises a Biotin modification (3', 5' or internal), Amine modification (3', 5' or internal), Carboxylic acid modification (3', 5' or internal), Alkyne modification (3', 5' or internal), or Azide modification (3', 5' or internal).

[0009] In some embodiments, the substrate comprises at least one substrate from Table 3.

[0010] In some embodiments, the hairpin region of the nucleic acid template molecule comprises a loop region comprising from about 4 nucleotides to about 100 nucleotides.

[0011] In some embodiments, the at least one nucleic acid processing site of the nucleic acid template molecule comprises a nickase site and / or a restriction site and / or a nickase site adjacent to or overlapping with a restriction site. In some embodiments, the nickase site comprises anynucleic acid sequence selected from Table 4. In some embodiments, the restriction site comprises any nucleic acid sequence from Table 5.

[0012] In some embodiments for RNA production, the nickase site is not needed.

[0013] In some embodiments, the single- or double-stranded region comprises a 3 ’ overhang. In some embodiments, the single- or double-stranded region comprises a 5’ overhang. In some embodiments, the single- or double-stranded region does not comprise an overhang.

[0014] In some embodiments, the single-or double-stranded region comprising the nucleic acid template sequence is from about 2 nucleotides to about 100 kb.

[0015] In some embodiments, the nucleic acid template sequence is the reverse complement of the sequence of the plurality of modified nucleic acid oligos to be generated.

[0016] In some embodiments, the at least one nucleic acid processing site is directly adjacent to the single- or double-stranded region comprising the nucleic acid template sequence.

[0017] In some embodiments, the nucleic acid template molecule further comprises an RNA polymerase promoter before or after the at least one nucleic acid processing site. In some embodiments, the nucleic acid template molecule further comprises an RNA polymerase promoter as part of the nucleic acid template sequence.

[0018] Embodiments of the present disclosure also include a composition for generating a plurality of modified nucleic acid oligos comprising the nucleic acid template molecule comprising a hairpin region of the nucleic acid template molecule that is chemically coupled to a substrate.

[0019] In some embodiments, the composition comprises at least one nickase enzyme, and / or at least one restriction enzyme. In some embodiments, the at least one nickase enzyme and / or the at least one restriction enzyme is chemically coupled to the substrate or to a separate substrate than the hairpin template is coupled to.

[0020] In some embodiments, the composition further comprises dNTPs. In some embodiments, the dNTPs comprise at least one modified dNTP.

[0021] In some embodiments for RNA production, the composition further comprises NTPs. In some embodiments, the NTPs comprise at least one modified NTP.

[0022] Embodiments of the present disclosure also include a method for enzymatically generating a plurality of modified nucleic acid oligos with any arbitrary combinations of modifications at specific sites, the method comprising (a) providing the composition, (b) adding a first solution comprising at least one unmodified nucleotide base and a DNA or RNA polymeraseto generate at least a portion of the modified nucleic acid oligo from the nucleic acid template sequence, (c) repeating step (b) until the oligo is synthesized up to the nucleic acid just before the first desired modified nucleic acid, (d) adding at least a solution comprising at least one modified dNTP or NTP, (e) repeating steps (b-d) until the modified nucleic acid oligo is generated, and (f) for DNA oligos, adding a nickase enzyme to release the modified nucleic acid oligo from the nucleic acid template molecule.

[0023] In some embodiments, the method further comprises a wash step.

[0024] In some embodiments, step (e) of the method is performed with at least a second modified dNTP / NTP that is the same as or is different from the first modified dNTP / NTP in step (d).

[0025] In some embodiments, the first modified dNTP / NTP and the at least second modified dNTP / NTP are from about 1 nucleotide to about 1000 nucleotides apart.

[0026] In some embodiments, if modifications are desired near the end of the template molecule, the oligo can be completed with unmodified dNTPs to reduce the number of cycles of steps (b-e) needed, and a restriction enzyme can be added after step (e) to generate a 5 ’ overhang of 1 -6 nucleotides in length at the end of the nucleic acid template molecule which can be filled in with modified dNTPs.

[0027] In some embodiments, a restriction enzyme can be added after step (e) to generate a blunt end, a 3 ’ overhang, or a 5 ’ overhang at the nuclei acid processing site next to the hairpin.

[0028] In some embodiments, the method further comprises ligating a different single-stranded or double-stranded nucleic acid sequence to the nucleic acid template molecule via the blunt end, the 3 ’ overhang, or the 5 ’ overhang.

[0029] In some embodiments, this new sequence may contain an RNA polymerase promoter.

[0030] In some embodiments, the method further comprises repeating steps (b) through (f) to generate a modified nucleic acid oligo.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 : Exemplary cyclic strategy. Relying on the absence of a specific dNTP / NTP to pause the reaction before incorporation of a modified dNTP / NTP at a specific site.

[0032] FIGS. 2A-2B: Exemplary template. A 90 nucleotide (“nt”) sequence with a BIOdT on the 75thnt. Top part of the strand forming the double-stranded region is a recognition site (GGATCNNNN) for the nickase Nt.alwl. The BIOdT in the loop interacts with streptavidin onthe magnetic beads (FIG. 2A). Continuous synthesis of codewords. The method works by the consecutive and repeated action of a polymerase and a nicking endonuclease. First, the polymerase binds to the hairpin sliding across the single stranded portion in the 5 ’ to 3 ’ direction, adding bases along the way. The nicking endonuclease then nicks the strand after the recognition site and in the next cycle of polymerization, the polymerase pushes the 60 nt long strand out, producing a single- stranded codeword. This cycle repeats (FIG. 2B).

[0033] FIGS. 3A-3B: Attaching hairpins to magnetic beads reduces non-specific interactions; Hairpin without attached magnetic beads; DI: Hairpin in water, D2: Polymerase + nickase + dNTPs, D3: Reaction mixture incubated for 5 minutes, D4: Reaction mixture incubated for 15 minutes D5: Reaction mixture incubated for 30 minutes D6: Reaction mixture incubated for 45 minutes, D7: Polymerase + hairpin + dNTPs, D8: Polymerase + hairpin + dNTPs+ codeword, D9: Polymerase + dNTPs+ codeword, D10: Codeword in water, D11: Blank, D12: DNA ladder. Samples D1 and D10 serve as positive controls for hairpin and codeword respectively. D2 is a negative control for hairpin in the reaction mixture. D3, D4, D5 and D6 represent reaction mixtures incubated for 5, 15, 30 and 45 minutes respectively while samples D7, D8 and D9 test for any non- specific interactions that may occur in reactions (FIG. 3A). Hairpin attached to magnetic beads; C1 : Hairpin in water, C2: Polymerase + nickase + dNTPs, C3: Reaction mixture incubated for 5 minutes, C4: Reaction mixture incubated for 15 minutes C5: Reaction mixture incubated for 30 minutes C6 : Reaction mixture incubated for 45 minutes, C7 : Polymerase + dNTPs, C8 : Polymerase + dNTPs+ codeword, D9: Polymerase + dNTPs+ codeword, D10: Codeword in water, Dl l: Blank, D12: DNA ladder. Samples D1 and D10 serve as positive controls for hairpin and codeword respectively. D2 is a negative control for hairpin in the reaction mixture. D3, D4, D5 and D6 represent reaction mixtures incubated for 5, 15, 30 and 45 minutes respectively while samples D7, D8 and D9 test for any non-specific interactions that may occur in reactions. Template was magnetically separated out from samples C3, C4, C5, C6, C7 and C8 before running on the fragment analyzer (FIG. 3B).

[0034] FIGS. 4A-4G: Optimization of Enzymatic synthesis method. Large Klenow polymerase produced maximum codewords; Samples were cycled for 30 minutes between 75C and 37C, 25C and 37C and 30C and 37C for Deep vent polymerase, Large Klenow fragment and phi29 polymerase respectively (FIG. 4A). Incubation at 25C for 1 hour was the optimal condition. Samples were subjected to incubation temperatures of 25C, 30C and 37C for 15, 30, 45 and 60minutes (FIG. 4B). Too much hairpin in the reaction reduces yield. Four hairpin concentrations were tested (14.8uM, 27.7uM, 41.5uM, 55.3uM). 41.5uM hairpin samples produced the maximum codeword. Interestingly, the codeword produced dipped for hairpin concentrations higher than 41.5uM possibly indicating that higher hairpin concentrations results in more non-specific interactions (FIG. 4C). High dNTP concentration inhibits the reaction. The reaction was run with varying dNTP concentrations- 83.3, 166.7, 333.4, 666.7 nM. dNTP concentrations higher than 333.4nM inhibited the reaction thereby reducing codeword production (FIG. 4D). A sample dilution of 2: 1 produced 5 times as much codeword as the 1:1 samples. Beads were linked to the hairpin according to bead binding sites to hairpin ratios of 1: 1, 2: 1, 5:1 and 10: 1. Samples were run for 1 hour at 25 C using the Large Klenow fragment (FIG. 4E). Increasing concentration of polymerase and nickase increases codeword yield. Reaction was run with varying enzyme concentrations between 0.75ul and 3ul of polymerase and nickase for 1 hour (FIG. 4F). Visual representation of the ratio 2:1 of bead binding sites to hairpin (FIG. 4G).

