Method for enzymatic synthesis of nucleic acids or polynucleotides and kits for carrying out said method

Abstract

Methods for enzymatic polynucleotide synthesis and kits for carrying out the methods are provided. The method includes an initiator having a free 3'-hydroxyl group, and a polymerase of a nucleic acid chain is formed from the free 3'-hydroxyl group by incorporating a nucleotide monomer into the initiator, the nucleic acid chain includes a guiding nucleotide recognized by an endonuclease, and the endonuclease cleaves the nucleic acid chain to release a predetermined polynucleotide and form a new free 3'-hydroxyl group at the 3'-end of the remaining nucleic acid chain, which serves as a reusable initiator. A kit for carrying out the polynucleotide synthesis method is also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 304,282, filed January 28, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Description of sequence listing) In accordance with 37 CFR § 1.831-835, this application has been submitted electronically in XML format, and the following submission is incorporated by reference in its entirety into this specification: Sequence Listing in computer readable form (XML file name: Sequence Listing.xml, text file creation date: January 18, 2023, size: 20.8 kb).

[0003] The present disclosure relates to methods for enzymatic nucleic acid or polynucleotide synthesis under different conditions and kits for carrying out the methods. [Background technology]

[0004] Enzymatic methods for the de novo synthesis of nucleic acids are being rapidly developed to meet the burgeoning demand in a variety of new applications (e.g., synthetic biology, next-generation sequencing, nucleic acid-based therapeutics, diagnostics, vaccines, DNA data storage, etc.) for producing nucleic acids or polynucleotides with user-defined sequences and lengths.

[0005] Currently, enzymatic nucleic acid synthesis methods must rely on template-independent polymerases, such as terminal deoxynucleotidyl transferase (TdT), which iteratively add nucleotides to an initiator. The initiator usually consists of a single stranded nucleic acid or polynucleotide as the starting point for new nucleic acid or polynucleotide synthesis. Typically, the synthetic initiator must be immobilized on a solid support. Enzymatic nucleic acid synthesis can then continue to extend the nucleic acid or polynucleotide chain from the initiator until the desired length and sequence is obtained.

[0006] When devising a practical implementation of such enzymatic synthesis of nucleic acids, one of the major challenges is to efficiently obtain the newly synthesized oligonucleotide / polynucleotide product from the initiator immobilized on a solid support. For example, U.S. Patent No. 10,683,536 B2 describes an enzymatic method for synthesizing polynucleotides that requires alkaline solutions, metal ions, and specific cleavage reagents, such as type II restriction endonucleases, to decouple the newly synthesized nucleic acid or polynucleotide from the nucleic acid initiator. However, the use of alkaline solutions to release the nascent nucleic acid or polynucleotide requires the use of a unique chemical linker between the newly synthesized nucleic acid and the initiator for a site-specific cleavage reaction, which is inconvenient for routine implementation. Meanwhile, the above description of cleaving the nascent nucleic acid or polynucleotide from the initiator using type II restriction endonucleases still lacks examples to prove the feasibility. Additionally, type II restriction endonucleases known in the art must rely on sequence-specific recognition sites to function, e.g., typically cleave DNA strands using palindromic sequences of 4-8 bases, limiting their broad application in enzymatic de novo nucleic acid synthesis.

[0007] Also, US Patent Application Publication No. 2021 / 0254114 A1 describes a method for cleaving a polynucleotide product of template-independent enzymatic nucleic acid synthesis, in which an initiator is designed to retain a site-specific 3'-end deoxyinosine (dI). The position of dI in this initiator is recognized by endonuclease V (EndoV), and therefore serves as a guide for EndoV to precisely cleave the newly synthesized DNA strand from the initiator. According to this disclosure, DNA strand cleavage activity is achieved only by E. coli endonuclease V (EcoEndoV), and the overall performance corresponds to a two-enzyme-based USER method that releases the DNA strand from the initiator by combining the excision of deoxyinosine by deoxyuridine of E. coli uracil-DNA glycosylase and DNA strand cleavage by endonuclease VIII. However, both EcoEndoV and USER methods are limited by the need to use deoxyinosine and deoxyuridine, respectively, in site-specific recognition of nucleic acid initiators. In addition, the enzyme reactions of EcoEndoV and USER need to be performed at moderate reaction temperatures (<50°C), and are not suitable for nucleic acid cleavage reactions at high temperatures (≥50°C). The cleavage capabilities of these two enzyme systems cannot meet the requirements of users to adopt de novo polynucleotide synthesis in different conditions and applications.

[0008] Therefore, there is an urgent need for a more efficient, more versatile, more cost-effective and thermostable method to obtain a newly synthesized nucleic acid or polynucleotide strand and regenerate in the same step an initiator with a free hydroxyl group to enable the next round of enzymatic nucleic acid synthesis. Summary of the Invention [Problem to be solved by the invention]

[0009] Disclosed herein are methods and kits for preparing synthetic polynucleotides for multiple uses, such as for preparing custom polynucleotides, DNA probes, or RNA probes.

[0010] This specification discloses a site-specific acquisition method and kit for obtaining a polynucleotide of a predetermined sequence and length according to the user's intention. By adopting the method disclosed herein, the user can set the sequence and length of the predetermined polynucleotide and the position of the guide nucleotide in the newly synthesized nucleic acid, and directly obtain the desired predetermined polynucleotide by site-specific polynucleotide cleavage using a single enzyme. Therefore, according to the method and kit disclosed herein, the user can accurately and efficiently obtain a polynucleotide of a predetermined sequence and length. [Means for solving the problem]

[0011] Provided herein is a method for enzymatic polynucleotide synthesis comprising the steps of providing an initiator comprising a 3'-terminal nucleotide having a free 3'-hydroxyl group, incorporating a nucleotide monomer into the initiator by a polymerase to extend a nucleic acid strand from the free 3'-hydroxyl group, the nucleotide monomer comprising a guide nucleotide that is recognized by an endonuclease such that the guide nucleotide is incorporated at a specific position in the newly synthesized nucleic acid strand, and allowing the endonuclease to cleave the nucleic acid strand according to the position of the guide nucleotide to release a polynucleotide of a predetermined sequence and length and to separate the remaining nucleic acid strand or the initiator having a free 3'-hydroxyl group. The endonuclease specifically recognizes the position of the guide nucleotide in the newly synthesized nucleic acid strand and cleaves the second phosphodiester bond at the 3'-end of the guide nucleotide, the first phosphodiester bond at the 5'-end of the guide nucleotide, the second phosphodiester bond at the 5'-end of the guide nucleotide, or the third phosphodiester bond at the 5'-end of the guide nucleotide, thereby obtaining a desired polynucleotide of a predetermined sequence and length, and the remaining nucleic acid strand has a free 3'-hydroxyl group, which serves as a new initiator to be used for the next round of polynucleotide synthesis.

[0012] In some embodiments, the methods provided herein can be used for template-independent or template-dependent (i.e., template-directed) polynucleotide synthesis. In template-independent polynucleotide synthesis, the initiator is a single-stranded nucleic acid with a free 3'-hydroxyl group for extending a nascent nucleic acid strand. In template-dependent polynucleotide synthesis, the initiator is a single-stranded primer that anneals with a complementary template nucleic acid to form a primer-template duplex and induces nucleic acid synthesis by a polymerase. The polymerase extends the primer according to the sequence information of the template.

[0013] In some embodiments, the guide nucleotide is a natural, non-natural or modified nucleotide, and the nucleobase of the nucleotide is, for example, uracil, xanthine, or hypoxanthine.

[0014] In some embodiments, the guide nucleotide is a natural, non-natural or modified nucleotide, such as a nucleotide containing uracil or inosine. In some embodiments, the methods provided herein include the use of a template-independent polymerase or a template-dependent polymerase, which can be, for example, a family B DNA polymerase.

[0015] Provided herein is a method for obtaining a polynucleotide by an enzyme, the method comprising the steps of providing a synthetic polynucleotide having a guide nucleotide that is specifically recognized by an endonuclease, and allowing the endonuclease to cleave the synthetic polynucleotide to release a polynucleotide of predetermined sequence and length, wherein the endonuclease recognizes the position of the guide nucleotide in the synthetic polynucleotide and cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, thereby obtaining a polynucleotide of predetermined sequence and length.

[0016] In some embodiments, the endonuclease is derived from Thermococcus barophilus (Tba), Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac), Bacillus pumilus (Bpu), Pyrococcus abyssi (Pab), Thermococcus kodakarensis (Tko), Thermococcus gammatolerans (Tga), or Bacillus subtilis (Bsu).

[0017] In some embodiments, the methods provided herein include the use of hyperthermophilic or mesophilic endonucleases that can specifically recognize the guide nucleotide incorporated into a newly synthesized nucleic acid or polynucleotide strand, and thus, the endonucleases cleave the nucleic acid or polynucleotide strand according to the position of the guide nucleotide in the nucleic acid or polynucleotide strand, such that the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide can be specifically cleaved by the corresponding endonuclease.

[0018] In some embodiments, the hyperthermophilic endonuclease is from the group consisting of Thermococcus barophilus endonuclease V (Tba Endo V), Pyrococcus furiosus endonuclease V (Pfu Endo V), Thermococcus kodakaraensis endonuclease V (Tko Endo V), Pyrococcus furiosus endonuclease Q (Pfu Endo Q), Methanosarcina acetivorans endonuclease Q (Mac Endo Q), Pyrococcus abyssi NucS endonuclease (Pab NucS), Thermococcus kodakaraensis EndoMS endonuclease (Tko EndoMS), Thermococcus gamma tolerance NucS endonuclease (Tga NucS), and enzyme variants or mutants thereof.

[0019] In some embodiments, the methods provided herein comprise the use of a mesophilic endonuclease from the group such as Bacillus subtilis endonuclease V (Bsu Endo V; SEQ ID NO: 11), Bacillus pumilus endonuclease Q (Bpu Endo Q; SEQ ID NO: 12) and variants of these enzymes on a newly synthesized nucleic acid or polynucleotide strand.

[0020] In some embodiments, the endonuclease is selected from the group consisting of Bacillus subtilis endonuclease V (Bsu Endo V), Escherichia coli endonuclease V (Eco Endo V; SEQ ID NO: 7), Pyrococcus furiosus endonuclease V (Pfu Endo V; SEQ ID NO: 8), Thermococcus barophilus endonuclease V (Tba Endo V; SEQ ID NO: 10), Thermococcus kodakaraensis endonuclease V (Tko Endo V; SEQ ID NO: 16), or a variant of these enzymes, and specifically cleaves the second phosphodiester bond located 3' to the guide nucleotide.

[0021] In some embodiments, the endonuclease is selected from the group consisting of Pyrococcus furiosus endonuclease Q (Pfu Endo Q; SEQ ID NO: 9), Methanosarcina acetivorans endonuclease Q (Mac Endo Q; SEQ ID NO: 14), Bacillus pumilus endonuclease Q (Bpu Endo Q; SEQ ID NO: 12), and variants thereof, and specifically cleaves the first phosphodiester bond located on the 5'-terminal side of the guide nucleotide in a newly synthesized nucleic acid or polynucleotide strand.

[0022] In some embodiments, the endonuclease is selected from the group consisting of Thermococcus gamma tolerance NucS endonuclease (Tga NucS; SEQ ID NO: 15), Pyrococcus abyssi NucS endonuclease (Pab NucS; SEQ ID NO: 13), and variants of these enzymes, and specifically cleaves the second phosphodiester bond 5' to the guide nucleotide in a newly synthesized nucleic acid or polynucleotide strand.

[0023] In some embodiments, the endonuclease is selected from the group consisting of Thermococcus kodakaraensis EndoMS endonuclease (Tko EndoMS, SEQ ID NO: 17) and variants thereof, and specifically cleaves the third phosphodiester bond located 5' to the guide nucleotide in a newly synthesized nucleic acid or polynucleotide strand.

[0024] In some embodiments, the endonuclease recognizes a guide nucleotide in a nascent nucleic acid strand or synthetic polynucleotide to cleave at a specific phosphodiester bond adjacent to the guide nucleotide. In some embodiments, the methods provided herein can be performed at higher temperatures than previously used methods. The temperature range for performance can be, for example, 10°C to 100°C, 50°C to 90°C, or 70°C to 90°C. In some embodiments, the nucleic acid initiator in the methods provided herein is free or forms a duplex synthetic polynucleotide that is attached and immobilized via its 5' end to a solid support according to the methods disclosed herein. The solid support can be, for example, a granule, a resin, a bead, a slide, a chip, an array, a membrane, a matrix, a flow cell, a well, a chamber, a microfluidic chamber, a channel, a microfluidic channel, a gel, a synthetic polymer, or any surface to which a synthetic nucleic acid strand or polynucleotide can be attached.

