Methods and kits for regenerating reusable initiators for nucleic acid synthesis - Patents.com

Abstract

A method for nucleic acid synthesis and regeneration of a reusable synthesis initiator, comprising the steps of incorporating a linked nucleotide into an immobilized initiator using a polymerase, synthesizing a nucleic acid immediately after the linked nucleotide using a polymerase, excising a substrate base of the linked nucleotide in the nucleic acid with a DNA glycosylase to generate an abasic site, cleaving the abasic site with an endonuclease to release the nucleic acid from the initiator, and converting the 3' end of the initiator back to its original form with an enzyme having 3' phosphatase activity. A kit based on the above method and a method for regenerating a reusable initiator are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 17 / 128,677, filed December 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to methods and kits for regenerating reusable initiators for enzymatic nucleic acid synthesis. [Background technology]

[0003] DNA synthesis methods, including template-dependent and template-independent DNA synthesis methods, require an initiator (i.e., a short polynucleotide) that serves as a primer for nucleotide addition. However, after DNA synthesis, such initiators are usually not reusable and are discarded. Thus, a new initiator is required for each new round of DNA synthesis, increasing its overall production cost and making such synthesis inconvenient.

[0004] To facilitate a cost-effective and robust DNA synthesis process, novel approaches need to be developed to synthesize DNA and yield reusable initiators for de novo synthesis. Summary of the Invention [Problem to be solved by the invention]

[0005] It is therefore an object of the present disclosure to provide methods and kits for nucleic acid synthesis and regenerating reusable initiators for such synthesis that can alleviate at least one of the drawbacks of the prior art. [Means for solving the problem]

[0006] Such a method comprises the steps of: exposing an initiator attached to a solid support for nucleic acid synthesis to a linking nucleotide having a substrate base, substrate sugar and a 3' hydroxyl group in the presence of a polymerase, such that the linking nucleotide becomes incorporated onto the initiator; exposing the initiator containing the linking nucleotide to nucleotide monomers in the presence of the polymerase such that a nucleic acid is synthesized and attached to the initiator immediately after the linking nucleotide; providing a monofunctional DNA glycosylase capable of recognizing and excising a linked nucleotide having said substrate base; subjecting the substrate base to an excision treatment with the monofunctional DNA glycosylase, such that the substrate base is excised by the monofunctional DNA glycosylase to generate an abasic site; providing an abasic site endonuclease capable of recognizing the resulting abasic site and cleaving said substrate sugar; subjecting the abasic site to cleavage with the abasic site endonuclease such that both the substrate sugar and the backbone of the nucleic acid are cleaved at the abasic site and the newly synthesized nucleic acid is released from the initiator such that the 3' terminal nucleotide of the initiator leaves a 3' phosphate group and such that the 5' terminal nucleotide of the newly synthesized nucleic acid has a 5' phosphate group; Providing an enzyme having 3' phosphatase activity; and A process of subjecting the 3'-terminal nucleotide of the initiator to a dephosphorylation treatment by the enzyme having 3' phosphatase activity, so that the 3' phosphate group of the 3'-terminal nucleotide of the initiator is converted back to the original 3' hydroxyl group so that the initiator can be reused for a new synthesis reaction.

[0007] The kit includes a polymerase, a linking nucleotide, a monofunctional DNA glycosylase, an abasic site endonuclease, and an enzyme having 3' phosphatase activity, and is used according to the method described above.

[0008] Another object of the present disclosure is to provide a method for regenerating a reusable initiator for nucleic acid synthesis, which can alleviate at least one of the shortcomings of the prior art.

[0009] Such a method comprises the steps of: Providing a monofunctional DNA glycosylase; providing an initiator linked to a solid support and a newly synthesized nucleic acid linked to said initiator immediately following a linked nucleotide having a substrate base and a substrate sugar, said linked nucleotide having said substrate base being recognizable and excisable by said monofunctional DNA glycosylase; subjecting the substrate base to an excision treatment with the monofunctional DNA glycosylase, such that the substrate base is excised by the monofunctional DNA glycosylase to generate an abasic site; providing an abasic site endonuclease capable of recognizing the resulting abasic site and cleaving said substrate sugar; subjecting the abasic site to cleavage with the abasic site endonuclease such that both the substrate sugar and the backbone of the nucleic acid are cleaved at the abasic site and the newly synthesized nucleic acid is released from the initiator such that the 3' terminal nucleotide of the initiator leaves a 3' phosphate group and such that the 5' terminal nucleotide of the newly synthesized nucleic acid has a 5' phosphate group; Providing an enzyme having 3' phosphatase activity; and A process of subjecting the 3'-terminal nucleotide of the initiator to a dephosphorylation treatment by the enzyme having 3' phosphatase activity, so that the 3' phosphate group of the 3'-terminal nucleotide of the initiator is converted back to the original 3' hydroxyl group so that the initiator can be reused for a new synthesis reaction. [Brief description of the drawings]

[0010] Other features and advantages of the present disclosure will become apparent with reference to the following detailed description of the embodiments and the accompanying drawings, in which:

[0011] [Figure 1] FIG. 1 is a schematic diagram showing non-template-dependent nucleic acid synthesis and reversion of the initiator back to its original form as applied in Example 1 below, where the symbol "U" represents linked deoxyuridine, the symbol "N" represents an incorporated nucleoside monomer, the symbol "UDG" represents uracil-DNA glycosylase, the symbol "Nei" represents endonuclease VIII, and the symbol "T4 PNKP" represents T4 polynucleotide kinase with 3' phosphatase activity.

[0012] [Diagram 2] 1 is a fluorescent image of a urea-polyacrylamide gel showing the feasibility of template-independent nucleic acid synthesis as demonstrated in Example 1 below.

[0013] [Diagram 3] FIG. 1 is a fluorescent image of a urea-polyacrylamide gel showing the results of Example 1 below, where the symbol "S" represents a polynucleotide containing an initiator and a newly synthesized nucleic acid containing a linked deoxyuridine, the symbol "U" represents treatment with UDG only, the symbol "N" represents treatment with Nei only, the symbol "U+N" represents treatment with UDG and Nei, and the symbol "U+N+P" represents treatment with UDG, Nei, and T4 PNKP.

[0014] [Figure 4] FIG. 1 is a schematic diagram showing template-independent nucleic acid synthesis and the reversion of the initiator to its original form as applied in Example 2 below, in which the symbol "I" represents linked deoxyinosine, the symbol "N" represents an incorporated nucleoside monomer, the symbol "AAG" represents alkyladenine DNA glycosylase, the symbol "Nei" represents endonuclease VIII, and the symbol "T4 PNKP" represents T4 polynucleotide kinase with 3' phosphatase activity.