[0035] FIGS. 5A-5B: Hairpin-bead template can be reused. After the first reaction run, the template was magnetically separated and reused for the consecutive run. This was repeated for 6 reaction runs (FIG. 5A). Cost analysis shows cost can be reduced by over 40%. Columns 1, 2, and 3 detail the components in the reaction mixture of the enzymatic synthesis method, the cost per 30ul run before and after optimization of polymerase, beads, hairpin and dNTPs. Column 4 represents reduction in cost after reusing the hairpin-bead template 6 times (FIG. 5B).

[0036] FIGS. 6A-6B: Overhang and full strand length. Six ssDNA codewords with varied overhang lengths (4bp, 8bp, 10bp and 20bp) were run with T7 DNA ligase, Taq DNA ligase and T4 DNA ligase. Strands were phosphorylated, annealed, ligated and amplified (FIG. 6A). Strands with 4bp overhangs ligated with T7 ligase had the best assembly efficiency. The samples were run on a standard agarose gel. Band intensity was measured using ImageJ and was normalized with the length of the resulting strand (FIG. 6B).

[0037] FIG. 7: Scheme for synthesis and assembly of DNA. Six strands of DNA are generated, 64bp in length using the enzymatic synthesis method and selectively annealed and ligated to form 2 separate full length data encoding strands. Both full length strands have a different order of codewords (codeword 1-blue and codeword 2-gray).

[0038] FIG. 8: Recycling the biotinylated DNA-bead complex. Recycling a ‘universal’ biotinylated DNA-bead complex using restriction enzymes nested within each other and cutting at the sites indicated by color-matched triangles.

[0039] FIGS. 9A-9B: HPLC purification of ssDNA generated via this enzymatic method. A peak corresponding to a 60 nt ssDNA is detected at the same position as a control ssDNA purified and ordered commercially as well the 60 nt position on a ssDNA ladder (FIG. 9A). Example fragment analyzer traces demonstrate that the enzymatic synthesis method creates the 60 nt ssDNA only when all dNTPs are provided but not when dCTP is missing (FIG. 9B).

[0040] FIGS. 10A-10B: Incorporation of diverse modified nucleotides. Methyl dCTP was incorporated into a 60 nt oligo and exhibit a shift from unmodified oligo (FIG. 10A). Additional examples of modified nucleotides that were successfully incorporated into a 60 nt oligo through two different DNA polymerases, Klenow and Bst, demonstrating the diversity and versatility of the method (FIG. 10B).

[0041] FIGS. 11A-11E: DNA Hairpin Bound to Magnetic Bead through Amide Bond Synthesizes ssDNA. AmC6 thymine modification; DNA hairpin was ordered from Eurofins genomics with a AmC6 modified thymine located at the loop of the hairpin (FIG. 11 A). Interaction between primary amine and carboxylic acid group; 4.43 pmol of the AmC6 modified hairpin was allowed to interact with 88.8ug of Dynabeads MyOne carboxylic acid functionalized magnetic beads in the presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (also known as EDC) to produce an amide linkage between the hairpin and magnetic bead (FIG. 11B). Hairpin-Bead construct can synthesize codeword; Hairpin-bead construct was added to a 30ul reaction mixture containing 0.61 pmol Large Klenow, 0.17 pmol Nt.Alwl, 0.33 mM dNTPs, reaction buffer and water. This mixture was incubated at 25 C for 1 hour. Post incubation, the hairpin-bead construct was magnetic separated and the supernatant was run on a fragment analyzer (FIG. 11C). Hairpin positive control; AmC6 modified hairpin was added to water and run on a fragment analyzer (FIG. 11D). ssDNA positive control; Expected DNA synthesis product was ordered from IDT, rehydrated, diluted and run on a fragment analyzer (FIG. 1 IE).

[0042] FIGS. 12A-12E: Fusion Klenow (NT_Klenow) is active and capable of enzymatic ssDNA synthesis. NT_Klenow design; Large klenow polymerase was recombinantly synthesized in E-coli with fusion tags (Avi-tag and HIS tag) and a linker (FIG. 12A(i)). Biotinylation of Avi- tag by BirA ligase; The lysine present in the Avi-tag was biotinylated in-vivo by a biotin ligase(BirA) present in the culture medium (FIG. 12A(ii). PAGE gel electrophoresis indicated presence of NT_Klenow; after cell lysis, protein was purified by Immobilized metal affinity chromatography (IMAC) (FIG. 12B(i)). IMAC products were run on polyacrylamide gel electrophoresis (PAGE) and the gel was imaged ( Protein ladder used for gel electrophoresis (FIG. 12B(ii)). Fusion Klenow (NT_Klenow) is capable of ssDNA synthesis; 10 ul of purified NT_Klenow was bound to 40 ug streptavidin magnetic bead and added to a reaction mixture containing 4.43 pmol Hairpin-bead construct, 0.17 pmol Nt.Alwl, 0.33 dNTPs, reaction buffer and water. This mixture was incubated at 25 C for 1 hour. Post incubation, the hairpin and NT_Klenow were magnetic separated and the supernatant was run on a fragment analyzer (FIG. 12C). Hairpin positive control; AmC6 modified hairpin was diluted with water and run on a fragment analyzer (FIG. 12D). ssDNA positive control; Expected DNA synthesis product was ordered from IDT, rehydrated, diluted and run on a fragment analyzer (FIG. 12E).

[0043] FIGS. 13A-13D: Nt.Alwl variants synthesized for enzymatic ssDNA synthesis. Nt.Alwl variants fragment design; The nicking endonuclease Nt.Alwl was recombinantly synthesized in E-coli with fusion tags (Avi-tag, HIS tag and Maltose binding protein tag) and a linker. The lysine present in the Avi-tag was biotinylated in-vivo by a biotin ligase (BirA) present in the culture medium (FIG. 13A). PAGE gel electrophoresis images indicated presence of different variants of Fusion Nt.Alwl ; After cell lysis, proteins were purified by Immobilized metal affinity chromatography (IMAC). IMAC products were run on polyacrylamide gel electrophoresis (PAGE) along with a protein ladder and the gel was imaged (FIG. 13B). Fusion endonucleases (Nt.Alwl variants) exhibit varying efficiencies of ssDNA synthesis; NT_Nt.Alwl is not capable of ssDNA synthesis (FIG. 13C(i)), Nt.Alwl_CT is capable of ssDNA synthesis (FIG. 13C(ii)), MBP_NT_Nt / Alwl is capable of ssDNA synthesis (FIG. 13C(iii)); 5ul of Nt.Alwl variant was added to a 30ul reaction mixture containing Hairpin-bead construct, NEB Large Klenow, dNTPs, reaction buffer and water. This mixture was incubated at 25 C for 1 hour. Post incubation, the hairpin and Nt.Alwl variant were magnetically separated and the supernatant was run on a fragment analyzer. Peaks in the graph indicate the length and amount of DNA present in the sample. Bound to a magnetic bead, fusion endonucleases (Nt.Alwl variants) exhibit varying efficiencies of ssDNA synthesis; NT_Nt.Alwl is not capable of ssDNA synthesis (FIG. 13D(i)), Nt.Alwl_CT is capable of ssDNA synthesis (FIG. 13D(ii)), MBP_NT_Nt / Alwl is capable of ssDNA synthesis (FIG. 13D(iii)); 5ul of Nt.Alwl variant was bound to streptavidin magnetic beadsand added to a 30ul reaction mixture containing Hairpin-bead construct, NEB Large KI enow, dNTPs, reaction buffer and water. This mixture was incubated at 25 C for 1 hour. Post incubation, the hairpins and Nt.Alwl variant were magnetically separated and the supernatant was run on a fragment analyzer. Peaks in the graph indicate the length and amount of DNA present in the sample.

[0044] FIGS. 14A-14F: Production of DNA oligos from an unmodified template using the scheme in FIG. 1. Six different reaction conditions were optimized to maximize yield including enzyme type (FIG. 14A), reaction temperature (FIG. 14B), concentration of the DNA template hairpin (FIG. 14C), amount of enzyme (FIG. 14D), monomer dNTP concentration (FIG. 14E), and the ratio of magnetic beads that the template DNA is immobilized on to the amount of template DNA (FIG. 14F).