[0025] The present disclosure further provides a kit for enzymatically synthesizing polynucleotides. In some embodiments, the sequence and length of the polynucleotide are predetermined or designed. The kit for synthesizing an oligonucleotide of a predetermined sequence and length of the present disclosure may include a nucleic acid initiator including a 3'-terminal nucleotide having a free 3'-hydroxyl group, and a polymerase for incorporating a plurality of nucleotide monomers into the initiator to extend a nucleic acid chain from the free 3'-hydroxyl group of the initiator, the nucleotide monomers including a guide nucleotide that is specifically recognized by an endonuclease, thereby incorporating the guide nucleotide at a designated position of the nucleic acid chain. The endonuclease then cleaves the nucleic acid chain according to the position of the guide nucleotide to release a polynucleotide of the desired sequence and length, while leaving a free 3'-hydroxyl group at the 3' end of the remaining nucleic acid chain. The endonuclease specifically recognizes the guide nucleotide and cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, respectively. Thus, a polynucleotide of the desired sequence and length is obtained, and the remaining nucleic acid strand with a new free 3'-hydroxyl group can be used as a new initiator for the next round of polynucleotide synthesis.

[0026] The present disclosure further provides a kit for obtaining a polynucleotide of a predetermined sequence and length by an enzyme. In some embodiments, the target polynucleotide to be cleaved is any synthetic polynucleotide prepared in advance. In some embodiments, the kit includes a synthetic polynucleotide having a site-specific guide nucleotide and an endonuclease that cleaves the synthetic polynucleotide to release a predetermined polynucleotide, and the endonuclease recognizes the position of the guide nucleotide in the polynucleotide and cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, respectively, to obtain a desired predetermined polynucleotide.

[0027] In some embodiments, guide nucleotides, including uracil or inosine, in a nucleic acid strand or polynucleotide chain may be pre-existing or may be synthesized de novo using chemical or enzymatic methods. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram according to at least one embodiment of the present disclosure illustrating template-independent de novo synthesis of single-stranded DNA (ssDNA) from an initiator (with a free 3'-hydroxyl group at its 3' end), cleavage of the newly synthesized DNA strand to obtain a polynucleotide of desired sequence and length, and regeneration of a new free 3'-hydroxyl group from the remaining nucleic acid strand after cleavage of the synthesized DNA strand to obtain a polynucleotide of interest, while using the remaining nucleic acid strand as a new reusable initiator in the next round of nucleic acid synthesis. Prior to the nucleic acid synthesis reaction, the 5' end of the initiator is fixed to a solid support (SS). X represents a guide nucleotide and is used as a starting point for numbering nucleotide positions in the nucleic acid strand. N represents the incorporated nucleotide monomer, the subscript numbers are in ascending order toward the 3' end or downstream of the nucleic acid chain (e.g., N1, N2, N3, N4, and N5), and the subscript numbers are in descending order toward the 5' end or upstream of the nucleic acid chain (e.g., N -1 , N -2 and N -3 E1 to E4 represent different endonucleases. E1, E2, E3, and E4 enzymes are responsible for the division between N2 and N1, and between X and N, respectively. -1 Between -1 and N -2 Between or N -2 and N -3 The present invention generally describes an enzymatic nucleic acid synthesis process in which the phosphodiester bond between the initiator and the 5' end of the initiator is cleavable, and the 5' end of the initiator is linked to a solid phase support (SS). 2A and 2B are schematic diagrams according to at least one embodiment of the present disclosure showing template-directed DNA synthesis from an initiator (i.e., a primer) (having a free 3'-hydroxyl group at its 3' end), cleavage of the nascent DNA strand to obtain a polynucleotide of desired sequence and length, and regeneration of the free 3'-hydroxyl group at the 3' end of the remaining nucleic acid strand to form a new reusable initiator for the next round of nucleic acid synthesis. The 5' end of the initiator is fixed to a solid support (SS) prior to the nucleic acid synthesis reaction. Thus, the template DNA is either linked to the solid support via its 3' end (FIG. 2A) or partially hybridized to the initiator by annealing or other known hybridization techniques (FIG. 2B). X represents a guide nucleotide and is used as a starting point for numbering the nucleic acid strand. N represents the incorporated nucleotide monomer, the subscript numbers are in ascending order toward the 3' end or downstream of the nucleic acid chain (e.g., N1, N2, N3, N4, and N5), and the subscript numbers are in descending order toward the 5' end or upstream of the nucleic acid chain (e.g., N -1 , N -2 and N -3 E1 to E4 represent different endonucleases. E1, E2, E3, and E4 enzymes are responsible for the division between N2 and N1, and between X and N, respectively. -1 Between -1 and N -2 Between or N -2 and N -3 This invention generally describes an enzymatic nucleic acid synthesis method that can cleave the phosphodiester bond between the nucleotides 1 and 2. FIG. 3 is a fluorescent image of a urea polyacrylamide gel showing the feasibility of template-independent nucleic acid synthesis according to at least one embodiment of the present disclosure. [Lane S shows initiator DNA only (biotin-FAM-45mer ssDNA). Lane 1 shows initiator DNA extended with 3'-O-(azidomethyl)-2'-deoxyuridine (U) by a polymerase. Lane 2 shows initiator DNA extended sequentially with 3'-O-(azidomethyl)-2'-deoxyuridine (U) and 3'-O-(azidomethyl)-2'-deoxyguanosine (G) after each nucleotide incorporation and 3'-deprotection step. Lane 3 shows the initiator DNA extended with 3'-O-(azidomethyl)-2'-deoxyuridine after a 3'-deprotection step, followed by extension with dNTPs (a mixture of dATP, dCTP, dGTP, and dTTP). 4A and 4B are fluorescence images of urea polyacrylamide gels showing the results of Examples 1 and 2, respectively, at 37° C. or 70° C., showing site-specific cleavage of free (FIG. 4A(1) and FIG. 4B(1)) or immobilized (FIG. 4A(2) and FIG. 4B(2)) single-stranded DNA (ssDNA), and free (FIG. 4A(3) and FIG. 4B(3)) or immobilized (FIG. 4A(4) and FIG. 4B(4)) double-stranded DNA (dsDNA) by preferentially or specifically recognizing deoxyinosine (I, FIG. 4A) or deoxyuridine (U, FIG. 4B), respectively, with exemplary enzymes. S means substrate DNA only. Eco EndoV means homemade E. coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu EndoV refers to Pyrococcus furiosus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and endonuclease VIII (EndoVIII). C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). 5A and 5B are fluorescence images of urea polyacrylamide gels showing the results of Examples 3 and 4, respectively, showing site-specific cleavage of free (FIG. 5A(1) and FIG. 5B(1)) or immobilized (FIG. 5A(2) and FIG. 5B(2)) single-stranded DNA (ssDNA), and free (FIG. 5A(3) and FIG. 5B(3)) or immobilized (FIG. 5A(4) and FIG. 5B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by the exemplary enzymes, respectively, by preferentially or specifically recognizing deoxyinosine (I, FIG. 5A) or deoxyuridine (U, FIG. 5B). Pfu EndoQ refers to Pyrococcus furiosus endonuclease Q. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylators (hAAG) and EndoVIII. C3 refers to a uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). 6A and 6B are fluorescence images of urea polyacrylamide gels showing the results of Examples 5 and 6, respectively, showing site-specific cleavage of free (FIG. 6A(1) and FIG. 6B(1)) or immobilized (FIG. 6A(2) and FIG. 6B(2)) single-stranded DNA (ssDNA), and free (FIG. 6A(3) and FIG. 6B(3)) or immobilized (FIG. 6A(4) and FIG. 6B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by preferentially or specifically recognizing deoxyinosine (I, FIG. 6A) or deoxyuridine (U, FIG. 6B) with exemplary enzymes, respectively. S means substrate DNA only. Eco EndoV means homemade Escherichia coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Tba Endo V means Thermococcus balophilus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII. C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). FIG. 7 shows the fluorescence images of urea polyacrylamide gels showing the results of Example 7, respectively [showing the site-specific cleavage of free (FIG. 7(1) single-stranded DNA (ssDNA) and free double-stranded DNA (dsDNA) (FIG. 7(2)) by preferentially or specifically recognizing deoxyuridine (U) with exemplary enzymes at 37° C. S refers to substrate DNA only. Eco EndoV refers to homemade E. coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Bsu Endo V refers to Bacillus subtilis endonuclease V. C1 refers to commercially obtained endonuclease V (NEB Eco EdoV) (Cat.#M0305S, Ipswitch, MA). C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA)]. 8A and 8B are fluorescence images of urea polyacrylamide gels showing the results of Example 8, showing site-specific cleavage of extended DNA strand products from free (FIGS. 8A(1), 8A(3), 8B(1), and 8B(3)) single-stranded DNA (ssDNA) or immobilized (FIGS. 8A(2), 8A(4), 8B(2), and 8B(4)) single-stranded DNA (ssDNA) by preferentially or specifically recognizing deoxyinosine (I, FIGS. 8A(1), 8A(2), 8B(1), and 8B(2)) and deoxyuridine (U, FIGS. 8A(3), 8A(4), 8B(3), and 8B(4)), respectively, by exemplary enzymes at 37° C., 55° C., and / or 60° C. S denotes substrate DNA only. Eco EndoV means homemade E. coli endonuclease V, which may be made with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu Endo V means Pyrococcus furiosus endonuclease V. Tba means Thermococcus barophilus endonuclease V. Pfu Endo Q means Pyrococcus furiosus endonuclease Q. Bpu Endo Q means Bacillus pumilus endonuclease Q.]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The following embodiments are provided to describe the present invention in detail. Those skilled in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and can implement and use it in other different embodiments. Therefore, in order to implement the disclosure of this specification, the following embodiments can be adjusted and / or modified for different aspects or applications without departing from the scope of the present invention, and any element or method within the scope of the contents disclosed in this specification can be combined with any other element or method in other embodiments of this specification.

[0029] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. The term "or" is used interchangeably with the term "and / or," unless the context clearly dictates otherwise.

[0030] As used herein, the terms "comprise," "include," "contain," and any variations thereof are intended to cover non-exclusive inclusions. For example, when an object is described as "comprising" a limitation, unless otherwise specified, other ingredients, elements, compositions / components, structures, regions, parts, devices, systems, steps, or connections, etc. may additionally be included and other limitations are not excluded.

[0031] The numerical ranges used in this specification are inclusive and combinable, and any numerical value within the numerical ranges of this specification can be set as a maximum or minimum value to derive subranges therefrom. For example, the numerical range "10°C to 100°C" includes any subrange from a minimum value of 10°C to a maximum value of 100°C, such as subranges of 10°C to 50°C, 60°C to 100°C, and 70°C to 90°C. Numerical ranges can be derived by arbitrarily selecting multiple numerical values ​​used in this specification as maximum and minimum values. For example, numerical ranges such as 37°C to 55°C, 37°C to 70°C, and 55°C to 70°C can be derived from the numerical ranges of 37°C, 55°C, and 70°C.

[0032] The term "about" as used herein generally means a numerical value that is intended to encompass, and the range covers a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from a given numerical value or a given numerical range. Such variations in numerical values ​​may occur, for example, due to experimental error, typical errors in measuring or handling procedures for producing a compound, composition, concentrate, or formulation, differences in source, manufacture, or purity of starting materials or components used in the present disclosure, and similar considerations. Alternatively, the term "about" means that the value is within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless otherwise specified, numerical ranges, amounts, values, and percentages, such as amounts of materials, lengths of time, temperatures, operating conditions, ratios of amounts, etc. disclosed herein should be understood to be modified in all cases by the term "about".

[0033] As used herein, the terms "nucleic acid", "nucleic acid sequence" and "nucleic acid fragment" refer to a nucleotide or ribonucleotide sequence in single- or double-stranded form, the source of which is not limited herein and generally includes natural nucleotides or artificial chemical mimetics. As used herein, the term "nucleic acid" can be used interchangeably with terms including natural or non-natural "oligonucleotides", "polynucleotides", "genes", "cDNA", "RNA" and "mRNA".