[0015] [Diagram 5] 1 is a fluorescent image of a urea-polyacrylamide gel showing the feasibility of template-independent nucleic acid synthesis as demonstrated in Example 2 below.

[0016] [Figure 6] FIG. 1 is a fluorescent image of a urea-polyacrylamide gel showing the results of Example 2 below, where the symbol "S" represents a polynucleotide containing an initiator and a newly synthesized nucleic acid containing a linked deoxyinosine, the symbol "A" represents treatment with AAG only, the symbol "N" represents treatment with Nei only, the symbol "A+N" represents treatment with AAG and Nei, and the symbol "A+N+P" represents treatment with AAG, Nei, and T4 PNKP.

[0017] [Figure 7] FIG. 1 is a schematic diagram showing template-dependent nucleic acid synthesis and the reversion of the initiator to its original form as applied in Example 3 below, in which the symbol "U" represents linked deoxyuridine, the symbol "N" represents a nucleoside, the symbol "UDG" represents uracil-DNA glycosylase, the symbol "Nei" represents endonuclease VIII, and the symbol "T4 PNKP" represents T4 polynucleotide kinase with 3' phosphatase activity.

[0018] [Figure 8] FIG. 1 is a fluorescent image of a urea-polyacrylamide gel showing the results of Example 3 below, in which the symbol "S" represents a duplex polynucleotide containing an initiator and a newly synthesized nucleic acid containing a linked deoxyuridine, the symbol "U" represents treatment with UDG only, the symbol "N" represents treatment with Nei only, the symbol "U+N" represents treatment with UDG and Nei, and the symbol "U+N+P" represents treatment with UDG, Nei, and T4 PNKP.

[0019] [Figure 9]1 is a schematic diagram showing template-dependent nucleic acid synthesis and the return of the initiator to its original form as applied in Example 4 below, in which the symbol "I" represents linked deoxyinosine, the symbol "N" represents an incorporated nucleoside monomer, the symbol "AAG" represents alkyladenine DNA glycosylase, the symbol "Nei" represents endonuclease VIII, and the symbol "T4 PNKP" represents T4 polynucleotide kinase with 3' phosphatase activity; and

[0020] [Figure 10] FIG. 1 is a fluorescent image of a urea-polyacrylamide gel showing the results of Example 4 below, where the symbol "S" represents a duplex polynucleotide comprising an initiator and a newly synthesized nucleic acid containing a linked deoxyinosine, the symbol "A" represents treatment with AAG only, the symbol "N" represents treatment with Nei only, the symbol "A+N" represents treatment with AAG and Nei, and the symbol "A+N+P" represents treatment with AAG, Nei, and T4 PNKP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Where any prior art publication is referred to in this specification, it should be understood that such reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Taiwan or any other country.

[0022] It will be clearly understood that for purposes of this specification the word "comprising" means "including but not limited to" and the word "comprises" has a corresponding meaning.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which this invention belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to implement this disclosure. Indeed, this disclosure is in no way limited to the methods and materials described.

[0024] The present disclosure provides a method for nucleic acid synthesis and regeneration of reusable initiators for such synthesis, the method comprising the steps of: exposing an initiator attached to a solid support for nucleic acid synthesis to a linking nucleotide having a substrate base, substrate sugar and a 3' hydroxyl group in the presence of a polymerase, such that the linking nucleotide becomes incorporated onto the initiator; exposing the initiator containing the linking nucleotide to nucleotide monomers in the presence of the polymerase such that a nucleic acid is synthesized and joined to the initiator immediately following the linking nucleotide; providing a monofunctional DNA glycosylase capable of recognizing and excising a linked nucleotide having said substrate base; subjecting the substrate base to an excision treatment with the monofunctional DNA glycosylase, such that the substrate base is excised from the linked nucleotide by the monofunctional DNA glycosylase to generate an abasic site; providing an abasic site endonuclease capable of recognizing the resulting abasic site and cleaving said substrate sugar; subjecting the abasic site to cleavage with the abasic site endonuclease such that both the substrate sugar and the backbone of the nucleic acid are cleaved at the abasic site and the newly synthesized nucleic acid is released from the initiator such that the 3' terminal nucleotide of the initiator leaves a 3' phosphate group and such that the 5' terminal nucleotide of the synthesized nucleic acid has a 5' phosphate group; Providing an enzyme having 3' phosphatase activity; and A process of subjecting the 3'-terminal nucleotide of the initiator to a dephosphorylation treatment by the enzyme having 3' phosphatase activity, so that the 3' phosphate group of the 3'-terminal nucleotide of the initiator is converted back to the original 3' hydroxyl group so that the initiator can be reused for a new synthesis reaction.

[0025] According to the present disclosure, the excision, cleavage and dephosphorylation processes may be performed simultaneously or sequentially.

[0026] As used herein, the terms "nucleic acid", "nucleic acid sequence" and "nucleic acid fragment" refer to a deoxyribonucleotide or ribonucleotide sequence in single- or double-stranded form, including naturally occurring nucleotides or artificial chemical mimetics. As used herein, the term "nucleic acid" is interchangeable with the terms "oligonucleotide", "polynucleotide", "gene", "DNA", "cDNA", "RNA" and "mRNA".

[0027] The term "initiator" refers to a mononucleoside, mononucleotide, oligonucleotide, polynucleotide, or modified analogues thereof by which a nucleic acid is synthesized. The term "initiator" may also refer to a xenonucleic acid (XNA) or peptide nucleic acid (PNA) having a 3'-hydroxyl group.

[0028] According to the present disclosure, the initiator may be template-independent or template-dependent (i.e., the initiator may not anneal or hybridize to a complementary template, or may anneal to a template to form a duplex or double strand).

[0029] When the initiator is template-independent, the initiator may have a sequence selected from non-self-complementary and non-self-complementary sequences. The term "self-complementary" means that a sequence (e.g., a nucleotide sequence, an XNA sequence, or a PNA sequence) folds back on itself (i.e., a region of the sequence binds or hybridizes to another region of the sequence) to generate a duplex, double-stranded-like structure that can serve as a template for nucleic acid synthesis. Depending on how close the complementary regions of the sequence are, the strands may form, for example, hairpin loops, junctions, bulges, or internal loops. The term "self-complementary" is used to describe a sequence (e.g., a nucleotide sequence, an XNA sequence, or a PNA sequence) that, when acting as a template, forms a complementary extension (i.e., a self-complementary sequence is formed based on such a sequence acting as a template). For example, the self-complementary sequence may be "ATCC". When the "ATCC" sequence serves as a template, a stretch of "GGAT" is formed from such sequence that is complementary to such sequence (ie, the self-complementary sequence "ATCCGGAT" is formed).

