Synthesis of oligonucleotides

Abstract

The present invention relates to a novel method of oligonucleotide synthesis using a catalytic primer template, e.g., a hairpin primer, in which multiple rounds of oligonucleotide synthesis and dissociation from the template are performed in the same reaction such that at the end of the reaction, the amount of oligonucleotide produced is greater than the amount of template provided to the reaction. The primer contains a cleavable site and the reaction is performed in a single mixture with a cleavage agent. The cleavable site can be inosine and the cleavage agent can be endonuclease V.

Description

[Technical field]

[0001] The present invention relates to a method for synthesizing oligonucleotides, particularly single stranded oligonucleotides. The present invention also relates to oligonucleotides produced by the method of the present invention. The present invention also relates to a kit of parts for synthesizing oligonucleotides and a support for synthesizing oligonucleotides. The present invention also relates to a reaction comprising a primer / template, a polymerase and a cleavage agent. The present invention also relates to a cell comprising a nucleic acid sequence or vector encoding the primer / template of the present invention. [Background technology]

[0002] Therapeutic oligonucleotides, which bind to mRNA and regulate the production of disease-related proteins, have emerged as a new drug discovery modality to treat a broad range of disease areas (Khvorova et al. Nat. Biotechnol. 2017, 35, 238). Following the Nobel Prize for the discovery of RNA interference technology and the FDA approval of several RNA-based therapeutics for the treatment of rare diseases, significant investments have been made in therapeutic oligonucleotides in recent years. Currently, over 160 oligonucleotide products, including those for population-based indications, are in clinical trials (Hugget et al. Nat. Biotechnol. 2017, 35, 708). The growing number of potential therapeutics, including those for common diseases, poses significant manufacturing challenges as existing chemical synthesis methods are limited to 10 kg batches and are not suitable for large-scale applications (>100 kg) (Tedebark et al. Methods Mol. Biol. 2011, 683, 505).

[0003] Therapeutic oligonucleotides are short DNA analogs that selectively bind to target mRNAs by Watson-Crick base pairing to regulate the production of disease-related proteins. Therapeutic oligonucleotides include double-stranded RNA molecules known as small interfering RNA (siRNA) and single-stranded molecules known as antisense oligonucleotides (ASO). Oligonucleotides have been investigated as potential drug molecules for over 40 years, but progress in translating oligonucleotides into effective therapeutics has been limited by poor biological efficacy, bioavailability, and off-pathway toxic effects. In recent years, a number of chemical modifications have been reported that significantly improve the delivery, metabolic stability and efficacy of oligonucleotides, including substitutions to the DNA backbone, pyrimidine bases and ribose sugars (Figure 1) (Khvorova et al supra; Rinaldi et al. Nat. Rev. Neurol. 2018, 1, 9;., and Smith et al. Annu. Rev. Pharmacol.Toxicol. 2019, 59, 605; Dowdy, Nat. Biotechnol. 2017, 35, 222).

[0004] Therapeutic oligonucleotides may require chemical modifications to confer improved efficacy, selectivity, metabolic stability, and toxicity profiles (Rinaldi et al. Nat. Rev. Neurol. 2018, 1, 9; Smith et al. Annu. Rev. Pharmacol. Toxicol. 2019, 59, 605). Although natural polymerases tolerate some nucleotide modifications (Nakamaye et al Nucleic Acids Res. 1988, 21, 9947; Yang et al. Nucleic Acids Res. 2007, 35, 3118; Romaniuk et al. J. Biol. Chem. 1982, 257, 7684), their activity may be reduced.

[0005] The rise of siRNAs and ASOs as novel drug modalities brings significant manufacturing challenges (Tedebark et al. Methods mol. Biol. 2011, 683, 505; Kosuri, nat. Methods 2014, 11, 499). Current synthesis methods generally use a four-step solid-phase phosphoramidite method developed in the 1980s (Beaucage et al. Tetrahedron left. 1981, 22, 1859). This method can be easily automated and has been adapted for the synthesis of modified DNA structures. Unfortunately, this method is not suitable for large-scale synthesis (>100 kg) and is currently limited to batches of oligomeric products less than 10 kg (Kim et al. Org. Process res. Dev. 2016, 20, 1439). Currently, kilo-scale syntheses are carried out using columns with diameters of 1 m or less with shallow beds (10-15 cm). Increasing the column diameter leads to non-linear flow rates and reduced product purity, which means that the synthesis cannot be intensified, but instead requires multiple parallel reactors. The reagents used in such methods are environmentally hazardous and pose handling and disposal problems.

[0006] Currently, there is no cost-effective method for in-line reaction monitoring that requires the use of excessive amounts of expensive phosphoramidites. These monomers contain multiple protecting groups (including 4'4-dimethoxytrityl-protected 5'-OH, benzoyl- and isopropyl-protected bases, and 2-cyanoethyl-protected phosphites) that further impair atom efficiency and generate by-products that must be separated during synthesis. The process uses extremely large amounts of acetonitrile (1000 kg per kg of oligonucleotide), necessitating chromatographic purification of the final product to remove impurities resulting from depurination, base modifications (resulting from the formation of cyanoethyl adducts), and truncated sequences. Due to limitations of existing synthetic methods, currently commercially available RNA-based therapeutics are typically supplied as complex mixtures of diastereoisomers (resulting from phosphorothioate modifications) at approximately 90% purity, and indeed our understanding of the biological activity of the individual stereoisomers remains highly limited. Although a number of alternative methods have been developed in recent years, including the use of P(V) reagents (Knouse et al. Science 2018, 361, 1234) and covalent attachment of nucleotide building blocks to deoxynucleotidyl transferase (Palluk et al. Nat. Biotechnol. 2018, 36, 645), existing methods share the same basic approach of using sequential coupling and deprotection reactions on a solid support.

[0007] Thus, there is a need for improved methods for producing oligonucleotides, particularly therapeutic oligonucleotides such as ASOs and miRNAs. Summary of the Invention

[0008] The present invention relates to a method for synthesizing oligonucleotides, such as therapeutic oligonucleotides. The present invention aims to provide a scalable approach for producing oligonucleotides with high purity at low cost. This establishes oligonucleotides as viable therapeutic agents for diseases. The present invention provides a method for simultaneous primer extension and release of oligonucleotides by cleavage of the extended primer. The primer template can be used multiple times during the reaction or incubation time to catalyze the oligonucleotide synthesis reaction.

[0009] In a first aspect, the present invention provides a method for generating a single stranded oligonucleotide comprising the steps of: i) providing a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; ii) incubating the primer template with a nucleic acid polymerase, one or more dNTPs, and a cleavage agent to form a reaction mixture; iii) maintaining the reaction mixture under conditions that allow: a) extension of the primer by a polymerase to form an extended primer template; and b) cleavage of the extended primer template at the cleavable site by a cleavage agent; and iv) optionally separating the oligonucleotide products from the reaction mixture.

[0010] In a second aspect, the present invention provides an oligonucleotide produced by the method of the first aspect.

[0011] In a third aspect, the present invention provides a kit for producing a single stranded oligonucleotide, comprising: a) a primer template comprising a primer for initiating oligonucleotide synthesis, b) a template for directing synthesis of an oligonucleotide product, and c) a cleavable site that allows release of the oligonucleotide product from the template; ii. a nucleic acid polymerase, and optionally one or more nucleotides; and iii. a cleavage agent.

[0012] In a fourth aspect, the present invention provides a support for generating single stranded oligonucleotides, comprising a population of primer templates immobilized to the support, each primer template comprising: a) a primer for initiating oligonucleotide synthesis, b) a template to direct synthesis, and c) a cleavable site that allows release of the oligonucleotide product from the template.

[0013] In a fifth embodiment, there is provided a method of generating a population of single stranded oligonucleotides, comprising the steps of: i) providing a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; ii) incubating the primer template with a nucleic acid polymerase, one or more dNTPs, and a cleavage agent to form a reaction mixture; iii) maintaining the reaction mixture under conditions that allow: a) extension of the primer by a polymerase to form an extended primer template; and b) cleavage of the extended primer template at the cleavable site by a cleavage agent; iv) optionally separating the oligonucleotide products from the reaction mixture; and v) maintaining the reaction mixture in step iii) for a suitable reaction time to produce a population of oligonucleotides; the nucleotide comprises a modified thiotriphosphate (NTPαS), and the oligonucleotide population comprises substantially the same stereoisomer; Methods are provided in which the polymerase is selected from the group consisting of polymerases from Thermococcus kodakaraensis (KOD), Stoffel, family B polymerase 9°N, Thermus filiformis (TfPol) and Marinithermus hydrothermalis (MhPol) as shown in Table 3; Klenow fragment from Escherichia coli, T4 and T7 polymerase, SFP1, Stoffel variant SF4-6, Stoffel homologues from Thermus filiformis and Marinithermus hydrothermalis (Mhpol), or variants of any of the above polymerases as shown in Table 3.

[0014] Also, in one aspect of the invention, a reaction mixture for producing a single stranded oligonucleotide is provided, the reaction mixture comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a primer template comprising a cleavable site that allows release of the product oligonucleotide from the template; i) a nucleic acid polymerase, and optionally one or more nucleotides; ii) a cleavage agent; and iii) a product oligonucleotide complementary to the template sequence.

[0015] Also provided is a cell for producing a single stranded oligonucleotide, the cell comprising a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template that directs synthesis of a product oligonucleotide; and c) a cleavable site that allows release of the product oligonucleotide from the template.

[0016] Embodiments of the invention are further described below with reference to the accompanying drawings.

[0017] In the figures and examples, T refers to the template sequence, E refers to the extension template, and P refers to the product. The sequences of the T, E and P sequences referred to herein can be found in Table 1. [Brief description of the drawings]