[0034] Nucleic acids as used herein also include nucleic acid analogs. The term nucleic acid analog is known to refer to compounds that are functionally or structurally equivalent to naturally occurring RNA and DNA or artificial nucleic acids. Nucleic acid analogs may have one or more modified nucleotide moieties (phosphate backbone, pentose, nucleobase). These modifications on the nucleotides change the structure, geometry, and interaction of the nucleic acid with nucleic acid polymerase. Nucleic acid analogs also cover new categories of artificial nucleic acids, such as xeno nucleic acids (XNAs), which are designed to have new-to-nature sugar backbones that are different from natural products.

[0035] Examples of nucleic acid analogs include universal bases such as inosine, 3-nitropyrrole, and 5-nitroindole, which can base pair with all four standard bases; phosphate-sugar backbone analogs such as peptide nucleic acids (PNAs), which affect the backbone properties of nucleic acids; and amine-reactive aminoallyl nucleotides. Chemical linkers or fluorophore-conjugated analogues such as nucleotides, thiol-containing nucleotides, biotin-conjugated nucleotides, rhodamine-conjugated nucleotides and cyanine-conjugated nucleotides; fluorescent base analogues such as 2-aminopurine (2-AP), 3-methylisoxanthopterin (3-MI), 6-methylisoxanthopterin (6-MI), 4-amino-6-methylisoxanthopterin (6-MAP) and 4-dimethylaminopyridine (DMAP); and oligonucleotides conjugated to fluorescent reporter dyes (ALEXA, FAM, TET, TAMRA, CY3, CY5, VIC, JOE, HEX, NED, PET, ROX, Texas Red). These include, but are not limited to, nucleic acid probes using various genes such as fluorophores and / or fluorescent quenchers (BHQ), molecular beacons (MB) that include stem-loop structures and single-stranded nucleic acid probes that are dual-labeled with a fluorophore and a quencher, and nucleic acid aptamers.

[0036] The term "oligonucleotide" as used herein is a general term for any type of polynucleotide, including polyribonucleotides, polydeoxyribonucleotides, or other polynucleotides consisting of N-glycosides with purine or pyrimidine bases. The terms "oligonucleotide" and "polynucleotide" as used herein do not refer to differences in length, but only to their molecular structure, and therefore can be used interchangeably herein. An oligonucleotide or polynucleotide may further include natural, damaged, or modified nucleotides.Examples of nucleic acid bases contained in an oligonucleotide or polynucleotide include adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, isoguanine, 5-fluorouracil, 5-hydroxymethyluracil, 5-formylcytosine, 5-carboxylcytosine, 3-methyladenine, 3-methylguanine, and the like. nine), 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytosine, 5-hydroxyluracil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine derivatives of adenine, formamidopyrimidine derivatives of guanine, adenine opposite guanine, uracil opposite guanine, uracil opposite adenine, thymine opposite guanine, ethenocytosine opposite guanine, adenine opposite 8-oxo-7,8-dihydroguanine and 2-hydroxyladenine opposite guanine.

[0037] The term "endonuclease activity" as used herein refers to an enzymatic activity that cleaves a bond at a specific recognizable nucleic acid site, resulting in a cleavage reaction of single-stranded or double-stranded DNA. Endonuclease activity can be provided by naturally occurring enzymes and their modified derivatives. Examples of modified derivatives include mutants / variants with enzymatic activity, enzyme fragments, and recombinant proteins derived from enzymes with endonuclease activity. For example, endonucleases can cleave single-stranded DNA on the one hand, releasing oligonucleotides / polynucleotides with 5'-monophosphate, and on the other hand, leave a free 3'-hydroxyl group on the remaining nucleic acid strand.

[0038] The terms "abasic", "apurinic / apyrimidinic" and D-spacer can be used interchangeably to refer to sites where no bases are present but the sugar-phosphate backbone remains intact. Abasic endonucleases are therefore also known as apurinic / apyrimidinic endonucleases.

[0039] The term "template" as used herein refers to a polynucleotide or polynucleotide mimic that contains a desired or unknown target nucleotide sequence. In some cases, the terms "target sequence", "template polynucleotide", "target nucleic acid", "target polynucleotide", "nucleic acid template", "template sequence", and variations thereof can be used interchangeably. For example, the term "template" refers to a nucleic acid strand from which a complimentary copy is synthesized from nucleotides or nucleotide analogs via replication by a template-dependent or template-directed nucleic acid polymerase. In a nucleic acid duplex structure, the template strand is depicted and described as the "bottom" strand by conventional definition. Similarly, the non-template strand is depicted and described as the "top" strand. The "template" strand may also be referred to as the "sense" or "plus" strand, and the non-template strand may also be referred to as the "antisense" or "minus" strand.

[0040] The term "initiator" as used herein refers to a mononucleoside, mononucleotide, oligonucleotide, polynucleotide, or modified analog at which a nucleic acid polymerase initiates de novo synthesis of a nucleic acid. The term "initiator" may also refer to XNA or peptide nucleic acid (PNA) with a 3'-hydroxyl group. The term "primer" as used herein refers to a short single-stranded oligonucleotide, polynucleotide, or modified nucleic acid analog that is used to combine with a template and initiate nucleic acid synthesis by a nucleic acid polymerase.

[0041] The nucleotide monomers disclosed herein include typical nucleotides and nucleotide analogs. The term "nucleotide analog" is known to those skilled in the art to refer to chemically modified or artificial nucleotides that are structural mimics of typical nucleotides. These nucleotide analogs can be used as substrates for nucleic acid polymerases to synthesize nucleic acids. Nucleotide analogs may have one or more modified nucleotide components (e.g., phosphate backbone, pentose, and nucleobase) of a nucleotide, which alter the structure and configuration of the nucleotide and affect the interaction of the nucleotide analog with other nucleobases and nucleic acid polymerases. For example, nucleotide analogs with modified nucleobases can impart different base-pairing and base-stacking properties to DNA or RNA. Also by way of example, base modifications can be made to nucleosides such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other analogs that allow hybridization. In other exemplary embodiments, modifications can be made at the sugar moiety (e.g., replacement of deoxyribose with an analog) and / or the phosphate group (e.g., boronate, alkylphosphonate, or phosphorothioate derivatives). Nucleotide analog monomers can be selected from phosphate groups having monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

[0042] Other examples of nucleotide analogs include nucleotides having removable blocking moieties, including, but not limited to, 3'-O-blocking moieties, base blocking moieties, and combinations thereof. Examples of 3'-O-blocking moieties include O-azido, 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, and O-[2-methyl,4-methoxy]-tetrahydropyranyl. Examples of base-blocking moieties include, but are not limited to, {O-[2-methyl,4-methoxy]-tetrahydropyranyl}, O-[5-methyl]-tetrahydropyranyl}, and O-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl. Examples of base-blocking moieties include reversible dye-terminators.Examples of reversible dye terminators include, but are not limited to, the Illumina MiSeq reversible dye terminator, the Illumina HiSeq reversible dye terminator, the Illumina Genome Analyzer IIX reversible dye terminator, the Helicos Biosciences Heliscope reversible dye terminator, and the LaserGen lightning terminator.

[0043] The term "polymerase" as used herein refers collectively to DNA polymerases, including naturally occurring enzymes and modified derivatives thereof. For example, sequence modifications can be used in polymerases to eliminate 5' to 3' or 3' to 5' exonuclease activity. Means for improving polymerase performance also include mutations or deletions to functional groups or sequences of the polymerase.

[0044] According to the present disclosure, the polymerase can be a template-dependent or template-independent polymerase. The polymerase can be selected from the group consisting of family A DNA polymerases (e.g., T7 DNA polymerase, Pol I, Pol γ, θ, and ν), 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 fragments having enzymatic activity thereof.

[0045] Non-limiting examples of widely used template-dependent polymerases include DNA-dependent DNA polymerases T7 DNA polymerase of T7 phage and T3 DNA polymerase of T3 phage, DNA-dependent RNA polymerases T7 RNA polymerase of T7 phage and T3 RNA polymerase of T3 phage, DNA-dependent DNA polymerase E. coli DNA polymerase I or a fragment thereof called Klenow fragment, thermostable DNA-dependent DNA polymerases Thermus aquaticus DNA polymerase, Tth DNA polymerase and Vent DNA polymerase, DNA-dependent DNA polymerases eukaryotic DNA polymerase β, RNA-dependent DNA polymerase telomerase, 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, polyA polymerase, DNA polymerase θ (θ), DNA polymerase μ (μ), DpoIV polymerase, and terminal deoxynucleotidyl transferase. Polymerases suitable for nucleic acid synthesis and addition of linking nucleotides should be within the expertise and prior art of the skilled artisan, and therefore a detailed description thereof will be omitted here for the sake of brevity.

[0047] In some embodiments, the methods provided herein include the use of a Family B polymerase. Examples of family B polymerases include DNA polymerase II from Escherichia coli (Eco), DNA polymerase II from Pseudomonas aeruginosa (Pae), DNA polymerase from Escherichia phage RB69 (RB69), DNA polymerase from Escherichia phage T4 (T4), DNA polymerase from Bacillus phage Phi29 (Phi29), DNA polymerase delta catalytic subunit from Saccharomyces cerevisiae (ScePOLD), human DNA polymerase delta catalytic p125 subunit from Sulfolobus solfataricus (Sso), DNA polymerase from Pyrobaculum islandicum (Pis), and the hyperthermophilic Thermococcus (strain 9°N-7) DNA polymerase (9°N), Thermococcus kodakaraensis DNA polymerase (Kod1), Methanococcus maripaludis DNA polymerase (Mma), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus gorgonarius DNA polymerase (Tgo), and Thermococcus litoralis DNA polymerase (Vent).

[0048] The nucleotide monomers described herein may have a removable blocking moiety. Examples of the removable blocking moiety include, but are not limited to, a 3'-O-blocking moiety, a base blocking moiety, and combinations thereof. The nucleotide monomers having the removable blocking moiety are also called reversible terminators. Thus, the nucleotide monomers having the 3'-O-blocking moiety are also called 3'-blocked reversible terminators or 3'-O-modified reversible terminators, and the nucleotide monomers having the base blocking moiety are also called 3'-unblocked reversible terminators or 3'-OH unblocked reversible terminators. The term "reversible terminator" as used herein refers to a chemically modified nucleotide monomer. When this reversible terminator is incorporated into a growing nucleic acid by a polymerase, it blocks the further incorporation of additional nucleotide monomers by the polymerase. Such "reversible terminator" bases and nucleic acids can be deprotected by chemical or physical treatment, and after such deprotection, the nucleic acid can be further extended by the polymerase.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-methylmethyl, and O-acylacyl. Examples of 3'-unblocked reversible terminators include 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, and Examples of suitable dUTPs include, but are not limited to, 1-[(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]-5-methyl-7-deaza-dGTP {1-[(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy]-5-methyl-7-deaza-dGTP}, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethyl-propyloxy] methyl-dUTP {5-[(S)-1-(5-methoxy-2-nitrophen-yl)-2,2-dimethyl-propyloxy] methyl-dUTP} and 5-[(S)-1-(2-nitrophenyl)-2,2-dimethyl-propyloxy] methyl-dUTP {5-[(S)-1-(2-nitrophenyl)-2,2-dimethyl-propyloxy] methyl-dUTP}.The base block portion may be a reversible dye-terminator. Examples of reversible dye-terminators include, but are not limited to, the reversible dye-terminator of Illumina NovaSeq, the reversible dye-terminator of Illumina NextSeq, the reversible dye-terminator of Illumina MiSeq, the reversible dye-terminator of Illumina HiSeq, the reversible dye-terminator of Illumina Genome Analyzer IIX, the Lightning terminator of LaserGen, and the reversible dye-terminator of Helicos Biosciences Heliscope.