[0030] Generally, a "template" is a polynucleotide that includes a target nucleotide sequence. In some instances, the terms "target sequence", "template polynucleotide", "target nucleic acid", "target polynucleotide", "nucleic acid template", "template sequence", and variations thereof are used interchangeably. Specifically, the term "template" refers to a strand of nucleic acid whose complementary copy is synthesized from nucleotides or nucleotide analogs through the activity of a template-dependent nucleic acid polymerase. Within a duplex, the template strand is, by convention, shown and described as the "bottom" strand. Similarly, the non-template strand is often shown and described as the "top" strand. The "template" strand is also referred to as the "sense" strand, and the non-template strand is also referred to as the "antisense" strand.

[0031] According to the present disclosure, the initiator has a 5' end linked to a solid support, and the linking nucleotide is attached to the 3' terminal nucleotide of the initiator and the 5' terminal nucleotide of the synthesized nucleic acid. The initiator may be directly attached to the support or may be attached to the support via a linker.

[0032] Examples of solid supports include, but are not limited to, microarrays, beads (coated or uncoated), columns, optical fibers, wipes, nitrocellulose, nylon, glass, quartz, diazotized membranes (paper or nylon), silicones, polyformaldehyde, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, magnetic particles, plastics (such as polyethylene, polypropylene and polystyrene), gel-forming materials (proteins (e.g., gelatin), lipopolysaccharides, silicates, agarose, polyacrylamide, methyl methacrylate polymers, etc.), sol-gels, porous polymer hydrogels, nanostructured surfaces, nanotubes (such as carbon nanotubes), and nanoparticles (such as gold nanoparticles, quantum dots, etc.).

[0033] According to the present disclosure, depending on the form of the initiator, the synthesized nucleic acid and the linked nucleotides can be template-independent or template-dependent, respectively.

[0034] As used herein, the terms "incorporated" or "incorporation" refer to becoming part of a nucleic acid. There is known flexibility in the terminology regarding incorporation of nucleic acid precursors.

[0035] For example, the nucleotide dGTP is a deoxyribonucleoside triphosphate. When incorporated into DNA, dGTP becomes the moiety dGMP, or deoxyguanosine monophosphate. Although DNA does not contain the dGTP molecule, we can say that dGTP is incorporated into DNA.

[0036] According to the present disclosure, the nucleotide monomer may be a natural nucleic acid nucleotide, consisting of a sugar, a phosphate group and a nitrogenous base. The sugar may be ribose in RNA or 2'-deoxyribose in DNA. Depending on whether the nucleic acid to be synthesized is DNA or RNA, the nitrogenous base is selected from adenine, guanine, uracil, cytosine and thymine. Alternatively, the nucleotide monomer may be a nucleotide in which at least one of the three components is modified. By way of example, the modification may occur at the level of the base (e.g., inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other modified base that allows hybridization) to generate a modified product, at the level of the sugar (e.g., replacement of deoxyribose by an analogue), or at the level of the phosphate group (e.g., boronate, alkylphosphonate or phosphorothioate derivatives). According to the present disclosure, a nucleotide monomer may have a removable blocking moiety. Examples of removable blocking moieties include, but are not limited to, a 3'-O-blocking moiety, a base-blocking moiety, and combinations thereof.

[0037] The nucleotide monomer having a removable blocking portion is also called a reversible terminator.Thus, the nucleotide monomer having a 3'-O-blocking portion is also called a 3'-blocked reversible terminator or a 3'-O-modified reversible terminator, and the nucleotide monomer having a base-blocking portion is also called a 3'-unblocked reversible terminator or a 3'-OH unblocked reversible terminator.

[0038] As used herein, the term "reversible terminator" refers to a chemically modified nucleotide monomer. When such a reversible terminator is incorporated into a growing nucleic acid by a polymerase, it blocks the further incorporation of another nucleotide monomer by the polymerase. Such a "reversible terminator" base and nucleic acid can be deprotected by chemical or physical treatment, and following such deprotection, the nucleic acid can be further extended by a polymerase.

[0039] Examples of 3'-O-blocking moieties include, but are not limited to, O-azidomethyl, O-amino, O-allyl, O-phenoxyacetyl, O-methoxyacetyl, O-acetyl, O-(p-toluene)sulfonate, O-phosphate, O-nitrate, O-[4-methoxy]-tetrahydrothiopyranyl, O-tetrahydrothiopyranyl, O-[5-methyl]-tetrahydrofuranyl, O-[2-methyl,4-methoxy]-tetrahydropyranyl, O-[5-methyl]-tetrahydropyranyl and O-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl.

[0040] Examples of 3' unblocked reversible terminators include, but are not limited to, 7-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-7-deaza-dATP, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-dCTP, 1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-7-deaza-dGTP, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-dUTP, and 5-[(S)-1-(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-dUTP.

[0041] According to the present disclosure, the base block portion can be a reversible dye terminator. Examples of reversible dye terminators include, but are not limited to, Illumina NovaSeq reversible dye terminator, Illumina NextSeq reversible dye terminator, Illumina MiSeq reversible dye terminator, Illumina HiSeq reversible dye terminator, Illumina Genome Analyzer IIX reversible dye terminator, LaserGen lightning terminator, and Helicos Biosciences Heliscope reversible dye terminator.

[0042] Reversible terminator is well known and commonly used by those skilled in the art, so for the sake of brevity, further details thereof are omitted herein.However, applicable 3'block reversible terminator, applicable 3'non-block reversible terminator, and applicable conditions for protection and deprotection (i.e., conditions for adding and removing removable blocking moiety) can be found in, for example, Gardner et al.(2012), Nucleic Acids Research,40(15):7404-7415, Litosh et al.(2011), Nucleic Acids Research,39(6):e39, and Chen et al.(2013), Genomics Proteomics Bioinformatics,11:34-40.

[0043] According to the present disclosure, the polymerase can be a template-dependent or template-independent polymerase.

[0044] According to the present disclosure, the polymerase may be selected from the group consisting of Family A DNA polymerases (e.g., T7 DNA polymerase, Pol I, Pol γ, θ, and v), Family B DNA polymerases (e.g., Pol II, Pol B, Pol ζ, Pol α, δ, and ε), Family C DNA polymerases (e.g., Pol III), Family D DNA polymerases (e.g., Pol D), Family X DNA polymerases (e.g., Pol β, Pol σ, Pol λ, Pol μ, and terminal deoxynucleotidyl transferase), Family Y DNA polymerases (e.g., Pol ι, Pol κ, Pol η, DinB, Pol IV, and Pol V), reverse transcriptases (e.g., telomerase and Hepatitis B virus), and enzymatically active fragments thereof.