[0018] [Figure 1] Chemical modifications of oligonucleotides are shown. [Diagram 2] The nucleotide sequence and sequence of Spinraza are shown (*=PS m=2'MOE). [Diagram 3] FIG. 1 is a schematic diagram showing scalable oligonucleotide production according to the present invention. [Figure 4A] KOD (Merck, 2.5 units) catalyzed elongation of T1 (Table 1, 20 μM) with dNTPs (1 mM) gives the extension product E1 (Table 1, 7.0 min, expected mass 18170.8) in greater than 99% yield. [Figure 4B] TmEndoV (ThermoFisher, 5 units) catalyzed cleavage of extension template E1 (20 μM) gives product P1 (Table 1, 4.85 min, expected mass 5544.6) and template T1 (6.54 min, expected mass 12644.2) in >99% yield. The UV trace is shown on the left and the mass spectrum on the right. [Figure 4C] One-pot KOD and TmEndoV cascade reaction using T1 (20 μM) and dNTPs (0.25 mM each) gives product P1 (5.45 min, expected mass 5544.6). [Diagram 5] Polymerase and endonuclease activity at different dNTP concentrations. A) PAGE analysis showing that KOD (0.2 μM) catalyzed the extension of T1 (20 μM) at dNTP concentrations up to 30 mM. B) PAGE analysis showing that TmEndoV (2 μM) catalyzed the cleavage of E1 (20 μM) at dNTP concentrations up to 2 mM. C) PAGE analysis showing that TnEndoV (2 μM) catalyzed the cleavage of E1 (20 μM) at dNTP concentrations up to 20 mM. [Figure 6]Temperature optimization experiments are shown. A) Thermal shift assay showing the melting temperatures of TnEndoV (highest peak), TmEndoV (middle peak) and PfEndoV (lowest peak). B) Polyacrylamide gel analysis of one-pot reactions catalyzed by KOD and TnEndoV at various temperatures. Lane 1: ladder, lane 2: E1 standard (top band), T1 standard (middle band) and P1 (bottom band), lanes 3-8: one-pot reactions performed at 60 °C, 65 °C, 70 °C, 75 °C, 80 °C and 85 °C (from left to right). C) Overlay of HPLC traces showing that KOD activity is not affected by pre-incubation at 70 °C for 24 h. Biotransformations were performed using T1 (20 μM), dNTPs (4 mM) and either fresh KOD (0.2 μM, green) or KOD pre-incubated at 70 °C for 24 h (0.2 μM, blue). Each HPLC trace shows the conversion to E1 after 30 min of reaction time. D) Overlay of HPLC traces showing that TnEndoV retains 85% activity after 24 h preincubation at 70 °C. Biotransformations were performed using E1 (20 μM) and either fresh TnEndoV (2 μM, green) or TnEndoV (0.2 μM, blue) preincubated at 70 °C for 24 h. Each HPLC trace shows the conversion to T1 and P1 after 30 min of reaction time. [Figure 7]Polymerase / endonuclease activity against self-priming templates with various sequences was examined. PAGE analysis shows the generation of P1 after cycles of polymerase-catalyzed template extension and endonuclease-catalyzed product cleavage. One-pot reactions were performed using KOD (0.2 μM), TnEndoV (2 μM), template (10 μM), and dNTPs (1 mM each). A DNA ladder is shown in lane 1, and T1 and E1 standards are shown in lane 2. The yield of product P1 was largely unaffected by 5'-biotinylation (lane 4), alternative hairpin sequences (lane 7), changing the nucleobase pair to inosine (lanes 8 & 9), or using a different primer from the template (lane 6). Placing biotin in the hairpin (lane 5) or changing the length of the hairpin (lanes 10 and 11) slightly reduced the product yield. [Figure 8] HPLC analysis of polymerase / endonuclease catalyzed P1 generation under optimized conditions. HPLC trace showing product P1 generated during a reaction catalyzed by KOD (2 μM) and TnEndoV (2 μM) with T1 (1 μM), dNTPs (4 mM each) in 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM arginine-HCl (pH 8), 100 mM glutamate-KOH (pH 8), DTT (10 mM), formamide (10%), trehalose (0.2 M), 1,2-propanediol (1 M), and acetylated BSA (0.01 mg / ml) after 12 h incubation at 70° C. Product was generated after 330 cycles of template extension and product cleavage, yielding 0.33 mM P1 (equivalent to 1.9 g / L). [Figure 9] Polymerase activity towards modified dNTPs is shown. Polymerase activity towards modified nucleotide triphosphates was assessed using a time-resolved FRET-based assay. Reactions were performed with three natural dNTPs and one modified NTP. Values ​​for each polymerase are reported relative to activity towards natural dNTPs and represent the average of triplicate measurements. [Figure 10]Stereoselective synthesis of oligonucleotides containing a single phosphorothioate bond. HPLC traces showing products generated during a one-pot polymerase-endonuclease reaction. Reactions were performed using mixtures of A) Sp-dATPαS, B) Sp-dCTPαS, C) Sp-dTTPαS, D) Sp-dGTPαS, E) Rp / Sp-dATPαS, F) Rp / Sp-dCTPαS, G) Rp / Sp-dTTPαS, H) Rp / Sp-dGTPαS, (0.7 mM) with T0-T13 (10 μM) and three unmodified NTPs (1.4 mM each). Comparison of the enzymatically synthesized products with the diastereomeric mixtures generated by chemical synthesis indicates that this biocatalytic reaction affords each product in greater than 99% diastereomeric excess. [Figure 11] LC-MS analysis of TnEndoV activity on modified oligonucleotides. UV traces showing products generated during cleavage reactions of extended templates (20 μM) A) E6, B) E2, C) E3, D) E4, and E) E5 catalyzed by TnEndoV (2 μM). Deconvoluted masses of each 18-mer product and template are shown in each table. Cleavage of phosphorothioate-modified oligonucleotide E6 by EndoV yielded the expected product (PA) and a 5'-dephosphorylated analog (PB). Incubation of PA standard (20 μM) in reaction buffer at 70°C resulted in 5'-dephosphorylation, confirming that side reactions are not catalyzed by the enzyme. [Figure 12] Substrate range of the EndoV panel. PAGE analysis showing EndoV (2 μM) catalyzed cleavage of extended templates (20 μM) with no modification (E1), 18 consecutive phosphorothioate (E6), 2'-fluoro (E2), 2'-methoxy (E3) or 2'-methoxyethoxy (E4) modifications, or one LNA modification 3' to the cleavage site (E8). Samples were analyzed after 2 and 4 hours of incubation at 70°C. Each standard is composed of extended template E1, template T1, and an 18-mer oligonucleotide (P). [Figure 13]Preparative scale synthesis of P1. HPLC trace showing the synthesis of P1 catalyzed by KOD (2 μM) and TnEndoV (2 μM) using T2 (4 μM) and dNTPs (4 mM each). A) HPLC of the reaction mixture after 12 hours of incubation. B) Isolated product. The table shows the observed masses and impurities present in the crude reaction mixture and isolated product. The sequences printed in bold correspond to the target product. [Figure 14] Substrate range of the polymerase panel. PAGE analysis showing polymerase (0.2 μM) activity towards modified NTPs. Reactions were performed using T22 (20 μM) and a mixture of three unmodified NTPs (0.25 mM each) and one modified NTP (0.25 mM) and incubated at 70° C. for 1 h. Each polymerase was able to transcribe the template and incorporate two copies of the modified NTP, resulting in a full-length extension. Control reactions were performed in the absence of modified NTPs to determine background misincorporation rates of non-complementary bases. A) Polymerase activity towards dNTPs (PO), Sp-dNTPαS (Sp), Sp-2'-fluoro-NTPαS (Sp-2'F) Rp-dNTPαS (Rp), 2'fluoro-NTP (2'F) and LNA-NTP (LNA). B) Polymerase activity towards 2'-methoxy modified NTPs. + indicates the addition of 2'MeO-NTP, - indicates the control reaction in the absence of 2'MeO-NTP. T22=5'-CTGACTGACACGTGCACCATTGGTGCACGIG-3' [Figure 15]Polymerase activity for sequential incorporation of 2'MeO-NTPs and LNA-NTPs. Templates encoding 8 copies of a single nucleobase (T18-T21, 20 μM) were extended using complementary 2'MeO-NTPs (1 mM) or LNA-NTPs (1 mM) and the optimal polymerases SF4-6 (0.2 μM) or KOD DGLNK (0.2 μM), respectively. LC-MS analysis showed that after 12 h of incubation, each reaction yielded a mixture of partially extended products. Labeled peaks in the UV spectrum were assigned to truncated products by MS analysis, no additional peaks were assigned. nt added = number of nucleotide bases incorporated by the polymerase. [Figure 16] Polymerase-catalyzed reactions using modified NTP mixes. Template T2 (20 μM) was extended using a mixture of Sp-dNTPαS (2, 0.25 mM, respectively) and 2'F-NTP (3, 0.25 mM, respectively), with KOD (0.2 μM) and TfPol* (0.2 μM), respectively. Template T7 (20 μM) was extended using a mixture of Sp-2'FA / GTPαS (4, 0.25 mM, respectively) and KOD (0.2 μM). PAGE analysis showed that each reaction reached full conversion to extension products. B) LC-MS analysis was used to confirm product type. Addition number = number of nucleotide bases incorporated by the polymerase. [Figure 17]Stereoselectivity of TnEndoV at phosphorothioate centers undergoing hydrolysis. Stereodefined extension templates were generated using KOD (0.2 μM) and Sp-dNTPαS (0.5 mM each). Cleavage of enzymatic synthesis products (20 μM) by TnEndoV (2 μM) was compared to cleavage of the corresponding stereoirregular oligonucleotides (20 μM). PAGE analysis shows that A) polymerase-catalyzed extension of templates T1 and T15-T17 (20 μM) proceeds to completion. Each template encodes an 18-mer oligonucleotide product with a different nucleobase at the first position. B) Cleavage of stereodefined oligonucleotides with Rp bonds by TnEndoV (2 μM) (lanes 9-12) proceeds to completion, whereas cleavage of the corresponding stereoirregular oligonucleotides is stalled at 50% conversion (lanes 13-16). Activity is not affected by the base 3' to the cleavage site. [Figure 18] One-pot polymerase / EndoV-catalyzed generation of P19–P32. UPLC chromatograms (recorded at 260 nm) show the product generation after one-pot polymerase-endonuclease-catalyzed reactions. Specific reaction conditions for each biotransformation are listed in Table S1. The masses of products and by-products were confirmed by mass spectrometry. T: template, E: extension template. Nucleotide sequences printed in bold correspond to the correct octamer products (+: LNA, *: phosphorothioate linkage, f: 2'-fluoro, P: 5' phosphate group, HO / OH: 5' / 3' hydroxyl groups). Sequences printed in bold correspond to the correct products. Bases highlighted in red correspond to untemplated over-extension. [Figure 19]Preparative-scale synthesis of bitrabene. A) Crude reaction mixture after 12 h incubation at 70 °C, B) UPLC chromatogram (recorded at 260 nm) showing isolated product after reaction workup. During the reaction, the product is 5'-dethiophosphorylated (peak a). Incubation of a product standard in reaction buffer results in 5'-dethiophosphorylation, confirming that side reactions are not catalyzed by the enzyme. Since the target product contains a 5' hydroxyl, any remaining 5' triphosphate groups were removed during reaction workup using alkaline phosphatase. [Figure 20] Polymerase activity towards Rp-dNTPαS in the presence of cobalt and manganese cofactors. A) PAGE analysis showing KOD-catalyzed extension of a template encoding eight copies of a single nucleobase using complementary Rp-dNTPαS (1 mM) in the presence of 0.5 mM COCl2 or MgCl2. B) LC-MS analysis showing KOD-catalyzed addition of eight Rp-dATPαS nucleotides to a self-priming template in the presence of a mixture of 1 mM CoCl2 and 0.4 mM MnCl2. The peak at 5.2 min corresponds to the extension product (observed mass 12141) and the peak at 0.5 min corresponds to unreacted NTPs. [Figure 21] Endonuclease activity in the presence of alternative nucleophiles. UV traces after LC-MS analysis of EndoV reactions performed in the presence of increasing concentrations of different nucleophiles. Nucleophilic attack of extension template E1 yielded "modified 18-mer products" containing a 5'-phosphate functionalized with glycerol (detected mass 5618.8), ethylene glycol (detected mass 5587.4), 1,2-propanediol (detected mass 5601.9) or 1,3-diaminopropanol (detected mass 5616.6). The major product was the canonical 18-mer with an unmodified 5'-phosphate. [Figure 22] Biocatalytic synthesis of oligonucleotide sequences. Oligonucleotides with various sequences and chemical modifications were generated by one-pot biotransformation using the polymerase of choice and TnEndoV. Conversion (%) represents the conversion of NTP starting material to product. See Table 3 for specific reaction conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention provides a method for synthesizing single-stranded oligonucleotides, such as therapeutic oligonucleotides, in a versatile and scalable manner.

[0020] The present invention is based on the novel concept of providing a reusable or catalytic primer template, referred to herein as an extended primer template, that can be extended by a polymerase to form a double-stranded nucleic acid molecule. A suitable cleavage system is used to cleave the double-stranded strand, allowing the release of the extended portion, which is the desired single-stranded oligonucleotide product. Advantageously, the double strand is cleaved at a cleavable site incorporated into the nucleic acid molecule, releasing the single-stranded oligonucleotide product and regenerating the primer template. Cleavage to regenerate the primer template allows repeated cycles of extension and cleavage, resulting in the accumulation of oligonucleotide products in the reaction mixture. Thus, the method of the present invention can provide the target oligonucleotide in a single operation, in contrast to the repeated rounds of chain extension, oxidation, and deprotection associated with chemical synthesis of oligonucleotides, which is well known in the art. In chemical synthesis, many repeated reactions are required to generate short oligomers. In the present invention, oligonucleotides can be produced in a single reaction. The method of the present invention has the advantage that it can be carried out under aqueous conditions without the need for large amounts of acetonitrile. Moreover, the method of the present invention is more atom-efficient than standard phosphoramidite chemistry, since no protective groups are required.The method of the present invention also has the advantage that it can provide oligonucleotide products with significantly higher purity than those produced by chemical synthesis, thus reducing the need for expensive chromatographic purification.In addition, the method of the present invention can be isothermal.This has the advantage that the energy required to change temperature at different parts of each cycle is reduced.

[0021] The present invention provides a method for the scalable production of therapeutic oligonucleotides by providing a method for the production of oligonucleotides that can contain one or more modified or non-natural nucleotides in a highly pure and scalable manner, thereby allowing for the large scale production of therapeutic oligonucleotides. The method of the invention can utilize a modified polymerase enzyme suitable for incorporating one or more modified nucleotides into the nucleotide chain.

[0022] The present invention is based on the simultaneous use of primer template, polymerase and cleavage agent to generate oligonucleotides, and after newly synthesized oligonucleotides are released from the template, the primer template is available for one or more additional rounds of oligonucleotide synthesis in the same reaction.Once the template is copied, the synthesized oligonucleotide is cleaved by the cleavage system and released from the template, and the primer template is available for the next round of oligonucleotide synthesis.Therefore, the combined use of primer template, polymerase and cleavage agent in the same reaction means that the reaction is not stoichiometric, allowing the production of an amount of product that exceeds the amount of template present in the reaction.

[0023] definition As used herein, a "nucleic acid molecule" refers to a chain of nucleotides having a specific sequence. A nucleic acid molecule can contain all four nucleotides (G, A, T / U and C), or any combination thereof (e.g., G, A, T / U or C, or any combination thereof).

[0024] An "oligonucleotide" is a short nucleic acid sequence, generally between 5 and 100 nucleotides in length.

[0025] As used herein, a "single-stranded nucleic acid" refers to a first nucleotide multimer that is not bound to (i.e., not base-paired with) a second, distinct nucleotide multimer that can form a duplex with the first nucleotide multimer. A "double-stranded nucleic acid" is a first nucleotide multimer that is base-paired with a second, distinct nucleotide multimer.

[0026] As used herein, an "oligonucleotide synthesis reaction" refers to a reaction in which an oligonucleotide is synthesized by adding monomers one at a time to a growing chain. Nucleotides can be added to the 3' end of the growing chain. The reaction can be carried out with a suitable polymerase.

[0027] As used herein, the term "nucleoside triphosphate" refers to a molecule that contains a nucleoside (i.e., a base bound to a deoxyribose or ribose sugar molecule) bound to three phosphate groups. A "nucleotide" or "nucleic acid residue" refers to a molecule that contains a nucleoside bound to one phosphate. A nucleotide generally refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. Examples of nucleoside triphosphates that contain deoxyribose are deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP) or deoxythymidine triphosphate (dTTP). Examples of nucleoside triphosphates that contain ribose are adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) or uridine triphosphate (UTP). Other types of nucleosides, including naturally occurring modified nucleosides and artificial nucleosides, can be linked with three phosphates to form nucleoside triphosphates (nucleotides).

[0028] "Modified" nucleic acids are nucleic acids in which the phosphodiester linkages, sugars, and / or bases have been altered.

[0029] "RNA" is a linear molecule composed of four small molecules called ribonucleotide bases: adenine (A), cytosine (C), guanine (G), and uracil (U). "DNA" is a linear molecule composed of four small molecules called deoxyribonucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T). RNA and DNA may each be single-stranded or double-stranded. RNA and DNA may each form secondary structures.

[0030] "MicroRNA" is a single-stranded RNA of about 17-25 nucleotides that may play a role in cell proliferation, apoptosis, or differentiation. miRNA may contain modified nucleotides. miRNA may be a therapeutic oligonucleotide.

[0031] "ASO" (antisense oligonucleotide) is a short synthetic DNA or RNA that can be complementary to an mRNA target. ASO can downregulate downstream targets. ASOs are usually modified, such as phosphorothioates.

[0032] An "aptamer" is a nucleic acid molecule that binds with high affinity and specificity to a target, such as a protein, a nucleic acid, an oligosaccharide, a small molecule, a hormone, a cytokine, a signaling molecule, a metal ion, or an organic molecule. Aptamers can be single-stranded or double-stranded, or partially single-stranded or partially double-stranded. Aptamers can contain modified nucleotides.

[0033] A "polymerase" catalyzes the formation of an oligonucleotide chain by the addition of successive nucleotides derived from nucleoside triphosphates or deoxynucleoside triphosphates. The polymerase reaction occurs only in the presence of an appropriate nucleic acid template. Each incoming nucleoside triphosphate first forms an appropriate base pair with a base in the template. The polymerase then combines the incoming base with the previous base in the chain. Thus, polymerases are template-directed enzymes.

[0034] As used herein, "primer" refers to a natural or synthetic oligonucleotide that acts as an initiation point for nucleic acid synthesis and can be extended from its 3' end along a template to form an extended double strand. Primer extension is usually carried out using a nucleic acid polymerase such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually, primers are extended by DNA polymerase.

[0035] As used herein, a "template" is a nucleic acid sequence that is copied by a polymerase. The template provides the complementary sequence of the desired oligonucleotide to be synthesized.

[0036] An "extended primer template" is a product formed during oligonucleotide synthesis such that the primer initiates synthesis of a nucleic acid strand that base-pairs with the template strand.

[0037] A "primed template" is a template that contains a primer and thus does not require a separate primer to initiate oligonucleotide synthesis.

[0038] A "self-priming template" is a nucleic acid molecule that contains a sequence that self-binds to form a double-stranded primer sequence and a single-stranded template sequence.

[0039] A "hairpin loop" or "stem loop" structure is an intramolecular base-pairing pattern that can occur in single-stranded DNA or RNA. A hairpin loop occurs when two regions of the same strand (which are usually complementary in sequence when read in opposite directions) base pair to form a double helix that ends in an unpaired loop.

[0040] "Cleavage" refers to the cleavage of a phosphodiester bond between two nucleic acid residues in a polynucleotide chain.

[0041] As used herein, a "cleavable site" is a bond or pair of bonds between nucleotides in a nucleic acid molecule that can be cleaved to cleave the nucleic acid molecule or release one strand of the nucleic acid molecule. A cleavable site can be defined by a specific nucleotide sequence recognized by an enzyme. A cleavable site can also be defined by the position of a modified nucleotide recognized by a specific nuclease.

[0042] As referred to herein, a "cleaving" enzyme or system is capable of cleaving a phosphodiester bond within a polynucleotide chain.

[0043] "Extension" refers to the synthesis of a new oligonucleotide strand, preferably by the action of a polymerase.

[0044] As used herein, "immobilization" refers to the reversible or irreversible binding of a nucleic acid molecule or a binding partner to a support.

[0045] A sequence that is "complementary" to another sequence has a nucleic acid sequence that base pairs with the nucleotides of the other sequence to form a stable duplex or double-stranded sequence. "Substantially complementary" means that not all of the base pairs between the two sequences match, but there is sufficient base pairing to form a stable duplex.