[0049] In some embodiments, the guide nucleotide is a natural, non-natural or modified nucleotide, and the nucleobase of the nucleotide is, for example, adenine, thymine, cytosine, guanine, uracil, xanthine, hypoxanthine, isocytosine, isoguanine, 5-fluorouracil, 5-hydroxymethyluracil, 5-formylcytosine, 5-carboxylcytosine, 3-methyladenine, 3-methylguanine, 7-methyladenine, 7-methylguanine, N6-methyladenine, 8-oxo-7,8-dihydroguanine, 5-hydroxylcytos ... The guiding nucleotide may be silacil, dihydroxyuracil, ethenocytosine, ethenoadenine, thymine glycol, cytosine glycol, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, formamidopyrimidine derivatives of adenine, formamidopyrimidine derivatives of guanine, adenine opposite guanine, uracil opposite guanine, uracil opposite adenine, thymine opposite guanine, ethenocytosine opposite guanine, adenine opposite 8-oxo-7,8-dihydroguanine, and 2-hydroxyladenine opposite adenine. In at least one embodiment, the guiding nucleotide contains a nucleobase selected from the group consisting of uracil, xanthine, hypoxanthine, cytosine, and guanine. In some embodiments, the guiding nucleotide contains an apurinic / apyrimidinic lesion, which is an abasic site or a D spacer.

[0050] Reversible terminators are well known to those skilled in the art and are commonly used, so further details are omitted here for the sake of brevity.Nevertheless, applicable 3'-blocked reversible terminators, applicable 3'-unblocked reversible terminators, and applicable conditions for protection and deprotection (i.e., conditions for adding or removing the removable blocking moiety) can be found in literature such as, 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.

[0051] According to the present disclosure, an initiator or primer-template duplex can be used as a starting material for nucleic acid synthesis. The 5' end of the initiator / primer-template duplex can be linked to a solid support. The initiator can be attached directly to the solid support or can be attached to the solid support via a linker. Examples of solid supports include 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 granules, plastics (e.g., polyethylene, polypropylene, and polystyrene), gel-forming materials (e.g., gelatin), lipopolysaccharides, silicates, agarose, polyacrylamides, methyl methacrylate polymers, sol-gels, porous polymers, hydrogels, nanostructured surfaces nanotubes (e.g., carbon nanotubes), and nanogranules (e.g., gold nanogranules or quantum dots). Examples of suitable holographic dots include, but are not limited to, holographic dots.

[0052] Specifically, the disclosed method is used under the scenario of enzymatic nucleic acid synthesis, where a polymerase can extend a nucleic acid chain by adding multiple nucleotide monomers to an initiator, including site-specific recognizable nucleotide monomers. A given nucleic acid base or apurinic / apyrimidinic lesion is defined as a guide nucleotide. During enzymatic nucleic acid synthesis, the guide nucleotide is incorporated into the nucleic acid chain at a designated position, followed by site-specific enzymatic cleavage to obtain a desired target polynucleotide from the newly synthesized nucleic acid chain. Thus, the sequence and length of the nucleic acid chain and the corresponding cleavage enzyme can be flexibly designed and customized in advance before effectively obtaining a polynucleotide of the desired sequence and length.

[0053] Depending on the user's design, the resulting desired nucleic acid strand or polynucleotide may include newly synthesized nucleic acids or may use commercially available ready-to-use synthetic nucleic acids. The cleavage enzymes disclosed herein preferentially or specifically cleave a designated nucleic acid strand at a particular position, thereby releasing a polynucleotide of desired sequence and length.

[0054] The present disclosure provides a method for enzymatic synthesis of polynucleotides of defined sequence and length, which includes the use of an initiator with a free 3'-hydroxyl group at its 3' end, a newly synthesized nucleic acid strand by a polymerase, and an enzyme / endonuclease with polynucleotide cleavage activity that cleaves a specific phosphodiester bond within the newly synthesized nucleic acid strand. Specifically, the polymerase incorporates a nucleotide monomer containing a guide nucleotide into the initiator to extend the nucleic acid strand from the free 3'-hydroxyl group. Thus, upon incorporation by the polymerase, the guide nucleotide is at a specific position on the extended nucleic acid strand. The selected endonuclease then recognizes the guide nucleotide and cleaves the newly synthesized nucleic acid strand according to the position of the guide nucleotide, thereby releasing a polynucleotide of desired or predetermined sequence and length.

[0055] A polynucleotide having a predetermined or desired sequence and length can be prepared by cleaving a newly synthesized nucleic acid strand with a selected endonuclease, which specifically recognizes the position of the guide nucleotide in the nascent nucleic acid strand and preferentially cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, respectively. Also, cleaving the nucleic acid strand with an endonuclease leaves the remaining nucleic acid strand with a free 3'-hydroxyl group, which can be used as a new initiator or usable initiator for the next round of nucleic acid synthesis.

[0056] It should be noted that thermophilic endonucleases can be used to perform site-specific nucleic acid cleavage by recognizing the position of a guide nucleotide in a nascent nucleic acid strand. Due to the thermostability of the thermophilic enzyme itself, the thermostabilized endonucleases can accelerate the cleavage of nucleic acids under various reaction conditions, such as increasing the reaction temperature. In some embodiments, the endonuclease of the present disclosure is endonuclease V, endonuclease Q, NucS endonuclease, or EndoMS endonuclease.

[0057] Since the method provided herein does not limit the nucleic acid strand that can be used for cleavage and the user has ready-to-use starting material for various scenarios, the present specification also provides a method for enzymatically obtaining a polynucleoside of a predetermined sequence and length. The method includes the steps of providing a synthetic nucleic acid containing a guide nucleotide arranged to be specifically recognized by a selected endonuclease, and allowing the endonuclease to cleave the synthetic nucleic acid according to the position of the guide nucleotide in the nucleic acid, releasing a polynucleotide of a predetermined or desired sequence and length. As described above, the endonuclease specifically recognizes the position of the guide nucleotide in the nucleic acid and preferentially cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, respectively, to obtain a predetermined polynucleotide.

[0058] According to the method disclosed herein, the present invention provides a kit for synthesizing and obtaining a predetermined polynucleotide. Under an enzymatic nucleic acid synthesis scenario, the kit may include an initiator having a free 3'-hydroxyl group at the 3'-terminal nucleotide, a polymerase for incorporating nucleotide monomers (including guide nucleotide monomers) into the initiator to extend the nucleic acid chain from the free 3'-hydroxyl group so that the guide nucleotide is incorporated into the newly synthesized nucleic acid chain at a designated position, and an endonuclease that recognizes the guide nucleotide in the newly synthesized nucleic acid chain and cleaves the nucleic acid chain according to the position of the guide nucleotide to release a polynucleotide of a predetermined or desired sequence and length, so that the remaining nucleic acid chain has a new free 3'-hydroxyl group and can be easily used as a new initiator for the next round of nucleic acid synthesis. Under an enzymatic polynucleotide obtaining scenario, the kit may include a synthetic polynucleotide having a designated guide nucleotide and an endonuclease that recognizes the guide nucleotide and cleaves the synthetic polynucleotide to release a polynucleotide of a predetermined or desired sequence and length. The endonuclease recognizes the guide nucleotide and cleaves a specific phosphodiester bond according to the position of the guide nucleotide in the synthetic polynucleotide, resulting in a polynucleotide of desired or predetermined sequence and length. In either scenario, the site specificity of the endonuclease included in the kit is as described above.

[0059] As exemplified in more detail below, the methods and kits provided by the present invention utilize Pfu Endo V, Pfu Endo Q or Bpu Endo Q, Bsu Endo V or Tba Endo V, and a nucleic acid initiator or primer-template duplex to generate nucleic acid strands containing different unique guide nucleotides, which are used for site-specific recognition, and the phosphodiester bond cleavage reaction can be carried out at a common enzyme reaction temperature (e.g., ambient temperature (e.g., 10°C to 40°C), 37°C), or at a high reaction temperature (e.g., 70°C). The following examples and corresponding results show that the methods and kits provided by the present invention can utilize a nucleic acid initiator or primer-template duplex to generate nucleic acid strands containing unique guide nucleotides, which are used for recognition and site-specific nucleic acid cleavage, and precisely release polynucleotide strands of a predetermined or desired sequence and length at a wide range of reaction temperatures, thereby broadening the application and utility of enzymatic nucleic acid synthesis.

[0060] Endonucleases are enzymes that cleave nucleic acids (e.g., DNA or RNA) at the phosphodiester bonds connecting nucleotides and play an important role in maintaining biological functions (e.g., nucleic acid mismatch repair). Endonucleases are also used in DNA manipulation applications. T7 endonuclease I is widely used for mismatch repair and genome editing (e.g., mutations and deletions). The enzyme recognizes DNA mismatches and cleaves the first, second, or third phosphodiester bond on the 5'-terminal side of the mismatched position. Escherichia coli endonuclease V (Eco Endo V) is another enzyme that can specifically recognize DNA sites and cleave nucleic acid strands. E. coli EndoV recognizes deoxyinosine (dI) lesions in DNA and cleaves the second phosphodiester bond on the 3'-terminal side of the dI lesion, resulting in DNA strand breaks. As mentioned above, E. coli Endo V has been used in enzymatic de novo DNA synthesis to cleave the phosphodiester bond between the initiator and newly synthesized DNA. However, E. coli Endo V has a narrow substrate specificity and limited thermostability, limiting its utility, making it difficult to be widely used in de novo enzymatic DNA synthesis.

[0061] Other endonucleases, such as endonuclease V from Pyrococcus furiosus (Pfu) or Thermococcus var. philus (Tba), endonuclease Q from Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac) and Bacillus pumilus (Bpu), respectively, NucS endonuclease from Pyrococcus abyssi (Pab) and Thermococcus gamma tolerans (Tga), respectively, and EndoMS endonuclease from Thermococcus kodakaraensis (Tko), have a broader substrate range than E. coli Endo V. These endonucleases recognize various deaminated or oxidized bases, such as deoxyuridine and deoxyinosine, in nucleic acids. These endonucleases are also capable of recognizing apurinic / apyrimidinic lesions in DNA, such as abasic sites and dSpacers (also called abasic furans), which are structurally similar to abasic sites.

[0062] In addition, these endonucleases have higher thermostability. The reaction temperature range for DNA strand cleavage by Pfu Endo V, Tba Endo V, Pfu Endo Q, Mac Endo Q, Pab NucS, Tko EndoMS, and Tga NucS is much wider than that of E. coli Endo V, for example, 10°C to 100°C, 20°C to 30°C, 30°C to 40°C, 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C, as shown in the exemplary results. Compared to the prior art, the methods and kits disclosed herein provide an improved method for endonuclease-based nucleic acid strand cleavage, utilizing nucleic acid initiators or primers containing different types of deaminated or oxidized bases (e.g., deoxyuridine or deoxyinosine for site-specific recognition and cleavage of nucleic acid strands). Furthermore, the methods and kits disclosed herein provide an improved method of thermostable endonuclease-based nucleic acid cleavage that can be performed at a wider range of reaction temperatures to broaden the applications and usefulness of enzymatic nucleic acid synthesis. Also, after the release of a nucleic acid or polynucleotide strand of a given or desired sequence and length, the nucleic acid initiator or primer can be regenerated with a normal 3'-hydroxyl group. Thus, the nucleic acid initiator or primer can be reused for a new round of enzymatic nucleic acid synthesis.

[0063] 1, 2A and 2B show exemplary schemes of the present invention for template-independent (FIG. 1) and template-directed / template-dependent (FIG. 2A and 2B) nucleic acid synthesis. As shown in FIG. 1, 2A and 2B, an initiator (ssDNA) attached to a solid support is provided. In contrast to template-independent nucleic acid synthesis, template-directed / template-dependent nucleic acid synthesis requires a nucleic acid template to be attached to a solid support (FIG. 2A) or hybridized to the initiator by adhesion or other known hybridization techniques (FIG. 2B). In some embodiments, under a template-dependent scenario, a primer is used in combination with a template to generate a primer / template duplex (abbreviated as P / T duplex) for DNA synthesis. In the example scheme, the extension of an initiator or primer uses a nucleotide monomer (N) containing a conventional 3'-hydroxyl or 3'-blocking chemical moiety, respectively, to produce the desired sequence (N -3 ~N n ) to synthesize an oligonucleotide / polynucleotide strand having a predetermined nucleic acid base or a predetermined apurinic / apyrimidinic (AP) lesion. The nucleic acid strand sequence is configured to include at least one guide nucleotide (X) having a predetermined nucleic acid base or a predetermined apurinic / apyrimidinic (AP) lesion for recognition by a selected endonuclease. In some embodiments, the predetermined nucleic acid base can be deoxyuridine or deoxyinosine, and the apurinic / apyrimidinic lesion can be an abasic site or dSpacer, and each recognizable deoxynucleotide residue can be specifically recognized by a different type of corresponding endonuclease. Thus, the method and kit provide a scheme for synthesizing customized polynucleosides by incorporating nucleotide monomers (including typical and atypical nucleotides) into an initiator by a polymerase for site-specific cleavage according to the needs of the user.