[0045] Non-limiting examples of widely used template-dependent polymerases include the DNA-dependent DNA polymerases T7 DNA polymerase and T3 DNA polymerase of phage T3, the DNA-dependent RNA polymerases T7 RNA polymerase and T3 RNA polymerase of phage T7, the DNA-dependent DNA polymerase Escherichia coli DNA polymerase I or a fragment thereof known as the Klenow fragment, the thermostable DNA-dependent DNA polymerases Thermophilus aquaticus DNA polymerase, Tth DNA polymerase and vent DNA polymerase, the DNA-dependent DNA polymerase eukaryotic DNA polymerase β, the RNA-dependent DNA polymerase telomerase, and non-protein catalytic molecules such as modified RNA (ribozymes; Unrau & Bartel, 1998) and DNA with template-dependent polymerase activity.

[0046] Non-limiting examples of template-independent polymerases include reverse transcriptase, poly(A) polymerase, DNA polymerase theta (θ), DNA polymerase mu (μ), and terminal deoxynucleotidyl transferase.

[0047] Suitable polymerases for nucleic acid synthesis, ligated nucleotide addition and nucleic acid synthesis are within the expertise and routine skill of one of ordinary skill in the art and therefore further details thereof are omitted herein for the sake of brevity.

[0048] As used herein, the term "monofunctional DNA glycosylase" refers to a naturally occurring monofunctional glycosylase that originally has only glycosylase activity. The term "monofunctional DNA glycosylase" also refers to a monofunctional glycosylase derived from a bifunctional DNA glycosylase by removing or inactivating the abasic site lyase domain of a bifunctional DNA glycosylase that originally has glycosylase activity and abasic site lyase activity.

[0049] According to the present disclosure, monofunctional DNA glycosylases include uracil-DNA glycosylase (UDG or UNG), alkyladenine DNA glycosylase (AAG; also known as methylpurine DNA glycosylase (MPG)), single-strand-selective monofunctional uracil DNA glycosylase 1 (SMUG1), methyl-binding domain glycosylase 4 (MBD4), thymine DNA glycosylase (TDG), mutY homolog DNA glycosylase (MYH), alkylpurine glycosylase C (AlkC), alkylpurine glycosylase D (AlkD). , 8-oxo-guanine glycosylase 1 with no abasic site lyase activity (OGG1), endonuclease III-like 1 with no abasic site lyase activity (NTHL1), endonuclease VIII-like glycosylase 1 with no abasic site lyase activity (NEIL1), endonuclease VIII-like glycosylase 2 with no abasic site lyase activity (NEIL2), endonuclease VIII-like glycosylase 3 with no abasic site lyase activity (NEIL3), and enzymatically active fragments thereof.

[0050] Removing or inactivating the abasic site lyase domain of a bifunctional DNA glycosylase to obtain a monofunctional glycosylase is within the expertise and routine skill of one of ordinary skill in the art, and therefore details thereof are omitted herein for the sake of brevity.

[0051] As used herein, the term "enzymatically active fragment" refers to a catalytically or enzymatically active fragment of a protein or polypeptide that contains at least 10%, preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, or even more preferably at least 95% of the activity of the protein or polypeptide from which the fragment is derived.

[0052] In one exemplary embodiment of the present disclosure, the monofunctional DNA glycosylase is uracil-DNA glycosylase. In another exemplary embodiment of the present disclosure, the monofunctional DNA glycosylase is alkyladenine-DNA glycosylase.

[0053] The terms "abasic," "apurinary / apyrimidinic," and D-spacer can be used interchangeably to refer to sites where a base is absent but the sugar-phosphate backbone remains intact. Thus, abasic endonucleases are also known as apurinic / apyrimidinic endonucleases.

[0054] According to the present disclosure, the abasic site endonuclease may be selected from the group consisting of endonuclease VIII (Nei), endonuclease III (EndoIII or Nth), and enzymatically active fragments thereof. In an exemplary embodiment, the abasic site endonuclease is endonuclease VIII.

[0055] According to the present disclosure, the enzyme having 3' phosphatase activity may be selected from the group consisting of polynucleotide kinase 3'-phosphatase, 3'-phosphoesterase, and enzymatically active fragments thereof. The enzyme having 3' phosphatase activity may be T4 polynucleotide kinase (PNK) having 3' phosphatase activity (also called T4 polynucleotide kinase / phosphatase (T4 PNKP)), and zinc finger DNA 3'-phosphoesterase (ZDP).

[0056] Applicable enzymes possessing 3' phosphatase activity are within the expertise and routine skill of the person skilled in the art and therefore further details thereof will not be given herein for the sake of brevity. However, applicable enzymes possessing 3' phosphatase activity can be found, for example, in Blondal et al. (2005), J. Bio. Chem., 280(7):5188-5194; Dobson et al. (2006), Nucleic Acids Research, 34(8):2230-2237; Blasius et al. (2007), BMC Molecular Biology, 8:69; Coquelle et al. (2011), PNAS, 108(52):21022-21027; Vance et al. (2001), J. Bio. Chem., 276(18):15703-15781, and the NCBI website (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=11284#general-protein-info).

[0057] According to the present disclosure, the substrate bases of the linked nucleotides attached to the initiator are uracil, hypoxanthine, thymine, cytosine, guanine, 5-fluorouracil, 5-hydroxymethyluracil, 5-formylcytosine, 5-carboxylcytosine, 3-methyladenine, 3-methylguanine, 7-methyladenine, 7-methylguanine, N 6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxyl cytosine, 5-hydroxyuracil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine derivatives of adenine, formamidopyrimidine derivatives of guanine, adenine paired with guanine, uracil paired with guanine, uracil paired with adenine, thymine paired with guanine, ethenocytosine paired with 8-oxo-7,8-dihydroguanine, and 2-hydroxyladenine paired with guanine. In an exemplary embodiment of the present disclosure, the substrate base of the linking nucleotide is uracil. In another exemplary embodiment of the present disclosure, the substrate base of the linking nucleotide is hypoxanthine.

