Polymerase variants for template-independent enzymatic nucleic acid synthesis and kits containing the same

Abstract

We provide a tailor-made modified nucleic acid polymerase that demonstrates its usefulness in various nucleic acid synthesis processes. [Solution] A DNA polymerase variant and a kit containing the same are provided, the DNA polymerase variant having improved functionality and activity for template-independent nucleic acid synthesis using standard nucleotides and non-standard nucleotide analogs in a heat-stable manner.

Description

[Technical Field]

[0001] cross reference This application claims priority and benefits of U.S. Provisional Application No. US63 / 249,819, filed on 29 September 2021, the contents of which are incorporated herein by reference.

[0002] This disclosure relates to B family DNA polymerase variants and kits containing these variants, particularly for use in the context of de novo enzymatic nucleic acid synthesis.

[0003] Sequence List This application is submitted together with an electronic sequence listing. The sequence listing is provided as a 98kb file titled 211019US-sequence listing.XML, created on July 12, 2022. The information of the electronic sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Enzymatic de novo nucleic acid synthesis is emerging as a non-toxic alternative to the toxic chemical phosphoramidite-based nucleic acid synthesis methods that have been used for decades.

[0005] All living organisms rely on nucleic acid polymerases to efficiently replicate their DNA. Due to their DNA replication function, most nucleic acid polymerases require a template to direct the synthesis of nucleotides and their incorporation into the growing nucleic acid chain. In the template-dependent method of nucleic acid synthesis, it is necessary for the nucleic acid polymerase to associate with the primer-template DNA before the polymerase adds nucleotides to the 3′ end of the primer. To ensure high-fidelity DNA synthesis, nucleic acid polymerases have developed robust nucleotide selection mechanisms to precisely select and incorporate the correct nucleotides corresponding to their complementary template bases during nucleic acid synthesis. The active site pocket of a nucleic acid polymerase is pre-configured to accommodate correctly matched standard nucleotides with a normal 3′-hydroxyl (3′-OH) group. Therefore, the removal of the 3′-OH group or its substitution with bulky chemical groups on nucleotides, such as 2′,3′-dideoxynucleotides (ddNTPs) and 3′-O-azidomethyl-dNTPs, can significantly alter the nucleotide composition within the active site pocket of nucleic acid polymerases, thereby reducing the nucleotide binding affinity and overall DNA synthesis efficiency of nucleic acid polymerases. Similarly, modifications to the nucleic acid base or 5′-triphosphate group of nucleotides disrupt the interaction between the nucleotide and the active site residue of nucleic acid polymerases, preventing these modified nucleotides from being effectively utilized in nucleic acid synthesis by polymerases.

[0006] Unlike most DNA polymerases, terminal deoxynucleotidyl transferase (TdT), a member of the X family of DNA polymerases, is a unique class of mesophilic enzymes that do not require a template for nucleotide addition during nucleic acid synthesis. TdT only requires a short initiator DNA or primer to direct nucleotide synthesis and incorporation into the growing initiator DNA or primer. TdT can perform template-independent DNA synthesis, and the active site pocket of TdT is also preconfigured in an appropriate shape to accommodate standard nucleotides with a normal 3'-OH group. Similar to other DNA polymerases, substituting the 3'-OH group on the nucleotide with a bulky chemical group causes steric hindrance of the nucleotide when entering the nucleotide-binding pocket of TdT, resulting in a decrease in both the nucleotide-binding affinity and overall DNA synthesis activity of TdT. Naturally, the template-independent DNA synthesis function of TdT is a major option for application in de novo enzymatic DNA synthesis. However, some of the unique properties of TdT, such as limited thermal stability (mesophilic enzyme), preference for incorporation of specific nucleotides, intolerance to larger substitutions of the 3'-OH group on the nucleotide, and synthetic inefficiency, have been barriers to the practical application of enzymatic DNA synthesis.

[0007] To expand its application in enzymatic DNA synthesis, alternative nucleic acid polymerases and their derivatives with thermal stability and the ability to accommodate various non-standard nucleotide analogs, such as reversible terminator and di-terminator nucleotides, are needed and remain an unmet need to date. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0008] Due to the diverse structural and functional relationships described above, naturally occurring nucleic acid polymerases cannot readily utilize standard nucleotides or nucleotide analogs as substrates for de novo nucleic acid synthesis. Therefore, tailor-made modified nucleic acid polymerases are essential for demonstrating their usefulness in various nucleic acid synthesis processes. [Means for solving the problem]

[0009] The inventors have discovered a novel location / region in the amino acid sequence of a B-family DNA polymerase variant that plays a crucial role in conferring template independence to the polymerase and enhancing its nucleotide substrate binding affinity to both standard and modified nucleotides, thereby improving the nucleic acid synthesis efficiency in template-independent nucleic acid synthesis methods.

[0010] In one embodiment, the Disclosure provides a B-family DNA polymerase variant comprising motifs ExoI, ExoII, ExoIII, A, B, and C, corresponding to positions 349-364, 450-476, 590-608, 706-730, 843-855, and 940-956 of the consensus sequence (SEQ ID NO: 1), respectively, and multiple amino acid substitutions at positions present in motifs selected from motifs ExoI, ExoII, ExoIII, A, B, C, or combinations thereof.

[0011] In one embodiment, the B family DNA polymerase variant is modified from a wild-type B family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

[0012] In one embodiment, the wild-type B family DNA polymerases include Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), and Methanosarcina acetylborans. DNA polymerases of various bacteria including: acetivorans (Mac), Pyrobaculum islandicum (Pis), Sulfolobus solfataricus (Sso), human DNA polymerase δ catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae (ScePOLD), Pseudomonas aeruginosa (Pae), Escherichia coli (Eco), Escherichia phage RB69 DNA polymerase (RB69), and Escherichia phage T4. This is either DNA polymerase (T4) or Bacillus phage Phi29 DNA polymerase (Phi29).