[0064] The inventors have unexpectedly discovered that the nucleic acid cleavage activity of the endonuclease disclosed herein can recognize a guide nucleotide at a cleavable region at a specific site (site-specific cleavable region). After sufficient cycles of nucleotide addition / incorporation to generate a desired nucleic acid strand, an endonuclease-based cleavage reaction is utilized to cleave and / or modify a nucleic acid or polynucleotide strand that is close to the guide nucleotide and included in the site-specific cleavable region. The guide nucleotide (X) used for endonuclease-based recognition in the site-specific cleavable region is subjected to nucleic acid cleavage using a corresponding endonuclease. In at least one embodiment of the present disclosure, the nucleic acid strand having the site-specific cleavable region contains deoxyinosine or deoxyuridine, respectively, and can be treated with an endonuclease to release a predetermined extended nucleic acid strand. The endonuclease used may be endonuclease Q from Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac) or Bacillus pumilus (Bpu), endonuclease V from Bacillus subtilis (Bsu), Escherichia coli (Eco), Pyrococcus furiosus (Pfu) or Thermococcus barophilus (Tba), NucS endonuclease from Thermococcus gamma tolerans (Tga) or Pyrococcus abyssi (Pab), or EndoMS endonuclease from Thermococcus kodakaraensis (Tko). In the example scheme, endonucleases E1 to E4 recognize the guide nucleotide (X) and also act to recognize the nucleotide between N2 and N1, between X and N, and between N and N. -1 Between -1 and N -2 Between and N -2 and N -3and cleave the phosphodiester bond between them, respectively. In some embodiments, X may be deoxyinosine or deoxyuridine, E1 may be Bsu EndoV, Eco EndoV, Pfu EndoV, or Tba EndoV, E2 may be Pfu EndoQ, Mac EndoQ, Bpu Endo Q, E3 may be Tga NucS, and E4 may be Tko EndoMS, but the disclosure is not limited thereto. In addition, the thermostable properties of the endonucleases disclosed herein allow the nucleic acid strand cleavage reaction to be carried out over a wide range of reaction temperatures. Combined with the advantageous properties of the endonucleases disclosed herein, the methods of the disclosure can be used in various situations and applications of enzymatic nucleic acid synthesis. Site-specific cleavage of newly synthesized nucleic acid or polynucleotide strands by endonucleases not only releases the desired polynucleotide fragment, but also generates a new free 3'-hydroxyl group at the 3' end of the remaining nucleic acid strand, which can be used in a new round of synthesis reaction. Thus, when combined with the proper design and configuration of nucleotide monomer addition, enzymatic synthesis can be performed in a cyclically efficient manner without interruption by additional enzymatic processing (such as dephosphorylation by phosphatases).The present disclosure thus provides an accurate, efficient, cost-effective, and thermostable method to obtain a desired nucleic acid or polynucleotide strand and, in the same step, regenerate reusable nucleic acid initiators with free hydroxyl groups to accelerate the synthesis of nucleic acids on demand for users. EXAMPLES

[0065] The present disclosure will be described in detail through the following examples.However, these examples are merely illustrative of the present disclosure and do not limit the scope and meaning of the present disclosure in any way.In fact, upon reading this specification, many adjustments and modifications of the present disclosure can be clearly understood by those skilled in the art, and adjustments and modifications can be made without departing from the scope thereof.

[0066] A. Nucleic acid synthesis by nucleic acid polymerase to incorporate nucleotide monomers into an initiator The following synthetic polynucleotide initiator, FAM-45-mer DNA initiator, is used for template-independent DNA synthesis in this example.

[0067] FAM-45-mer DNA initiator:

[0068] [ka]

[0069] This single-stranded 45-mer polynucleotide initiator is modified with a biotin group at the 5' end, contains a fluorescein amidite (FAM) dye at the 23rd thymine base (underlined T) and has a free 3'-hydroxyl group at its 3' end. TM Nevertheless, in other embodiments, the initiator can be internally labeled with hexachlorofluorescein (HEX) or other fluorescent reporter dyes as disclosed herein, and the initiator can be in free form or can be immobilized at the 5' end on Dynabeads as disclosed herein. TM It may be immobilized on a solid phase carrier other than -280 streptavidin beads.

[0070] Also, template-independent nucleic acid synthesis reactions were carried out using family B type DNA polymerase (1 μM) to incorporate linked 3'-O-(azidomethyl)-2'-deoxyuridine triphosphate (100 μM) into the 3' end of the initiator, and the reaction was allowed to proceed for 15 min. To demonstrate that template-independent nucleic acid synthesis is possible immediately after incorporation of deoxyuridine monophosphate (dUMP) into the 3' end of the synthetic initiator, family B type DNA polymerase (1 μM) was used to stepwise incorporate 3'-O-(azidomethyl)-2'-deoxyguanosine triphosphate (100 μM) or a dNTP mixture (dATP, dCTP, dGTP, and dTTP) (100 μM) into an initiator containing deoxyuridine (U) at the 3' end. The synthesis reaction was started by adding 10 mM manganese cations, followed by incubation at 75° C. for 15 min. The reaction was stopped by adding 10 μL of 2× quench solution (95% deionized formamide and 25 mM EDTA) and heat denatured for 10 min at 98° C. The reaction products were analyzed on a 15% denaturing urea polyacrylamide gel and the gel analysis results were displayed on an Amersham Typhoon scanner (GE Healthcare Life Sciences, Marlborough, Mass., United States).

[0071] As shown in Figure 3, lane S shows only the initiator, lane 1 shows that deoxyuridine triphosphate (dUTP) was efficiently incorporated into the 3' end of the initiator by the family B DNA polymerase, and lanes 2 and 3 show that the family B DNA polymerase can incorporate deoxyguanosine triphosphate (dGTP) and dNTPs (N1, N2, etc.), respectively, immediately after incorporation of deoxyuridine (U) into the 3' end of the initiator. The polynucleotide samples in lanes 1 and 2 have the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0072] Thus, the examples demonstrate how a polymerase can incorporate typical (e.g., dNTPs) or atypical nucleotides (e.g., deoxyuridine or deoxyguanosine triphosphate) into an initiator to synthesize a customized polynucleotide chain containing at least one exemplary guide nucleotide (located at a user-desired position for recognition by an endonuclease) depending on the needs of the user.

[0073] While the above examples demonstrate the results of template-independent nucleic acid synthesis, in other embodiments, similar results can be achieved by designing an initiator for template-dependent nucleic acid synthesis. For example, the initiator's complementary template nucleic acid can be attached to a solid support (FIG. 2A) or hybridized to the initiator to form a duplex or double strand (FIG. 2B).

[0074] B. Endonucleases cleave or denature newly synthesized polynucleotides to release the desired nucleic acid fragment and regenerate initiators for the next round of nucleic acid synthesis. To demonstrate the feasibility of endonuclease cleavage / denaturation of newly synthesized polynucleotides and regeneration of synthetic initiators, single-stranded 38-mer polynucleotides containing deoxyuridine (U) or deoxyinosine (I) labeled with Hex dye were synthesized using the method described in section A above, and are called Hex-Top-U38-mer and Hex-Top-I38-mer, respectively. These two polynucleotides represent the nucleic acid products obtained by template-independent nucleic acid synthesis, which contain the initiator and the newly synthesized polynucleotide. To illustrate the nucleic acid products obtained by template-dependent nucleic acid synthesis (containing the initiator and the newly synthesized polynucleotide), the Hex-Top-U38-mer, Hex-Top-I38-mer, and a complementary single-stranded 38-mer nucleic acid (Bot-A38-mer) were hybridized at a molar ratio of 1:1.5 in 1xTE buffer containing 100 mM NaCl. DNA annealing reactions were carried out in a Bio-Rad thermal cycler machine, where the sample mixture was heated to 95° C. for 3 min and gradually cooled (5° C. / 30 sec) to 4° C. to form duplex and double-stranded 38-mer nucleic acids. These two nucleic acid products were used as DNA substrates in the following examples.

[0075] The polynucleotide sequence, buffer and solution series of the example are shown in the table below. Hex-Top-U38-mer and Hex-Top-I38-mer have a hexachlorofluorescein (HEX) label at the 5' end and a free 3'-hydroxyl group at the 3' end, but the present invention is not limited to this aspect. In other embodiments, the 5' end of the initiator can be labeled with fluorescein amidite (FAM) or other fluorescent reporter dyes disclosed herein. In addition, the initiator can be free or immobilized on a solid support of different embodiments disclosed herein.

[0076] [Table 1]

[0077] [Table 2]

[0078] To test whether newly synthesized polynucleotides, resulting from template-independent or template-dependent nucleic acid synthesis, containing guide nucleotides bearing defined nucleobases or apurinic / apyrimidinic (AP) lesions, can be recognized and cleaved to regenerate initiators for the next round of nucleic acid synthesis, the following experimental procedure was performed.

[0079] Example 1: Deoxyinosine recognition and DNA strand cleavage by Pfu Endo V at two different reaction temperatures The samples included (1) DNA substrate (S) alone as a negative control, (2) Escherichia coli endonuclease type V (Eco EndoV) (SEQ ID NO: 7) prepared in-house as described in U.S. Patent Application Publication No. 2021 / 0254114 A1, (3) Pyrococcus furiosus endonuclease V (Pfu Endo V) (SEQ ID NO: 8), (4) Escherichia coli endonuclease V (C1) obtained from New England BioLabs (Ipswich, MA), and (5) an enzyme mixture (C2) containing human alkyl adenine DNA glycosylase (hAAG) and EndoVIII as a positive control, representing the results of deoxyinosine excision and DNA strand cleavage at the guide nucleotide position in the nucleic acid product. For each sample group, a sample mixture (10 μL) containing 100 nM of single-stranded Hex-Top-I38-mer DNA substrate or double-stranded Hex-Top-I38-mer / Bot-A38-mer DNA substrate and 400 nM of endonuclease were incubated in the enzyme reaction buffer. The sample mixture was incubated at 37°C and 70°C for 20 min, respectively, and each enzyme reaction was stopped by adding an equal volume (10 μL) of 2× quench solution.

[0080] A total of 20 μL of samples were denatured at 95° C. for 10 min, and each sample mixture was analyzed by 20% denaturing polyacrylamide gel electrophoresis (4 μL of each sample) in 1× TBE buffer (90 mM Tris-alkali, 90 mM boric acid, and 2 mM EDTA) containing 8 M urea. The gel imaging results were then analyzed with an Amersham Typhoon scanner.

[0081] As shown in FIG. 4A and Table 3 below, Eco EndoV, Pfu Endo V, and C1 efficiently recognize deoxyinosine (I) at a reaction temperature of 37° C. and cleave the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) counting from deoxyinosine (I) toward the 3' end (or downstream) of DNA, releasing a 15-mer single-stranded or double-stranded DNA, which has a free 3'-hydroxyl group at the end of the remaining 23-mer single-stranded or double-stranded DNA and can immediately be used as a new (or reusable) initiator for nucleic acid synthesis in the next round. In contrast, C2 only excised deoxyinosine (I) and cleaved the DNA strand at a reaction temperature of 37° C. to release a 15-mer single-stranded or double-stranded DNA, but was unable to generate a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single-stranded or double-stranded DNA, which could not be used for a new round of nucleic acid synthesis. This example also demonstrated that only Pfu Endo V was able to efficiently cleave or denature DNA strands as described above at a reaction temperature of 70°C.

[0082] [Table 3]

[0083] Example 2: Deoxyuridine recognition and DNA strand cleavage by Pfu Endo V at two different reaction temperatures The samples included (1) DNA substrate only (S) as a negative control, (2) Escherichia coli endonuclease type V (Eco EndoV) prepared in-house as described in U.S. Patent Application Publication No. 2021 / 0254114 A1, (3) Pyrococcus furiosus endonuclease V (Pfu Endo V), (4) Escherichia coli endonuclease V (C1) obtained from New England BioLabs (Ipswich, MA), and (5) Uracil-specific excision reagent (Cat.#M5505S, Ipswich, MA) (C3) obtained from New England Biolabs as a positive control. The sample mixture (10 μL) contained 100 nM of single-stranded Hex-Top-U38-mer DNA substrate or double-stranded Hex-Top-U38-mer / Bot-A38-mer DNA substrate. Samples were processed and analyzed as described in Example 1, the detailed description of which is omitted here for the sake of brevity.