[0058] Suitable monofunctional DNA glycosylases and their corresponding substrate bases are within the expertise and routine skill of a person skilled in the art, so for the sake of brevity, the details thereof are omitted herein.However, suitable monofunctional DNA glycosylases and their corresponding substrate bases can be found, for example, in Jacobs et al. (2012), Chromosoma, 121:1-20, Krokan et al. (1997), Biochem. J., 325:1-16, and Kim et al. (2012), Current Molecular Pharmacology, 5:3-13.

[0059] The term "linking nucleotide" refers to the first nucleotide that is incorporated into the initiator on a newly synthesized nucleic acid.

[0060] The term "substrate base" refers to the base of a linked nucleotide that serves as a substrate for an enzyme. The term "substrate sugar" refers to the nucleoside sugar portion of a linked nucleotide that serves as a substrate for an enzyme.

[0061] The present disclosure further provides a kit for nucleic acid synthesis and regeneration of a reusable initiator for such synthesis, comprising said polymerase, said monofunctional DNA glycosylase, said linked nucleotides that serve as substrates for the monofunctional DNA glycosylase, said abasic site endonuclease, and said enzyme possessing 3' phosphatase activity, for use in accordance with the aforementioned methods of the present disclosure.

[0062] Further disclosed herein is a method for regenerating a reusable initiator for nucleic acid synthesis, comprising: providing the monofunctional DNA glycosylase; providing an initiator for said nucleic acid synthesis and a synthesized nucleic acid linked to said initiator immediately following said linking nucleotide; subjecting the substrate bases to the above-mentioned excision treatment using a monofunctional DNA glycosylase; providing said abasic site endonuclease; subjecting the abasic site to the cleavage treatment using the abasic site endonuclease; providing an enzyme having 3' phosphatase activity as described above; and subjecting the 3'-terminal nucleotide of the initiator to the above-mentioned dephosphorylation treatment using an enzyme having 3' phosphatase activity; The present invention provides a method comprising:

[0063] The present disclosure is further illustrated by the following examples, however, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the present disclosure in any way. EXAMPLES

[0064] Example 1. Template-independent nucleic acid synthesis and reversion of the synthesis initiator to its original form by T4 polynucleotide kinase (T4 PNKP), which has uracil-DNA glycosylase (UDG), endonuclease VIII (Nei) and 3' phosphatase activity

[0065] To test whether the initiator used in template-independent nucleic acid synthesis can be converted back to its original form after nucleic acid synthesis, the following experimental steps were performed. The detailed scheme of template-independent nucleic acid synthesis using linked deoxyuridine nucleotides and enzyme-assisted conversion of the initiator to its original form, as applied in this example, is illustrated in FIG. A. Template-independent nucleic acid synthesis initiated using tethered deoxyuridine triphosphate (dUTP)

[0066] The initiator (a single-stranded 21-mer polynucleotide of SEQ ID NO: 1) with a 5'-hexachloro-fluorescein (HEX) label at the 5' end and a hydroxyl group at the 3' end was synthesized by Integrated DNA Technologies (Coralville, Iowa, United States). Template-independent nucleic acid synthesis reactions were performed using 3' to 5' exonuclease-deficient Pfu DNA polymerase (Pfu exo- ) (200 nM) to incorporate linked deoxyuridine triphosphate (dUTP) (100 μM) at the 3′ end of the initiator.

[0067] Specifically, Pfu exo- The DNA polymerase (having the amino acid sequence of SEQ ID NO: 8) was prepared as follows: A gene construct encoding the intein-free Pfu DNA polymerase was synthesized by Genomics BioSci and Tech Co. (New Taipei City, Taiwan). exo- The DNA polymerase was used to catalyze the Asp mutation on the gene backbone using the Q5 Site-directed Mutagenesis Kit from New England Biolabs (Ipswich, MA, United States). 141 to Ala (D141A), and the Glu 143 This was generated by changing Pfu exo-The DNA polymerase was expressed in E. coli BL21(DE3) cells and purified by Sepharose-Q and heparin columns using an Akta FPLC system from GE Healthcare Life Sciences (Marlborough, MA, United States). As illustrated in FIG. 2, deoxyuridine monophosphate (dUMP) was purified by the Pfu exo- It was efficiently incorporated at the 3' end of the initiator by DNA polymerase. B. Template-independent nucleic acid synthesis immediately following the tethered dUMP at the 3' end of the initiator

[0068] To demonstrate template-independent nucleic acid synthesis immediately following the tethered dUMP at the 3' end of the synthesis initiator, Pfu exo- DNA polymerase (200 nM) was used to stepwise incorporate 3'-O-azidomethyl-dATP and 3'-O-azidomethyl-dTTP (100 μM) (Jena Bioscience, Erfurt, Germany) into the initiator containing tethered dUMP at the 3' end. The synthesis reaction was started by the addition of 10 mM manganese cations and then incubated at 75 °C for 30 min. The reaction was stopped by adding 10 μL of 2x quench solution (95% deionized formamide and 25 mM EDTA) and subjected to heat denaturation at 98 °C for 10 min. The reaction products were analyzed by 15% denaturing urea-polyacrylamide gel and visualized by Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0069] As illustrated in FIG. exo-Template-independent nucleic acid synthesis using a DNA polymerase can sequentially incorporate dAMP and dTMP immediately after the linked dUMP at the 3' end of the initiator (the resulting product containing the initiator, linked dUMP, and dAMP and dTMP has SEQ ID NO:2). Thus, the template-independent nucleic acid synthesis reaction can continue to synthesize a 16-mer nucleic acid polynucleotide of SEQ ID NO:3, thus generating a 38-mer nucleic acid (SEQ ID NO:4) containing the initiator, linked dUMP, and the newly synthesized 16-mer nucleic acid polynucleotide.

[0070] It should be noted that template-independent nucleic acid synthesis is within the expertise and routine skill of one of ordinary skill in the art, and thus the nucleic acid of the 16-mer nucleic acid of SEQ ID NO: 3 can be synthesized de novo by one of ordinary skill in the art using the information provided herein. In this example, to simplify the experimental procedure, the 16-mer nucleic acid was synthesized by Integrated DNA Technologies (Coralville, Iowa, United States) and linked to an initiator using linked dUMP as described in Section C below to represent template-independent nucleic acid synthesis of the 16-mer nucleic acid. C. Combined treatment with UDG, Nei and T4 PNKP releases newly synthesized nucleic acids and reverts synthesis initiators back to their original form.

[0071] To demonstrate the feasibility of releasing newly synthesized nucleic acids and enzymatically regenerating synthesis initiators, a 38-mer nucleic acid (SEQ ID NO: 4) was prepared containing an initiator, linked dUMP, and a newly synthesized 16-mer nucleic acid polynucleotide. Specifically, the 16-mer nucleic acid polynucleotide was synthesized using Pfu exo- DNA polymerase was used to ligate the initiator with the ligated dUMP.