[0013] In one embodiment, the B-family DNA polymerase variant provided herein has a deficiency in exonuclease activity in the 3′ to 5′ direction.

[0014] In one embodiment, amino acid L or M corresponding to position 715 of SEQ ID NO: 1 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y corresponding to position 716 of SEQ ID NO: 1 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P corresponding to position 717 of SEQ ID NO: 1 is either unsubstituted or substituted with A, G, S, or T.

[0015] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, wherein amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 2 is either unsubstituted or substituted with A, G, S, or T.

[0016] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, wherein amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 2 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0017] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, wherein amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 3 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 3 is either unsubstituted or substituted with A, G, S, or T.

[0018] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, wherein amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 3 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 3 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0019] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, wherein amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 4 is either unsubstituted or substituted with A, G, S, or T.

[0020] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, wherein amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 4 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0021] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, wherein amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 410 of SEQ ID NO: 5 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 411 of SEQ ID NO: 5 is either unsubstituted or substituted with A, G, S, or T.

[0022] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, wherein amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 410 of SEQ ID NO: 5 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 411 of SEQ ID NO: 5 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0023] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, wherein amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 412 of SEQ ID NO: 6 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 413 of SEQ ID NO: 6 is either unsubstituted or substituted with A, G, S, or T.

[0024] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, wherein amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 412 of SEQ ID NO: 6 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 413 of SEQ ID NO: 6 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0025] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Methanosarcina acetivorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, wherein amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 486 of SEQ ID NO: 7 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 487 of SEQ ID NO: 7 is either unsubstituted or substituted with A, G, S, or T.

[0026] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Methanosarcina acetivorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, wherein amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 486 of SEQ ID NO: 7 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 487 of SEQ ID NO: 7 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0027] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, wherein amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 427 of SEQ ID NO: 8 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 428 of SEQ ID NO: 8 is either unsubstituted or substituted with A, G, S, or T.

[0028] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, wherein amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 427 of SEQ ID NO: 8 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 428 of SEQ ID NO: 8 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0029] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, wherein amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 519 of SEQ ID NO: 9 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 520 of SEQ ID NO: 9 is either unsubstituted or substituted with A, G, S, or T.

[0030] In one embodiment, a B-family DNA polymerase variant having exonuclease activity deficiency in the 3′ to 5′ direction is derived from Sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, wherein amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 519 of SEQ ID NO: 9 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 520 of SEQ ID NO: 9 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0031] In some embodiments, the B-family DNA polymerase variants provided herein exhibit template-independent activity for the synthesis of nucleic acids by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator.

[0032] In some embodiments, the extendable initiator includes a single-strand oligonucleotide initiator, a blunt-ended double-strand oligonucleotide initiator, or a mixture thereof.

[0033] In some embodiments, the extendable initiator is a free form of nucleic acid, as opposed to immobilized nucleic acid, to be reacted in a liquid phase such as a liquid culture medium or other aqueous solution.

[0034] In some embodiments, the extendable initiator is immobilized on a solid support, the solid support including particles, beads, slides, array surfaces, membranes, flow cells, wells, microwells, nanowells, chambers, microfluidic chambers, channels, microfluidic channels, or any other surface.

[0035] In some embodiments, at least one nucleotide is linked to a detectable label.

[0036] In some embodiments, B-family DNA polymerase variants exhibit nucleotide incorporation activity at reaction temperatures ranging from 10°C to 100°C.

[0037] In another embodiment, the disclosure further provides a kit for de novo enzymatic nucleic acid synthesis comprising a B-family DNA polymerase variant derived from wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17. The B-family DNA polymerase variant exhibits template-independent activity to synthesize nucleic acids by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator, thereby synthesizing a desired nucleic acid sequence.

[0038] Accordingly, the present invention relates to a specific B-family DNA polymerase variant that exhibits improved performance in incorporating various nucleotides for nucleic acid synthesis at various reaction temperatures in the absence of a nucleic acid template. More specifically, the de novo nucleic acid synthesis method can be efficiently performed using a wide range of nucleotides and nucleotide analogs with the heat-stable B-family DNA polymerase variant.

[0039] This disclosure will be more readily understood by referring to the following description in conjunction with the attached drawings. [Brief explanation of the drawing]

[0040] [Figure 1] Figures 1-1 to 1-9 show the amino acid sequence alignment and consensus sequence of wild-type B-family DNA polymerase (PolB) related to the present invention. [Figure 2A] Figure 2A shows the results of the reaction described in Example 3. [Figure 2B] Figure 2B shows the results of the reaction described in Example 3. [Figure 3A] Figure 3A shows the results of the reaction described in Example 4. [Figure 3B] Figure 3B shows the results of the reaction described in Example 4. [Figure 4] Figure 4 shows the results of the reaction described in Example 5.1. [Figure 5A] Figure 5A shows the results of the reaction described in Example 5.2. [Figure 5B] Figure 5B shows the results of the reaction described in Example 5.2. [Figure 5C] Figure 5C shows the results of the reaction described in Example 5.2. [Figure 5D] Figure 5D shows the results of the reaction described in Example 5.2. [Figure 6] Figure 6 shows the results of the reaction described in Example 5.3. [Figure 7A] Figure 7A shows the results of the reaction described in Example 5.4. [Figure 7B]Figure 7B shows the results of the reaction described in Example 5.4. [Figure 8] Figure 8 shows the results of the reaction described in Example 5.5. [Figure 9] Figure 9 shows the results of the reaction described in Example 6. [Figure 10] Figure 10 shows the results of the reaction described in Example 7.1. [Figure 11] Figure 11 shows the results of the reaction described in Example 7.2. [Figure 12] Figure 12 shows the results of the reaction described in Example 8. Detailed description of the invention

[0041] ■Definition All terms used herein, including descriptive and technical terms, should be interpreted as having meanings understandable to those skilled in the art. However, the meaning of terms may differ depending on the intent of the explainer, case law, the emergence of new technologies, etc. Furthermore, some terms may be arbitrarily selected by the applicant, in which case the meaning of the selected terms will be explained in detail in this disclosure. Accordingly, terms used herein are defined in accordance with their meanings as described throughout this specification. In addition, titles and subtitles may be added to the content to facilitate understanding, but these titles do not affect the scope of the invention.