[0084] As shown in Figure 4B and Table 4 below, Pfu Endo V efficiently recognizes deoxyuridine (U) at reaction temperatures of 37°C or 70°C and cleaves the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) counting from deoxyuridine (U) toward the 3' end (or downstream) of DNA, releasing a 15-mer single-stranded or double-stranded DNA, and the remaining 23-mer single-stranded or double-stranded DNA has a free 3'-hydroxyl group at its end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. In contrast, Eco EndoV and C1 did not show such site-specific cleavage or modification. In addition, C3 released a 17-mer single- or double-stranded DNA by cleaving only the phosphodiester bond between deoxyuridine (U) and the first nucleotide (G) counting from deoxyuridine (U) to the 5' end (or upstream) of DNA at a reaction temperature of 37°C, but was unable to generate a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single- or double-stranded DNA, which could not be used for a new round of nucleic acid synthesis.

[0085] [Table 4]

[0086] Example 3: Deoxyinosine recognition and DNA strand cleavage by Pfu Endo Q at two different reaction temperatures The samples included (1) Pyrococcus furiosus endonuclease Q (Pfu Endo Q) (SEQ ID NO: 9), (2) Escherichia coli endonuclease V (C1) obtained from New England BioLabs (Ipswich, MA), and (3) an enzyme mixture (C2) containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII, which represents the results of deoxyinosine excision and DNA strand cleavage at the guide nucleotide position in the nucleic acid product and serves as a positive control. The sample mixture (10 μL) contained 100 nM of single-stranded Hex-TOP-I38-mer DNA substrate or double-stranded Hex-Top-I38-mer / Bot-A38-mer DNA substrate. The samples were processed and analyzed as described in Example 1, and detailed description thereof will be omitted here for brevity.

[0087] As shown in FIG. 5A and Table 5 below, Pfu Endo Q efficiently recognizes deoxyinosine (I) at reaction temperatures of 37° C. or 70° C. and cleaves the phosphodiester bond between deoxyinosine (I) and the first nucleotide (C) counting from deoxyinosine (I) to the 5' end of the DNA, releasing a 17-mer single- or double-stranded DNA, and the remaining 21-mer single- or double-stranded DNA has a free 3'-hydroxyl group at the 3' end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. C1 can also recognize deoxyinosine (I) and cleaves the phosphodiester bond between the first nucleotide (G) and the second nucleotide (G) from deoxyinosine (I) toward the 3' end of DNA at a reaction temperature of 37°C, releasing a 15-mer single-stranded or double-stranded DNA, which has a free 3'-hydroxyl group at the 3' end of the remaining 23-mer single-stranded or double-stranded DNA and can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. However, C1 did not show site-specific cleavage in such DNA at a reaction temperature of 70°C. Also, C2 was only shown to excise deoxyinosine (I) and cleave the DNA strand to release a 15-mer single-stranded or double-stranded DNA, but was unable to generate a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single-stranded or double-stranded DNA and therefore could not be used for a new round of nucleic acid synthesis.

[0088] [Table 5]

[0089] Example 4: Deoxyuridine recognition and DNA strand cleavage by Pfu Endo Q at two different reaction temperatures The samples included (1) Pyrococcus furiosus endonuclease Q (Pfu Endo Q), (2) Escherichia coli endonuclease V (C1) from New England BioLabs (Ipswich, MA), and (3) a positive control, Uracil-specific excision reagent (Cat.#M5505S, Ipswich, MA) (C3) from New England Biolabs. Sample mixtures (10 μL) contained 100 nM of single-stranded Hex-Top-U38-mer DNA substrate or double-stranded Hex-Top-U38-mer / Bot-A38-mer DNA substrate. Samples were processed and analyzed as described in Example 1, and detailed descriptions thereof are omitted here for brevity.

[0090] As shown in FIG. 5B and Table 6 below, Pfu Endo Q efficiently recognizes deoxyuridine (U) at reaction temperatures of 37° C. or 70° C. and cleaves the phosphodiester bond between deoxyuridine (U) and the first nucleotide (C) counting from deoxyinosine (I) to the 5' end of DNA, releasing a 17-mer single-stranded or double-stranded DNA, which has a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single-stranded or double-stranded DNA and can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. The ability of C3 to site-specifically cleave DNA is similar to that of Pfu Endo Q, which can release a 17-mer single-stranded or double-stranded DNA, but C3 could not generate a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single-stranded or double-stranded DNA and therefore could not be used for a new round of nucleic acid synthesis. In contrast, C1 failed to demonstrate site-specific cleavage on DNA at reaction temperatures of 37°C or 70°C.

[0091] [Table 6]

[0092] Example 5: Deoxyinosine recognition and DNA strand cleavage by Tba Endo V at two different reaction temperatures The samples included (1) only the DNA substrate (S) as a negative control, (2) a homemade Escherichia coli endonuclease type V (Eco EndoV) as described in US Patent Application Publication No. 2021 / 0254114 A1, (3) Thermococcus barophilus endonuclease V (Tba Endo V) (SEQ ID NO: 10), and (4) an enzyme mixture (C2) containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII as a positive control, representing the results of deoxyinosine excision and DNA strand cleavage at the guide nucleotide position in the nucleic acid product. The sample mixture (10 μL) contained 100 nM of single-stranded Hex-TOP-I38-mer DNA substrate or double-stranded Hex-Top-I38-mer / Bot-A38-mer DNA substrate. The samples were processed and analyzed as described in Example 1, and detailed descriptions thereof will be omitted here for brevity.

[0093] As shown in FIG. 6A and Table 7 below, Eco EndoV, Tba Endo V, and C1 efficiently recognize deoxyinosine (I) at a reaction temperature of 37° C. and cleave the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) from deoxyinosine (I) toward the 3′-end of DNA, releasing a 15-mer single-stranded or double-stranded DNA, which has a free 3′-hydroxyl group at the 3′-end of the remaining 23-mer single-stranded or double-stranded DNA and can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. In contrast, C2 only showed excision of deoxyinosine (I) and DNA strand cleavage at the reaction temperature, releasing a 15-mer single-stranded or double-stranded DNA, but was unable to generate a free 3′-hydroxyl group at the 3′-end of the remaining 21-mer single-stranded or double-stranded DNA and therefore could not be used for a new round of nucleic acid synthesis. Furthermore, it was found that, compared with Eco EndoV and C1, only Tba Endo V efficiently cleaved or denatured DNA strands at a reaction temperature of 70°C as described above.

[0094] [Table 7]

[0095] Example 6: Deoxyinosine recognition and DNA strand cleavage by Tba Endo V at two different reaction temperatures The samples included (1) DNA substrate (S) only as a negative control, (2) Escherichia coli endonuclease type V (Eco EndoV) prepared in-house as described in US Patent Application Publication No. 2021 / 0254114 A1, (3) Thermococcus balophilus endonuclease V (Tba Endo V), and (4) Uracil-specific excision reagent (Cat.#M5505S, Ipswich, MA) (C3) obtained from New England Biolabs as a positive control. The sample mixture (10 μL) contained 100 nM of single-stranded Hex-Top-U38mer DNA substrate or double-stranded Hex-Top-U38-mer / Bot-A38-mer DNA substrate. The samples were processed and analyzed as described in Example 1, and detailed description thereof will be omitted here for brevity.

[0096] As shown in Figure 6B and Table 8 below, Tba Endo V efficiently recognizes deoxyuridine (U) at a reaction temperature of 70°C and cleaves the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) counting from deoxyinosine (I) toward the 3' end of DNA, releasing a 15-mer single- or double-stranded DNA, and the remaining 23-mer single- or double-stranded DNA has a free 3'-hydroxyl group at the 3' end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. In contrast, Eco EndoV and C1 did not show site-specific cleavage of DNA at a reaction temperature of 70°C. In addition, C3 exhibited site-specific cleavage of DNA similar to that of Pfu Endo Q, releasing 17-mer single-stranded or double-stranded DNA, but C3 was unable to generate a free 3′-hydroxyl group at the 3′ end of the remaining 21-mer single-stranded or double-stranded DNA, which could not be used for a new round of nucleic acid synthesis.

[0097] [Table 8]

[0098] The experimental results of Tba Endo V are similar to those of Pfu Endo V and Pfu Endo Q. As shown in Example 5 and Example 6 (see Figures 6A and 6B, respectively), Tba Endo V efficiently and site-specifically cleaved DNA strands from DNA initiators / primers containing deoxyinosine or deoxyuridine. Conversely, E. coli Endo V cleaved DNA strands only from DNA initiators / primers containing deoxyinosine. Tba Endo V also efficiently and site-specifically cleaved DNA strands from DNA initiators / primers containing deoxyinosine or deoxyuridine at both temperatures of 37°C and 70°C, whereas E. coli Endo V cleaved DNA strands only at 37°C. Similarly, Tba Endo V also efficiently cleaved primer-template DNA duplexes containing deoxyinosine or deoxyuridine. Based on the individual results of hAAG plus EndoVIII and NEB USER herein, and the results of the commercially available NEB Eco Endo V enzyme, the relative positions on the gel of the intact DNA fragments and the DNA fragments generated by Tba Endo V cleavage were confirmed.

[0099] As shown in Examples 1 to 6 (see Figures 4A to 6B, respectively), Pfu Endo V, Pfu Endo Q, and Tba Endo V efficiently and site-specifically cleaved the DNA strands of synthesized single-stranded or double-stranded DNA containing deoxyinosine or deoxyuridine. Conversely, Eco Endo V cleaved DNA strands only from DNA containing deoxyinosine. In addition, Pfu Endo V, Pfu Endo Q, and Tba Endo V can efficiently and site-specifically cleave DNA at both temperatures of 37°C and 70°C, while E. coli Endo V cleaved DNA strands only at 37°C. In addition, whether the initiator was free or immobilized did not affect the DNA cleavage activity of Pfu Endo V, Pfu Endo Q, and Tba Endo V. In addition, both of the positive control groups C2 and C3 could cleave DNA, but could not generate a free 3'-hydroxyl group at the 3'-end of the remaining single-stranded or double-stranded DNA, and the remaining single-stranded or double-stranded DNA could not be used for a new round of nucleic acid synthesis. In summary, Pfu Endo V, Pfu Endo Q, and Tba Endo V can site-specifically recognize guide nucleotides at a wide range of reaction temperatures (including ultra-high temperature reaction temperatures), cleave nucleic acid strands according to the position of the guide nucleotide, and effectively regenerate initiators with free 3'-hydroxyl groups at the 3'-end.

[0100] Example 7: Deoxyuridine recognition and DNA strand cleavage by Bsu Endo V The samples included (1) only the DNA substrate (S) as a negative control, (2) homemade E. coli endonuclease type V (Eco EndoV) as described in U.S. Patent Application Publication No. 2021 / 0254114 A1, (3) Bacillus subtilis endonuclease V (Bsu Endo V) (SEQ ID NO: 11), (4) E. coli endonuclease V (C1) obtained from New England BioLabs (Ipswich, MA), and (5) a positive control uracil-specific excision reagent (Cat. #M5505S, Ipswich, MA) (C3) obtained from New England Biolabs. The sample mixture (10 μL) contained 100 nM of single-stranded Hex-Top-U38-mer DNA substrate or double-stranded Hex-Top-U38-mer / Bot-A38-mer DNA substrate. Samples were processed and analyzed as described in Example 1, the detailed description of which is omitted here for the sake of brevity.