[0046] As used herein, "release" refers to the dissociation of a synthesized oligonucleotide strand from the template.

[0047] "Kit" refers to any presentation system for providing materials or reagents for carrying out the methods of the invention.

[0048] reagent The present invention uses reagents to synthesize the desired oligonucleotide in the same reaction, which may include primers, templates, polymerases, cleavage systems, nucleotide residues, cofactors, and reagents for reaction optimization.

[0049] I) Primer template The primer template may be RNA or DNA, and may comprise modified RNA or DNA.

[0050] A primer template has the ability to perform three functions: as a primer to initiate polymerase-mediated extension, as a template for the synthesis of a new oligonucleotide strand, and to cleave the new oligonucleotide from the nucleic acid molecule. Thus, a primer template can contain a primer to initiate oligonucleotide synthesis, a template to direct oligonucleotide synthesis, and a cleavable site to allow release of the oligonucleotide product from the template.

[0051] The primer template induces extension by successively adding nucleotides to the 3' end of the primer to form an extended primer template. The primer is positioned relative to the template sequence such that the template sequence is copied, preferably in its entirety, as the extended portion of the primer. The primer sequence can be positioned upstream of the template sequence. The primer sequence can be such that it binds to an upstream sequence of the template sequence to form a double-stranded (duplex) sequence upstream of the single-stranded template sequence from which polymerase activity is initiated. Thus, the primer template comprises a single-stranded nucleic acid template sequence and a double-stranded (or duplex) portion comprising the primer sequence. Thus, the single-stranded template sequence extends beyond the double-stranded portion comprising the primer to form a 5' tail or sticky end available to be copied by extension of the 3' end of the primer sequence. The template sequence can be a 5' to 3' sequence extending from the double-stranded portion.

[0052] The double-stranded portion of the primer template containing the primer is formed from a pair of complementary nucleic acid sequences. The double-stranded portion containing the primer may be formed from a single-stranded nucleic acid molecule containing a pair of complementary sequences that bind to form a secondary structure by folding the single-stranded nucleic acid molecule. A primer template formed from a single-stranded nucleic acid molecule containing a pair of complementary sequences may be referred to herein as a self-priming template. The pair of complementary sequences may be arranged within the single-stranded nucleic acid molecule such that when combined to form a duplex, the nucleic acid molecule folds asymmetrically on itself to form a double-stranded portion with a crease or loop at one end and an overhang of the single-stranded sequence that forms the template at the other end. Suitably, the single-stranded molecule may fold asymmetrically such that the overhanging single-stranded template sequence ends at the 5' end and the sequence that forms the double-stranded portion ends at the 3' end. This structure provides a free 3'OH end for extension to form a new oligonucleotide sequence by base pairing with the template sequence. Suitably, the single-stranded sequence is folded back such that the 3'-terminal nucleotide base pairs with the nucleotide immediately adjacent (upstream) to the first nucleotide of the template sequence, thus forming a single-stranded overhang beginning with the template sequence to be copied.

[0053] Suitably, the secondary structure formed by pairing of complementary sequences of the nucleic acid molecule is a hairpin loop. A hairpin loop is formed by a single-stranded nucleic acid molecule that adopts a secondary structure in which the sequence folds back on itself to form a double-stranded stem and a loop at one end. A hairpin loop single-stranded nucleic acid molecule adopts a hairpin structure due to the presence of a pair of complementary sequences separated by a third sequence, such that the complementary sequences base pair to form a double-stranded stem, and the third intervening sequence forms a loop at one end of the double-stranded stem. The 3' end of one of the pair of complementary sequences serves as a primer for transcription of the nucleic acid sequence. Suitably, the length of the stem and / or the size of the loop are of an appropriate length to allow a polymerase to bind to the hairpin loop and initiate transcription from the free 3' hydroxyl group at one end of the stem.

[0054] The length of the stem portion is determined by the length of the complementary sequence in the single stranded sequence that base pairs to form the hairpin loop. The size of the loop is generally determined by the length of the non-complementary sequence between the two portions of the complementary sequence that base pair to form the stem portion. A hairpin loop typically comprises a 5' end and a 3' end at the end of the double stranded stem portion. The 3' end has a free OH group at which a polymerase can initiate primer extension. The hairpin loop can be of any suitable size such that it can function as a primer for transcription. Suitably, the loop portion of the hairpin comprises more than three nucleotides such that a loop structure is formed. Suitably, the loop portion of the hairpin loop comprises more than four nucleotides, suitably 4-30 nucleotides, more suitably 4-25, 4-20 or 10-20 nucleotides, or any range or integer therebetween. The double-stranded stem portion may be of any suitable length, for example, 1-200 nucleotides in length, or 1-190, 1-180, 1-170, 1-150, 1-120, 1-100, or 10-90, 20-80, or 30-50 nucleotides in length, or any range using any of the above starting and ending points, or any length therebetween. The most suitable length of the stem portion of the hairpin loop is 10-30 nucleotides in length, most suitable is 10-20 nucleotides in length.

[0055] The stem portion of the hairpin loop is generally double-stranded and is formed from a single strand of nucleic acid folded back on itself as described herein. The stem portion of the hairpin loop has a free 5' end and a free 3' end. The two strands of the stem portion may be the same length to form a blunt end or may be of different lengths to form a sticky end on one strand. The presence of a blunt or sticky end depends on the location of a complementary sequence in the single-stranded nucleic acid sequence that forms the hairpin loop. Suitably, the stem portion includes a blunt end. A nucleic acid molecule that includes a template sequence (for a polymerase to copy) that extends beyond the double-stranded stem portion may be referred to as having a sticky end or a sequence that lacks a complementary base to bind to it.

[0056] Examples of complementary sequences that can be placed in a single-stranded nucleic acid molecule to allow for the formation of a hairpin loop are shown in Table 1. Examples of non-complementary sequences that form loop structures are shown in Table 1. Single-stranded nucleic acid sequences that form hairpin loops for use in the present invention can include any combination of complementary (stem) and loop sequences as shown in Table 1.

[0057] Alternatively, the primer template may comprise separate primers and templates that are not formed from a single self-complementary nucleic acid sequence. Instead, the primer template may comprise i) a single-stranded nucleic acid molecule template, ii) another primer sequence that base pairs to the template, and iii) a cleavable site. Suitably, the primer sequence is shorter than the single-stranded molecule template. Suitably, the primer base pairs to the 3' end of the single-stranded nucleic acid molecule template, thereby providing a single-stranded template that extends downstream of the duplex formed by the primer and the single-stranded nucleic acid molecule template. Suitably, the primer comprises a free 3' hydroxyl group for extension by a polymerase. Thus, such a primer template may be equivalent to the self-priming template described herein, except that it does not have a secondary structure such as a hairpin loop.

[0058] The primer and the template may be bound by other means than base pairing. For example, the primer may be immobilized on the template, or the primer and the template may be chemically bound. Any suitable means may be used to bind the primer to the template. If the primer and the template are separated during the reaction, the primer may be used to synthesize separately from or in the same way as the template (i.e., the primer may be copied), which may result in the production of by-products rather than the desired oligonucleotide. Using a means to bind the primer and the template so that they are separated during the reaction reduces the possibility of producing by-products and makes it easier to isolate the downstream product.

[0059] When the primer portion of the primer template is provided as a separate nucleic acid molecule, the primer portion can be an RNA molecule or a DNA molecule. The primer portion can include one or more natural and / or modified nucleotide residues. The primer can be a 3' ribonucleotide primer, which includes a DNA sequence having a ribonucleotide residue at the 3' end. Such a primer retains its function after the oligonucleotide is cleaved from the template sequence and can be reused. Suitable methods for preparing 3' ribonucleotide primers will be well known to those skilled in the art.

[0060] The primer is appropriately hybridized to the template prior to oligonucleotide synthesis. If a self-priming template is used, a secondary structure is formed prior to oligonucleotide synthesis. The hybridization conditions required to form a primer template depend on factors including the specificity of the pair of complementary primer sequences or the specificity of the primer and template sequences.

[0061] Hybridization of the primer with the template can occur during or prior to the oligonucleotide synthesis reaction. If hybridization occurs during the synthesis reaction in the presence of polymerase enzymes, cleavage systems, and other reagents, the hybridization conditions must be such that they do not affect the function of the other reagents.

[0062] The template portion of the primer template comprises a single-stranded sequence that provides the sequence to be copied for the synthesis of the desired oligonucleotide sequence. Suitably, the sequence of the template is such that it does not anneal or base-pair with any portion of the primer template, so that the template remains single-stranded and can be used as a template for polymerase-mediated extension. Suitably, the sequence of the template portion of the primer template is therefore not sufficiently complementary with any portion of the remainder of the primer template sequence to allow the formation of a duplex of the template. Thus, the template sequence has no more than 1, 3, 5, 10, 15 or 20% sequence complementarity with the remainder of the primer template nucleic acid molecule. "Sequence complementarity" means that the sequences can base-pair with each other to form a double-stranded sequence.

[0063] The template will contain a sequence that is complementary to the sequence of the oligonucleotide of interest that is to be synthesized.Therefore, the template can contain a sequence that is complementary to a therapeutic oligonucleotide, such as an aptamer, an mRNA, or an siRNA.The template sequence is suitably 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 95% or 99% complementary to the oligonucleotide sequence that is to be synthesized.

[0064] A template may contain two or more portions, each portion directing the synthesis of a separate or different oligonucleotide of interest. Thus, a single template can be used to generate two or more different oligonucleotides. Thus, the template sequence directs the synthesis of one oligonucleotide product, which can then be cleaved into individual oligonucleotides of interest. The template sequence can be a sequence suitable for incorporating one or more restriction enzyme sites into the product, so that the product can be cleaved into individual oligonucleotides of interest.

[0065] The template can include sequences that direct the synthesis of functional elements or motifs, such as, but not limited to, termination sequences, spacer sequences, polyadenylation sequences, tags, and / or cleavable sites. When a spacer or transcription termination sequence is included in the template, a cleavable site (or its complement) that allows cleavage of the synthesized oligonucleotide can be included to generate the desired product without excess sequence.

[0066] The template sequence may suitably be the same length as the length of the oligonucleotide of interest, or may be longer, for example if it comprises template sequences for two or more oligonucleotides, or templates for one or more functional elements or motifs, as described above.

[0067] The template can be of any suitable length. The template may be 3-200 nucleotides in length, preferably 5-200, 5-150, 5-100, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 20-50, 20-40, or 20-30 nucleotides in length, or any range using any of the above start and end points, or any length therebetween. The most suitable length of the template sequence is 10-30 nucleotides in length, most suitable is 10-20 nucleotides in length.

[0068] The extended primer template can be RNA, DNA, or a hybrid of RNA and DNA.For example, if the primer template is DNA and the extension portion is RNA, the extended primer template is a hybrid of RNA and DNA.The extended primer template can include one or more modified nucleotides.

[0069] The primer template contains a cleavable site that allows the synthesized oligonucleotide to be released from the template so that the primer template can be reused in one or more additional rounds of oligonucleotide synthesis.

[0070] Any suitable cleavable site may be used.

[0071] The cleavable site is suitably located in the stem portion of the nucleic acid molecule, upstream of the primer start site. The cleavable site may be located in the stem portion immediately adjacent to the primer start site, or may be within 1-5 nucleotides of the primer start site, preferably within 1-2 nucleotides of the primer start site, more preferably with one residue between the cleavage site and the primer start site. The cleavable site may be suitably selected such that cleavage does not alter either the oligonucleotide product or the primer template. In other words, apart from cleavage of the oligonucleotide from the primer template, cleavage is traceless and undetectable in the oligonucleotide product or in the primer template from which the oligonucleotide is released.

[0072] The cleavable site may comprise a cleavable bond, a cleavable nucleotide, or a specific sequence motif.

[0073] Cleavable linkages include modified 3'-5' internucleotide linkages replacing one of the phosphodiester groups, such as, for example, dialkoxysilane, phosphorothioate, phosphorodithioate, methylphosphonate, mesyl phosphoramidate and phosphoramidate internucleotide linkages.

[0074] Cleavable nucleotides may include, but are not limited to, deaminated bases, base analogs, abasic or urea sites, or mismatched nucleosides.Cleavable oligonucleotide analogs may also include a substituent or replacement at one of the bases or sugars, such as 7-deazaguanosine, 5-methylcytosine, inosine, uridine, etc.Cleavable nucleotides include base analogs that can be cleaved by endonucleases, preferably those that leave a free 3' hydroxyl group to allow for further rounds of extension. In some embodiments, cleavable nucleotides include nucleotides containing base analogs cleavable by formamidopyrimidine DNA glycosylase, including but not limited to 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoinosine, 7,8-dihydro-8-oxoadenine, 7,8-dihydro-8-oxonebularine, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, 5-hydroxycytosine, 5-hydroxyuracil. In some embodiments, cleavable nucleotides include nucleotides containing base analogs cleavable by thymine DNA glycosylase, including but not limited to 5-formylcytosine and 5-carboxycytosine. In some embodiments, cleavable nucleotides include nucleotides containing base analogs cleavable by human alkyladenine DNA glycosylase, including, but not limited to, 3-methyladenine, 3-methylguanine, 7-methylguanine, 7-(2-chloroethyl)-guanine, 7-(2-hydroxyethyl)-guanine, 7-(2-ethoxyethyl)-guanine, 1,2-bis-(7-guanyl)ethane, 1,N6-ethenoadenine, 1,N2-ethenoguanine, N2,3-ethenoguanine, N2,3-ethanoguanine, 5-formyluracil, 5-hydroxymethyluracil, and hypoxanthine.In some embodiments, the cleavable nucleotides include 5-methylcytosine, which is cleavable by 5-methylcytosine DNA glycosylase. Nucleotide analogs such as deoxyuridine or 8-oxo-deoxyguanosine, which are recognized by certain glycosylases (e.g., uracil deoxyglycosylase followed by endonuclease VIII and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, the cleavable nucleotides include nucleotides containing base analogs, such as inosine, uracil, hypoxanthine, xanthine, abasic sites, urea sites, base pair mismatch insertions and deletions, which are recognized by endonuclease V. A suitable cleavable nucleotide is inosine, which is cleaved by endonuclease V.

[0075] A cleavable sequence motif may be any sequence recognized by an enzyme that cleaves a sequence in a sequence-specific manner. For example, a cleavable sequence may be a specific sequence recognized by an enzyme, such as an endonuclease. Cleavage may be upstream, downstream, or within the sequence motif. By way of example, the sequence motif may be GAGTCNNNN*N (where N is any base and * is the cleavage site) recognized by NtBstNBI, GGATCNNNN*N recognized by NtAlwl, or GCTCTTCN*N recognized by NtBspQI. Other examples will be known to those skilled in the art. Preferably, the cleavage site (e.g., nucleotides) is located upstream of the primer start site, preferably within 1-5 nucleotides, preferably within 1-2 nucleotides of the polymerase start site, or immediately adjacent to the polymerase start site.

[0076] Preferably, a single cleavable site is positioned within the nucleic acid molecule to allow for reuse of the primer / template after release of the oligonucleotide product.Preferably, the oligonucleotide product produced in the reaction does not contain a cleavable site recognized by the cleavage system used in the reaction.