[0045] FIGS. 15A-15B: Exemplary hairpin designed with nested restriction site facilitates synthesis of long ssDNA. Biotinylated hairpin loop is designed with nested Bbsl and Nt.Alwl sites (FIG. 15A). Short hairpin loop with a 4 bp overhang is ligated to long strands of dsDNA digested with Bbsl (FIG. 15B). Subsequently, the hairpin is pulled out with a magnet and introduced into an enzymatic synthesis reaction to produce multiple single stranded copies of the template strand.

[0046] FIGS. 16A-16B: Verification of ssDNA product by digestion with Exonuclease 1. Arrows point to the ssDNA product (FIG. 16A) Lane 1&5: 1 kb plus ladder; Lane 2: Enzymatic synthesis product treated with Exonuclease 1; Lane 3: Enzymatic synthesis product; Lane 4: dsDNA (of length -350 bp) digested with Bbsl that is introduced into the ligation reaction. (FIG. 16B) Lane 1&5: 1 kb plus ladder; Lane 2: Enzymatic synthesis product treated with Exonuclease 1; Lane 3: Enzymatic synthesis product; Lane 4: dsDNA (of length ~1100 bp) digested with Bbsl that is introduced into the ligation reaction.

[0047] FIG. 17: Exemplary HPLC traces of dNTP monomers and products of the DNA production reactions of the present disclosure. The first column (left) shows HPLC traces of the indicated unmodified or modified dNTPs. The second column (right) shows HPLC traces of the product resulting from the synthesis process, where the dNTP indicated was complemented with the other remaining unmodified versions of the dNTPs (e.g., dATP was added along with dGTP, dCTP, dTTP; Biotin- 14-dATP was added along with dGTP, dCTP, dTTP).

[0048] FIG. 18: Exemplary HPLC traces of the products from each of three DNA production reactions. From left to right, Bst, Klenow(exo-), or DNA polymerase I (Large Klenow) Fragment, were added to a bead-linked DNA hairpin encoding for a 24nt ssDNA product. Unmodified dNTPs and nickase were also added. The blue peaks indicate the expected product, with the indicated yields showing that Klenow (exo-) had the highest yield and DNA polymerase I (Large Klenow) Fragment having the lowest yield.

[0049] FIG. 19: Exemplary results from experiments indicating that Taq polymerase can incorporate unmodified dNTPs, 5-methyl-dCTP, and 5 -hydroxymethyl- dCTP, respectively, without a drop in efficiency, using the molecules and methods of the present disclosure (Lanes 2- 4).DETAILED DESCRIPTION

[0050] The proportion of the economy reliant on biological systems is rapidly growing and is driven by the ability to synthesize DNA and RNA. However, almost all naturally occurring nucleic acids, and a large proportion of nucleic acids used in commercial applications, are not just bare As, Cs, Ts, and Gs. Rather, nucleic acids exist with a variety of distinct types of modifications. Yet, current nucleic acid synthesis technologies exclude the central importance of nucleic acid modifications. For example, the harsh chemical conditions of current synthesis methods and the high cost and long turnaround times of synthesizing modified nucleic acid products restrict the creation of many forms of modified nucleic acids. The presently disclosed subject matter provides in some embodiments compositions and methods related to the generation of a plurality of modified nucleic acid oligos. This platform of technologies can support storage systems with extreme-scale synthesis requirements by enabling faster, cheaper, and longer synthesis and sequencing of arbitrary and customizable modified nucleic acid oligos.1. Definitions

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents andother references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0052] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0053] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, some embodiments includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms an embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed, then “less than or equal to 10” as well as “greater than or equal to 10” are also disclosed.

[0054] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0055] “Correlated to” as used herein refers to compared to.

[0056] The terms “oligomers”, “oligos,” or “oligonucleotides” include RNA or DNA sequences of more than one nucleotide in either single chain or duplex form and specifically includes short sequences such as dimers and trimers, in either single chain or duplex form, which can be intermediates in the production of the specifically binding oligonucleotides. “Modified” formsused in candidate pools contain at least one non-native residue. “Oligonucleotide,” “oligo,” or “oligomer” is generic to poly deoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), such as DNA, to polyribonucleotides (containing D-ribose or modified forms thereof), such as RNA, and to any other type of polynucleotide which is an N-glycoside or C- glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base or abasic nucleotides. “Oligonucleotide” or “oligomer” can also be used to describe artificially synthesized polymers that are similar to RNA and DNA, including, but not limited to, oligos of peptide nucleic acids (PNA).

[0057] The terms “binding activity” and “binding affinity” generally refer to the tendency of a ligand molecule to bind or not to bind to a target. The energetics of these interactions are significant in “binding activity” and “binding affinity” because they can include definitions of the concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free molecules in a solution.

[0058] “Complementary” refers to the characteristic of two or more structural elements (e.g., peptide, polypeptide, nucleic acid, small molecule, etc.) of being able to hybridize, dimerize, or otherwise form a complex with each other. For example, a “complementary peptide and polypeptide” are capable of coming together to form a complex. Complementary elements may require assistance to form a complex (e.g., from interaction elements), for example, to place the elements in the proper conformation for complementarity, to co-localize complementary elements, to lower interaction energy for complementation, etc.

[0059] As used herein, the terms “nucleotide sequence identity” or “nucleic acid sequence identity” refers to the presence of identical nucleotides at corresponding positions of two polynucleotides. Polynucleotides have “identical” sequences if the sequence of nucleotides in the two polynucleotides is the same when aligned for maximum correspondence (e.g., in a comparison window). Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. The “percentage of sequence identity” for polynucleotides, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or 100 percent sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparisonwindow can include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. In some embodiments, the percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100. Optimal alignment of sequences for comparison can also be conducted by computerized implementations of known algorithms, or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (e.g., the website of the EMBL-EBI). Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG Package available from Accelrys, Inc. of San Diego, Calif., United States of America. See also Smith & Waterman, 1981; Needleman & Wunsch, 1970; Pearson & Lipman, 1988; Ausubel et al., 1988; and Sambrook & Russell, 2001.

[0060] The term “and / or”, when used in the context of a list of entities, refers to the entities being present singly or in combination.

[0061] The term “ligase” or “ligase enzyme,” as used herein, generally refers to any enzyme capable of catalyzing a ligase reaction, i.e., enzymes which catalyze the formation of a bond. Various ligases may be used for ligation. The ligases can be naturally occurring or synthesized. Examples of ligases include T4 DNA Ligase, T7 DNA Ligase, 13 DNA Ligase, Taq DNA Ligase, 9oNTM DNA Ligase, E. coli DNA Ligase, and SplintR DNA Ligase. Different ligases may be stable and function optimally at different temperatures. For example, Taq DNA Ligase is thermostable and T4 DNA Ligase is not. Moreover, different ligases have different properties. For example, T4 DNA Ligase may ligate blunt ended dsDNA while T7 DNA Ligase may not.

[0062] The term “polymerase” or “polymerase enzyme,” as used herein, generally refers to any enzyme capable of catalyzing a polymerase reaction. Examples of polymerases include, without limitation, a nucleic acid polymerase. The polymerase can be naturally occurring or synthesized. An example polymerase is a Φ29 polymerase or derivative thereof. In some cases, a ligase is used (i.e., enzymes which catalyze the formation of a bond) in conjunction with polymerases or as an alternative to polymerases to construct new nucleic acid sequences. Examples of polymerases include a DNA polymerase, a RNA polymerase, a thermostable polymerase, a wild-type polymerase, a modified polymerase, E. coli DNA polymerase I, T7 DNA polymerase,bacteriophage T4 DNA polymerase, Φ29 DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase Pwo polymerase, VENT polymerase, DEEPVENT polymerase, Ex- Taq polymerase, LA-Taw polymerase, Sso polymerase Poe polymerase, Pab polymerase, Mth polymerase ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tea polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Tbr polymerase, polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Tbr polymerase thereof. Different DNA polymerases have different capacity to displace an existing strand that is “in the way” of the polymerase. For example, after the hairpin undergoes one polymerization, the overhang of the hairpin is then filled in. The nickase then makes a nick, but the single stranded oligomer (codeword) that results is still bound to the hairpin. To make another codeword, a polymerase then has to polymerase through and displace that codeword (knock it off the hairpin). Thus, in some embodiments, the presently disclosed subject matter employs a DNA polymerase having this activity. By way of elaboration and not limitation, enzymes with different abilities to displace ssDNA strands during polymerization have greater activity / ability to make codewords. By way of particular example, Large Klenow fragment DNA polymerase has that activity, as does Bst DNA Polymerase, Large Fragment. Examples of RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.2. Compositions

[0063] The provides compositions and methods related to enzymatic nucleic acid synthesis. In particular, the present disclosure provides novel compositions and methods for generating a plurality of modified nucleic acid oligos using a nucleic acid template molecule. In accordance with these embodiments, the present disclosure includes a nucleic acid template molecule for generating a plurality of modified nucleic acid oligos comprising a hairpin region chemically configured for attachment to a substrate, a double-stranded region comprising at least one nucleic acid processing site, and a single- or double-stranded region comprising a nucleic acid template sequence.