[0101] As shown in Figure 7 and Table 9 below, Bsu Endo V efficiently recognizes deoxyuridine (U) and cleaves the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) counting from deoxyuridine (U) toward the 3' end of the DNA, releasing a 15-mer single- or double-stranded DNA, and the remaining 23-mer single- or double-stranded DNA has a free 3'-hydroxyl group at the 3' end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. In contrast, Eco EndoV and C1 did not show such site-specific cleavage or modification. In addition, C3 only cleaved the phosphodiester bond between deoxyuridine (U) and the first nucleotide (G) counting from deoxyuridine (U) to the 5' end of DNA to release a 17-mer single-stranded or double-stranded DNA, but was unable to generate a free 3'-hydroxyl group at the 3' end of the remaining 21-mer single-stranded or double-stranded DNA, which could not be used for a new round of nucleic acid synthesis. Therefore, Bsu Endo V can site-specifically recognize the guide nucleotide, cleave the nucleic acid chain depending on the position of the guide nucleotide, and effectively regenerate an initiator having a free 3'-hydroxyl group at the 3' end.

[0102] [Table 9]

[0103] Example 8: Recognition of deoxyinosine and deoxyuridine and DNA strand cleavage by Endo V and Endo Q at three different reaction temperatures The sample groups included (1) only the DNA substrate (S) as a negative control group, (2) homemade Escherichia coli endonuclease type V (Eco EndoV) which can be carried out with reference to US Patent Application Publication No. 2021 / 0254114 A1, (3) Pyrococcus furiosus endonuclease V (Pfu Endo V), (4) Pyrococcus furiosus endonuclease Q (Pfu Endo Q), (5) Thermococcus barophilus endonuclease V (Tba Endo V), and (6) Bacillus pumilus endonuclease Q (Bpu Endo Q) (SEQ ID NO: 12). For each sample group, a sample mixture (10 μL) containing 100 nM of single-stranded Hex-Top-U38mer DNA substrate or Hex-Top-I38mer DNA substrate and 200 nM of endonuclease were incubated in the enzyme reaction buffer. The sample mixtures were incubated for 20 min at 37° C., 55° C. or 60° C., respectively. Each enzymatic reaction was stopped by adding an equal volume (10 μL) of 2× quench solution.

[0104] A total of 20 μL of samples were denatured at 95°C for 10 min, and each sample mixture was analyzed (4 μL of each sample) by 20% denaturing polyacrylamide gel electrophoresis in 1× TBE buffer (90 mM Tris-alkaline, 90 mM boric acid, and 2 mM EDTA) containing 8 M urea. The gel imaging results were then analyzed with an Amersham Typhoon scanner (Cytiva, Marlborough, MA).

[0105] The results show that Pfu Endo V and Pfu Endo Q site-specifically cleaved both free and immobilized DNA strands containing deoxyinosine or deoxyuridine at 55° C. and 60° C., Tba Endo V site-specifically cleaved both free and immobilized DNA strands containing deoxyuridine at 55° C., and Bpu Endo Q site-specifically cleaved both free and immobilized DNA strands containing deoxyinosine or deoxyuridine at 37° C. Conversely, E. coli Endo V cleaved only DNA strands containing deoxyinosine but not DNA strands containing deoxyuridine at 37° C.

[0106] As shown in Figures 8A and 8B and Tables 10 and 11 below, Pfu Endo V and Tba Endo V efficiently recognize deoxyinosine (I) or deoxyuridine (U) and cleave the phosphodiester bond between the first nucleotide (G) and the second nucleotide (C) counting from deoxyinosine (I) or deoxyuridine (U) to the 3'-end of DNA, releasing a 15-mer single-stranded or double-stranded DNA, and the remaining 23-mer single-stranded or double-stranded DNA has a free 3'-hydroxyl group at the 3'-end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis. In contrast, Eco EndoV was able to show such activity only for DNA containing deoxyinosine. In addition, both Pfu Endo Q and Bpu Endo Q efficiently recognize deoxyinosine (I) or deoxyuridine (U) and cleave the phosphodiester bond between deoxyinosine (I) or deoxyuridine (U) and the first nucleotide (C) counting from deoxyinosine (I) or deoxyuridine (U) to the 5' end of DNA, releasing a 17-mer single-stranded or double-stranded DNA, and the remaining 21-mer single-stranded or double-stranded DNA has a free 3'-hydroxyl group at the 3' end, which can immediately be used as a new (or reusable) initiator for the next round of nucleic acid synthesis.

[0107] Furthermore, both Pfu Endo V and Pfu Endo Q site-specifically and efficiently cleaved DNA strands containing deoxyinosine or deoxyuridine at 55° C. and 60° C., Tba Endo V site-specifically and efficiently cleaved DNA strands containing deoxyinosine or deoxyuridine at 55° C. and 60° C., and Bpu Endo Q site-specifically and efficiently cleaved DNA strands containing deoxyinosine or deoxyuridine at 37° C. In contrast, E. coli EndoV cleaved only DNA strands containing deoxyuridine at 37° C.

[0108] In summary, Pfu Endo V, Pfu Endo Q, Tba Endo V, and Bpu Endo Q can site-specifically recognize guide nucleotides over a wide range of reaction temperatures (including ambient reaction temperature), cleave the nucleic acid chain according to the position of the guide nucleotide, and effectively regenerate initiators with a free 3'-hydroxyl group at the 3' end.

[0109] [Table 10]

[0110] [Table 11]

[0111] Although the present invention has been described by its embodiments, it should be understood that the embodiments of the present disclosure can be modified in various ways without departing from the scope of the present disclosure. Therefore, the described embodiments are intended to cover modifications within the scope of the present disclosure, and are not intended to limit the present invention. Therefore, the scope of the claims should be interpreted in the broadest possible manner to include all such modifications. [Brief description of the drawings]

[0112] [Figure 1]FIG. 1 is a schematic diagram according to at least one embodiment of the present disclosure illustrating template-independent de novo synthesis of single-stranded DNA (ssDNA) from an initiator (having a free 3'-hydroxyl group at its 3' end), cleaving the newly synthesized DNA strand to obtain a polynucleotide of desired sequence and length, and after cleaving the synthesized DNA strand, regenerating a new free 3'-hydroxyl group from the remaining nucleic acid strand to obtain a polynucleotide of interest, while using the remaining nucleic acid strand as a new reusable initiator in the next round of nucleic acid synthesis. Prior to the nucleic acid synthesis reaction, the 5' end of the initiator is fixed to a solid support (SS). X represents a guide nucleotide, which is used as a starting point for numbering nucleotide positions in the nucleic acid strand. N represents the incorporated nucleotide monomer, the subscript numbers are in ascending order toward the 3' end or downstream of the nucleic acid chain (e.g., N1, N2, N3, N4, and N5), and the subscript numbers are in descending order toward the 5' end or upstream of the nucleic acid chain (e.g., N-1, N-2, and N-3). E1 to E4 represent different endonucleases. The enzymatic nucleic acid synthesis process is outlined, including the cleavage at the phosphodiester bond between N2 and N1, between X and N-1, between N-1 and N-2, or between N-2 and N-3 by E1, E2, E3, or E4 enzymes, respectively, and the 5' end of the initiator is linked to a solid support (SS). [Figure 2A]2A-2C are schematic diagrams according to at least one embodiment of the present disclosure showing template-directed DNA synthesis from an initiator (i.e., a primer) (having a free 3'-hydroxyl group at its 3' end), cleavage of the nascent DNA strand to obtain a polynucleotide of desired sequence and length, and regeneration of the free 3'-hydroxyl group at the 3' end of the remaining nucleic acid strand to form a new reusable initiator for the next round of nucleic acid synthesis. The 5' end of the initiator is fixed to a solid support (SS) prior to the nucleic acid synthesis reaction. Thus, the template DNA is either linked to the solid support via its 3' end (FIG. 2A) or partially hybridized to the initiator by annealing or other known hybridization techniques (FIG. 2B). X represents a guide nucleotide and is used as a starting point for numbering the nucleic acid strand. N represents the incorporated nucleotide monomer, the subscript numbers are in ascending order toward the 3' end or downstream of the nucleic acid chain (e.g., N1, N2, N3, N4, and N5), and the subscript numbers are in descending order toward the 5' end or upstream of the nucleic acid chain (e.g., N-1, N-2, and N-3). E1 to E4 represent different endonucleases. The enzymatic nucleic acid synthesis means are outlined, including the ability to cleave the phosphodiester bond between N2 and N1, between X and N-1, between N-1 and N-2, or between N-2 and N-3 by E1, E2, E3, or E4 enzymes, respectively. [Figure 2B]2A-2C are schematic diagrams according to at least one embodiment of the present disclosure showing template-directed DNA synthesis from an initiator (i.e., a primer) (having a free 3'-hydroxyl group at its 3' end), cleavage of the nascent DNA strand to obtain a polynucleotide of desired sequence and length, and regeneration of the free 3'-hydroxyl group at the 3' end of the remaining nucleic acid strand to form a new reusable initiator for the next round of nucleic acid synthesis. The 5' end of the initiator is fixed to a solid support (SS) prior to the nucleic acid synthesis reaction. Thus, the template DNA is either linked to the solid support via its 3' end (FIG. 2A) or partially hybridized to the initiator by annealing or other known hybridization techniques (FIG. 2B). X represents a guide nucleotide and is used as a starting point for numbering the nucleic acid strand. N represents the incorporated nucleotide monomer, the subscript numbers are in ascending order toward the 3' end or downstream of the nucleic acid chain (e.g., N1, N2, N3, N4, and N5), and the subscript numbers are in descending order toward the 5' end or upstream of the nucleic acid chain (e.g., N-1, N-2, and N-3). E1 to E4 represent different endonucleases. The enzymatic nucleic acid synthesis means are outlined, including the ability to cleave the phosphodiester bond between N2 and N1, between X and N-1, between N-1 and N-2, or between N-2 and N-3 by E1, E2, E3, or E4 enzymes, respectively. [Diagram 3]FIG. 2 is a fluorescent image of a urea polyacrylamide gel showing the feasibility of template-independent nucleic acid synthesis according to at least one embodiment of the present disclosure. [Lane S shows initiator DNA only (biotin-FAM-45mer ssDNA). Lane 1 shows initiator DNA extended with 3'-O-(azidomethyl)-2'-deoxyuridine (U) by a polymerase. Lane 2 shows initiator DNA extended sequentially with 3'-O-(azidomethyl)-2'-deoxyuridine (U) and 3'-O-(azidomethyl)-2'-deoxyguanosine (G) after each nucleotide incorporation and 3'-deprotection step. Lane 3 shows the initiator DNA extended with 3'-O-(azidomethyl)-2'-deoxyuridine after a 3'-deprotection step, followed by extension with dNTPs (a mixture of dATP, dCTP, dGTP, and dTTP). [Figure 4A]Fluorescence images of urea polyacrylamide gels showing the results of Examples 1 and 2, respectively, at 37° C. or 70° C., showing site-specific cleavage of free (FIG. 4A(1) and FIG. 4B(1)) or immobilized (FIG. 4A(2) and FIG. 4B(2)) single-stranded DNA (ssDNA), and free (FIG. 4A(3) and FIG. 4B(3)) or immobilized (FIG. 4A(4) and FIG. 4B(4)) double-stranded DNA (dsDNA) by preferentially or specifically recognizing deoxyinosine (I, FIG. 4A) or deoxyuridine (U, FIG. 4B), respectively, with exemplary enzymes. S means substrate DNA only. Eco EndoV means homemade Escherichia coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu EndoV means Pyrococcus furiosus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and endonuclease VIII (EndoVIII). C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 4B]Fluorescence images of urea polyacrylamide gels showing the results of Examples 1 and 2, respectively, at 37° C. or 70° C., showing site-specific cleavage of free (FIG. 4A(1) and FIG. 4B(1)) or immobilized (FIG. 4A(2) and FIG. 4B(2)) single-stranded DNA (ssDNA), and free (FIG. 4A(3) and FIG. 4B(3)) or immobilized (FIG. 4A(4) and FIG. 4B(4)) double-stranded DNA (dsDNA) by preferentially or specifically recognizing deoxyinosine (I, FIG. 4A) or deoxyuridine (U, FIG. 4B), respectively, with exemplary enzymes. S means substrate DNA only. Eco EndoV means homemade Escherichia coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu EndoV means Pyrococcus furiosus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and endonuclease VIII (EndoVIII). C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 5A]Fluorescence images of urea polyacrylamide gels showing the results of Examples 3 and 4, respectively, [showing site-specific cleavage of free (FIG. 5A(1) and FIG. 5B(1)) or immobilized (FIG. 5A(2) and FIG. 5B(2)) single-stranded DNA (ssDNA), and free (FIG. 5A(3) and FIG. 5B(3)) or immobilized (FIG. 5A(4) and FIG. 5B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by the exemplary enzymes, respectively, by preferentially or specifically recognizing deoxyinosine (I, FIG. 5A) or deoxyuridine (U, FIG. 5B). Pfu EndoQ refers to Pyrococcus furiosus endonuclease Q. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylators (hAAG) and EndoVIII. C3 refers to a uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 5B]Fluorescence images of urea polyacrylamide gels showing the results of Examples 3 and 4, respectively, [showing site-specific cleavage of free (FIG. 5A(1) and FIG. 5B(1)) or immobilized (FIG. 5A(2) and FIG. 5B(2)) single-stranded DNA (ssDNA), and free (FIG. 5A(3) and FIG. 5B(3)) or immobilized (FIG. 5A(4) and FIG. 5B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by the exemplary enzymes, respectively, by preferentially or specifically recognizing deoxyinosine (I, FIG. 5A) or deoxyuridine (U, FIG. 5B). Pfu EndoQ refers to Pyrococcus furiosus endonuclease Q. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylators (hAAG) and EndoVIII. C3 refers to a uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 6A]Fluorescence images of urea polyacrylamide gels showing the results of Examples 5 and 6, respectively, [showing site-specific cleavage of free (FIG. 6A(1) and FIG. 6B(1)) or immobilized (FIG. 6A(2) and FIG. 6B(2)) single-stranded DNA (ssDNA), and free (FIG. 6A(3) and FIG. 6B(3)) or immobilized (FIG. 6A(4) and FIG. 6B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by the exemplary enzymes, respectively, by preferentially or specifically recognizing deoxyinosine (I, FIG. 6A) or deoxyuridine (U, FIG. 6B). S means substrate DNA only. Eco EndoV means homemade Escherichia coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Tba Endo V means Thermococcus balophilus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII. C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 6B]Fluorescence images of urea polyacrylamide gels showing the results of Examples 5 and 6, respectively, [showing site-specific cleavage of free (FIG. 6A(1) and FIG. 6B(1)) or immobilized (FIG. 6A(2) and FIG. 6B(2)) single-stranded DNA (ssDNA), and free (FIG. 6A(3) and FIG. 6B(3)) or immobilized (FIG. 6A(4) and FIG. 6B(4)) double-stranded DNA (dsDNA) at 37° C. or 70° C. by the exemplary enzymes, respectively, by preferentially or specifically recognizing deoxyinosine (I, FIG. 6A) or deoxyuridine (U, FIG. 6B). S means substrate DNA only. Eco EndoV means homemade Escherichia coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Tba Endo V means Thermococcus balophilus endonuclease V. C1 refers to endonuclease V (NEB Eco EdoV) commercially obtained from New England Biolabs (Cat.#M0305S, Ipswitch, MA). C2 refers to a homemade enzyme mixture containing human alkyladenine DNA glycosylase (hAAG) and EndoVIII. C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA). [Figure 7]Fluorescence images of urea polyacrylamide gels showing the results of Example 7, respectively [showing the site-specific cleavage of free (FIG. 7(1) single-stranded DNA (ssDNA) and free double-stranded DNA (dsDNA) (FIG. 7(2)) by preferential or specific recognition of deoxyuridine (U) with exemplary enzymes at 37° C. S refers to substrate DNA only. Eco EndoV refers to homemade E. coli endonuclease V, which can be performed with reference to U.S. Patent Application No. 2021 / 0254114 A1. Bsu Endo V refers to Bacillus subtilis endonuclease V. C1 refers to commercially obtained endonuclease V (NEB Eco EdoV) (Cat.#M0305S, Ipswitch, MA). C3 refers to uracil-specific excision reagent (NEB USER) from New England Biolabs (Cat.#M5505S, Ipswich, MA)]. [Figure 8A] 8A(1), 8A(3), 8B(1), and 8B(3)) single-stranded DNA (ssDNA) or immobilized (FIGS. 8A(2), 8A(4), 8B(2), and 8B(4)) single-stranded DNA (ssDNA) at 37° C., 55° C., and / or 60° C., respectively, by the exemplary enzymes, showing site-specific cleavage of extended DNA strand products from free (FIGS. 8A(1), 8A(3), 8B(1), and 8B(3)) single-stranded DNA (ssDNA) at 37° C., 55° C., and / or 60° C. S denotes substrate DNA only. Eco EndoV means homemade E. coli endonuclease V, which may be made with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu Endo V means Pyrococcus furiosus endonuclease V. Tba means Thermococcus barophilus endonuclease V. Pfu Endo Q means Pyrococcus furiosus endonuclease Q. Bpu Endo Q means Bacillus pumilus endonuclease Q.]. [Figure 8B]8A(1), 8A(3), 8B(1), and 8B(3)) single-stranded DNA (ssDNA) or immobilized (FIGS. 8A(2), 8A(4), 8B(2), and 8B(4)) single-stranded DNA (ssDNA) at 37° C., 55° C., and / or 60° C., respectively, by the exemplary enzymes, showing site-specific cleavage of extended DNA strand products from free (FIGS. 8A(1), 8A(3), 8B(1), and 8B(3)) single-stranded DNA (ssDNA) at 37° C., 55° C., and / or 60° C. S denotes substrate DNA only. Eco EndoV means homemade E. coli endonuclease V, which may be made with reference to U.S. Patent Application No. 2021 / 0254114 A1. Pfu Endo V means Pyrococcus furiosus endonuclease V. Tba means Thermococcus barophilus endonuclease V. Pfu Endo Q means Pyrococcus furiosus endonuclease Q. Bpu Endo Q means Bacillus pumilus endonuclease Q.].