[0077] The cleavable site can be a deaminated base that is cleaved by endonuclease V, or the associated sequence for a nicking endonuclease.

[0078] Deaminated bases should not be used in therapeutic oligonucleotide products because they can form Watson-Crick base pairs with multiple bases and are therefore not specific.

[0079] The cleavable sequence may be a consensus sequence recognized by a restriction enzyme, e.g., an endonuclease enzyme. If the cleavable site is an enzyme binding site, the cleavable site may be generated during synthesis of the oligonucleotide molecule by locating the complement of the cleavable site in the template portion, such that the cleavable site is located within the newly generated sequence.

[0080] The primer template may include one or more motifs or sequences, including, for example, but not limited to, binding sites, labels, immobilization sites, and cleavable sites. Any one or more of such motifs or sequences may be present in or operably linked to the primer sequence, including the hairpin loop or single-stranded nucleic acid template, or both.

[0081] Immobilization sites or moieties may be provided for binding the nucleic acid molecules to a support, e.g., a solid support, for example, in forming an array. The immobilization site or binding site may be any suitable sequence or binder / moiety that mediates direct binding to the support or binding with a binding partner disposed on the support. Examples of binding partners include nucleic acid molecules, analytes / antibodies, oligonucleotide pairs, nucleic acid molecules and complementary sequences, aptamers, affinity binding proteins and oligonucleotide adaptors configured to bind with their binding partners to receptors or binding proteins, lipids, carbohydrates, etc. A suitable example is streptavidin, which can bind to biotinylated nucleic acid molecules.

[0082] The immobilization site may be located either in the loop, the stem, or downstream (5') of the template sequence of the nucleic acid molecule. Preferably, the immobilization site is located in a position such that immobilization of the nucleic acid molecule does not sterically interfere with the binding and / or activity of the polymerase or the cleavage system to the nucleic acid molecule. In a suitable embodiment, the immobilization site may be located within the loop of the hairpin loop, preferably at the midpoint of the loop. In a suitable embodiment, the immobilization site may be located within the template sequence, provided that the presence of the immobilization site does not affect the activity of the polymerase. In a suitable embodiment, the immobilization site may be located at the 5' end or downstream of the template sequence.

[0083] Primers and templates can be designed using any suitable technique available in the art, such as Primer-BLAST, and implemented using Primer 3, BLASTN, Melting, pandas, and the Python standard library.

[0084] Primer and template design parameters include the length and melting temperature of the annealing sequence. For a given sequence, increasing length can increase the strength of the specific binding interaction, but can also increase inappropriate ligation due to non-specific binding to off-target sequences and / or decrease the effective concentration of the probe in the reaction.

[0085] Self-priming templates can be prepared using any suitable method, such as, for example, enzymatic or chemical synthesis. Self-priming templates can be prepared using available recombinant or cloning methods well known to those of skill in the art. Suitable methods include enzymatic amplification of nucleic acids to provide multiple copies of the primer template.

[0086] II) Nucleotides Nucleotides are provided so that a polymerase can extend the 3' end of the stem portion to generate the desired oligonucleotide to obtain a sequence using the template. Nucleotides can include adenine, cytosine, thymine, guanine, and uracil. Nucleotides can be natural (unmodified) nucleotides, or modified or non-natural nucleotides, or nucleotide analogs, or combinations thereof. Non-naturally occurring analogs (or modified nucleotides) can include nucleotides that contain modified bases, sugars, or internucleoside linkages, such as phosphorothioate internucleoside linkages, 5'-N-phosphoramidite linkages, or bases with linking groups that allow for the attachment of labels, such as fluorophores or haptens. If the use of the oligonucleotide or polynucleotide requires enzymatic processing, such as extension by a polymerase or ligation by a ligase, it will be understood by those skilled in the art that the oligonucleotide or polynucleotide in such cases does not contain a particular analog of an internucleoside linkage, sugar moiety, or base at any or particular positions.

[0087] In some embodiments, the nucleic acid analog is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, , 2-thiocytidine, 2'-deoxynucleoside 5'-triphosphate (dNTP) analogs (including Sp-dNTPαS, Rp-dNTPαS, Sp-2'F-dNTP, and dNTP-αS), methylated bases, inserted bases, and combinations thereof. In some embodiments, the nucleic acid analog contains one or more modified sugars (e.g., 2'-fluororibose, 2'-methoxyribose, 2'-methoxyethoxyribose, ribose, 2'-deoxyribose, arabinose, hexose, or locked nucleic acids) compared to those found in commonly occurring, naturally occurring nucleic acids.

[0088] Suitably, a plurality (or population) of nucleotides is provided. The plurality of nucleotides may or may not be of the same type of base (e.g., A, T, G, C, U, etc.). For example, the solution may or may not include only one type of base. The solution may include at least one type of base, or at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten types of base. For example, the solution may include all possible mixtures of A, T, C, U, and G. All possible mixtures of A, T, C, and G may be provided. The plurality of natural and non-natural nucleotides may or may not be of the same type of base (e.g., A, T, G, C). In some cases, the solution may include a plurality of natural and non-natural nucleotides. The plurality of natural and non-natural nucleotides may or may not be of the same type of base (e.g., A, T, G, C). As described herein or known to those skilled in the art, the plurality of nucleotides may be substantially all natural nucleotides or substantially all modified nucleotides. The plurality of nucleotides may be a mixture of natural and modified nucleotides. In such a mixture, the ratio of modified nucleotides:natural nucleotides may depend on the nature of the oligonucleotide to be produced and the proportion of modified nucleotides therein. Suitable ratios of modified nucleotides:natural nucleotides may be 1:99, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50. 60:40, 70:30, 80:20, 90:10, 95:5 or 99:1, or any range between any of the upper and lower ranges listed above, or any integer therebetween.

[0089] One or more of the nucleotides of the plurality of nucleotides may be labeled with a dye, a fluorophore, or a quantum dot. For example, the solution may include labeled nucleotides. In another example, the solution may include unlabeled nucleotides. In another example, the solution may include a mixture of labeled and unlabeled nucleotides.

[0090] The nucleotides can be provided in any suitable concentration or amount, which can be calculated based on the length of the template and the number of rounds of oligonucleotide synthesis expected in a single reaction. The nucleotides can be provided in a suitable amount such that at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, or substantially all, of the provided nucleotides are used in the reaction. Thus, the amount of nucleotides can be calculated for each reaction based on the knowledge of the skilled artisan. Alternatively, the nucleotides can be provided in excess, for example when the number of rounds of extension is not predetermined. The nucleotides can be provided in 1.5-fold, 2-fold, 3-fold, 4-fold or 5-fold excess compared to the calculated amount of nucleotides required for the desired number of oligonucleotides to be produced in the reaction. As an example, the "optimized" reaction shown in FIG. 8 uses 4 μM template and completes 53 cycles to produce 212 μM product. This oligonucleotide product contains 18 nucleotides and 3.78 mM dNTPs were used in the reaction. This reaction condition resulted in 16 mM nucleotides, i.e., a 4-fold excess.

[0091] III) Polymerase The polymerase used in the present invention may be any suitable enzyme capable of synthesizing a chain of nucleotides. The polymerase may be an RNA polymerase or a DNA polymerase. Suitably, the polymerase used in the present invention may be capable of incorporating modified or non-natural nucleotides into a nucleic acid chain. The polymerase may be naturally occurring or engineered. Suitable polymerases may be of eukaryotic or prokaryotic origin.

[0092] Suitable polymerases may be those that can tolerate high concentrations of dNTPs without substantially affecting their activity or performance. Examples of such polymerases are described herein. Examples of polymerases that can tolerate high concentrations of dNTPs include KOD or its variants.

[0093] A suitable polymerase may be a thermostable polymerase, such as Taq polymerase, pfu from Pyrococcus furiosus, or KOD from Thermococcus kodakaraensis (Tkod-pol), or variants or modified forms thereof. Other suitable polymerases that may be suitable for use in the present invention include the Klenow fragment from Escherichia coli, T4 and T7 polymerases, SFP1, Stoffel variant SF4-6, Stoffel homologue (Mhpol) from Thermus filiformis and Marinithermus hydrothermalis, and Family B polymerase 9°N, Q5 DNA polymerase, and variants or modified forms thereof. A list of modified polymerases that may be suitable for use in the present invention is provided in Trends in Biotechnology, October 2019, Vol. 37, No. 10 https: / / doi.org / 10.1016 / j.tibtech.2019.03.011 or are known to those skilled in the art. Variants may have one or more improved properties suitable for the present invention, such as improved fidelity, stability, or the ability to provide a substantially single stereoisomer. Suitable variants include, for example, SF4-6, TfPol*, and MhPol*, as provided herein. Most preferably, SF4-6, TfPol*, and MhPol* may be useful for generating a population of oligonucleotides of substantially the same stereoisomer.

[0094] Also provided herein are polymerases having a sequence defined in Table 3 or a sequence having at least 80%, 85%, 90% or 95% or more sequence identity thereto. Suitable modified polymerases are capable of extending RNA or DNA sequences, capable of accepting one or more modified nucleotides into a nucleotide chain, and having one or more of the nucleotide mutations set forth in Table 3. Such polymerases can be used in the methods of the invention or provided in the kits of the invention. The polymerases of the invention can tolerate high concentrations of nucleotides such that concentrations of nucleotides greater than 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mM do not significantly inhibit or affect polymerase activity.

[0095] Any suitable amount of polymerase can be used in the reaction. The amount of polymerase can be selected based on the characteristics of the reaction, such as the amount of product produced, the concentration of nucleotides, the properties and efficiency of the polymerase, and the properties of the cleavage agent. A person skilled in the art can determine the appropriate amount of primer using the information provided herein and the general knowledge of the person skilled in the art. Suitably, 10 mol%, 9 mol%, 8 mol%, 7 mol%, 6 mol%, 5 mol%, 4 mol%, 3 mol%, 2 mol% or 1 mol% or less of polymerase can be used in the method of the present invention. A suitable range can be 0.1 to 5 μM.

[0096] The polymerase used in the present invention can be produced by any suitable method, for example, the polymerase can be recombinantly produced in a host cell. Suitable host cells are well known in the art and can be microbial cells, mammalian cells, or cell lines. Suitable host cells can be bacterial cells, such as E. coli.

[0097] V Cleavage Agent The reagents of the present invention comprise a suitable cleavage agent capable of mediating cleavage at a cleavable site in the extended nucleic acid molecule to allow dissociation of the synthesized oligonucleotide from the nucleic acid molecule template. Suitably, the cleavage agent is site-specific so as to cleave the extended primer template at a specific site.

[0098] The term "cleavage agent" as used herein refers to a substance that can cleave the bond between the newly formed oligonucleotide and the primer. The cleavage agent suitable for use in the present invention can appropriately cleave the oligonucleotide from the primer template, leaving a free 3'OH group at the end, so that the primer template can be recycled and used again as a template for oligonucleotide synthesis. The cleavage agent can be a chemical cleavage agent, an enzymatic cleavage agent, or a photocleavage agent, or any other suitable reagent or system. Preferably, the cleavage agent is an enzymatic cleavage agent.

[0099] A suitable cleavage agent may be one that can tolerate high concentrations of dNTPs without substantially affecting its activity or performance, examples of such cleavage agents are described herein, such as an enzymatic cleavage agent, such as endonuclease V, preferably TnEndoV.

[0100] Examples of suitable enzyme cleavage systems include any enzyme capable of cleaving a single strand of nucleic acid, such as a restriction enzyme or an endonuclease. Examples of endonucleases suitable for use in the present invention include TmEndoV (TmEndoV) from Thermotoga maritima, TmEndoV from Thermotoga neapolitana, PtEndoV from Pseudothermotoga thermarum, TmeEndoV from Thermosipho melanesiensis, and TaEndoV from Thermosipho atlanticus, or the nicking endonucleases Nt BstNBI, NtAlwl, and Nt BspQI. Protein sequences are found in Table 2. Also included are endonucleases that are substantially identical to the endonucleases in Table 2.

[0101] The enzymatic cleavage agent can be one that can tolerate high concentrations of nucleotides such that concentrations of nucleotide greater than 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM do not significantly inhibit or affect enzyme activity.

[0102] Preferred endonucleases are specific for deaminated bases such as inosine. Endonuclease V cleaves / hydrolyzes the second phosphodiester bond 3' to the inosine.

[0103] The cleavage can be done in a single step or in a multi-step process.

[0104] If the cleavage agent requires binding of an enzyme or reagent to the nucleic acid molecule, it is expected that the endonuclease will not bind to the template until the polymerase is released, to avoid steric constraints due to the size of the polymerase.

[0105] In the case of endonucleolytic cleavage, the product must be double-stranded in order to be bound and cleaved. In a further embodiment, the cleavage solution comprises one or more buffers. It will be appreciated by those skilled in the art that the choice of buffer will depend on the exact cleavage chemistry and cleavage agent required.

[0106] Cleavage can usually be performed in an aqueous system. Alternatively, cleavage can occur in the presence of an alternative nucleophile, which is incorporated into the 5' phosphate group of the oligonucleotide product. This incorporation of the nucleophile results in a modified oligonucleotide suitable for conjugation with a delivery vehicle, such as an antibody, peptide, protein, or sugar, for targeted delivery to a specific organ. Any suitable nucleophile can be used, such as, for example, but not limited to, glycerol, ethylene glycol, 1,2-propanediol, or 1,3-diaminopropanol. The appropriate concentration can be determined to achieve the desired incorporation. For example, at least 30%, 40%, 50%, 60%, 70%, or 80% w / v, or any suitable range between the integers listed above, can be used.

[0107] VI) Other Reagents For example, but not limited to, washes, buffers, primers, enzymes, catalysts, quenchers, dyes, probes, tags, labels, cofactors, fluidic components (e.g., detergents, buffers, triton X-100, nonidet P-40, DMSO, etc.), and / or optical components (e.g., reference beads, dyes, etc.), and magnesium ions (Mg 2+ ), manganese ion (Mn2 + ), glutamine, arginine, tetramethylammonium chloride, betaine, formamide, bovine serum albumin, or any combination thereof may be provided.

[0108] A widely used variant of the dNTP backbone is phosphorothioate (also called S-oligo), in which the phosphodiester bond is converted to a phosphorothioate bond. The sulfurization process produces R and S diastereomers. Various cofactors can be applied to the reaction mixture to induce the synthesis of R or S stereoisomers. For example, if the polymerase naturally accepts the S isomer, it may be appropriate to apply a cofactor to induce the polymerase toward the R isomer, so that both forms will be used. For example, if KOD is used, it may be appropriate to use a cobalt cofactor in combination. A suitable reagent for use in the reaction may be a cobalt salt, such as cobalt chloride. Other suitable combinations of polymerases and cofactors can be determined using the teachings herein and are available in the art.