[0064] In some embodiments, the chemically configured region of the hairpin comprises a Biotin modification (3’, 5’ or internal), Amine modification (3’, 5’ or internal), Carboxylic acid modification (3’, 5’ or internal), Alkyne modification (3’, 5’ or internal), or Azide modification (3’, 5’ or internal). In some embodiments, the chemically configured region of the hairpin comprises a covalent linkage between the hairpin and the substrate or to a solid support / columnthrough an NHS-ester linkage. In some embodiments, the linkage can also be a non-covalent stable binding interaction, e.g., through streptavidin-biotin. In some embodiments, the linkage can also be a covalent primary amine-carboxylic acid reaction. In some embodiments, the hairpin nucleic acid is linked to the substrate.

[0065] In some embodiments, the substrate comprises a Streptavidin substrate, Carboxylic substrate, Amine substrate, Azide substrate, or Alkyne substrate. In some embodiments, the substrate comprises at least one of the substrates selected from Table 3.

[0066] In some embodiments, the hairpin region of the nucleic acid template molecule comprises a loop region comprising about 2 nucleotides to about 100 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 90 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 70 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 60 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 50 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 40 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 30 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 20 nucleotides. In some embodiments, the loop region comprises about 2 nucleotides to about 10 nucleotides. In some embodiments, the loop region comprises about 10 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 20 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 30 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 40 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 50 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 60 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 70 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 25 nucleotides to about 50 nucleotides. In some embodiments, the loop region comprises about 10 nucleotides to about 80 nucleotides. In some embodiments, the loop region comprises about 20 nucleotides to about 60 nucleotides. In some embodiments, the loop region comprises about 40 nucleotides to about 80 nucleotides.

[0067] In some embodiments, the concentration of the hairpin in a composition ranges from about 5 uM to about 100 uM. In some embodiments, the concentration of the hairpin in acomposition ranges from about 5 uM to about 75 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 5 uM to about 50 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 5 uM to about 25 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 10 uM to about 100 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 25 uM to about 100 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 50 uM to about 100 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 75 uM to about 100 uM. In some embodiments, the concentration of the hairpin in a composition ranges from about 25 uM to about 75 uM.

[0068] In some embodiments, the hairpin region is chemically configured for attachment to a substrate. In some embodiments, the ratio of substrate binding sites to hairpins is about 1 :1, about 2:1, about 3: 1, about 4: 1, about 5:1, about 6: 1, about 7:1, about 8: 1, about 9: 1, about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14:1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, or about 20:1.

[0069] In some embodiments, the at least one nucleic acid processing site of the nucleic acid template molecule comprises a nickase site and / or a restriction site. In some embodiments, the at least one nucleic acid processing site of the nucleic acid template molecule comprises a nickase site adjacent to or overlapping with a restriction site. In some embodiments, the nickase site comprises any nucleic acid sequence selected from Table 4. In some embodiments, the restriction site comprises any nucleic acid sequence selected from Table 5. In some embodiments, the at least one nucleic acid processing site of the nucleic acid template molecule comprises a second restriction site. In some embodiments, this site can be a redundant site that facilitates removal of the template sequence.

[0070] In some embodiments, the single- or double-stranded region of the nucleic acid template molecule comprises a 3’ overhang. In some embodiments, the single- or double-stranded region of the nucleic acid template molecule comprises a 5’ overhang. In some embodiments, the single- or double-stranded region of the nucleic acid template molecule does not comprise an overhang.

[0071] In some embodiments, the single-or double-stranded region of the nucleic acid template molecule comprises from about 2 nucleotides (or basepairs / bp) to about 100 kb. In some embodiments, the single-or double-stranded region comprising the nucleic acid template sequence is about 2 nucleotides (or basepairs / bp), about 4 nucleotides (or basepairs / bp), about 6 nucleotides(or basepairs / bp), about 8 nucleotides (or basepairs / bp), about 10 nucleotides (or basepairs / bp), about 15 nucleotides (or basepairs / bp), about 20 nucleotides (or basepairs / bp), about 25 nucleotides (or basepairs / bp), about 30 nucleotides, (or basepairs / bp) about 35 nucleotides (or basepairs / bp), about 40 nucleotides(or basepairs / bp), about 45 nucleotides (or basepairs / bp), about 50 nucleotides (or basepairs / bp), about 55 nucleotides (or basepairs / bp), about 60 nucleotides (or basepairs / bp), about 65 nucleotides (or basepairs / bp), about 70 nucleotides (or basepairs / bp), about 75 nucleotides (or basepairs / bp), about 80 nucleotides (or basepairs / bp), about 85 nucleotides (or basepairs / bp), about 90 nucleotides (or basepairs / bp), about 95 nucleotides (or basepairs / bp), about 100 nucleotides (or basepairs / bp), about 110 nucleotides (or basepairs / bp), about 120 nucleotides (or basepairs / bp), about 130 nucleotides (or basepairs / bp), about 140 nucleotides (or basepairs / bp), about 150 nucleotides (or basepairs / bp), about 160 nucleotides (or basepairs / bp), about 170 nucleotides (or basepairs / bp), about 180 nucleotides (or basepairs / bp), about 190 nucleotides (or basepairs / bp), about 200 nucleotides (or basepairs / bp), about 220 nucleotides (or basepairs / bp), about 240 nucleotides (or basepairs / bp), about 260 nucleotides (or basepairs / bp), about 280 nucleotides (or basepairs / bp), about 300 nucleotides(or basepairs / bp), about 320 nucleotides(or basepairs / bp), about 340 nucleotides (or basepairs / bp), about 360 nucleotides (or basepairs / bp), about 380 nucleotides (or basepairs / bp), about 400 nucleotides (or basepairs / bp), about 420 nucleotides (or basepairs / bp), about 440 nucleotides (or basepairs / bp), about 460 nucleotides (or basepairs / bp), about nucleotides (or basepairs / bp), about 480 nucleotides (or basepairs / bp), about 500 nucleotides (or basepairs / bp), about 520 nucleotides (or basepairs / bp), about 540 nucleotides (or basepairs / bp), about 560 nucleotides (or basepairs / bp), about 580 nucleotides (or basepairs / bp), about 600 nucleotides (or basepairs / bp), about 620 nucleotides (or basepairs / bp), about 640 nucleotides (or basepairs / bp), about 660 nucleotides (or basepairs / bp), about 680 nucleotides (or basepairs / bp), about 700 nucleotides (or basepairs / bp), about 720 nucleotides (or basepairs / bp), about 740 nucleotides (or basepairs / bp), about 760 nucleotides (or basepairs / bp), about 780 nucleotides (or basepairs / bp), about 800 nucleotides (or basepairs / bp), about 820 nucleotides (or basepairs / bp), about 840 nucleotides (or basepairs / bp), about 860 nucleotides (or basepairs / bp), about 880 nucleotides (or basepairs / bp), about 900 nucleotides (or basepairs / bp), about 920 nucleotides (or basepairs / bp), about 940 nucleotides (or basepairs / bp), about 960 nucleotides (or basepairs / bp), about 980 nucleotides (or basepairs / bp), about 1000 nucleotides (or basepairs / bp).

[0072] As described further herein, the nucleic acid template molecules of the present disclosure comprise a single- or double-stranded region that comprises a nucleic acid template sequence that corresponds to a plurality of modified nucleic acid oligos that can be generated using the methods and compositions of the present disclosure. In some embodiments, the nucleic acid template sequence is the reverse complement of the sequence of the plurality of modified nucleic acid oligos to be generated. In some embodiments, the at least one nucleic acid processing site is directly adjacent to the single- or double-stranded region comprising the nucleic acid template sequence. In some embodiments, an RNA polymerase promoter is positioned before or after the at least one nucleic acid processing site.

[0073] In some embodiments, the identity and / or purity of the modified nucleic acid oligos is determined by MALDI-TOF, electrospray ionization mass spectrometry, reverse-phase HPLC, ultra-performance liquid chromatography, and any other means known in the art. In some embodiments, the modified nucleic acid oligos generated using the compositions and methods of the present disclosure are substantially stable (e.g., without undesired truncations, deletions, chemical degradation, and / or chemical conversions) for hours and at temperatures ranging from about -5 C to about 40 C.