[0042] In this specification, the terms “a,” “an,” or “the” include multiple references unless explicitly and obviously limited to a single reference. The term “or” is used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0043] Furthermore, where a part or method "includes" or "comprises" a component or step, unless otherwise stated, that part or method may further include other components or steps, and does not exclude other components or steps.

[0044] As used herein, “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term “amino acid” includes the following 20 natural or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid or aspartate (Asp or D), cysteine ​​(CyS or C), glutamine (Gln or Q), glutamic acid or glutamate (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). If the "X" residue is undefined, it should be defined as "any amino acid".

[0045] The terms “functionally equivalent” or “equivalent” are used to describe specific B-family DNA polymerase (PolB) variants that have substitutions or mutations that are expected to occur at the amino acid positions of other PolB, PolB variants, according to sequence alignment or reference sequence, and that have the same functional or structural role in the enzyme. Such equivalent positions may be defined according to homologs, conserved motifs, user-defined, or derived consensus sequences.

[0046] Generally, homologous PolBs have similar or identical amino acid sequences and functional structures; therefore, equivalent amino acid substitution mutations between different PolBs generally occur at homologous amino acid positions. The terms “functionally equivalent” or “equivalent” as used herein also encompass mutations that are “homologous” or “positionally equivalent” to a given mutation in terms of protein sequence or structural alignment, regardless of the actual function of the mutated amino acid. Indeed, “functionally equivalent,” “homologous,” and / or “positionally equivalent” amino acid residues of different polymerases can be identified according to protein sequence or structural alignment. Thus, as shown in Figures 1-1 to 1-9, interspecies alignments were performed for multiple wild-type PolBs, with the consensus sequence (SEQ ID NO: 1) used as the positional reference sequence.

[0047] For example, the substitution of the amino acid aspartic acid (D) with alanine (A) at position 141 of the amino acid sequence of wild-type Thermococcus kodakarensis (Kod1) (D141A) is considered functionally equivalent to the amino acid substitution mutation D114A at a conserved residue in the amino acid sequence of the wild-type Escherichia phage RB69 DNA polymerase (RB69). When using a positional reference sequence to describe these equivalent amino acid substitutions, the functionally equivalent positions of both amino acid residue 141 in Kod1 and amino acid residue 114 in RB69 correspond to position 354 of the consensus sequence (SEQ ID NO: 1).

[0048] The term "conserved" refers to a polymerase segment having the same amino acid residues at homologous or equivalent positions in different PolBs from various sources. As used herein, the term "semi-conserved" refers to a polymerase segment having similar or identical amino acid residues at homologous positions in different PolBs from various sources.

[0049] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “polynucleotide,” and “nucleic acid fragment” mean a deoxyribonucleotide or ribonucleotide sequence in single-stranded or double-stranded form, the source and length of which are not limited herein, and generally include naturally occurring nucleotides or artificial chemical mimics. As used herein, the term “nucleic acid” is used interchangeably with terms including “oligonucleotide,” “polynucleotide,” “DNA,” “RNA,” “gene,” “complementary DNA” (cDNA), and “messenger RNA” (mRNA), whether natural or unnatural.

[0050] As used herein, “nucleic acid,” “oligonucleotide,” or “polynucleotide” means polymers or analogues that are equivalent to ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymers. This includes polymers of nucleotides such as RNA and DNA, as well as synthetic forms, modified (e.g., chemically or biochemically modified) forms, and mixed polymers (e.g., containing RNA and DNA subunits). Examples of modifications include methylation, substitution with one or more naturally occurring nucleotide analogues, nucleotide-nucleotide modifications such as uncharged linking (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates), pendant molecules (e.g., polypeptides), intercalators (e.g., acridines, psoralens), chelators, alkylators, and modified linkages (e.g., α-anomeric nucleic acids). Furthermore, synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence via hydrogen bonding or other chemical interactions are also included. Typically, nucleic acids are linked via phosphodiester bonds, although other linkages are possible as synthetic forms of nucleic acids (e.g., peptide nucleic acids, as described in Nielsen et al. (Science 254:1497-1500, 1991)). Nucleic acids can be, or may include, chromosomes or chromosomal segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, polymerase chain reaction (PCR) products, oligonucleotides, probes, and primers. Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to a specific length. Unless otherwise noted, a particular nucleic acid sequence may optionally include or encode complementary sequences in addition to the explicitly indicated sequence.

[0051] The term nucleic acids as used herein also includes nucleic acid analogs. The term nucleic acid analog is known to refer to compounds or artificial nucleic acids that are functionally or structurally equivalent to naturally occurring RNA or DNA. In nucleic acid analogs, one or more parts of a nucleotide (phosphate backbone, pentose, or nucleic acid base) may be modified. These modifications of nucleotides alter the structure and shape of the nucleic acid and change its interaction with nucleic acid polymerase. Nucleic acid analogs also include a new category of artificial nucleic acids, such as xeno nucleic acids (XNAs), which are designed to have novel sugar backbones not found in nature.

[0052] Examples of nucleic acid analogs include universal bases that can form base pairs with all four standard bases, such as inosine, 3-nitropyrrole, and 5-nitroindole; phosphate-sugar skeleton analogs such as peptide nucleic acids (PNA) that affect the skeletal properties of nucleic acids; analogs with chemical linkers or fluorophores attached, such as amine-reactive aminoallyl nucleotides, thiol-containing nucleotides, biotin-binding nucleotides, rhodamine-binding nucleotides, and cyanine-binding nucleotides; fluorescent base analogs 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 fluorescent reporter dyes (ALEXA, FAM, TET, TAMRA, CY3, CY5, VIC, JOE, HEX, NED, PET, ROX, Texas) Nucleic acid probes for various genetic applications include nucleic acids such as oligonucleotides conjugated with a fluorescent quencher (BHQ), such as Red; molecular beacons (MBs), which are single-stranded nucleic acid probes containing stem-loop structures and bifluorophore-quencher labeling; and nucleic acid aptamers, among others.