[0109] A suitable buffer for use in the present invention is one that provides aqueous conditions. Thus, the buffer is suitably an aqueous buffer. A suitable buffer may comprise one or more reagents selected from Tris-HCl, Hepes, ammonium sulfate, potassium chloride, calcium chloride, magnesium sulfate, glutamine, arginine, formamide, nonidet, and Triton X-100. A suitable buffer may comprise Tris-HCl, (NH4)2SO4, KCl, Triton X-100, and MgSO4. For example, a suitable buffer may comprise 20 mM Tris-HCl, 10 mM (NH4)2SC4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, pH 8.8. Another suitable buffer may comprise Tris-HCl, MgCl2, potassium glutamate, arginine-HCl, acetylated BSA, DTT, trehalose, and 1,2-propanediol. For example, a suitable buffer can include 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM potassium glutamate, 100 mM arginine-HCl, 0.01 mg / ml acetylated BSA, 10 mM DTT, 0.2 M trehalose, and 1 M 1,2-propanediol. Another suitable buffer can include Tris-AcOH, MgSO4, KOAc, potassium glutamate, arginine-HCl, acetylated BSA, Triton X-100, and DTT. For example, a suitable buffer can include 50 mM Tris-AcOH (pH 8), 20 mM MgSO4, 50 mM KOAc, 100 mM potassium glutamate, 100 mM arginine-HCl, 0.01 mg / ml acetylated BSA, 0.1% Triton X-100®, and 30 mM DTT. Another suitable buffer can include Tris-HCl (pH 8.8), (NH4)2SO4, KCl, MgSO4, Triton X-100, and NiCl220. For example, a suitable buffer can include 20 mM Tris-HCl (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-100, and 2 mM NiCl2.Another suitable buffer can include Tris-HCl (pH 8), MgCl2, potassium glutamate, arginine-HCl, acetylated BSA, DTT. For example, a suitable buffer can include 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM potassium glutamate, 100 mM arginine-HCl, 0.01 mg / ml acetylated BSA, 10 mM DTT. Alternative buffers can be identified by one of skill in the art using the teachings herein and are available in the art.

[0110] Suitable combinations of buffers and enzymes can be determined using the teachings herein and are available in the art. For example, any one of the above buffers can be used in combination with a polymerase selected from KOD or its variants, such as KOD DGLNK, or TfPol or its variants.

[0111] Suitably, the reagents used in the methods of the present invention are aqueous based.

[0112] Suitably, the reagents used in the methods of the present invention are substantially free of acetonitrile.

[0113] combination In the present invention, suitable reagents include enzyme combinations (i.e., polymerase and endonuclease) appropriately combined with any reagent, such as one or more of the reagents defined herein, that are appropriately optimized for generating the desired oligonucleotide. Preferably, the reaction includes a primer template, a polymerase, an endonuclease, dNTPs, and a buffer. Preferably, the reaction includes a self-priming template, a polymerase, an endonuclease, dNTPs, and a buffer. The polymerase may be KOD, TfPol*, KOD DGLNK, or SFM4-6. The endonuclease may be endonuclease V, preferably TnEndoV.

[0114] In one embodiment, the present invention may comprise a polymerase selected from the group in Table 3 and an endonuclease selected from the group in Table 2. All such combinations are included within the scope of the present invention, together with the primer template, or specifically the self-priming template. Preferably, the endonuclease is TnEndoV. Preferably, the nucleotide may comprise one or more types of modified nucleotides. Preferably, the cleavable site is a cleavable nucleotide, such as inosine.

[0115] In one embodiment, the polymerase may be a modified polymerase as shown in Table 3 and the endonuclease may be endonuclease V. Suitably, the endonuclease is TnEndoV. One or more nucleotides may be modified nucleotides. Suitably, the cleavable site may be an inosine residue. Suitably, the buffer may be as provided herein.

[0116] method The present invention provides a method for producing a single stranded oligonucleotide, comprising the steps of: i) providing a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; ii) incubating the primer template with a nucleic acid polymerase, one or more dNTPs, and a cleavage agent to form a reaction mixture; iii) maintaining the reaction mixture under conditions that allow for extension of the primer by a polymerase to form an extended primer template and cleavage of the extended primer template at the cleavable site by a cleavage agent; and iv) optionally separating the oligonucleotide products from the reaction mixture.

[0117] The primer template, polymerase, dNTPs, and / or cleavage agent can each be individually as described herein. Any suitable combination of primer templates, polymerases, dNTPs, and / or cleavage agents described herein can be used in the methods of the invention.

[0118] The nucleotides may be naturally occurring nucleotides or may include one or more modified nucleotides, e.g., as described herein. The nucleotides may be provided in excess of the amount required, e.g., as described herein.

[0119] In a suitable embodiment, the method is for producing a therapeutic oligonucleotide. Suitably, the cleavage agent is TnEndoV and the nucleotide comprises one or more modified nucleotides.

[0120] The method of the invention may be a single-step method or may comprise two or more consecutive steps. In a single-step method, all reagents can be added in a single step under suitable conditions for the extension and cleavage reactions to proceed. The method of the invention may be a one-pot method, where extension and cleavage occur in a single reaction vessel.

[0121] Maintaining the reaction mixture as defined in step iii) can be carried out for a reaction time, preferably a defined reaction time. During the defined reaction time, both extension and cleavage can occur in the reaction mixture, preferably within the same reaction time. Thus, the reaction can include repeated cycles of extension and cleavage within the same reaction mixture during the reaction time. In this way, the primer template can be continuously reused.

[0122] Suitably, extension and cleavage may occur under the same set of reaction conditions, which may include the presence of a suitable buffer and one or more additional reagents as defined herein, at a suitable temperature for a defined reaction time.

[0123] Suitably, the stretching and cleavage may occur in any preceding or subsequent step iii) above, in separate steps and / or vessels, or in the same step and / or vessel as steps i) to iv), and the conditions, or part of the conditions, of the preceding or subsequent steps may be the same or different.

[0124] The methods of the invention may include two or more methods carried out in parallel, for example on one array or on multiple arrays. Each reaction may include the same or different templates.

[0125] The method of the present invention can be carried out under any suitable conditions to extend the primer sequence and maintain the primer-template duplex appropriately to generate new oligonucleotide sequences. If a self-priming template is used, the conditions are suitable to allow the maintenance of secondary structures, such as hairpin loops, during the reaction time. Suitable conditions include suitable temperature, pH, buffer, incubation time and salt concentration. The conditions of any step of the method can be the same or different from the conditions of one or more other steps.

[0126] Step iii) can be performed at a temperature equal to or greater than the melting temperature of the template and cleavage product. At such a temperature, the extension product (oligonucleotide product) is released from the primer template after cleavage, and the primer template is available for another extension cycle. However, the primer remains bound and does not dissociate during the reaction conditions. The appropriate temperature can be calculated by the skilled artisan based on factors such as the length of the template and extension product, and their sequences, as well as the optimum temperature of the polymerase and cleavage agent. Longer oligonucleotide products have higher melting temperatures than shorter products. A suitable temperature when using a self-priming template for the synthesis of longer oligonucleotide products, such as 18-mers, can be 60-85°C, or any integer or range therebetween. A more suitable temperature range can be 65-80°C, or any integer therebetween, such as about 70°C. If the targeted therapeutic oligonucleotide is shorter, a lower temperature can be used, such as 45-60°C, or any integer or range therebetween. Alternatively, the temperature can be cycled between a first temperature suitable for primer extension and a second temperature suitable for cleavage.

[0127] The combination of providing a polymerase and a cleavage agent in the same reaction under conditions that allow dissociation of the oligonucleotide product from the primer template allows the primer template to be used repeatedly in a reaction. The primer template can be referred to as catalytic because it mediates an increase in product over the usual 1:1 ratio of template:product in a conventional extension reaction and remains unchanged by the extension or cleavage steps.

[0128] Thus, the methods of the invention allow for the production of more than 1-fold, or at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, or 200-fold, 300-fold, 500-fold, or 1000-fold amounts of oligonucleotide products compared to the amount of primer template at the start of the reaction. The amounts can be expressed as mol %.

[0129] The extension reaction may occur at a suitable pH, e.g., 8.0 to 9.5, representing a pH suitable for the activity of the polymerase. A suitable buffer, such as Tris-HCl, or a suitable buffer with one or more buffering agents described herein, or equivalents known to those skilled in the art, may be provided to stabilize the pH.

[0130] The reaction time is determined by the amount of product required and the number of extension cycles. Typical extension times can be 1-2 minutes per cycle. Reactions can be incubated for minutes to hours, or up to a day or more, to obtain suitable yields. The optimal incubation time for extension can be calculated for each reaction. Optimal reaction times can be 12 hours or more. Reaction times can be based on the number of extension cycles, which can be 2 or more, 10 or more, 50 or more, 100 or more, 200 or more, 300 or more, 500 or more, or 1000 or more, and this method can allow for accumulation of oligonucleotide products in the reaction mixture.

[0131] The conditions under which the extension reaction is carried out can be aqueous, hi one embodiment, the conditions are substantially free of acetonitrile.

[0132] The method may include providing a suitable buffer in which reagents are combined for performance of the extension reaction. The suitable buffer may include one or more components selected from buffer salts (e.g., Tris-HCl, Tris-AcOH, HEPES-KOH, bicine-HCl), metal ions (e.g., MgSO4, MgCl2, MgOAc2, CaCl2), amino acids (e.g., glutamic acid, arginine, proline, glycine, serine, aspartic acid), and additives (e.g., sodium citrate, (NH4)2SO4, tetramethylammonium chloride (TMAC), KCl, KOAc, glycerol, 1,2-propanol, sucrose, trehalose, acetylated BSA, Tween-20, Triton X-100, DTT, formamide, DMSO, and / or PEG-3000). Suitable buffers may include one or more cosolvents including magnesium, potassium chloride, Tris-HCl, ammonium sulfate, e.g., DMSO, glycerol, formamide, BSA, PEG, gelatin, non-ionic detergents (TWEEN 20 or Triton-X-100), and N,N,N trimethylglycine. The most suitable buffer may include 20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8. Another suitable buffer may include 50 mM Tris AcOH, 20 mM MgSO4, 50 mM KOAc, 100 mM KGIu, 100 mM ArgHCL, 0.01 mg / ml AcBSA, 0.1% Triton X-100, 30 mM DTT, 5% formamide. Other suitable buffers may be described herein or available as known to those skilled in the art.

[0133] When the polymerase is KOD, the cleavage agent can be endonuclease V, e.g., TnEndoV (2 μM). The amplification catalyzed is 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM arginine-HCl (pH 8), 100 mM glutamic acid-KOH (pH 8), DTT (10 mM), formamide (10%), trehalose (0.2 M), 1,2-propanediol (1 M), and acetylated BSA (0.01 mg / ml). An example of optimized conditions for large-scale reactions is 50 mM Tris AcOH, 20 mM MgSO4, 50 mM KOAc, 100 mM KGIu, 100 mM ArgHCL, 0.01 mg / ml AcBSA, 0.1% Triton X-100, 30 mM DTT, 5% formamide, pH 8.0, S p -dGTPαS (2.5 mM), S p -dCTPαS (2.5 mM), S p - The reaction was stopped by incubating at 70°C for 12 hours or more in a buffer containing dTTPαS (5 mM), template (4 μM), KOD (2 μM), TnEndoV (6 μM) and 0.012 U / μl TIPP (inorganic pyrophosphatase), and heating at 98°C for 2 hours.

[0134] The amounts of enzyme and nucleotides can be determined empirically based on, for example, the properties of the polymerase, as well as the concentrations of nucleotides, chelators and proteins present. Either reagent can be provided in excess.

[0135] Cleavage can be achieved enzymatically or by light-based cleavage or any other suitable mechanism. The conditions required for cleavage will depend on the nature of the cleavage system used and are well known to those skilled in the art. The most suitable conditions are those that do not affect or do not substantially affect the function of reagents such as enzymes in the reaction, thereby allowing multiple cycles.

[0136] The methods of the invention can be used, for example, to generate double-stranded DNA by performing the methods of the invention to generate two complementary strands of a single-stranded nucleic acid and carrying out an annealing step to generate a double-stranded oligonucleotide.

[0137] The methods of the invention can also include one or more additional steps, such as stopping, terminating or quenching the reaction, extracting the protein, extracting the product, washing the product, purifying the product, modifying or adapting the oligonucleotide product, for example, by ligation to another molecule or cleavage into different oligonucleotides. The methods can include preserving the product, for example, by lyophilizing, packaging and / or storing the product.

[0138] Suitable methods and conditions for purification will be known to those skilled in the art.Any suitable method can be used to extract the oligonucleotide product, preferably without the use of a primer / template nucleic acid molecule.Examples of suitable methods are described herein and include selective crystallization, ethanol / chloroform precipitation, or the use of established filtration systems.

[0139] Solid Supports / Arrays The present invention provides one or more primer templates of the present invention provided on a support, for example, a solid substrate.The immobilization of nucleic acid molecules on a surface can be reversible or irreversible.Any suitable immobilization method can be used, such as by the nucleic acid sequence that binds to the surface or by incorporating a suitable moiety that mediates binding to the surface.For example, suitable binding motifs can include sequences that bind to complementary sequences on solid support, such as polyA sequences.

[0140] Binding can be mediated through an immobilization site within the nucleic acid molecule and a complementary site or binding moiety provided on the solid support. Alternatively, the nucleic acid sequence can be non-covalently bound to the support, for example by physical adsorption.

[0141] The substrate may be a solid substrate. The substrate may comprise, in whole or in part, one or more of rubber, glass, silicon; metals such as aluminum, copper, titanium, chromium, or steel; ceramics such as titanium oxide or silicon nitride; plastics such as polyethylene (PE), low density polyethylene (LDPE), high density polyethylene (HDPE), polypropylene (PP), polystyrene (PS), high impact polystyrene (HIPS), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC), acrylonitrile butadiene styrene (ABS), polyacetylene, polyamide, polycarbonate, polyester, polyurethane, polyepoxide, polymethyl methacrylate (PMMA), polytetrafluoroethylene (PTFE), phenol formaldehyde (PF), melamine formaldehyde (MF), urea formaldehyde (UF), polyether ether ketone (PEEK), polyetherimide (PEI), polyimide, polylactic acid (PLA), furan, silicone, polysulfone, any mixture of any of the above materials, or any other suitable material. The substrate may be wholly or partially coated with one or more layers of metals such as aluminum, copper, silver, gold, oxides such as silicon oxide (SixOy, where x, y can take any possible value), photoresists such as SU8, surface coatings such as aminosilanes or hydrogels, polyacrylic acid, polyacrylamide dextran, polyethylene glycol (PEG), or any combination of any of the above materials, or any other suitable coating.

[0142] The surface of the substrate may be modified to include any of the immobilization reagents or binding partners described herein. The surface of the substrate may be modified to include active chemical groups such as amines, esters, hydroxyls, epoxides, etc., or combinations thereof. In some cases, such binding partners, chemicals, proteins, nucleic acid sequences, or surface modifications may be applied to the support as an additional layer or coating.

[0143] The substrate may have the general shape of a cylinder, a cylindrical shell or disk, a cuboid, or any other geometric shape. The surface of the substrate may be planar. The surface of the substrate may be uncoated and exposed to the atmosphere. Alternatively, or in addition, the surface of the substrate may be textured or patterned. For example, the substrate may include grooves, valleys, peaks, and / or pillars. The substrate may define one or more cavities (e.g., microscale cavities or nanoscale cavities). The substrate may define one or more grooves. The substrate may have a regular texture and / or pattern across the surface of the substrate. For example, the substrate may have regular geometric structures (e.g., wedges, cuboids, cylinders, spheroids, hemispheres, etc.) above or below a reference level of the surface. Alternatively, the substrate may have an irregular texture and / or pattern across the surface of the substrate. For example, the substrate may have any structure above or below a reference level of the substrate.