[0074] In some embodiments, the compositions of the present disclosure can further include at least one nickase enzyme, and / or at least one restriction enzyme. In some embodiments, suitable nickase enzymes are selected from Table 4. In some embodiments, suitable restriction enzymes are selected from Table 5. In some embodiments, the at least one nickase enzyme and / or the at least one restriction enzyme is chemically coupled to the substrate. In some embodiments, the nickase enzyme and / or restriction enzyme is covalently linked to the substrate or is linked to the substrate via a non-covalent stable binding interaction.

[0075] In some embodiments, the composition further comprises dNTPs or NTPs. In some embodiments, the dNTP or NTP concentration is from about 50 uM to about 1000 uM. In some embodiments, the dNTP or NTP concentration is from about 50 uM to about 750 uM. In some embodiments, the dNTP or NTP concentration is from about 50 uM to about 500 uM. In some embodiments, the dNTP or NTP concentration is from about 50 uM to about 250 uM. In some embodiments, the dNTP or NTP concentration is from about 50 uM to about 100 uM. In some embodiments, the dNTP or NTP concentration is from about 100 uM to about 1000 uM. In some embodiments, the dNTP or NTP concentration is from about 250 uM to about 1000 uM. In someembodiments, the dNTP or NTP concentration is from about 500 uM to about 1000 uM. In some embodiments, the dNTP or NTP concentration is from about 750 uM to about 1000 uM. In some embodiments, the dNTP or NTP concentration is from about 250 uM to about 750 uM. In some embodiments, the dNTP or NTP concentration is from about 200 uM to about 500 uM. In some embodiments, the dNTP or NTP concentration is from about 600 uM to about 800 uM.

[0076] In some embodiments, one or more dNTPs or NTPs used in the compositions and methods of the present disclosure include at least one modified dNTP or NTP. For example, a modified dNTP or NTP that can be incorporated into a modified nucleic acid oligo that can be generated using the methods and compositions of the present disclosure can include any dNTP or NTP modification known in the art, including but not limited to, the modifications listed in Table 6.3. Methods

[0077] Embodiments of the present disclosure also include a method for enzymatically generating a plurality of modified nucleic acid oligos. In accordance with these embodiments, the method includes (a) providing the composition, (b) adding a first solution comprising at least one unmodified nucleotide base and a DNA or RNA polymerase to generate at least a portion of the modified nucleic acid oligo from the nucleic acid template sequence, (c) adding at least a second solution comprising at least one modified dNTP or NTP, (d) repeating steps (b) and (c) until the modified nucleic acid oligo is generated, and (e) in the case of DNA, adding a nickase enzyme to release the modified nucleic acid oligo from the nucleic acid template molecule.

[0078] In some embodiments, suitable buffer conditions are employed, including acid or base aqueous solutions. In some embodiments, the buffering agent is selected from polyprotic acids and bases, monoprotic acids and bases, KH2PO4, CHES, Borate, TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, MES. In some embodiments, the method further comprises a wash step that includes application of a wash solution or wash buffer to remove, for example, unincorporated dNTPs.

[0079] In some embodiments, the DNA or RNA polymerase is provided at an enzyme concentration of about 2 U / 25 pL reaction. In some embodiments, the polymerase is a Deep Vent DNA polymerase, a phi29 DNA polymerase, a Large Klenow fragment DNA polymerase, a T7 RNA polymerase, aT3 RNA polymerase, or a SP6 RNA polymerase. In some embodiments, LargeKlenow fragment DNA polymerase produces the most oligo compared to Deep Vent and phi29 DNA polymerases.

[0080] In some embodiments, step (d) of the method is performed with at least a second modified dNTP or NTP that is the same as or is different from the first modified dNTP or NTP in step (c). In some embodiments, the first modified dNTP or NTP and the at least second modified dNTP or NTP are from about 1 nucleotide to about 100 nucleotides apart. In some embodiments, the first modified dNTP or NTP and the at least second modified dNTP or NTP are about 1 nucleotide to about 10 nucleotides apart, about 1 nucleotide to about 20 nucleotides apart, about 1 nucleotide to about 30 nucleotides apart, about 1 nucleotide to about 40 nucleotides apart, about 1 nucleotide to about 50 nucleotides apart, about 1 nucleotide to about 60 nucleotides apart, about 1 nucleotide to about 70 nucleotides apart, about 1 nucleotide to about 80 nucleotides apart, about 1 nucleotide to about 90 nucleotides apart.

[0081] In some embodiments, the method further comprises adding a restriction enzyme after step (d) to generate a blunt end, a 3 ’ overhang, or a 5 ’ overhang at the end of the nucleic acid template molecule.

[0082] In some embodiments, the method further comprises ligating a different single-stranded or double-stranded nucleic acid template sequence to the nucleic acid template molecule via the blunt end, the 3 ’ overhang, or the 5’ overhang. In some embodiments, the ligase is T7 DNA ligase, Taq DNA ligase, or T4 DNA ligase. In some embodiments, the overhang length is about 2bp, 4bp, 8bp, 10bp, 20bp, 40bp, 60bp, 80bp, or 100bp.

[0083] In some embodiments, the oligos produced have phosphorylated 5’ ends compatible with ligase and / or polymerase enzymes for downstream DNA assembly reactions. In some embodiments, the process is repeated multiple times by incubating isothermally. In some embodiments, the method further comprises repeating steps (b) through (e) to generate a second modified nucleic acid oligo.4. Sequences

[0084] The various embodiments of the present disclosure include polynucleotides and polypeptides having the following sequences.

[0085] Table 1: List of polynucleotides and polypeptides sequences.

[0086] Table 2: List of recombinantly synthesized enzymes with fusion tags and their amino acid sequences.5. Materials and Methods

[0087] Oligonucleotides, enzymes and other reagents: All oligonucleotides were purchased from Genewiz Inc. Nicking enzymes, polymerases and ligases including Nt.AlwI (NEB, R0627S), Klenow fragment (3’ -> 5’ exo) (NEB, #M0212S), Large (Klenow) Fragment (NEB, #M0210S), phi29 DNA polymerase (NEB, #M0269S), Deep Vent DNA polymerase (NEB, #M0258S), Q5 High-fidelity DNA Polymerase (NEB, #M0515),t PNK (NEB, #M0201L), T7 Ligase (M0318S), T4 Ligase (NEB, #M0202S) and Taq Ligase (NEB, #M0208S) were acquired from New England Biolabs (NEB).

[0088] Enzymatic synthesis hairpin design: The Hairpin was designed in such a way that it could self-prime with a binding interaction of 15nt. The double-stranded region contains a recognition site for the Nickase (NEB, #R0627S) (GGATCNNNN) and is Biotinylated at the loop. Details of the sequence can be found in Table 1. To create the hairpin from a single strand, 2ug of the oligo was added to a PCR tube and reconstituted with 50ml of IX TE buffer. A slow annealing protocol was run on a thermocycler. First temperature at 95C for 3 minutes followed by a slow ramp down of 2C per minute until temperature reached 25C.

[0089] Template: Hairpin structure specific for synthesis of a particular sequence (‘codeword’) was ordered with an internal Biotin modification. A 22ml solution of streptavidin magnetic beads (NEB #S1420S) was pipetted into a low retention tube and washed twice with 100ml Bind-wash buffer (containing 20nM Tris-HCl pH7.4, 2M NaCl, 2mM EDTA pH8), each time placing the tube on a magnetic stand to separate out the beads. The beads were then added to 2.67ml (Concentration: 1E12 strands / ml) of Hairpin and 50ml bind-wash buffer before being incubated at room temperature on a rotisserie for 30 minutes. After further washing twice with bind-wash buffer, the bead-hairpin (‘template’) was eluted in 25.5ml of water.

[0090] Codeword synthesis: 0.5ml DNA polymerase, 0.5ml Nickase (NEB, #R0627S), 2.5ml polymerase buffer and dATP (NEB, #N0440S), dCTP (NEB, #N0441S), dGTP (NEB, #N0442S), dTTP (NEB, #N0443S) each at 10mM were added to 25.5ml of template, bringing the total reaction volume up to 30ml, and incubated at 25C for 1 hour. Following this, the template was magnetically separated using a magnetic stand, and the supernatant was collected for analysis.