[0072] The 38-mer nucleic acid (25 nM) was subjected to uracil excision, abasic site / nucleic acid backbone cleavage and dephosphorylation reactions by adding 10 units each of UDG, Nei and T4 PNKP purchased from New England Biolabs (Ipswich, MA, United States) in 1x cleavage buffer [10 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol (DTT), and 50 mM Tris-HCl, pH 7.5] for 15 minutes at 37° C. Preparation of such a 38-mer nucleic acid (SEQ ID NO: 4) was confirmed by 15% denaturing urea-polyacrylamide gel as described above. In a control experiment, a 38-mer nucleic acid (SEQ ID NO: 4) was also subjected to treatment with UDG, Nei or a mixture of UDG and Nei under the same experimental conditions described above. Each reaction was then stopped by adding 10 μL of 2x quenching solution (95% formamide and 25 mM EDTA) and the enzyme was inactivated by heating at 98° C. for 10 min. The reaction products were analyzed by 20% denaturing urea-polyacrylamide gel and visualized by an Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0073] As illustrated in Figure 3, treatment with a mixture of UDG, Nei and T4 PNKP resulted in the removal of linked dUMP from a single-stranded 38-mer nucleic acid, the release of a newly synthesized 16-mer polynucleotide (SEQ ID NO:3), and the regeneration of an initiator with a hydroxyl group at the 3' end (SEQ ID NO:1). Nei alone, UDG alone, or the combination of UDG and Nei are unable to efficiently and completely release the newly synthesized nucleic acid and simultaneously regenerate an initiator with a hydroxyl group at the 3' end. Example 2. Template-independent nucleic acid synthesis and the reversion of the synthesis initiator to its original form by alkyladenine DNA glycosylase (AAG), Nei, and T4 PNKP.

[0074] To test whether the initiator used in template-independent nucleic acid synthesis can be converted back to its original form after nucleic acid synthesis, the following experimental procedure was performed. The detailed scheme of template-independent nucleic acid synthesis using tethered deoxyinosine triphosphate (dITP) and enzyme-assisted reversion of the initiator to its original form, as applied in this example, is illustrated in FIG. A. Template-independent nucleic acid synthesis initiated with ligated dITP

[0075] An initiator (SEQ ID NO: 1) having a 5'-hexachloro-fluorescein (HEX) label at the 5' end and an unprotected hydroxyl group at the 3' end was used. Template-independent nucleic acid synthesis reactions were carried out using Pfu as described in Example 1. exo- The reaction was carried out using DNA polymerase (200 nM) to incorporate tethered dITP (100 μM) at the 3′ end of the initiator. As illustrated in FIG. 5, deoxyinosine monophosphate (dIMP) was synthesized by the method of Pfu exo- It was efficiently incorporated at the 3' end of the initiator by DNA polymerase. B. Template-independent nucleic acid synthesis immediately following the tethered dIMP at the 3' end of the initiator

[0076] To demonstrate template-independent nucleic acid synthesis immediately following the tethered dIMP at the 3' end of the synthesis initiator, Pfu exo-DNA polymerase (200 nM) was used to stepwise incorporate 3'-O-azidomethyl-dATP and 3'-O-azidomethyl-dTTP (100 μM) (Jena Bioscience, Erfurt, Germany) into the initiator containing tethered dIMP at the 3' end. The synthesis reaction was started by the addition of 10 mM manganese cations and then incubated at 75 °C for 30 min. The reaction was stopped by adding 10 μL of 2x quench solution (95% deionized formamide and 25 mM EDTA) and subjected to heat denaturation at 98 °C for 10 min. The reaction products were analyzed by 15% denaturing urea-polyacrylamide gel and visualized by Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0077] As shown in FIG. exo- Template-independent nucleic acid synthesis using DNA polymerase can sequentially incorporate dAMP and dTMP immediately after the linked dIMP at the 3' end of the initiator (the resulting product containing the initiator, linked dIMP, and dAMP and dTMP has SEQ ID NO:5). Thus, the template-independent nucleic acid synthesis reaction can continue to synthesize the 16-mer nucleic acid polynucleotide of SEQ ID NO:3, and generate a 38-mer nucleic acid (SEQ ID NO:6) containing the initiator, linked dIMP, and the newly synthesized 16-mer nucleic acid polynucleotide. The preparation of such a 38-mer nucleic acid (SEQ ID NO:6) was confirmed by 15% denaturing urea-polyacrylamide gel as described above.

[0078] It should be noted that template-independent nucleic acid synthesis is within the expertise and routine skill of one of ordinary skill in the art, and thus the nucleic acid of the 16-mer nucleic acid of SEQ ID NO: 3 can be synthesized de novo by one of ordinary skill in the art using the information provided herein. In this example, to simplify the experimental procedure, the 16-mer nucleic acid was linked to an initiator using linked dIMP as described in Section C below to represent template-independent nucleic acid synthesis of the 16-mer nucleic acid. C. Combined treatment with AAG, Nei and T4 PNKP releases newly synthesized nucleic acids and reverts synthesis initiators back to their original form.

[0079] To demonstrate the feasibility of releasing newly synthesized nucleic acid and enzymatically regenerating the synthesis initiator, a single-stranded 38-mer nucleic acid (SEQ ID NO:6) was prepared containing the initiator (SEQ ID NO:1), linked dIMP, and a newly synthesized 16-mer polynucleotide (SEQ ID NO:3). exo- DNA polymerase was used to ligate the initiator with the ligated dUMP.

[0080] Single-stranded 38-mer nucleic acids (25 nM) were subjected to inosine excision, abasic site / nucleic acid backbone cleavage and dephosphorylation reactions by adding 10 units each of AAG, Nei and T4 PNKP purchased from New England Biolabs (Ipswich, MA, United States) in 1x cleavage buffer [10 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol (DTT), and 50 mM Tris-HCl; pH 7.5] at 37°C for 15 min.