[0144] The substrate may include an array. For example, the array may be disposed on a side of the substrate. The array may be a planar array. The array may have the general shape of a circle, ring, rectangle, or any other shape. The array may include linear and / or non-linear rows. The array may be equally spaced or distributed. The array may be arbitrarily spaced or distributed. The array may have regular spacing. The array may have irregular spacing. The array may be a textured array. The array may be a patterned array. The array may include a plurality of individually addressable locations.

[0145] The primer template can be immobilized on an array. Thus, the array can include one or more binding partners or means for mediating the immobilization of the primer template, as described herein. Such means can include one or more physical or chemical linkers or adapters. Alternatively, or in addition, the primer template can be bound to a bead, and the bead can be immobilized on the array.

[0146] Any number of primer templates can be immobilized on the support, for example, the support can immobilize at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 samples, and can immobilize thousands, tens of thousands, hundreds of thousands, millions, hundreds of millions, thousands, or more primer templates.

[0147] The primer template can be immobilized through non-specific interactions, such as one or more of hydrophilic interactions, hydrophobic interactions, electrostatic interactions, physical interactions (e.g., adhesion), and the like. The nucleic acid molecule can be immobilized by specific interactions, for example, via a binding partner. For example, the binding partner can include a nucleic acid molecule or an oligonucleotide adaptor configured to bind to one or more of an antibody, an oligonucleotide, a nucleic acid molecule, an aptamer, an affinity binding protein, a lipid, a carbohydrate, and the like. A suitable example is streptavidin, which can bind to a biotinylated nucleic acid molecule. The binding partner can immobilize the biological analyte by any possible combination of interactions, for example, by a combination of physical interactions and chemical interactions, a combination of protein and nucleic acid interactions, and the like.

[0148] The support can include one or more features, and multiple binding partners can be present on the same feature on the support, for example, thousands, tens of thousands, hundreds of thousands, millions, hundreds of millions, thousands, or more. The array can have a number of binding agents within a range defined by any two of the above values. In some cases, a single binding partner can bind to a single primer template. In some cases, a single binding partner can bind to multiple primer templates. In some cases, multiple binding partners can bind to a single primer template.

[0149] kit One aspect of the present invention provides a kit. The kit may allow for the storage, transport, or provision of one or more of the reaction reagents described herein, together with any additional elements, such as substrates, supports, and / or instructions for carrying out the method and / or for supporting materials from one location to another, preferably in a suitable container. For example, the kit may include one or more enclosed members (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. Such contents may be provided to the intended recipient together or separately. For example, a first container may contain the nucleic acid molecules used in the present invention, and a second, or more containers may contain the nucleotides, polymerases, and / or buffers described herein.

[0150] The kit may further comprise a support to which the primer template as described herein is preferably bound. The support may comprise a population of nucleic acid molecules bound to the support, for example as described herein. The support provided in the kit may be a support as described above. The kit may comprise one or more supports as described herein. In each support or in each kit, the nucleic acid molecule may comprise different templates for synthesizing different oligonucleotide products.

[0151] Oligonucleotide Products The methods of the invention are primarily directed to the production of a single-stranded oligonucleotide that is cleaved and released from the template sequence. Downstream processing can use the single-stranded oligonucleotide to produce a double-stranded oligonucleotide product.

[0152] The oligonucleotides produced by the method of the first embodiment are substantially the same length as the template sequence. Thus, the length of the oligonucleotides produced by steps i) to iv) of the method of the first embodiment is 2 to 500 nucleotides, more preferably 2 to 200 nucleotides. The oligonucleotides may be, for example, 5 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 250 nucleotides in length.

[0153] The length of the oligonucleotide can be increased or decreased as a result of downstream processing, such as cleavage, splicing, or recombination.

[0154] Products of the method of the first aspect of the invention comprising oligonucleotides may suitably be at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% pure. Thus, products produced by the method of the first aspect of the invention may suitably contain less than 20, 15, 10 or 5% contaminants. Contaminants may include products comprising oligonucleotides containing base substitutions, truncated sequences and / or over-extended sequences (including additional bases), or products containing salts.

[0155] When oligonucleotide is mentioned herein, it includes one or more oligonucleotides.Thus, the method of the present invention can generate one or more target oligonucleotides, but requires multiple cycles of extension and cleavage.Thus, the product of the method of the present invention can be a population of oligonucleotides.Preferably, the population includes single-stranded oligonucleotides.

[0156] One of the most common modifications of nucleic acid sequences is the introduction of phosphorothioate (PS) bonds, in which one of the non-bridging oxygen atoms in the phosphate group is replaced by sulfur. This PS modification renders oligonucleotides more stable against nucleolytic degradation and offers significant pharmacokinetic advantages due to increased protein binding. Substitution of phosphate with phosphorothioate results in the formation of a chiral center at the phosphorus atom (Sp or Rp). Thus, the introduction of all PS bonds into oligonucleotides would generate two sets of diastereoisomers, and in the absence of stereocontrolled synthesis, N-mer phosphorothioate oligonucleotides would be 2N-mers, each of which would have different and potentially opposing physical and biochemical properties. -1 The two stereoisomers are designated S and R. Given an Sp / Rp mixture of nucleotides, the polymerases defined herein can use only Sp-dNTPs to give the product as a single diastereoisomer.

[0157] The population of oligonucleotides may be a population of mixed diastereoisomers (S and R) or may be substantially a single stereoisomer. The population may be substantially the S or R stereoisomer. Substantially a single isomer means that the population contains at least 80%, 85%, 90%, 95% or 99% of a single stereoisomer.

[0158] The oligonucleotides synthesized by the methods of the invention may be any oligonucleotide, for example, those with industrial or therapeutic applications, or those with applications or roles in research. In some embodiments, the oligonucleotides synthesized by the methods of the invention have enzymatic activity. In some embodiments, the oligonucleotides synthesized by the methods of the invention perform a mechanistic function, for example, in a ribonucleoprotein complex or transfer RNA. In some embodiments, the oligonucleotides synthesized by the methods of the invention can function as aptamers. In some embodiments, the oligonucleotides synthesized by the methods of the invention can be used for data storage. In some embodiments, the oligonucleotides synthesized by the methods of the invention may be therapeutic oligonucleotides, for example, RNAs that target nucleic acids (either RNA or DNA), RNAs that target proteins, or RNAs that code for therapeutic proteins.Therapeutic RNA oligonucleotides include: i) aptamers, which are short, single-stranded nucleic acids that can bind to various targets such as proteins, peptides, carbohydrates, and other molecules due to the tertiary structure of the aptamer rather than its sequence, e.g., pegaptanib (Macugen, Bausch+Lomb Pharmaceutical Retina Portfolio), Emapticap pegol (NOXXON Pharma), olaptesed pegol (NOXXON Pharma), and REG1; ii) mRNAs, e.g., mRNA as replacement therapy, where mRNA is administered to a patient to compensate for a missing gene / protein (e.g., but not limited to, AZD8601 (Moderna), mRNA-3704 (Moderna), MRT5005 (Translate iii) single-stranded antisense oligonucleotides (ASOs), including either RNase H-dependent ASOs or RNase H-independent (stereoblock) ASOs, such as, but not limited to, nusinersen (also known as Spinraza), BNT131 (SAR441000), CV8102 (CureVac) or MEDI1191, (or mRNA is administered to deliver a therapeutic protein); vaccination, in which mRNA encoding a specific antigen is administered to elicit protective immunity (e.g., but not limited to, COVID-19 vaccine or cancer vaccine); or cell therapy, in which cells are transfected with mRNA ex vivo to alter cell phenotype or function and these cells are then administered to a patient (e.g., but not limited to, TriMix-based immunotherapy (ECI-006), MCY-M11 (MaxCyte), or iv)lonis Pharmaceuticals), eteplirsen (Sarepta Therapeutics) and inotersen (lonis Pharmaceuticals and Akcea Therapeutics); and RNAi (miRNA or siRNA) (short single-stranded DNA, phosphorothioate DNA, RNA analogs, conformationally restricted nucleosides (locked nucleic acids, LNA), or oligonucleotides complementary to specific regions of targeted RNA to promote protein degradation or inhibit translation). MicroRNAs (miRNAs) are small non-coding RNA molecules that control the expression of multiple mRNAs by inhibiting the translation of target mRNAs or promoting their degradation. siRNAs are small non-coding RNA duplexes derived from precursor siRNAs. The methods of the invention can be used to generate double-stranded oligonucleotides, e.g., they can be synthesized separately and annealed under appropriate conditions.

[0159] cell A cell or population of cells can be provided that contains a nucleic acid molecule of the invention, or a nucleic acid sequence encoding a nucleic acid molecule of the invention. Suitable mammalian and bacterial cells are well known to those skilled in the art.

[0160] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations thereof mean "including, but not limited to," and are not intended to (and do not) exclude, for example, other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating the plural as well as the singular, unless the context requires otherwise.

[0161] An element, integer, property, compound, chemical moiety, or group described in connection with a particular aspect, embodiment, or example of the invention should be understood as being applicable to any other aspect, embodiment, or example described herein, unless specifically incompatible. All of the elements disclosed in this specification (including all accompanying claims, abstracts, and drawings) and / or all of the steps of any method or process disclosed herein can be combined in any combination, except combinations in which at least some of these elements and / or steps are mutually exclusive. The invention is not limited to the details of any of the above embodiments. The scope of the invention extends to any novel one or any novel combination of the elements disclosed in this specification (including all accompanying claims, abstracts, and drawings) or to any novel one or any novel combination of the steps of any method or process disclosed herein.

[0162] The reader's attention is directed to all articles and documents related to this application that have been filed contemporaneously or prior to this application and that are hereby open to public inspection, the contents of all such articles and documents being incorporated herein by reference.

[0163] Features of any one embodiment described herein apply to other embodiments mutatis mutandis. EXAMPLES

[0164] Working Example We first evaluated the extension and cleavage reactions using commercially available enzymes alone, and characterized the products by LC-MS analysis. Polymerase from Thermococcus kodakaraensis (KOD) efficiently catalyzed the extension of the hairpin template (T1, Table 1a, 20 μM) with unmodified dNTPs (1 mM) to generate a double-stranded extension product (E1, Table 1a) with quantitative conversion (Figure 4A). Cleavage reactions performed using chemically synthesized E1 standards (Table 1a, 20 μM) and EndoV from Thermotoga maritima (TmEndoV) were completed to yield an 18-mer product (P1, Table 1) and template (T1, Table 1a) (Figure 4B). A one-pot polymerase endonuclease reaction using KOD, TmEndoV, T1 (Table 1a, 20 μM) and dNTPs (1 mM) was then performed to yield 29 μM P1, corresponding to 1.4 cycles of template extension and cleavage (Figure 4C).

[0165] The effect of increasing amounts of substrate (dNTP) added on the polymerase extension and endonuclease cleavage reactions was examined. KOD was able to efficiently extend T1 in the presence of 30 mM dNTPs (Figure 5). A panel of TmEndoV homologs identified from a BLAST search of the NCBI database (Table 2) was screened for improved activity in the presence of high concentrations of dNTPs. TnEndoV from Thermotoga neapolitana performed best at high dNTP concentrations, effectively hydrolyzing E1 (Table 1, 20 μM) in the presence of 30 mM dNTPs (Figure 5B). To effectively dissociate the cleaved product from the template and allow multiple cycles of template extension and cleavage, the reaction is preferably performed at a temperature higher than the melting temperature of the product. KOD and TnEndoV have optimum temperatures of >100°C and 80.5°C, respectively (Figure 6A) and retained 100% and 62% of their extension and cleavage activities after 24 h preincubation at the optimum reaction temperature of 70°C (Figures 6C-6D). A panel of templates with different hairpin sequences (T1-9) were also evaluated in one-pot polymerase-endonuclease reactions (Figure 7). The yield of product P1 (Table 1a) was largely unaffected by altering the nucleobase pairing to inosine (T1, T6 and T7, Table 1a), the hairpin sequence (T5, Table 1a), or 5'-biotinylation (T2, Table 1a). However, placing biotin in the hairpin (T3) or altering the length of the hairpin (T8 and T9) slightly reduced the product yield. Finally, the effect of buffer composition on the efficiency of the one-pot reaction was investigated, and 20 mM Tris pH 8, 50 mM KCl, 12 mM MgSO4, 10 mM DTT, 0.1% BSA, 100 mM arginine, 100 mM glutamic acid was selected as the optimal reaction buffer. The one-pot reaction was performed using the optimized reaction conditions, and the production of P1 was monitored by HPLC. After 18 h, the system completed 330 cycles of template (1 μM, 0.3 mol%) elongation and product cleavage, yielding 0.33 mM of P1 (equivalent to 1.9 g / L) (Figure 8).

[0166] To further exploit this approach in the production of therapeutic oligonucleotides, polymerases with activity on nucleotide triphosphate building blocks with pharma- ceutical relevant modifications are needed. A panel of polymerases including Stoffel, KOD, and 9oN were selected for evaluation. We also included a Stoffel variant (SF4-6) previously engineered to partially amplify 2'-methoxy and 2'-fluoro modified oligonucleotides (Chen et al. Nat. Chem. 2016, 8, 556). We identified Stoffel homologs in Thermus filiformis (TfPol) and Marinithermus hydrothermalis (MhPol) by BLAST searching of the NCBI database, and introduced point mutations shown to increase the substrate promiscuity of SF4-6 into TfPol and MfPol to obtain TfPol* and MhPol*, respectively. Polymerase activity towards modified nucleotide triphosphates was assessed using a time-resolved FRET-based assay utilizing templates modified with a FRET donor (fluorescein) and a FRET acceptor (rhodamine). Reactions were performed with three natural dNTPs and one modified NTP. Enzymes capable of transcribing the template and incorporating four copies of the modified NTP disrupt the secondary structure of the template, resulting in spatial separation of the donor and acceptor, generating a fluorescent signal. Control reactions performed in the absence of modified nucleotides did not generate fluorescence, indicating a high level of enzyme fidelity. All polymerases accepted 2'-fluoro modified NTPs (Figure 9). 2'-MeO-NTPs are known to be substrates for SF4-6 (Chen et al. Nat. Chem. 2016, 8, 556), and LNA-NTPs are known to be accepted by KOD and 9oN (Veedu et al. Mol. BioSyst., 2009, 5, 787). The activity relative to natural dNTPs is below the detection limit of the assay.

[0167] A panel of polymerases was screened for activity towards a single diastereoisomer of the nucleotide thiophosphate (dNTPαS). All polymerases screened showed activity towards S p Diastereoisomers (S p The variants SF4-6, TfPol*, and MhPol* showed activity toward the S of 2'-F-ATP and 2'-F-GTP, but neither Rp-dCTPαS nor Rp-dTTPαS was accepted (Figure 9). p We also demonstrated activity towards diastereoisomers. To demonstrate the potential of our approach in the synthesis of stereochemically pure therapeutic oligonucleotides, we performed a one-pot polymerase endonuclease reaction using three natural dNTPs and Sp-dNTPaS to generate products containing a single phosphorothioate bond (P2-P5). Comparison of the enzymatically synthesized products (P2-P5) with the diastereomeric mixture generated by chemical synthesis showed that this biocatalytic reaction afforded each product with a diastereomeric excess of >99% (Figure 10a-d). Since polymerases are known to proceed with the reaction with inversion of stereochemistry, S p -The product generated from dNTP is R p We were able to exploit the stereospecificity of the polymerase to perform kinetic resolution to generate oligonucleotide products as single stereoisomers from diastereomeric mixtures of dCTPαS or dATPαS (Figure S10e-h).