[0091] Ligation assembly: Assembly strands 1 and 6 were 60bp in length, strands 2,3,4 and 5 were single stranded and 64 bp in length, strands 7,8,9 and 10 were 68bp in length, strands 11,12,13 and 14 were 70bp in length and strands 15, 16,17,18 were 80bp in length. 12ul of strandconcentration IE 13 strands / ul of each strand 2-5,7-18 and 48ul of strand concentration lE13strands / ul of strand 1 and 6 (since strands 1 and 6 are common to all 4 sets) were phosphorylated using PNK enzyme and incubated at 37C for 30 minutes, 65C for 20 minutes and cooled to 4C . Strands were then annealed by addition of annealing buffer and incubation at 95 C for 5 minutes and gradual temperature drop to 25C to form double stranded DNA molecules with different length of overhang. 3ul of each dsDNA section with strand concentration 3E12 strands / ul were mixed together and run on a ligation reactions with 3 different ligases T7, T4 and Taq ligase and incubated at 25C for 30 minutes, 37C for 1 hour and 45C for 15 minutes respectively. The ligation product was then mixed with 5ul 5X Q5 reaction buffer, 0.6ul 10mM dNTPs and 0.3ml Q5 enzyme (NEB #M0515) and 1.5ul of lE13strands / up of strands 1 and 6 each (as forward and reverse primers). The reaction mixture was eluted to 30ul with water and cycled through temperatures for a standard PCR. After amplification the strands were sent for analysis.

[0092] Analysis: Samples were diluted to fit the recommended concentration range of the fragment analyzer of 5pg / ml - 500 pg / ml. 2ml of each diluted sample was pipetted into an unused well along with 22ml of the High Sensitivity NGS Diluent Marker (lbp-6000bp, part #DNF-373) and run on the 12 capillary Fragment Analyzer (Advanced Analytical) using the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, DNF-474).

[0093] Table 3: DNA Hairpin Modifications and Substrates.

[0094] Table 4: Nickase enzymes and corresponding recognition sites.

[0095] Table 5: Restriction enzymes and corresponding recognition sites.

[0096] Table 6: Representative dNTP and NTP Modifications.6. ExamplesExample 1

[0097] Generate site-specific and combinatorial modifications through automated enzymatic synthesis with cycles of selectively limiting reagents. Site-specific and combinatorial control of DNA modification, presented herein, is achieved by leveraging the templated synthesis technology and by halting the polymerization with removal of one of four of dNTPs (FIG. 1). Thefirst step is linking the unmodified DNA hairpin template to magnetic beads. Separate solutions each comprising only 3 of 4 of the dNTPs plus DNA polymerase I, large (Klenow) fragment, and a solution containing nickase only, are generated. These solutions are added and washed out in cycles, thereby adding nucleotides to the growing oligo strand until a specific position is reached where it is desired to incorporate a modified nucleotide. Then, a solution containing the modified dNTP, and any other dNTPs that can be added without disrupting the proper modification pattern, are added. If no other modifications are desired to be added, then all four dNTPs are added simultaneously to complete polymerization of the oligo. The nickase is then added to release the oligo. Subsequent rounds are repeated until a desired yield is reached. The speed of this overall process depends on where modifications are desired, with a maximum cycle number equal to the length of the oligo and a minimum cycle number equal to two (a single addition of a modified nucleotide at the 5 ’ end followed by addition of all four dNTPs simultaneously to fully polymerize the rest of the oligo.

[0098] Three parameters provide evidence that the technology is generalizable, flexible, and operationally practical for a broad range of oligo modification patterns. The first parameter is turnaround time or speed. Typical DNA polymerization rates measured using single molecule methods range from roughly 10 to 20 nt / s, with synthesis involving simultaneous strand displacement (which is the case for the second round of synthesis and on) closer to 1.2 nt / s. Synthesis of 28 nucleotide oligomers with the repeating motif (A-5hmC-T-5mC)? is, therefore, possible. This complex pattern provides evidence that the second parameter, complexity of combinatorial modifications, is achievable. The oligomer is synthesized where each cycle incorporates three nucleotides (i.e., A-5hmC-T without 5mC, then 5mC-A-5hmC without T, then T-5mC-A without 5hmC, etc.). Overall, 10 cycles generate one complete oligo, scaled by the number of template molecules present. The process is automated using an OpenTrons OT-2 system. Incubation and wash times vary from 0.1s (the limit of the pipetting action) to 40s, and subsequent yields are quantified using a fragment analyzer. The milestone target is 20 seconds for ten cycles, subtracting out the time it takes the OT-2 liquid handler to change pipets and translate its stage. The actual molecular synthesis time may be negligible relative to the OT-2 mechanical actuation time which could be replaced by much faster microfluidic actuation in the future. The third parameter is synthesis scale. Two experimental knobs may be tuned: the amount of template and the number of synthesis rounds. The greater the amount of template, the greater the potentialreaction and reagent volumes but the fewer rounds of synthesis cycles needed. A matrix of conditions may be explored ranging from 10 ng template and 100 rounds of synthesis cycles (-200 minutes maximum) to 1 pg of template and just 1 or 2 rounds of synthesis cycles. The milestone target is I pg synthesis scale typical for commercially available modified DNA oligos, quantified via fragment analyzer.

[0099] Incorporation of 5hmC and 5mC has been demonstrated. However, multiple synthesis cycles may introduce error. To confirm the accuracy of the synthesis, capillary electrophoresis via the fragment analyzer reveals if multiple different sized products are being generated. Furthermore, the oligo products are sent to the UNC-Chapel Hill mass spectrometry core facility (or commercial service vendors like Bioinfor, BGI Americas, and Emery Pharma) for analysis via MALDI-TOF and electrospray ionization mass spectrometry. HPLC is also performed. These methods were chosen to match the quality control methods used by synthetic DNA vendors. Finally, as this process involves multiple cycles including wash steps, the stability of the DNA template linkage to the magnetic beads is important. The stability of this linkage over each cycle is assessed and, if it drops, the linking of amine-functionalized DNA templates to carboxylated magnetic beads may be explored instead.Example 2

[0100] Development of an enzymatic method of synthesis of short DNA oligos. Presented herein is a template -based enzymatic method that allows continuous synthesis of short DNA strands and a system that employs a magnetically retrievable DNA hairpin, a polymerase, a nicking endonuclease, and dNTPs to produce 60 nucleotide long strands, termed “codewords”. First, the polymerase binds to the hairpin sliding across the single stranded portion in the 5 ’ to 3 ’ direction, adding bases along the way. The nicking endonuclease then nicks the strand after the recognition site and, in the next cycle of polymerization, the polymerase pushes the 60 nucleotide long strand out, producing a single-stranded codeword. This cycle repeats to continuously generate codewords, allows low energy (isothermal) synthesis, and produces oligos that have lower error probabilities (FIG. 2).

[0101] Hairpins attached to magnetic beads provide specific interactions. To demonstrate proof of concept, hairpin molecules were added to a reaction mixture containing polymerase, nickase, and dNTPs and then incubated for varied times (5, 15, 30, and 45 minutes) to ascertainI l lthe timeframe for codeword production. The results showed strong bands at 60bp length confirming the production of codewords (FIG. 3). However, a striated banding pattern suggested some non-specific interaction between DNA molecules, ft was hypothesized that this might be due to the hairpins unfolding and hybridizing during the reaction. To avoid this issue, the hairpins were bound to magnetic beads, through a standard biotin-streptavidin interaction, essentially tethering the hairpin molecules to avoid hairpin hybridization and allowing pullout and reusability. On repeating the experiment with tethered molecules, it was noticed that a significant reduction in non-specific interaction had occurred and the samples exhibited a single band at codeword length.

[0102] Optimization. To achieve the best design and maximize efficiency and utilization of the system, each parameter was optimized and the optimal composition was used in the next experiment. To optimize codeword production experiments were conducted with varying concentrations of polymerase, beads, hairpin and dNTPs. For the polymerase assay, samples with 3 blunt end producing polymerases, Deep Vent DNA polymerase, Large Klenow fragment, and the phi29 polymerase cycling samples were run between the incubation temperatures of the polymerase and that of nicking endonuclease. Results showed clear superiority of Large Klenow fragment polymerase in codeword production in the system over the other polymerases tested. Naturally, the optimum temperature and incubation time was then tested for the Large (Klenow) Fragment for this system as well as the codeword production at varied temperatures and times. Samples incubated at 25 C for 60 minutes produced maximum codeword yield, while samples kept at 30 C and 37 C seemed to plateau after 45 minutes (FIG. 4).