[0053] Generally, as used herein, “template” refers to a polynucleotide or polynucleotide mime containing a desired or unknown target nucleotide sequence. In some examples, the terms “target sequence,” “template polynucleotide,” “target nucleic acid,” “target polynucleotide,” “nucleic acid template,” and “template sequence and its variations” are used interchangeably. Specifically, the term “template” refers to a nucleic acid chain from which a complementary copy is synthesized from a nucleotide or nucleotide analog through replication by a template-dependent or template-directed nucleic acid polymerase. In a nucleic acid duplex, the template chain is conventionally depicted and described as the “bottom” strand. Similarly, the non-template chain is often depicted and described as the “top” strand. The “template” strand is sometimes called the “sense” strand or “plus” strand, and the non-template strand is sometimes called the “antisense” strand or “minus” strand.

[0054] The term "initiator" refers to mononucleosides, mononucleotides, and oligonucleotides, polynucleotides, or modified analogs thereof, from which nucleic acids are synthesized de novo by nucleic acid polymerase. The term "initiator" may also refer to xenonucleic acid (XNA) or peptide nucleic acid (PNA) having a 3′-hydroxyl group.

[0055] The terms “nucleotide incorporation,” “analogous incorporation,” “incorporation nucleotide,” and “incorporation analog” are known to those skilled in the art and are used to describe processes or reactions of nucleic acid synthesis. Therefore, as used herein, the term “incorporation” is known to flexibly mean the addition of one or more nucleotides, or any specified nucleic acid precursor, to the 3′-hydroxyl terminus of a nucleic acid initiator or primer. For example, nucleoside triphosphates such as deoxyguanosine triphosphate (dGTP) are substrates or precursors for DNA synthesis by DNA polymerase. When dGTP is incorporated into an extended DNA strand, it becomes the deoxyguanosine monophosphate (dGMP) portion of the newly synthesized DNA. In other words, when a dGTP nucleotide is converted into a dGMP portion of DNA, those skilled in the art may say that “one dGTP is incorporated into DNA.”

[0056] The term "nucleotide analogue" is known to those skilled in the art and refers to chemically modified nucleotides or artificial nucleotides that are structural mimics of standard nucleotides. These nucleotide analogues can serve as substrates for nucleic acid polymerases to synthesize nucleic acids. In nucleotide analogues, one or more components of the nucleotide (e.g., phosphate backbone, pentose sugar, nucleic acid base) may be altered, which changes the structure and arrangement of the nucleotide and affects its interaction with other nucleic acid bases and nucleic acid polymerases. For example, nucleotide analogues with altered nucleic acid bases may confer alternative base-pairing and base-stacking properties in DNA or RNA. Furthermore, modifications to the bases may, for example, produce a variety of nucleosides such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine, or bromo-5-deoxyuridine, and any other analogues that enable hybridization. In other exemplary embodiments, modifications may be made at the level of the sugar moiety (e.g., substitution of deoxyribose with an analog) and / or at the level of the phosphate group (e.g., boronate, alkylphosphonate, or phosphorothioate derivative). The nucleotide analog monomer may have a phosphate group selected from monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

[0057] Other examples of nucleotide analogs include nucleotides having removable blocking moieties. Examples of removable blocking moieties include, but are not limited to, 3′-O-blocking moieties, base blocking moieties, and combinations thereof. Examples of 3′-O-blocking moieties include, but are not limited to, O-azide (O-N3), 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, O-tetrahydrothiofuranyl, O-2-nitrobenzyl, O-methyl, and O-acyl. Examples of base blocking moieties include reversible di-terminators. Examples of reversible die-terminators include, but are not limited to, the reversible die-terminators in Illumina MiSeq, Illumina HiSeq, Illumina Genome Analyzer IIX, Helicos Biosciences Heliscope, and LaserGen Lightning Terminators. As used herein, “B-family DNA polymerase (PolB)” refers to the most common template-dependent nucleic acid polymerase or replicase in all life domains and many DNA viruses. Like most nucleic acid polymerases, natural PolB catalyzes the nucleotidyl transferase reaction by adding nucleotides to the 3′-OH ends of primers. This requires a double-stranded primer with a free 3′-hydroxyl (3′-OH) group at the primer end, template DNA, all four nucleoside triphosphates (dATP, dTTP, dCTP, dGTP), and a catalytic divalent cation (Mg 2+ or Mn 2+PolB enzymes, such as bacterial PolII and archaeal B-family DNA polymerases, are replication and repair polymerases that inherently contain a catalytic polymerase domain and a 3′-to-5′ exonuclease or proofreading domain for removing nucleotides mistakenly incorporated from the primer chain growing during nucleic acid replication. The term "3′-to-5′ exonuclease domain (Exo domain)" refers to the region of the polymerase amino acid sequence that exerts nucleolytic activity from the 3′ end of the primer or polynucleotide chain. Coordinatingly, the term "catalytic polymerase domain" (Pol domain) refers to the region of the polymerase amino acid sequence that exerts catalytic DNA / RNA polymerase activity for adding nucleotides to the 3′ end of the primer or polynucleotide chain.

[0058] All known structures of the PolB catalytic polymerase domain resemble the shape of a human right hand, with the primary functional regions characterized as finger, palm, and thumb subdomains. The most conserved region is the palm subdomain, which contains residues essential for catalytic reactions. Protein sequence alignments between various B-family DNA polymerases from different biological kingdoms and DNA viruses have revealed that PolB polymerase generally possesses six semi-conservative or conserved motifs (I-VI) for essential exonuclease and polymerase functions. The first three sequence motifs (ExoI, ExoII, ExoIII) are located in the Exo domain, while the other three motifs (referred to as motifs A, B, and C, respectively) are located in the Pol domain (Hopfner et al, Proc. Natl. Acad. Sci. USA 96, 3600-3605, 1999).