[0168] Next, a panel of EndoVs was screened for activity against chemically synthesized extension templates (20 μM) containing 2'-fluoro (E2, Table 1a), 2'-methoxy (E3, Table 1a), and 2'-methoxyethoxy (E4, Table 1) ribose modifications, locked nucleic acids (E5, Table 1a), and phosphorothioate linkages (E6, Table 1). All EndoVs evaluated catalyzed the cleavage of DNA with 2'-ribose modifications at the 3' end of the cleavage site to generate the expected products P6–P10 (Table 1a), which were confirmed by LC-MS or PAGE analysis (Figures 11 and 12). TmEndoV, TnEndoV, and PfEndoV generated P1, P6–P9 with >99% conversion within 18 h, but the reaction catalyzed by TnEndoV was the fastest, with nearly complete conversion (>96%) within 1 h (Figure 12). Long LNA sequences are known to distort secondary structure, therefore therapeutic oligonucleotides rarely contain more than three consecutive LNAs, and the LNA-modified extension template (E5) contains only one LNA at the 3' end of the cleavage site.

[0169] Stereochemistry was not controlled during the chemical synthesis of the phosphorothioate-modified extension template (E6), resulting in a complex mixture of stereoisomers. EndoV-catalyzed cleavage of phosphorothioate-modified DNA afforded the expected product P6 (Table 1a) in approximately 50% yield (Figures 12 and 17). To test the stereospecificity of TnEndoV, KOD and S p -dNTPαS is enzymatically used to generate R p The extended DNA template (E11–E14) containing the ligation was generated. The extended template was isolated using an oligonucleotide cleanup kit (Monarch) and then incubated with TnEndoV. PAGE analysis showed that the cleavage reaction had proceeded to completion, indicating that the endonuclease was R p This suggested that phosphorothioate bonds could be cleaved (Figure 17).

[0170] Materials and Methods material All chemicals and biological materials were obtained from commercial suppliers. Kanamycin, ammonium persulfate, IPTG, N,N,N',N'-tetramethylethylenediamine, and boric acid were purchased from Sigma-Aldrich; LB agar, 2xYT medium, arabinose, and Tris base were purchased from Formedium; E. coli 5α, Q5 DNA polymerase, T4 DNA ligase, restriction enzymes, and ThermoPol reaction buffer were purchased from New England BioLabs; E. coli BL21(AI) and DH10B were purchased from ThermoFisher; and S p - and R p LNA-NTP, 2'F-NTP, and dNTPαS (S p / R p ) 1:1) was purchased from Jena Bioscience, 2'MeO-NTP was purchased from Trilink Biotechnologies, each oligonucleotide was synthesized by Integrated DNA Technologies (IDT), acrylamide / bisacrylamide 19:1 solution was purchased from Severn Biotech Ltd., EDTA was purchased from National Diagnostics, urea, formamide, and sodium dodecyl sulfate were purchased from Fisher Scientific, and bromophenol blue was purchased from Alpha Aesar.

[0171] Protein production and purification: Genes encoding polymerase and endonuclease V (endoV) were codon-optimized for expression in E. coli and ordered as g-blocks from IDT. TfPol* and MhPol* genes were cloned into pET28 using the NdeI and XhoI restriction sites. All other genes were cloned into pET29 using the NdeI and XhoI restriction sites. To express polymerase and EndoV, chemically competent E. coli BL21AI cells transformed with the appropriate plasmid DNA were used to express the polymerase and EndoV at 50 μg / ml. -1 After incubation at 37°C for 18 h, the starter culture (4 mL) was used to inoculate 5 ml of 2xYT medium containing 50 μg ml -1 The culture was inoculated into 400 ml of 2xYT medium supplemented with kanamycin. The culture was analyzed by optical density at 600 nm (OD 600 The cultures were incubated at 37°C and 200 rpm until the chromatin density (KD) reached 0.6. Protein expression was induced by adding IPTG (1 mM) and L-arabinose (3.33 mM) and the cultures were incubated at 37°C for 4 h. Cells were pelleted by centrifugation (8000 rpm, 20 min) and the supernatant was discarded. Cells were resuspended in lysis buffer (50 mM Hepes, 300 mM NaCl, pH 7.5 with 20 mM imidazole) and lysed by sonication. Cell lysates were heated at 70°C for 30 min to denature endogenous proteins and then clarified by centrifugation (18,000 rpm, 20 min). His-tagged proteins were subjected to affinity chromatography using Ni-NTA agarose (Qiagen) and eluted using 50 mM HEPES, 300 mM NaCl, pH 7.5 with 250 mM imidazole. The purified protein was desalted using a 10DG desalting column (Bio-Rad) and eluted with 2x storage buffer (20 mM Tris-HCL, 200 mM KCl, 0.2 mM EDTA, 2 mM DTT, pH 8.0). Protein concentration was measured at 280 nm using the extinction coefficient (Supplementary Tables 1 and 2). Aliquots of protein were added with 1x volume of glycerol and then stored at -20°C.

[0172] The general steps of a polymerase-catalyzed extension reaction (Figure 4A): To compare the activity of polymerases towards different modified NTPs, analytical scale biotransformations were performed using template (20 μM), dNTPs (0.25 mM) and polymerase (0.2 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8). Reactions were incubated at 70 °C for 12 h and each sample was analyzed by HPLC, LC-MS or PAGE unless otherwise stated. To examine the sequential incorporation of modified NTPs, templates T18-T21 were extended as described except that 1 mM modified NTPs were used. The first assay was performed as described except that commercially available KOD (Merck, 2.5 units) was used. General procedure for endonuclease-catalyzed cleavage reactions (Figure 4B): To compare the activity of each EndoV towards different modified oligonucleotides, analytical scale biotransformations were performed using extended template (20 μM) and EndoV (2 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8). Reactions were incubated at 70 °C for 12 h and samples were analyzed by HPLC, LC-MS or PAGE unless otherwise stated. To examine the effect of increasing amounts of substrate (dNTPs) on EndoV activity, biotransformations were performed as described with the addition of dNTPs (0–60 mM total). The first assay was performed as described except that commercially available TmEndoV (ThermoFisher, 5 units) was used.

[0173] General procedure for one-pot polymerase endonuclease reaction: To compare the influence of template sequence, analytical-scale biotransformations were performed using template (20 μM), dNTPs (1 mM), KOD polymerase (0.2 μM), and TnEndoV (2 μM) in ThermoPol buffer (20 mM Tris-HCL, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8). Unless otherwise stated, reactions were incubated at 70 °C for 12 h and each sample was analyzed by HPLC, LC-MS or PAGE. The first assay was performed as described except that commercially available KOD (Merck, 2.5 units), TmEndoV (ThermoFisher, 5 units), and dNTPs (0.25 mM each) were used. The reaction conditions for the synthesis of P19–P32 are shown in Table 4.

[0174] Synthesis of PS-modified oligonucleotides as single stereoisomers One-pot KOD (0.1 μM) and TnEndoV (2 μM) reactions were performed with templates T10–T13 (10 μM), three dNTPs (1.4 mM each), and one S p -dNTPαS (0.7 mM) or Rp / Sp dNTPαS mix (0.7 mM). Reactions were incubated at 70° C. for 12 h and analyzed by HPLC and compared with chemically synthesized standards.

[0175] Optimization of P1 synthesis conditions A series of buffers of different composition and concentration were evaluated with respect to product yield. The following components were varied in the reactions: buffer salts (Tris-HCl, Tris-AcOH, HEPES-KOH, Bicine-HCL, pH 8.0), metal ions (MgSO4, MgCl2, MgOAc2, CaCl2), amino acids (glutamic acid, arginine, proline, glycine, serine, aspartic acid, pH 8) and additives (sodium citrate, (NH4)2SO4, tetramethylammonium chloride (TMAC), KCl, KOAc, glycerol, 1,2-propanol, sucrose, trehalose, acetylated BSA, Tween-20, TritonX-100, DTT, formamide, DMSO and PEG-3000). The optimized reaction conditions for P1 amplification catalyzed by KOD (2 pM) and TnEndoV (2 pM) with T1 (1 μM) and dNTPs (4 mM each) were 20 mM Tris-HCL (pH 8), 20 mM MgCl2, 100 mM arginine-HCL (pH 8), 100 mM glutamate-KOH (pH 8), DTT (10 mM), formamide (10%), trehalose (0.2 M), 1,2-propanediol (1 M), and acetylated BSA (0.01 mg / ml). Under these conditions, 330 cycles of template extension and product cleavage were achieved (Figure 8).

[0176] Preparative-scale synthesis of P1 Biotransformations at 5 ml scale were carried out in optimized reaction buffer (20 mM TrisHCl, 20 mM MgSO4, 100 mM KGIu, 100 mM ArgHCl, 0.01 mg / ml AcBSA, 10 mM DTT, 10% formamide, 0.2 M trehalose, 1 M 1,2-propanediol, pH 8.0) using dNTPs (4 mM each), T2 (4 μM), KOD (2 μM) and TnEndoV (2 μM). After 12 h incubation at 70 °C, the reaction was stopped by adding 0.5 M EDTA pH 8 (final concentration 40 mM). Proteins were denatured by heating at 98 °C for 2 h and pelleted by centrifugation (13,300 rpm for 15 min). The supernatant containing the oligonucleotides was collected and the protein pellet was washed with water (1000 μl). The combined aqueous fractions were extracted with 6 ml of Tris-saturated phenol:chloroform:isoamyl solution (Sigma Aldrich) and washed with chloroform. Residual protein was removed using a 10K MWCO ultrafiltration device (Sartorius). The flow-through was collected, concentrated, and desalted using a 3K MWCO ultrafiltration device (Merck Millipore). The final product was lyophilized. Figure 13).

[0177] Results: The final product P1 was obtained with 88% purity (11.5 mg) without chromatographic purification.

[0178] Stereoselectivity of TnEndoV at phosphorothioate centers undergoing hydrolysis. The stereodefined extension templates were prepared by PCR using ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8) containing templates T14 to T17 (20 μM), S p-dNTPαS (0.25 mM each) and KOD (0.2 μM). Reactions were incubated at 70°C for 12 h and samples were analyzed by PAGE. Extended oligonucleotide products were purified using the Monarch 5 μg DNA Cleanup Kit (New England Biolabs) according to the manufacturer's protocol. Enzymatically synthesized oligonucleotides (20 μM) were incubated with TnEndoV (2 μM) in ThermoPol buffer for 12 h at 70°C. For comparison, chemically synthesized stereoirregular oligonucleotides were also incubated with TnEndoV as described. Samples were analyzed by PAGE.

[0179] Chromatographic analysis: For chromatographic analysis, the reaction was stopped by adding 20 mM EDTA, one volume of water was added, and each sample was heated at 98°C for 2 h to denature the proteins. Precipitated proteins were removed by centrifugation (14,000g, 5 min), and the supernatant was transferred to a sample vial.

[0180] Ion-pairing reversed-phase chromatography was performed at 60 °C using a 1290 Infinity II Agilent LC system with an AdvanceBio Oligonucleotide 2.7 μm column, 50 × 2.1 mm (Agilent). After a 2 min hold with 5% buffer B, 0.6 mL of each aliquot were added. -1 Oligonucleotides were eluted using a gradient of 5-50% buffer B over 10 min at 20 C, followed by 5 min of re-equilibration to starting conditions before the next sample was injected. Each peak was designated by comparison to a chemically synthesized standard, and peak areas were integrated using AgilentOpenLab software. Relative molar extinction coefficients were calculated from reactions that proceeded to completion (Supplementary Table 3), and these values ​​were used to calculate the percent conversion of incomplete reactions. For reactions with phosphorothioate modifications, the relative molar extinction coefficient of the corresponding phosphorylated product was used.

[0181] Buffer B: 100 mM triethylammonium acetate containing 25% acetonitrile Buffer A: 100mM triethylammonium acetate LC-MS analysis was performed using a Waters Vion IMS QTOF operated in negative ESI mode with a 2.2 kV capillary, acquiring up to 2000 m / z. This was connected to a Waters Acquity I Class UPLC equipped with a DNAPac™ RP 4 μm column, 50 × 2.1 mm (ThermoFisher) at 65 °C. After a 2 min hold at 30% buffer B, oligonucleotides were eluted over 8 min using a gradient of 30–80% buffer B at 0.3 mL min−1, followed by a 5 min re-equilibration to starting conditions before the next sample was injected. The resulting multiply charged spectra were analyzed using Waters Unifi software and deconvoluted between 4000 and 20000 Da using the MaxEnt1 algorithm.

[0182] Buffer B: 50% methanol, 400 mM hexafluoroisopropanol, 15 mM triethylamine in water Buffer A: 400 mM hexafluoroisopropanol, 15 mM triethylamine in water Analysis by polyacrylamide gel electrophoresis (PAGE): For analysis of oligonucleotides longer than 18 nucleotides, TBE-urea acrylamide gels were prepared containing 15% acrylamide / bis-acrylamide and 8 M urea in 1× TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2 mM EDTA). For analysis of oligonucleotides shorter than 18 nucleotides, TBE-urea acrylamide gels were prepared containing 20% ​​acrylamide / bis-acrylamide, 8 M urea in 1× TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2 mM EDTA). Polymerization was initiated by adding ammonium persulfate (final concentration 0.1% w / v) and N,N,N',N'-tetramethylethylenediamine (final concentration 4.7 μM). A volume of 2 μl of sample was mixed with 2× denaturing RNA loading dye (95% v / v formamide, 0.02% w / v sodium dodecyl sulfate, 1 mM EDTA, 0.02% w / v bromophenol blue) and water to a final volume of 10 μl. Before loading, samples were denatured at 95° C. for 1 min. Gels were run at room temperature in 1× TBE at 200 V for 50 min and visualized with 200 mg / L methylene blue in water.

[0183] Effect of increasing amounts of substrate (dNTP) on enzyme activity (Figure 5A-C): To investigate the effect of dNTP concentration on KOD activity, biotransformation was performed as described in "General procedure for polymerase-catalyzed extension reactions" except that dNTP concentrations were varied (10-40 mM). To investigate the effect of dNTP concentration on TnEndoV and TmEndoV, reactions were performed as described in "General procedure for endonuclease-catalyzed cleavage reactions" except that various concentrations of dNTPs were added (0-50 mM and 0-3 mM for TnEndoV and TmEndoV, respectively). Samples were analyzed by PAGE.

[0184] Protein thermal shift assay (Figure 6A) SYPRO Orange was added to protein (5 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8) to a final concentration of 50x. Samples were incubated at 25°C for 45 min and then heated to 95°C in 0.5°C increments using a CFX96Touch real-time PCR (BioRad). Fluorescence derivatives were calculated using CFX Maestro software.

[0185] Effect of reaction temperature on the one-pot polymerase endonuclease reaction (Figure 6B) To investigate the effect of temperature on product yield, biotransformation was performed as described in “General procedure for one-pot polymerase endonuclease reaction” except that the reactions were incubated at different temperatures (60–85°C).