[0081] In control experiments, a single-stranded 38-mer nucleic acid (SEQ ID NO: 6) was also subjected to treatment with AAG, Nei, or a mixture of AAG and Nei under identical experimental conditions. Each reaction was then stopped by adding 10 μL of 2x quenching solution (95% formamide and 25 mM EDTA), and the enzyme was inactivated by heating at 98° C. for 10 min. The reaction products were analyzed by 20% denaturing urea-polyacrylamide gel and visualized by an Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0082] As illustrated in Figure 6, treatment with a mixture of AAG, Nei and T4 PNKP resulted in the removal of linked dIMP from a single-stranded 38-mer nucleic acid, the release of a newly synthesized 16-mer polynucleotide (SEQ ID NO:3), and the regeneration of an initiator (SEQ ID NO:1) bearing a hydroxyl group at its 3' end. Nei alone, AAG alone, or the combination of AAG and Nei are unable to efficiently and completely sever the newly synthesized nucleic acid and simultaneously regenerate an initiator bearing a hydroxyl group at its 3' end. Example 3. Template-dependent nucleic acid synthesis and reversion of synthesis initiators to their original forms by UDG, Nei, and T4 PNKP

[0083] To test whether the initiator used in template-dependent nucleic acid synthesis can be converted back to its original form after nucleic acid synthesis, the following experimental procedure was performed. The detailed scheme of template-dependent nucleic acid synthesis using tethered dUTP and enzyme-assisted reversion of the initiator to its original form, as applied in this example, is illustrated in Figure 7. A. Combined treatment with UDG, Nei and T4 PNKP releases newly synthesized nucleic acids and reverts synthesis initiators back to their original form.

[0084] To demonstrate the feasibility of releasing newly synthesized nucleic acid and regenerating synthesis initiator by enzyme, a single-stranded 38-mer nucleic acid (SEQ ID NO: 4) containing initiator, ligated dUMP and newly synthesized 16-mer polynucleotide was prepared as described in Example 1. To illustrate template-dependent nucleic acid synthesis, the single-stranded 38-mer nucleic acid (SEQ ID NO: 4) was hybridized with a complementary single-stranded 38-mer nucleic acid (SEQ ID NO: 7) by heating at 95°C for 10 minutes, followed by slow cooling to 4°C to form a duplex blunt-ended double-stranded 38-mer nucleic acid. The complementary single-stranded 38-mer nucleic acid (SEQ ID NO: 7) was obtained from Integrated DNA Technologies (Coralville, Iowa, United States).

[0085] 25 nM of the duplex 38-mer nucleic acid was subjected to uracil excision, abasic site / nucleic acid backbone cleavage and dephosphorylation reactions by adding 10 units each of UDG, Nei and T4 PNKP purchased from New England Biolabs (Ipswich, MA, United States) in 1x cleavage buffer [10 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol (DTT), and 50 mM Tris-HCl; pH 7.5] at 37°C for 15 min.

[0086] In control experiments, duplex 38-mer nucleic acids were subjected to treatment with UDG, Nei or a mixture of UDG and Nei under identical experimental conditions. Each reaction was then stopped by adding 10 μL of 2x quenching solution (95% formamide and 25 mM EDTA), the enzyme was inactivated by heating at 98°C for 10 min, and duplex 38-mer nucleic acids were denatured. The reaction products were analyzed by 20% denaturing urea-polyacrylamide gel and visualized by Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0087] As shown in Figure 8, treatment with a mixture of UDG, Nei and T4 PNKP resulted in the removal of linked dUMP from the 38-mer nucleic acid, the release of the newly synthesized 16-mer polynucleotide (SEQ ID NO: 3) after thermal denaturation of the duplex 38-mer nucleic acid, and the regeneration of the initiator (SEQ ID NO: 1) with a hydroxyl group at the 3' end. Nei alone, UDG alone, or the combination of UDG and Nei are unable to efficiently and completely release the newly synthesized nucleic acid after thermal denaturation of the duplex 38-mer nucleic acid and simultaneously regenerate the initiator with a hydroxyl group at the 3' end. Example 4. Template-dependent nucleic acid synthesis and reversion of synthesis initiators to their original forms by AAG, Nei, and T4 PNKP

[0088] To test whether the initiator used in template-dependent nucleic acid synthesis can be converted back to its original form after nucleic acid synthesis, the following experimental procedure was performed. The detailed scheme of template-dependent nucleic acid synthesis using ligated dITP and enzyme-assisted reversion of the initiator to its original form, as applied in this example, is illustrated in Figure 9. A. Combined treatment with AAG, Nei and T4 PNKP releases newly synthesized nucleic acids and reverts synthesis initiators back to their original form.

[0089] To demonstrate the feasibility of releasing newly synthesized nucleic acid and regenerating synthesis initiator by enzyme, a single-stranded 38-mer nucleic acid (SEQ ID NO: 6) containing initiator, linked dIMP and newly synthesized 16-mer polynucleotide was prepared as described in Example 2. To illustrate template-dependent nucleic acid synthesis, the single-stranded 38-mer nucleic acid (SEQ ID NO: 6) was hybridized with a complementary 38-mer nucleic acid (SEQ ID NO: 7) by heating at 95°C for 10 minutes, followed by slow cooling to 4°C to form a duplex blunt-ended double-stranded 38-mer nucleic acid. The complementary single-stranded 38-mer nucleic acid (SEQ ID NO: 7) was obtained from Integrated DNA Technologies (Coralville, Iowa, United States). 25 nM of the duplex 38-mer nucleic acid was subjected to inosine excision, abasic site / nucleic acid backbone cleavage and dephosphorylation reactions by adding 10 units each of AAG, Nei and T4 PNKP purchased from New England Biolabs (Ipswich, MA, United States) in 1x cleavage buffer [10 mM MgCl 2 , 50 mM KCl, 5 mM dithiothreitol (DTT), and 50 mM Tris-HCl; pH 7.5] at 37°C for 15 min.

[0090] In control experiments, duplex 38-mer nucleic acids were subjected to treatment with AAG, Nei or a mixture of AAG and Nei under identical experimental conditions. Each reaction was then stopped by adding 10 μL of 2x quenching solution (95% formamide and 25 mM EDTA), the enzyme was inactivated by heating at 98°C for 10 min, and the duplex nucleic acids were denatured. The reaction products were analyzed by 20% denaturing urea-polyacrylamide gel and visualized by Amersham Typhoon Imager, GE Healthcare Life Sciences (Marlborough, MA, United States).

[0091] As shown in Figure 10, treatment with a mixture of AAG, Nei and T4 PNKP resulted in the removal of linked dIMP from the duplex 38-mer nucleic acid, the release of a newly synthesized 16-mer polynucleotide (SEQ ID NO: 3) after thermal denaturation of the duplex 38-mer nucleic acid, and the regeneration of an initiator (SEQ ID NO: 1) with a hydroxyl group at the 3' end. Nei alone, AAG alone, or the combination of AAG and Nei cannot efficiently and completely release the newly synthesized nucleic acid after thermal denaturation of the duplex 38-mer nucleic acid and simultaneously regenerate an initiator with a hydroxyl group at the 3' end.