[0059] In this specification, the term "mutant" in the context of the DNA polymerase of the present invention means a polypeptide comprising one or more amino acid substitutions relative to the corresponding functional DNA polymerase, typically a recombinant.

[0060] In this specification, the expression “corresponding to another sequence” (e.g., region, fragment, nucleotide position, or amino acid position) in the context of DNA polymerase variants is based on the convention of numbering according to nucleotide or amino acid position numbers and performing sequence alignment to maximize the probability of sequence identity. An amino acid “corresponding to position X of a particular sequence” means an amino acid in the target polypeptide that matches the equivalent amino acid of a particular sequence. Generally, as described herein, the amino acid corresponding to a polymerase position can be determined using alignment algorithms such as BLAST and other currently available tools for performing amino acid sequence alignment. Not all positions within a given “corresponding region” do not need to be identical; therefore, non-matching positions within a corresponding region can be considered or defined as “corresponding positions.” Accordingly, in this specification, “an amino acid position corresponding to amino acid position X of a particular DNA polymerase” means an alignment-based equivalent position in other DNA polymerases and structural homologs and families.

[0061] In this specification, the term "consensus sequence of SEQ ID NO: 1" refers to a reference sequence containing conserved amino acids of B-family DNA polymerases across species. The consensus sequence of Sequence ID No. 1 is a hypothetical sequence, and to obtain the conserved amino acids, the following 16 wild-type B-family DNA polymerases are used: namely, Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), and Methanosarcina acetylborans. acetivorans DNA polymerase (Mac), human DNA polymerase δ catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase δ catalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 The DNA polymerase was created by aligning either DNA polymerase (T4) or Bacillus phage Phi29 DNA polymerase (Phi29).These PolB sequences are aligned to obtain an alignment sequence that serves as a functionally equivalent positional reference.

[0062] The positions of motifs ExoI, ExoII, ExoIII, A, B, and C are defined by the inventors using the consensus sequence of Sequence ID No. 1 of the present invention, and it should be noted that the positions of these motifs as defined in the present invention are not exactly identical to those described in the literature or prior art.

[0063] the purpose The inventors have discovered PolB variants with improved functionality and activity for utilizing standard nucleotides, nucleotide analogs, and initiators in template-independent polynucleotide synthesis. These PolB variants efficiently add the aforementioned standard nucleotides or nucleotide analogs to the aforementioned initiators in the absence of a replication template, enabling the synthesis of polynucleotides with random or defined sequences.

[0064] More specifically, the inventors have discovered that PolB variants can efficiently catalyze the addition of native nucleotides and nucleotide analogs to the 3′-OH terminus of single-stranded nucleic acid initiators or blunt-ended double-stranded nucleic acid initiators in the absence of a replication template, thereby generating polynucleotides having a desired nucleic acid sequence. Furthermore, the PolB variants provided herein generally exhibit broader substrate specificity. This means that PolB variants can be used for de novo nucleic acid synthesis not only with naturally occurring nucleotides but also with various modified nucleotides and nucleic acid analogs. Thus, modified nucleotides can also be designed to be incorporated into initiators to produce specific functional polynucleotides. Consequently, these PolB variants expand the range and utility of template-independent enzymatic nucleic acid synthesis applications for synthesizing polynucleotides with desired sequences and characteristics.

[0065] Protein sequence alignment of B family DNA polymerasesFigures 1-1 to 1-9 show the amino acid sequence alignments of 16 wild-type B-family DNA polymerases (PolB) used by the inventors, and the results of the aligned consensus sequences are listed below (SEQ ID NO: 1).The 16 aligned wild-type PolB strains include: Thermococcus gorgonarius DNA polymerase (Tgo, SEQ ID NO: 2), Thermococcus kodakarensis DNA polymerase (Kod1, SEQ ID NO: 3), Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N, SEQ ID NO: 4), Pyrococcus furiosus DNA polymerase (Pfu, SEQ ID NO: 5), Thermococcus litoralis DNA polymerase (Vent, SEQ ID NO: 6), Methanosarcina acetivorans DNA polymerase (Mac, SEQ ID NO: 7), and Pyrobaculum islandicum. * *Islandicum* DNA polymerase (Pis, SEQ ID NO: 8), *Sulfolobus solfataricus* DNA polymerase (Sso, SEQ ID NO: 9), *Methanococcus maripaludis* DNA polymerase (Mma, SEQ ID NO: 10), *Human* DNA polymerase δ catalytic p125 subunit (hPOLD, SEQ ID NO: 11), *Saccharomyces cerevisiae* DNA polymerase δ catalytic subunit (ScePOLD, SEQ ID NO: 12), *Pseudomonas aeruginosa* DNA polymerase II (Pae, SEQ ID NO: 13), *Escherichia coli* DNA polymerase II (Eco, SEQ ID NO: 14), *Escherichia* phage RB69 These include DNA polymerase (RB69, SEQ ID NO: 15), Escherichia phage T4 DNA polymerase (T4, SEQ ID NO: 16), and Bacillus phage Phi29 DNA polymerase (Phi29, SEQ ID NO: 17).

[0066] As shown in Figures 1-1 to 1-9, various sequence regions among these exemplary wild-type PolBs are highly conserved, while other regions are more variable. Those skilled in the art will readily recognize and understand that, in addition to those specifically identified and discussed herein, further mutations can be introduced into the variable regions of wild-type PolB without altering, or substantially altering, the polymerase activity of the mutated enzyme. Similarly, conserved mutations at any conserved residue / position of PolB can be made without altering, or substantially altering, the polymerase activity of the mutated enzyme. Enzyme engineering based on comparative structural analysis with other functionally related enzymes or homologs is a useful technique in the field of molecular biology, which allows the inventors to reasonably predict the effect of a given mutation on the catalytic activity of an enzyme. Based on this disclosure, those skilled in the art can use sequence, structural data, and known physical properties of amino acids to mutate enzymes such as DNA polymerases encompassed in the present invention without altering, or substantially altering, the essential intrinsic properties of the enzyme.