[0103] Magnetic beads and hairpin. Since magnetic beads were the biggest contributors to cost in the systems, it was essential to optimize binding interaction between hairpin and the beads. Varying ratios of bead binding sites to hairpins (1: 1,2:1,5:1,10:1) were tested. Higher ratios appeared to generate higher codeword concentration, ft was hypothesized that low concentration of codewords in lower ratio samples could be due to crowding of hairpin at bead binding sites, which would result in ineffective Biotin-streptavidin interactions and lead to more unbound template. A significant jump in codeword production is observed between ratios 2: 1 and 1:1, with the latter producing 5 times as much codeword as the former. Maintaining the bead binding site to hairpin ratio at 2: 1, varied hairpin concentration varied from 14.8 uM to 55.3 uM. Codeword production peaks at 41.5uM hairpin concentration and is observed to dip at concentrations higher,possibly indicating that higher hairpin concentrations result in more non-specific interactions amongst the hairpin molecules (FIG. 4).Bead binding site calculations;1 mg of bead = 500 pmol binding sitesDensity of bead solution = 4mg / μlHairpin per run = 2.67E12 strandsBeads per reaction = 2.67E12 binding sites

[0104] Enzymes and dNTPs. Higher dNTP concentration was shown to inhibit the reaction. Four dNTP concentrations were tested ranging from 83.3nM to 666.7nM with 333.4 nM dNTPs producing maximum codewords. Interestingly, the system observed a linear trend between the concentration of enzymes (polymerase and nickase) added to the system and codewords produced. However, since enzymes are the second largest cost contributors in the system, it is important to be able to reuse them to maintain or reduce cost (FIG. 4).

[0105] Cost analysis and reusability. To further drive down cost, the reusability of the template was tested. Codewords for 6 reaction runs were consistently obtained with reused bead- hairpin constructs. A cost analysis prior to and following reaction optimization indicated a reduction in cost by 20%. Since magnetic beads are the major cost contributors, reusing the template six times further reduced cost by another 20% indicating that further optimization can be centered around reusability of the components of the system (FIG. 5).Example 3

[0106] Optimize ligation reactions to be able to generate long DNA strands from short enzymatically synthesized DNA oligos. Block-wise synthesis methods like Ligase cycling, Gibson assembly, and CPEC have an obvious advantage over base-by-base synthesis methods due to their speed and accuracy. Typically used in molecular biology , assembly methods join sticky- end (2-4bp long) overhangs generated from endonuclease digested DNA fragments. Since ligation efficiencies have only been studied through plasmid transformations and colony growth, ligations were optimized for in-vitro assembly requirements. Additionally, a combined scheme of enzymaticoligo synthesis and optimized assembly was developed to provide a high-throughput synthesis platform that reduces cost and synthesis latency.

[0107] Ligation efficiency was tested for 3 different ligases (T7 DNA ligase, Taq DNA ligase, and T4 DNA ligase) for varied overhang lengths (4bp, 8bp, 10bp and 20bp) for a system of 6 single stranded codewords. The codewords were phosphorylated, annealed, ligated, and amplified. Results indicated that across ligases the 4bp overhang strands provided the most consistent results while the T7 ligase produced higher yields of full length strands across all tested overhang lengths (FIG. 6).

[0108] The experimental scheme is to continuously produce codewords using the enzymatic synthesis method provided herein and selectively assemble them (with the optimized assembly protocol) into full length data encoding strands. To demonstrate proof of concept, short oligos that represent 2 different codewords are synthesized and annealed and then assemble in two different ways. Specifically, 6 codewords are synthesized, 64bp in length with varying sequences of overhang region using the enzymatic synthesis method described herein. The strands contain a 60bp payload region and 4bp overhangs as optimized from the experiment described herein. The strands are designed to contain 2 different codewords in their payload regions and are annealed and ligated to form two different full length strands with a different order of codewords. This setup operates as a proof of concept and can be scaled to continuously and simultaneously produce multiple data encoding strands (FIG. 7).Example 4

[0109] Develop a molecular strategy for recycling the biotinylated DNA template substrate. It has been shown that a biotinylated hairpin, when linked to magnetic beads, can be used multiple times to generate an oligo. However, the entire hairpin, including the biotin modification, is ordered / synthesized when a new oligo sequence is desired. This synthesis is a significant cost because the nucleotides comprising the base hairpin as well as the higher cost of incorporating a biotin modification via phosphoramidite synthesis are needed.

[0110] Presented herein is the design of a new hairpin that creatively harnesses restriction enzymes and ligases to ‘recycle’ the base hairpin. This base hairpin comprises nickase (Nt.AlwI) and restriction endonuclease (BstXI) recognition sequences nested within each other. This hairpin can first be used to synthesize modified oligos according to the initial template sequence and can then be recycled for new sequences through digestion by BstXI, which will leave a 4 nt long 3 ’overhang with, importantly, a 5 ’ phosphate group intact to support a subsequent ligation reaction. New unmodified DNA templates may be synthesized with a 4 nt sequence complementarity to that overhang as well as an additional 4 nt to reconstitute the BstXl recognition sequence, ft is directly ligated to the digested hairpin with T4 DNA ligase. This allows incorporation of any new sequence simply with cheap unmodified DNA. Long double stranded DNA templates created through DNA assembly are also compatible simply by incorporating the BstXl recognition sequence and the 4 nt overhang sequence on the end of the dsDNA template. This template may then be first digested by BstXl to create a compatible end for ligation to the base hairpin (FIG. 8).

[0111] The key metrics for a recyclable DNA template are the efficiencies with which it can be regenerated by the restriction endonuclease and ligated to a new sequence by T4 DNA ligase. A target of 90% recovery and recycling of biotinylated DNA template substrates is commercially viable and achievable given the efficiencies of modem enzymes.

[0112] Loss of the hairpin from the bead impacts the efficiency of the system and may be addressed by exploring other linkages, including covalent primary amine-carboxylic acid reactions. If undigested hairpins remain after incubation with the endonuclease, creation of incorrect contaminating oligos in subsequent synthesis rounds may occur. Incorporation of a redundant second restriction enzyme site would help ensure removal of the prior template sequence. The use of alternative nickases, restriction endonucleases, and ligases to achieve maximum efficiencies may also be explored.Example 5

[0113] Map the incorporation efficiency of diverse chemically modified dNTPs with multiple polymerases. An important advantage of technology platform provided herein is that it has the potential to create diverse products using the same system without substantial re- engineering for each different type of modification. However, it is likely that the efficiencies with which different types of modifications are incorporated by DNA polymerases into oligos will differ depending on different polarities, sizes, and geometries. Therefore, 17 distinct modified dNTPs (Jena Biosciences, Trilink Biosciences, etc.) sampling across these parameter spaces are purchased, including dNTPs linked to fluorescent dyes, biotin, azide, primary amine, halogen atoms, heavy metals, alkyne, and photo-caged groups.

[0114] Measurements of oligo yield in triplicate are generated for 17 distinct dNTPs and 2 DNA polymerases (FIG. 10). This database provides available options and their efficiencies. Theefficiencies of incorporation into oligos are assessed by capillary electrophoresis (to detect quantity and distribution of any premature truncations) and by MADLI-TOF and electrospray ionization mass spectrometry.

[0115] The breadth of modifications that can be readily synthesized is quantitatively established. The primary risk is that bulky modifications such as fluorescent dyes would exhibit low efficiency of incorporation. This risk is mitigated by initially sampling a set of 10 different DNA polymerases, including several that are either naturally accommodating of bulky monomers like archaeal B-family polymerases (i.e., KOD polymerase) or have been used in applications like sequencing by synthesis that requires incorporation of dye-linked nucleotides.Example 6

[0116] Experiments were conducted to test two additional capabilities of the molecules and methods of the present disclosure. FIG. 15A provides a representative schematic of a magnetic bead linked to a DNA hairpin through a streptavidin molecule on the bead and a biotin group on the DNA hairpin. The hairpin contains encoded in it a type IIS restriction enzyme site (e.g., BbsI; however, any type IIS can be used) and a nickase site (e.g., Nt.Alwl; however, any nickase can be used). FIG. 15B then shows the experimental protocol, where double stranded DNA (e.g., generated through PCR or from a DNA plasmid) is digested to have a ssDNA overhang complementary to the ssDNA portion of the DNA hairpin. This digested dsDNA is then ligated to the DNA hairpin. This provides evidence of a first capability, where the ligation of new dsDNA to the DNA hairpin enables much cheaper routes to obtain diverse sequences of DNAs that can be produced using the method. This is because a single DNA hairpin sequence can be generated in large amounts in bulk and used in many different ligation reactions. The products of the ligation can also be digested again to regenerate the original DNA hairpin template.

[0117] The next step is to add a polymerase, a nickase, and dNTPs to produce ssDNA by the method described herein. Briefly, the nickase nicks based upon its location indicated by the green arrowhead in FIG. 15A, and the polymerase polymerizes from the nick, displacing a ssDNA off of the hairpin. This process is repeated multiple cycles until the reaction is stopped. FIG. 15B indicates two different DNAs ligated to the hairpin, one that produces 1100 nucleotide long ssDNA, and one that produces a 350nt ssDNA. This illustrates the second capability demonstrated by this experiment, namely, the ability to produce long ssDNAs.