[0092] All patent and literature references cited herein are incorporated herein by reference in their entirety. In the event of a conflict, the present description, including definitions, will control.

[0093] While the present disclosure has been described in connection with what are considered to be the exemplary embodiments, it will be understood that the disclosure is not intended to be limited to the disclosed embodiments, but rather to cover the various configurations falling within the broadest spirit and scope of the disclosure and to encompass all such modifications and equivalent configurations.

Claims

1. 1. A method for nucleic acid synthesis and regeneration of a reusable initiator for said nucleic acid synthesis, comprising the steps of: providing an initiator having a 3' hydroxyl group; incorporating a linking nucleotide into said initiator by a polymerase, said initiator being selected from the group consisting of a mononucleoside, a mononucleotide, an oligonucleotide, a polynucleotide, and a peptide nucleic acid, said linking nucleotide having a substrate base and a substrate sugar; incorporating a plurality of nucleotide monomers into the initiator immediately following the linking nucleotide with the polymerase to synthesize a nucleic acid, the polymerase being selected from the group consisting of Family A DNA polymerase, Family B DNA polymerase, Family C DNA polymerase, Family D DNA polymerase, Family Y DNA polymerase, and reverse transcriptase; cleaving the substrate base with a monofunctional DNA glycosylase to generate an abasic site; cleaving the substrate sugar and the backbone of the nucleic acid at the abasic site with an abasic endonuclease to form a 3' phosphate group at the 3' terminal nucleotide of the initiator; dephosphorylating the 3' terminal nucleotide of the initiator with an enzyme having 3' phosphatase activity to convert the 3' phosphate group back to a 3' hydroxyl group; The method includes:

2. The method of claim 1 , wherein the initiator is attached to a solid support.

3. 1. A method for regenerating an initiator for nucleic acid synthesis, comprising the steps of: providing a nucleic acid synthesized by a polymerase and an initiator attached to a solid support, said initiator being selected from the group consisting of a mononucleoside, a mononucleotide, an oligonucleotide, a polynucleotide, and a peptide nucleic acid, said initiator comprising a linked nucleotide having a substrate base and a substrate sugar, and said polymerase being selected from the group consisting of a Family A DNA polymerase, Family B DNA polymerase, Family C DNA polymerase, Family D DNA polymerase, Family Y DNA polymerase, and a reverse transcriptase; cleaving the substrate base with a monofunctional DNA glycosylase to generate an abasic site; cleaving the substrate sugar and the backbone of the nucleic acid at the abasic site with an abasic endonuclease to release the nucleic acid from the initiator and form a 3' phosphate group at the 3' terminal nucleotide of the initiator; and dephosphorylating the 3'-terminal nucleotide of the initiator with an enzyme having 3' phosphatase activity, whereby the 3' phosphate group is converted to a hydroxyl group on the 3'-terminal nucleotide, regenerating the initiator for reuse for further nucleic acid synthesis; The method includes:

4. 4. The method of claim 1, wherein the monofunctional DNA glycosylase is selected from the group consisting of uracil-DNA glycosylase, alkyladenine DNA glycosylase, single-strand-selective monofunctional uracil DNA glycosylase 1, methyl-binding domain glycosylase 4, thymine DNA glycosylase, mutY homolog DNA glycosylase, alkylpurine glycosylase C, alkylpurine glycosylase D, 8-oxo-guanine glycosylase 1 without abasic site lyase activity, endonuclease III-like 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 1 without abasic site lyase activity, endonuclease VIII-like glycosylase 2 without abasic site lyase activity, and endonuclease VIII-like glycosylase 3 without abasic site lyase activity.

5. 5. The method of claim 4, wherein the monofunctional DNA glycosylase is one of a uracil-DNA glycosylase and an alkyladenine DNA glycosylase.

6. 4. The method of claim 1, wherein the abasic site endonuclease is selected from the group consisting of endonuclease VIII and endonuclease III.

7. 7. The method of claim 6, wherein the abasic site endonuclease is endonuclease VIII.

8. The method according to any one of claims 1 to 3, wherein the enzyme possessing 3' phosphatase activity is selected from the group consisting of polynucleotide kinase 3'-phosphatase and 3'-phosphoesterase.

9. The method according to any one of claims 1 to 3, wherein the enzyme having 3' phosphatase activity is selected from the group consisting of T4 polynucleotide kinase having 3'-phosphatase activity, and zinc finger DNA 3'-phosphoesterase.

10. The substrate base of the linked nucleotide is uracil, hypoxanthine, thymine, cytosine, guanine, 5-fluorouracil, 5-hydroxymethyluracil, 5-formylcytosine, 5-carboxylcytosine, 3-methyladenine, 3-methylguanine, 7-methyladenine, 7-methylguanine, N ６ 4. The method of claim 1, wherein the 2-hydroxyl adenine is selected from the group consisting of 5-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxyl cytosine, 5-hydroxyuracil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine of adenine, formamidopyrimidine of guanine, adenine paired with guanine, uracil paired with guanine, uracil paired with adenine, thymine paired with guanine, ethenocytosine paired with 8-oxo-7,8-dihydroguanine, and 2-hydroxyladenine paired with guanine.

11. 11. The method of claim 10, wherein the substrate base of the linked nucleotide is uracil or hypoxanthine.

12. 4. The method of claim 1, wherein the substrate base is uracil, thereby forming deoxyuridine.

13. 4. The method of claim 1, wherein the substrate base is hypoxanthine, thereby forming deoxyinosine.

14. 4. The method of claim 1, wherein the initiator, the nucleic acid and the linking nucleotide are each one of template-independent and template-dependent.

15. The method of any one of claims 1 to 3, wherein the polymerase is a Family B DNA polymerase.

16. 3. A kit for nucleic acid synthesis and regeneration of reusable nucleic acids for said nucleic acid synthesis, comprising a polymerase selected from the group consisting of Family A DNA polymerase, Family B DNA polymerase, Family C DNA polymerase, Family D DNA polymerase, Family Y DNA polymerase, and reverse transcriptase; linking nucleotides; a monofunctional DNA glycosylase; an abasic site endonuclease; and an enzyme possessing 3' phosphatase activity; for use according to the method of claim 1 or 2.

17. 4. A kit for regenerating an initiator for nucleic acid synthesis, comprising: a monofunctional DNA glycosylase; an abasic site endonuclease; and an enzyme possessing 3' phosphatase activity, for use in accordance with the method of claim 3.

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