[0186] Temperature profiles of KOD and TnEndoV (Figure 6C-D) To evaluate the thermostability of KOD and TnEndoV in the reactions, analytical scale biotransformations were performed using enzyme solutions preincubated at 70°C for 0 or 24 h in a thermocycler (BioRad). Extension reactions catalyzed by KOD (0.2 μM) were performed using T1 (20 μM) and dNTPs (4 mM) in ThermoPol buffer. Cleavage reactions catalyzed by TnEndoV (2 μM) were performed using E1 (20 μM) in ThermoPol buffer. Reactions were incubated at 70°C and samples were taken at 15, 30, 45 min and 1 h, stopped by adding 20 μM EDTA and analyzed by HPLC.

[0187] Effect of temperature hairpin sequence on one-pot polymerase endonuclease reaction (Figure 7) To evaluate the effect of the hairpin sequence on product yield, biotransformations were performed as described in “General procedure for one-pot polymerase-endonuclease reactions” except that templates T1–T9 (20 μM) were used.

[0188] Optimization procedure for the one-pot polymerase endonuclease reaction (Figure 8) Template (4 μM), dNTPs (16 mM), polymerase (0.2 μM) and EndoV (2 μM) were incubated in buffer (20 mM Tris pH 8, 50 mM KCl, 12 mM MgSO4, 10 mM DTT, 0.1% BSA, 100 mM arginine, 100 mM glutamic acid) for 12 hours at 70° C. Samples were taken and analyzed by HPLC.

[0189] Time-resolved FRET-based polymerase assay (Figure 9) The assay was carried out in black 384-well plates (Greiner) containing FRET template (400 nM), FRET primer (500 nM) and dNTPs (2.5 mM each) in Thermopol buffer (15 μL).

[0190] Air bubbles were removed by centrifugation at 4000 rpm for 2 min. Samples were incubated at 37°C for 20 min before the reaction was initiated by the addition of polymerase (5 μl, final concentration 0.25 μM). Fluorescence was measured for 60 min on a Clariostar plate reader (BMG) with excitation at 485 nm and emission at 518 nm. Initial reaction rates were measured in triplicate and reaction rates for modified substrates were reported relative to activity for native dNTPs.

[0191] Stereoselective synthesis of oligonucleotides containing a single phosphorothioate linkage (Figure 10).

[0192] KOD (0.1 μM), TnEndoV (2 μM), 10 μM template, and 5 mM dNTP mix (S p -dNTP or R p / S p -dNTP mix) was used in a one-pot reaction. The reaction was incubated at 70°C for 12 hours and analyzed by HPLC.

[0193] Substrate profiling of the endonuclease V panel (Figures 11 and 12) Analytical scale biotransformations were performed using extension template (20 μM) and EndoV (5 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8) and reactions were incubated at 70 °C. Samples were taken at 1 and 18 h and the reaction was stopped by adding 20 mM EDTA. Samples were analyzed by PAGE, HPLC, and LC-MS analysis.

[0194] Polymerase Substrate Profiling (Figures 14 and 15) To compare the activity of the polymerases towards different modified NTPs, analytical scale biotransformations were performed using templates T18-T21 (20 μM), NTPs (0.25 mM each) and polymerase (0.2 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8). Reactions were incubated at 70 °C and samples were analyzed by LC-MS and denaturing urea PAGE.

[0195] Results: Polymerases with activity towards phosphorothioate, 2'-fluoro, 2'-methoxy, and locked nucleic acid modified NTPs were identified.

[0196] Polymerase activity towards Rp-dNTPαS using cobalt and manganese cofactors (Figure 20) Analytical-scale extension reactions were performed in 20 mM Tris-HCl pH 8, 10 mM KCl, 10 mM (NH4)2SO4 supplemented with CoCl2 or MgCl2 salts. 4 , the corresponding R p -dNTPαS was performed using templates T18-T21 (10 μM) and KOD (6 μM). Reactions were incubated at 70° C. for 12 h and samples were analyzed by denaturing urea PAGE and LC-MS analysis.

[0197] Results: In the presence of CoCl2, KOD was p -dNTPαS building blocks.

[0198] Endonuclease-catalyzed cleavage using alternative nucleophiles to water (Figure 21) Analytical-scale biotransformations were performed using TnEndoV (2 μM) and extension template E1 (20 μM) in ThermoPol buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8) supplemented with 60% w / v glycerol, 40% w / v ethylene glycol, 60% w / v 1,2-propanediol, or 60% w / v 1,3-diaminopropanol. Reactions were incubated at 70 °C for 2 h and analyzed by LC-MS.

[0199] Results: MS analysis showed that in the presence of high concentrations of nucleophiles, EndoV catalyzed the addition of alternative nucleophiles to the 5'-phosphate group of the 18-mer product. This afforded modified oligonucleotides suitable for conjugation with delivery vehicles such as antibodies, peptides, proteins, or sugars for targeted delivery to specific organs. Although the modified 18-mer was a minor product, the yield could be improved by engineering EndoV using directed evolution.

[0200] General protocol for one-pot polymerase / EndoV-catalyzed oligonucleotide synthesis (Figures 18 and 22) Analytical-scale biotransformations were performed using template (20 μM), dNTPs (1 mM each), KOD (0.2 μM) and TnEndoV (2 μM) in ThermoPol buffer (20 mM Tris-HCL, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% TritonX-100, pH 8.8). Reactions were incubated at 70 °C for 12 h and samples were analyzed by LC-MS and denaturing urea PAGE. The reaction conditions for the synthesis of P19–P32 are shown in Supplementary Table 3.

[0201] Results: Different base sequences and various 2'F, R p - P.S., R. p A series of 8-mer oligonucleotide products bearing -2'F-PS and LNA modifications were successfully amplified, with up to 238 cycles of template extension and product cleavage achieved in the best case, consuming 76% of the NTP starting material.

[0202] Preparative-scale synthesis of bitrabene (Figure 19) Biotransformations at a 0.75 ml scale were carried out in a buffer containing 50 mM TrisAcOH, 20 mM MgSO4, 50 mM KOAc, 100 mM KGIu, 100 mM ArgHCl, 0.01 mg / ml AcBSA, 0.1% TritonX-100, 30 mM DTT, 5% formamide, pH 8.0. p -dGTPαS (1.4 mM), S P -dCTPαS (1.7 mM), S p-dTTPαS (2.9 mM), template T25 (4 μM), KOD (4 μM) and TnEndoV (4 μM). After 12 hours of incubation at 70°C, the reaction was stopped by heating at 98°C for 2 hours. The reaction mixture was desalted with a 1K MWCO MicrosepAdvance (Pall). The template was removed by adding DNAse I (final concentration 60 U / ml) (New England Biolabs) and DNAse I 10× buffer (final concentration 1×) and incubated at 37°C for 1 hour. DNAse I was inactivated by heating at 75°C for 10 minutes and the reaction mixture was desalted again with a 1K MWCO MicrosepAdvance. Subsequently, the 5'-terminal phosphate group was removed by adding quickCIP (final concentration 250 U / ml) (New England Biolabs) and 10× QuickCIP buffer (final concentration 1×) and incubated at 37°C for 3 hours. Proteins were heat denatured by incubating samples at 95°C for 20 min and precipitated proteins were pelleted by centrifugation (13,300 rpm for 15 min). The supernatant containing the oligonucleotides was collected and the protein pellet was washed with water (200 pl). The combined aqueous fractions were extracted with Tris-saturated phenol-chloroform-isoamyl solution (SigmaAldrich) and washed with chloroform to remove residual protein. Organic impurities and traces of salts were removed by washing the product with a 1K MWCO MicrosepAdvance. The final product was lyophilized.

[0203] Results: After 65 cycles of template extension and product cleavage, the final product (0.26 mM) was generated, which was equivalent to 100 mM of the available S p -dNTPαS The final product was isolated with a purity of 87% without chromatographic purification, correlating with 90% consumption of the starting material.

[0204] array [Table 1-1] [Table 1-2] [Table 1-3] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 4] [Table 5]

[0205] Buffer A: 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM potassium glutamate, 100 mM arginine HCl, 0.01 mg / ml acetylated BSA, 10 mM DTT, 0.2 M trehalose, and 1 M 1,2-propanediol. Buffer B: 50 mM Tris-AcOH (pH 8), 20 mM MgSO4, 50 mM KOAc, 100 mM potassium glutamate, 100 mM arginine HCl, 0.01 mg / ml acetylated BSA, 0.1% Triton X-100®, and 30 mM DTT. Buffer C was 20 mM Tris-HCl (pH 8.8 at 25° C.), 10 mM (NH4)2SO4, 10 mM KCI, 2 mM MgSO4, 0.1% Triton® X-100 and 2 mM NiCI2. Buffer D: 20 mM Tris-HCl (pH 8), 20 mM MgCl2, 100 mM potassium glutamate, 100 mM arginine HCl, 0.01 mg / ml acetylated BSA, 10 mM DTT

Claims

1. 1. A method for producing a single-stranded oligonucleotide, comprising: i. providing a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; ii. Incubating the primer template with a nucleic acid polymerase, one or more dNTPs, and a cleavage agent to form a reaction mixture; iii. maintaining the reaction mixture under conditions that allow for extension of the primer by the polymerase to form an extended primer template and cleavage of the extended primer template at the cleavable site by the cleavage agent; The method comprising:

2. 2. The method of claim 1, wherein the polymerase is a polymerase from Thermococcus kodakaraensis (KOD), Stoffel, Family B polymerase 9°N, Thermus filiformis (TfPol), and Marinethermus hydrothermalis (MhPol) as shown in Table 3; Klenow fragment from Escherichia coli, T4 and T7 polymerase, SFP1, Stoffel variants SFM4-6, Stoffel homologs from Thermus filiformis and Marinethermus hydrothermalis (MhPol), and variants of any of the above polymerases as shown in Table 3 with the mutations described.

3. 1. A method for generating a population of single-stranded oligonucleotides, comprising: i. providing a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; ii) incubating the primer template with a nucleic acid polymerase, one or more dNTPs, and a cleavage agent to form a reaction mixture; iii) maintaining the reaction mixture under conditions that allow for extension of the primer by a polymerase to form an extended primer template, and cleavage of the extended primer template at the cleavable site by a cleavage agent; the nucleotides comprise modified thiotriphosphates (NTPαS), and the population of oligonucleotides comprises substantially the same stereoisomer; The method, wherein the polymerase is selected from the group consisting of polymerases from Thermococcus kodakaraensis (KOD), Stoffel, Family B polymerase 9°N, Thermus filiformis (TfPol) and Marineithermus hydrothermalis (MhPol) shown in Table 3; Klenow fragment from Escherichia coli, T4 and T7 polymerase, SFP1, Stoffel variant SF4-6, Stoffel homologs from Thermus filiformis and Marineithermus hydrothermalis (MhPol), and variants of any of the above polymerases shown in Table 3.

4. The method of any one of claims 1 to 3, wherein the reagent is substantially free of acetonitrile.

5. 4. The method of claim 1 or 3, wherein the cleavage is mediated by endonuclease V, TmEndoV from Thermotoga maritima (TmEndoV), TmEndoV from Thermotoga neapolitana, PtEndoV from Pseudothermotoga thermorum, TmeEndoV from Thermosipho melanesiensis, and TaEndoV from Thermosipho atlanticus, or variants thereof.

6. 4. The method of claim 1 or 3, wherein maintaining the reaction mixture in step iii) comprises maintaining the reaction mixture for a predetermined reaction time, the reaction time being a length that allows time for two or more cycles of extension and cleavage in the same reaction mixture.

7. 4. The method of claim 1 or 3, wherein the reaction conditions of step iii) comprise a reaction temperature equal to or greater than the melting temperature of the template and extension product, and the reaction conditions of step iii) are isothermal.

8. 4. The method of claim 1 or 3, further comprising one or more of the steps of dissociating the oligonucleotide product from the template, isolating the oligonucleotide product from the reaction mixture, and purifying the oligonucleotide product.

9. The method described in claim 1 or 3, further comprising separating the oligonucleotide product from the reaction mixture.

10. 1. A kit for generating single-stranded oligonucleotides, comprising: i. a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template for directing synthesis of a product oligonucleotide; and c) a cleavable site for allowing release of the product oligonucleotide from the template; The kit comprises:

11. i) a nucleic acid polymerase; and ii) a nucleotide; and iii) a cleavage agent; 11. The kit of claim 10, further comprising one or more of:

12. 11. The kit of claim 10, comprising a first container containing a primer template and a second or further container containing nucleotides, a polymerase, and / or a buffer.

13. A reaction mixture for producing a single-stranded oligonucleotide, the reaction mixture comprising: a) a primer for initiating oligonucleotide synthesis; b) a template that directs synthesis of a product oligonucleotide; and c) a cleavable site that allows release of the product oligonucleotide from the template; i. a nucleic acid polymerase; ii. a cleavage agent; iii. a product oligonucleotide complementary to the template sequence.

14. A support for producing single-stranded oligonucleotides, comprising a population of primer templates immobilized on the support, each nucleic acid molecule comprising: a) a primer for initiating oligonucleotide synthesis; b) a template that directs synthesis of an oligonucleotide product; and c) a cleavable site that allows release of the oligonucleotide product from the template.

15. The support of claim 14, wherein the support is an array, the array comprising 100, 1000, 10,000, 100,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or more nucleic acid molecules immobilized thereon.

16. 15. The method, kit, reaction, or support of claim 1, 3, 10, 13, or 14, wherein the cleavage site is a deaminated base.

17. 15. The method, kit, reaction, or support of claim 1, 3, 10, 13, or 14, wherein the template sequence is the complement of a therapeutic RNA.

18. 15. The method, kit, reaction, or support of claim 1, 3, 10, 13, or 14, wherein the oligonucleotide product is a therapeutic RNA, miRNA, or antisense oligonucleotide (ASO), or an aptamer.

19. 15. The method, kit, reaction, or support of claim 1, 3, 10, 13, or 14, wherein the oligonucleotide product comprises one or more modified oligonucleotides.

20. 14. The method, kit or reaction of claim 1, 3, 11 or 13, wherein the polymerase is as defined in Table 3 or a variant thereof, the cleavage system comprises endonuclease V and the cleavable site is an ionosine residue.

21. 14. The method, kit or reaction of claim 1, 3, 11 or 13, wherein the nucleotides include natural and / or modified nucleotides, and the modifications include nucleotides containing modified bases, sugars, or internucleoside linkages, such as phosphorothioate internucleoside linkages, 5'-N-phosphoramidite linkages, bases having linkage groups that allow for attachment of a label, such as a fluorophore or a hapten.

22. 15. The method, kit, reaction, or support of claim 1, 3, 10, 13, or 14, wherein the nucleic acid molecule comprises an immobilization moiety.

23. A population of oligonucleotides produced by the method of claim 1 or 3.

24. 24. The population of oligonucleotides of claim 23, wherein the population is at least 70% pure.

25. 1. A cell for producing a single-stranded oligonucleotide, the cell comprising a primer template comprising: a) a primer for initiating oligonucleotide synthesis; b) a template that directs synthesis of a product oligonucleotide; and c) a cleavable site that allows release of the product oligonucleotide from the template.

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