[0118] In accordance with the above experiments, FIG. 16 includes representative results. The hairpin ligated to the 350nt ssDNA (FIG. 16A) and the 1100nt ssDNA (FIG. 16B) are shown in the expected locations on an agarose gel (fourth lane from left). The products of the polymerization reaction using each of the ligated hairpin DNAs are shown in the second and third lanes from the left for both agarose gels. The third lane from the left shows the ssDNA products while the second lane from the left shows that upon treatment with Exonuclease 1 , the ssDNA products no longer appears. Exonuclease 1 selectively digests / destroys ssDNA, providing evidence that these bands represent the ssDNA products and not non-specific dsDNAs.Example 7

[0119] In this example, HPLC traces were used to verify dNTP monomers and products of the DNA production reactions of the present disclosure. Briefly, a hairpin encoding for the production of a 24nt long ssDNA was incubated with the DNA Polymerase I, Large (Klenow) Fragment, along with dNTPs and nickase. Different types of dNTPs were used to test their ability to be incorporated into synthesized ssDNAs. Referring to FIG. 17, the first column (left) shows HPLC traces of the indicated unmodified or modified dNTPs; and the second column (right) shows HPLC traces of the product resulting from the synthesis process, where the dNTP indicated was complemented with the other remaining unmodified versions of the dNTPs (e.g., dATP was added along with dGTP, dCTP, dTTP; Biotin- 14-dATP was added along with dGTP, dCTP, dTTP). All reactions produced 24 nt long ssDNA products, demonstrating that this polymerase (the DNA Polymerase I, Large (Klenow) Fragment) is able to incorporate Biotin- 14-dATP, Fluorescein- 12-dATP, 5- Propargylamino-dCTP, and AminoAllyl-dUTP. 5 -Propargylamino and AminoAllyl are both common linkers for more complex dNTP modifications like ATTO### and Cy3 / 5 dyes, where ### indicates a fluorescent dye wavelength such as 488nm or 546nm.Example 8

[0120] Experiments were also conducted to evaluate the ability of different polymerases to produce ssDNA using the molecules and methods of the present disclosure. Referring to FIG. 18, each plot is an exemplary HPLC traces of the product from each of three DNA production reactions. From left to right, Bst, Klenow(exo-), or DNA polymerase I (Large Klenow) Fragment, were added to a bead-linked DNA hairpin encoding for a 24nt ssDNA product. Unmodified dNTPs and nickase were also added. The blue peaks indicate the expected product, with the indicatedyields showing that Klenow (exo-) had the highest yield and DNA polymerase I (Large Klenow) Fragment having the lowest yield. These polymerases can be used for producing unmodified ssDNAs. Results not shown indicate that Bst and Klenow (exo-) do not incorporate the modified dNTPs: FL-dCTP, FL-dGTP, FL-dUTP, TxRed-dCTP, or TxRed-dGTP; so, DNA polymerase I (Large Klenow) Fragment can be used in those applications.Example 9

[0121] Experiments were also conducted to determine whether Taq DNA polymerase could incorporate modified dNTPs. Due to the relative ease of testing, these experiments were conducted outside the context of the specific ssDNA synthesis system. The experiments included performed PCR reactions of a 200bp dsDNA template with and without modified dNTPs. As shown in FIG. 19, Lanes 2-4 indicate that Taq can incorporate unmodified dNTPs, 5-methyl-dCTP, and 5- hydroxymethyl-dCTP, respectively, without a drop in efficiency. Therefore, this enzyme can be used in the ssDNA production system of the present disclosure. Lane 5 indicates that 5- propargylamino-dCTP was not incorporated as efficiently.

Claims

ClaimsWhat is claimed is:

1. A nucleic acid template molecule for generating a plurality of modified nucleic acid oligos comprising: a hairpin region chemically configured for attachment to a substrate; a double-stranded region comprising at least one nucleic acid processing site; and a single- or double-stranded region comprising a nucleic acid template sequence.

2. The nucleic acid template molecule of claim 1, wherein the chemically configured region of the hairpin comprises a Biotin modification (3', 5' or internal), Amine modification (3', 5' or internal), Carboxylic acid modification (3', 5' or internal), Alkyne modification (3', 5' or internal), or Azide modification (3', 5' or internal).

3. The nucleic acid template molecule of claim 1 or claim 2, wherein the substrate comprises at least one substrate from Table 3.

4. The nucleic acid template molecule of any one of claims 1 to 3, wherein the hairpin region comprises a loop region comprising from about 4 nucleotides to about 100 nucleotides.

5. The nucleic acid template molecule of any one of claims 1 to 4, wherein the at least one nucleic acid processing site comprises a nickase site.

6. The nucleic acid template molecule of any one of claims 1 to 5, wherein the at least one nucleic acid processing site comprises a restriction site.

7. The nucleic acid template molecule of any one of claims 1 to 6, wherein the at least one nucleic acid processing site comprises a nickase site adjacent to or overlapping with a restriction site.

8. The nucleic acid template molecule of any one of claims 5 to 7, wherein the nickase site comprises any nucleic acid sequence from Table 4.

9. The nucleic acid template molecule of any one of claims 5 to 7, wherein the restriction site comprises any nucleic acid sequence from Table 5.

10. The nucleic acid template molecule of any one of claims 1 to 9, wherein the single- or double-stranded region comprises a 3 ’ overhang.

11. The nucleic acid template molecule of any one of claims 1 to 9, wherein the single- or double-stranded region comprises a 5’ overhang.

12. The nucleic acid template molecule of any one of claims 1 to 11, wherein the single-or double-stranded region comprising the nucleic acid template sequence is from about 2 nucleotides to about 100 kb.

13. The nucleic acid template molecule of any one of claims 1 to 12, wherein the nucleic acid template sequence is the reverse complement of the sequence of the plurality of modified nucleic acid oligos to be generated.

14. The nucleic acid template molecule of any one of claims 1 to 13, wherein the at least one nucleic acid processing site is directly adjacent to the single- or double-stranded region comprising the nucleic acid template sequence.

15. The nucleic acid template molecule of any one of claims 1 to 14, further comprising a RNA polymerase promoter before or after the at least one nucleic acid processing site, or as part of the nucleic acid template sequence.

16. A composition for generating a plurality of modified nucleic acid oligos comprising the nucleic acid template molecule of any one of claims 1 to 14, wherein the hairpin region of the nucleic acid template molecule is chemically coupled to a substrate.

17. The composition of claim 16, comprising at least one nickase enzyme.

18. The composition of claim 16 or claim 17, comprising at least one restriction enzyme.

19. The composition of claim 17 or claim 18, wherein the at least one nickase enzyme and / or the at least one restriction enzyme is chemically coupled to the substrate.

20. The composition of any one of claims 16 to 19, further comprising dNTPs or NTPs.

21. The composition of claim 20, wherein the dNTPs or NTPs comprise at least one modified dNTP or NTP.

22. A method for enzymatically generating a plurality of modified nucleic acid oligos, the method comprising:(a) providing the composition of claim 16;(b) adding a first solution comprising at least one unmodified nucleotide base and a DNA or RNA polymerase to generate at least a portion of the modified nucleic acid oligo from the nucleic acid template sequence;(c) adding at least a second solution comprising at least one modified dNTP or NTP;(d) repeating steps (b) and (c) until the modified nucleic acid oligo is generated; and(e) adding a nickase enzyme to release the modified nucleic acid oligo from the nucleic acid template molecule.

23. The method of claim 22, wherein the method further comprises a wash step.

24. The method of claim 22 or 23, wherein step (d) is performed with at least a second modified dNTP or NTP that is the same as or is different from the first modified dNTP or NTP in step (c).

25. The method of claim 24, wherein the first modified dNTP or NTP and the at least second modified dNTP or NTP are from about 1 nucleotide to about 1000 nucleotides apart.

26. The method of any one of claims 22 to 25, wherein the method further comprises adding a restriction enzyme after step (d) to generate a 5 ’ overhang at the end of the nucleic acid template molecule and a modified dNTP can then be added by a DNA polymerase.

27. The method of any one of claims 22 to 26, wherein the method further comprises adding a restriction enzyme after step (d) to generate a blunt end, a 3 ’ overhang, or a 5’ overhang at the end of the nucleic acid template molecule.

28. The method of any one of claims 22 to 27, wherein the method further comprises ligating a different single-stranded or double-stranded nucleic acid template sequence to the nucleic acid template molecule via the blunt end, the 3’ overhang, or the 5’ overhang.

29. The method of any one of claims 22 to 28, wherein the method further comprises repeating steps (b) through (e) to generate a second modified nucleic acid oligo.

Images