[0067] In addition, this disclosure focuses on motifs ExoI, ExoII, ExoIII, A, B, and C corresponding to positions 349-364, 450-476, 590-608, 706-730, 843-855, and 940-956, respectively, of the consensus sequence of SEQ ID NO: 1. More specifically, the polymerase variants in the present invention are based on substitutional mutations in one or more residues present in each of the aforementioned motifs.

[0068] B family DNA polymerase variant From the above perspective, this specification provides a modified polymerase described based on the amino acid sequence of the consensus sequence of SEQ ID NO: 1. The modified polymerase contains substitutional mutations in one or more residues compared to the consensus sequence of SEQ ID NO: 1. The substitutional mutations may be at the same or homologous position, or functionally equivalent position, compared to the consensus sequence of SEQ ID NO: 1. Those skilled in the art will readily understand that the modified polymerases described herein do not exist in nature. Therefore, the modified polymerases described herein are based on the consensus sequence of SEQ ID NO: 1 and further contain substitutional mutations in one or more residues of the corresponding wild-type polymerase (parent polymerase). In one embodiment, at least one substitutional mutation is at a functionally equivalent position to an amino acid in the consensus sequence of SEQ ID NO: 1. "Functionally equivalent" means that the modified polymerase has amino acid substitutions at amino acid positions according to the consensus sequence of SEQ ID NO: 1 that have the same functional or structural role in both the consensus sequence and the modified polymerase.

[0069] Generally, functionally equivalent substitution mutations in two or more different polymerases occur at homologous amino acid positions in the polymerase amino acid sequences. Therefore, "functionally equivalent" includes mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutated amino acid is known. It is possible to identify regions of functionally equivalent and positionally equivalent amino acid residues in the amino acid sequences of two or more different polymerases based on sequence alignment and / or molecular modeling. For example, positionally equivalent and / or functionally equivalent residues are identified using amino acid sequence alignments of 16 exemplary wild-type B-family DNA polymerases from different biological domains. Figures 1-1 to 1-9 show the results of protein sequence alignments between these PolBs. In other words, the exemplary residue 141 of Tgo, Kod1, 9°N, Pfu, and Vent polymerases is functionally and positionally equivalent to residue 171 of Pis, residue 231 of Sso, and residue 198 of Mac polymerase. Similarly, the exemplary residue 143 of Tgo, Kod1, 9°N, Pfu, and Vent polymerases is functionally and positionally equivalent to residue 173 of Pis, residue 233 of Sso, and residue 200 of Mac polymerase. Those skilled in the art can easily identify functionally equivalent residues of DNA polymerases.

[0070] According to some embodiments, a B-family DNA polymerase variant is provided comprising motifs ExoI, ExoII, ExoIII, A, B, and C corresponding to positions 349-364, 450-476, 590-608, 706-730, 843-855, and 940-956 of the consensus sequence of SEQ ID NO: 1, respectively; at least one amino acid substitution (one or more amino acid substitutions, or a combination of amino acid substitutions) at the positions present in motifs ExoI, ExoII, and ExoIII; and at least one amino acid substitution (one or more amino acid substitutions, or a combination of amino acid substitutions) at the positions present in motifs A, B, and C.

[0071] In accordance with the objectives of the present invention, PolB variants are provided having sequences functionally or positionally equivalent to the amino acid sequence described in Sequence ID No. 1 and any amino acid substitutions or combinations described in Table 1. In Table 1, “essential substitutions” include substitutional motifs that, by themselves, confer de novo nucleic acid synthesis activity to the above-mentioned PolB variants, and “enhancing substitutions” include substitutional residues that, in a secondary capacity, can, by themselves, confer the above-mentioned activity. Therefore, preferably, “essential substitutions” can be used alone or in combination with other mutations, and “enhancing substitutions” can optionally be used in combination with “essential substitutions” or other mutations.

[0072] [Table 1] JPEG2026102853000002.jpg195142JPEG2026102853000003.jpg195142JPEG2026102853000004.jpg195142JPEG2026102853000005.jpg195142JPEG2026102853000006.jpg195142JPEG2026102853000007.jpg195142JPEG2026102853000008.jpg195142JPEG2026102853000009.jpg195142JPEG2026102853000010.jpg195142JPEG2026102853000011.jpg195142JPEG2026102853000012.jpg195142JPEG2026102853000013.jpg195142JPEG2026102853000014.jpg195142JPEG2026102853000015.jpg195142JPEG2026102853000016.jpg195142JPEG2026102853000017.jpg195142JPEG2026102853000018.jpg195142JPEG2026102853000019.jpg195142JPEG2026102853000020.jpg195142JPEG2026102853000021.jpg195142JPEG2026102853000022.jpg195142JPEG2026102853000023.jpg195142JPEG2026102853000024.jpg133142

[0073] In some embodiments, the B family DNA polymerase variants have amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, including Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), and Methanosarcina acetylborans. acetivorans DNA polymerase (Mac), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), Human DNA polymerase δ catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase δ catalytic subunit (SecPOLD), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 It is modified from wild-type B-family DNA polymerases derived from DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), and Bacillus phage Phi29 DNA polymerase (Phi29).

[0074] In some embodiments, the polymerase is substantially lacking in 3′ exonuclease or other editing activity, and therefore the PolB variants provided herein have a deficiency in 3′-5′ exonuclease activity. The deficiency in 3′-5′ exonuclease activity may be induced by any means. For example, the 3′-5′ exonuclease activity can be reduced, weakened, removed or inactivated by modifying the 3′-5′ exonuclease domain of the polymerase to produce a polymerase that is deficient in or lacks 3′-5′ exonuclease activity. Preferably, the means of amino acid substitution are adapted to modify the 3′-5′ exonuclease domain. For example, the PolB variant may have functionally or positionally equivalent substitutions in motif ExoI of sequence number 1, such as A at position 354 in native D (D354A) and A at position 356 in native E (E356A). This results in exonuclease deficiency in the 3′ to 5′ direction.