Claims

1. providing an initiator comprising a 3'-terminal nucleotide having a free 3'-hydroxyl group; incorporating a nucleotide monomer into the initiator by a polymerase to extend a nucleic acid strand from the free 3'-hydroxyl group, the nucleotide monomer comprising a guide nucleotide that is recognized by an endonuclease such that the guide nucleotide is incorporated at a specific position in the nucleic acid strand; causing said endonuclease to cleave said nucleic acid strand according to the position of said guide nucleotide to release a polynucleotide of interest and leave a remaining nucleic acid strand with a new free 3'-hydroxyl group, The endonuclease specifically recognizes the position of the guide nucleotide and cleaves the second phosphodiester bond at the 3'-end of the guide nucleotide, the first phosphodiester bond at the 5'-end of the guide nucleotide, the second phosphodiester bond at the 5'-end of the guide nucleotide, or the third phosphodiester bond at the 5'-end of the guide nucleotide, thereby obtaining the desired polynucleotide, and the remaining nucleic acid strand having the new free 3'-hydroxyl group is reserved and used as a new initiator for polynucleotide synthesis in the next round.

2. The method of claim 1 , wherein the nucleic acid strand is synthesized in a template-independent manner.

3. The method of claim 1 , wherein the nucleic acid strand is synthesized in a template-directed manner.

4. 2. The method of claim 1, wherein the nucleobase of the guide nucleotide is uracil, xanthine, or hypoxanthine.

5. 2. The method of claim 1, wherein the guide nucleotide comprises uracil or inosine.

6. The method of claim 1 , wherein the polymerase is a family B DNA polymerase.

7. The endonuclease may be selected from the group consisting of Thermococcus barophilus (Tba), Pyrococcus furiosus (Pfu), Methanosarcina acetivorans (Mac), Bacillus pumilus (Bpu), Pyrococcus abyssi (Pab), Thermococcus kodakarensis (Tko), Thermococcus gamma tolerans (Tko), and Thermococcus spp.

2. The method of claim 1, wherein the microbial strain is derived from Bacillus subtilis (Bsu), Bacillus gammatolerans (Tga), or Bacillus subtilis (Bsu).

8. The endonuclease is selected from the group consisting of Thermococcus balophilus endonuclease V (Tba Endo V), Bacillus subtilis endonuclease V (Bsu Endo V), Pyrococcus furiosus endonuclease V (Pfu Endo V), Thermococcus kodakaraensis endonuclease V (Tko Endo V), Pyrococcus furiosus endonuclease Q (Pfu Endo Q), Methanosarcina acetivorans endonuclease Q (Mac Endo Q), Bacillus pumilus endonuclease Q (Bpu Endo Q), Pyrococcus abyssi NucS endonuclease (Pab NucS), Thermococcus kodakaraensis 8. The method of claim 7, wherein the nucleic acid is selected from the group consisting of EndoMS endonuclease (Tko EndoMS, SEQ ID NO: 17), Thermococcus gamma tolerance NucS endonuclease (Tga NucS) and mutants thereof.

9. The method of claim 1, wherein the endonuclease for cleaving the second phosphodiester bond on the 3'-terminal side of the guide nucleotide is selected from the group consisting of Bacillus subtilis endonuclease V (Bsu Endo V), Escherichia coli endonuclease V (Eco Endo V), Pyrococcus furiosus endonuclease V (Pfu Endo V), Thermococcus barophilus endonuclease V (Tba Endo V), Thermococcus kodakaraensis endonuclease V (Tko Endo V), and enzyme mutants thereof.

10. The method of claim 1, wherein the endonuclease for cleaving the first phosphodiester bond on the 5'-terminal side of the guide nucleotide is selected from the group consisting of Pyrococcus furiosus endonuclease Q (Pfu Endo Q), Methanosarcina acetivorans endonuclease Q (Mac Endo Q), Bacillus pumilus endonuclease Q (Bpu Endo Q), and enzyme mutants thereof.

11. 2. The method of claim 1, wherein the endonuclease for cleaving the second phosphodiester bond 5' to the guide nucleotide is selected from the group consisting of Thermococcus gamma tolerance NucS endonuclease (Tga NucS), Pyrococcus abyssi NucS endonuclease (Pab NucS), and mutants thereof.

12. 2. The method of claim 1, wherein the endonuclease for cleaving the third phosphodiester bond on the 5'-terminal side of the guide nucleotide is selected from the group consisting of Thermococcus kodakaraensis EndoMS endonuclease (Tko EndoMS) and enzyme variants thereof.

13. The process of claim 1, carried out at a temperature in the range of 10°C to 100°C.

14. The method of claim 1 , wherein the initiator is immobilized at its 5′ end to a solid support.

15. providing a synthetic polynucleotide having a guide nucleotide that is specifically recognized by an endonuclease; and c) cleaving the synthetic polynucleotide with an endonuclease according to the position of said guide nucleotide to release a polynucleotide of interest, The method for obtaining a polynucleotide by an enzyme, in which the endonuclease recognizes the guide nucleotide and cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, thereby obtaining a predetermined polynucleotide.

16. a nucleic acid initiator comprising a 3'-terminal nucleotide having a free 3'-hydroxyl group; a polymerase for incorporating a nucleotide monomer into said initiator to extend a nucleic acid strand from said free 3'-hydroxyl group, said nucleotide monomer comprising a guide nucleotide, said polymerase causing said guide nucleotide to be incorporated into said nucleic acid strand; and an endonuclease that cleaves the nucleic acid strand according to the position of the guide nucleotide to release the polynucleotide of interest and leaves a new free 3'-hydroxyl group at the 3' end of the remaining nucleic acid strand, The kit for enzymatic polynucleotide synthesis, wherein the endonuclease specifically recognizes the guide nucleotide and cleaves the second phosphodiester bond at the 3'-end of the guide nucleotide, the first phosphodiester bond at the 5'-end of the guide nucleotide, the second phosphodiester bond at the 5'-end of the guide nucleotide, or the third phosphodiester bond at the 5'-end of the guide nucleotide, thereby obtaining the desired polynucleotide, and the remaining nucleic acid strand having the new free 3'-hydroxyl group is reserved and used as a new initiator for the next round of polynucleotide synthesis.

17. a synthetic polynucleotide having a guide nucleotide; and an endonuclease for cleaving the synthetic polynucleotide to release a polynucleotide of interest, The kit for obtaining a polynucleotide by an enzyme, wherein the endonuclease recognizes the position of the guide nucleotide and cleaves the second phosphodiester bond at the 3' end of the guide nucleotide, the first phosphodiester bond at the 5' end of the guide nucleotide, the second phosphodiester bond at the 5' end of the guide nucleotide, or the third phosphodiester bond at the 5' end of the guide nucleotide, thereby obtaining the predetermined polynucleotide.

18. 18. The kit of claim 16 or 17, wherein the guide nucleotide comprises uracil or inosine.

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