[0075] According to a particular embodiment, amino acid L or M corresponding to position 715 of SEQ ID NO: 1 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y corresponding to position 716 of SEQ ID NO: 1 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P corresponding to position 717 of SEQ ID NO: 1 is either unsubstituted or substituted with A, G, S, or T.

[0076] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from hermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, wherein amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 2 is either unsubstituted or substituted with A, G, S, or T.

[0077] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from hermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, wherein amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 2 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0078] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, wherein amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 3 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 3 is either unsubstituted or substituted with A, G, S, or T.

[0079] According to several embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, wherein amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 3 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 3 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0080] According to several embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, wherein amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 410 of SEQ ID NO: 4 is either unsubstituted or substituted with A, G, S, or T.

[0081] According to several embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, wherein amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 409 of SEQ ID NO: 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 410 of SEQ ID NO: 4 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0082] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, wherein amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 410 of SEQ ID NO: 5 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 411 of SEQ ID NO: 5 is either unsubstituted or substituted with A, G, S, or T.

[0083] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, wherein amino acid L at position 409 of SEQ ID NO: 5 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 410 of SEQ ID NO: 5 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 411 of SEQ ID NO: 5 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0084] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, wherein amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 412 of SEQ ID NO: 6 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 413 of SEQ ID NO: 6 is either unsubstituted or substituted with A, G, S, or T.

[0085] According to several embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, wherein amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 412 of SEQ ID NO: 6 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 413 of SEQ ID NO: 6 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0086] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from methanosarcina acetivorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, wherein amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 486 of SEQ ID NO: 7 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 487 of SEQ ID NO: 7 is either unsubstituted or substituted with A, G, S, or T.

[0087] According to several embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from methanosarcina acetivorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, wherein amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 486 of SEQ ID NO: 7 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 487 of SEQ ID NO: 7 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0088] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, wherein amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 427 of SEQ ID NO: 8 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 428 of SEQ ID NO: 8 is either unsubstituted or substituted with A, G, S, or T.

[0089] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, wherein amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 427 of SEQ ID NO: 8 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 428 of SEQ ID NO: 8 is either unsubstituted or substituted with A, G, S, or T; and amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0090] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, wherein amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 519 of SEQ ID NO: 9 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; and amino acid P at position 520 of SEQ ID NO: 9 is unsubstituted or substituted with A, G, S, or T.

[0091] According to some embodiments, B-family DNA polymerase variants having exonuclease activity deficiency in the 3′ to 5′ direction are derived from Sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, wherein amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y; amino acid Y at position 519 of SEQ ID NO: 9 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; amino acid P at position 520 of SEQ ID NO: 9 is unsubstituted or substituted with A, G, S, or T; and amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

[0092] According to some embodiments, B-family DNA polymerase variants exhibit template-independent activity in synthesizing nucleic acids by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator.

[0093] In certain embodiments, the extendable initiator includes a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof. In certain embodiments, the extendable initiator is a free form of nucleic acid that can be reacted in the liquid phase.

[0094] In certain embodiments, the extensible initiator is immobilized on a solid support, the solid support comprising particles, beads, slides, array surfaces, membranes, flow cells, wells, microwells, nanowells, chambers, microfluidic chambers, channels, microfluidic channels, or any other surface.

[0095] In certain embodiments, at least one nucleotide is linked to a detectable label such as a fluorophore, enzyme, radiophosphate, digoxigenin, or biotin.

[0096] According to several embodiments, B-family DNA polymerase variants exhibit template-independent nucleic acid synthesis activity at reaction temperatures ranging from 10°C to 100°C. For example, reaction temperatures in the range of 10°C to 20°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, 90°C to 95°C, 95°C to 100°C, or 15°C, 20°C, 25°C, 30°C, 35°C, 37°C. This refers to a temperature range with an upper limit of 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C, and a lower limit of 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C.

[0097] Polymerase variant creation To modify polymerases and produce variants of this application, various types of mutagenesis techniques are optionally used in this disclosure, for example, using random or semi-random mutagenesis approaches. In general, any available mutagenesis technique can be used to produce polymerase variants. Such mutagenesis techniques optionally include the selection of nucleic acids and polypeptides modified for one or more activities of interest. Available techniques include, but are not limited to, site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combinatorial overlap PCR), mutagenesis using uracil with templates, oligonucleotide-specific mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped double-strand DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restricted selection and restricted purification, deletion mutagenesis, mutagenesis by whole gene synthesis, degenerate PCR, double-strand break repair, and many others known to those skilled in the art.

[0098] Kits for template-independent nucleic acid synthesis reactions The present invention also provides a kit comprising the PolB variant described herein for performing a de novo enzymatic nucleic acid synthesis reaction, the kit comprising the above-mentioned B-family DNA polymerase variant. The PolB variant exhibits template-independent activity to synthesize nucleic acids by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator, thereby synthesizing a desired or predetermined nucleic acid sequence.

[0099] Optionally, other reagents such as buffers and solutions required for PolB variants and nucleotide solutions may also be included. Instructions for use for the combined or packaged components are usually included, but not necessarily.

[0100] Use of B family DNA polymerase variants In some embodiments, the PolB variants described herein can be used in a template-independent synthetic manner to add native nucleotides or 3′-modified nucleotide analogs to the 3′-hydroxyl (3′-OH) termini of single-stranded or blunt-ended, double-stranded nucleic acid initiators in order to produce polynucleotides having a desired or predetermined sequence.

[0101] In some embodiments, the PolB variants described herein can be used to add nucleotides or nucleotide analogs to the 3′-OH ends of arrays of isolated, clustered single-stranded or blunt-ended, double-stranded nucleic acid initiators immobilized or physically confined on a solid support, as described above, the solid support being preferably made of glass and implemented in the form of a silicon wafer. In this way, multiplexed parallel de novo nucleic acid synthesis can be performed, enabling the synthesis of large quantities of various polynucleotides or nucleic acids with different sequences.

[0102] Large-scale parallel de novo enzymatic nucleic acid synthesis methods based on PolB variants can reduce the overall cost of de novo nucleic acid synthesis and simultaneously shorten the time required to produce oligonucleotides, synthetic gene constructs, or genomes for new bioeconomic applications such as nucleic acid-based molecular diagnostics, vaccine and drug development, genome editing, synthetic biology applications, and DNA-based digital data storage. In certain embodiments, the PolB variants described herein can be used in a template-independent synthetic manner to add native nucleotides or 3'-modified nucleotide analogs to the 3'-OH terminus of an extensible initiator or polynucleotide chain to produce polynucleotides having a desired sequence.

[0103] In certain embodiments, the PolB variants described herein can be used to incorporate a nucleotide conjugate (one of the types of nucleotide analogs defined above) covalently bonded to an enzyme, antibody, nucleotide base, phosphate moiety, or chemical moiety / group such as biotin, desthiobiotin, or fluorophore on a pentose sugar, into the 3′ end of a nucleic acid initiator in a template-independent synthetic manner.

[0104] The incorporation of these nucleotide analogs into nucleic acids by PolB variants during nucleic acid synthesis allows for the simultaneous addition of desired components, such as enzymes, antibodies, or chemical moieties / groups, to the newly synthesized nucleic acid in a base-specific, site-specific, or sequence-specific manner. Common components used to label or generate nucleic acid probes and conjugates are known in the art and include, but are not limited to, radiolabeled nucleotides and nucleotide analogs, modified linkers such as biotin, thiols, azides, or amine groups, fluorophores, enzymes, and antibodies.

[0105] Alternatively, in other embodiments, post-synthesis modification of nucleic acids for labeling or generating nucleic acid probes can be achieved by covalently or non-covalently bonding enzymes, antibodies, chemical moieties / groups, or fluorophores to the base, phosphate moiety, or pentose of the synthetic nucleotide via a modified linker. As a result, desired components can be covalently or non-covalently bonded to specific bases or to newly synthesized nucleic acids.

[0106] In some embodiments, for broader applications of de novo enzymatic nucleic acid synthesis, PolB variant-dependent incorporation of linker-modified nucleotide analogs may be used to facilitate the attachment, immobilization, or physical confinement of newly synthesized polynucleotides or nucleic acids onto various solid surfaces. Retrospectively, in other embodiments, newly synthesized sequence-specific nucleic acids having unique labels, tags, or fluorophores can be used in a variety of nucleic acid-based molecular detections, including, but not limited to, fluorescence in situ hybridization (FISH), TaqMan real-time PCR (RT-PCR), real-time fluorescence ligase chain reaction (RT-LCR), real-time fluorescence recombinase-polymerase amplification (RPA) assays, and real-time fluorescence loop-mediated isothermal amplification assays. [Examples]

[0107] Example 1: Preparation of PolB variant Gene synthesis approaches and mutagenesis techniques are adapted to construct exemplary PolB variants according to the conserved / consensus amino acid characteristics of the conserved and semi-conserved regions of selective PolB disclosed herein. For example, known site-directed mutagenesis approaches are performed to alter the amino acid residues of the motif ExoI, motif ExoII, motif ExoIII, motif A, motif B, and motif C regions of the exemplary wild-type PolB listed herein.

[0108] In some embodiments, the procedure for obtaining a PolB variant generally involves step 1: wild-type PolB and its exonuclease deficiency in the 3′ to 5′ direction (Exo - Step 1: Gene synthesis of the mutant, Step 2: Construction of a specific PolB variant having a predetermined mutation in the desired region, and Step 3: Wild-type PolB, Exo - The process is divided into three steps, including the expression and purification of mutants and PolB variants. The techniques used in the above procedure are well known to those skilled in the art, as detailed below.

[0109] In Step 1, a codon-optimized gene fragment encoding wild-type and intein-free PolB polymerase is synthesized by Genomics BioSci&Tech Co. (New Taipei City, Taiwan). The 3′ to 5′ exonuclease-deficient (Exo - -referred to as) PolB polymerase is also provided by the same vendor. The superscript letter "exo - " following all abbreviations of PolB listed in this specification means that the wild-type PolB in question has been modified to remove its intrinsic 3′ to 5′ exonuclease activity, indicating that PolB is exonuclease-deficient PolB from 3′ to 5′. Preferably, in the examples of the present disclosure, Exo - means a PolB variant having a combined mutation at positions corresponding to D354 (D354A) of SEQ ID NO: 1 and E356 (E356A) of SEQ ID NO: 1, which are each substituted with an alanine residue.

[0110] In Step 2, the synthesized wild-type and Exo - PolB genes are subcloned into the pET28b vector using NdeI and NotI restriction sites, respectively. The sequence of the recombinant plasmid is confirmed by DNA sequencing. The polymerase variant is designated PolB Exo -Site-directed mutagenesis is performed to create the desired motif region of the protein backbone. Briefly, site-directed mutagenesis PCR is performed using the Q5 site-directed mutagenesis kit from New England Biolabs (Ipswich, MA) along with a recombinant plasmid to introduce amino acid substitutions. The resulting product is first analyzed on a 1% agarose gel to confirm the amplicon size, and the remainder of the PCR reaction mixture is treated with DpnI at 37°C for 1 hour. The mixture is further incubated at 70°C for 10 minutes to inactivate the function of DpnI. Next, the DpnI-treated PCR reaction mixture is purified using the Qiagen QIAquick PCR purification kit (Whatman, MA). The purified DNA fragments are treated with a mixture of T4 PNK and T4 DNA ligase. The recirculated PCR-amplified DNA is then transformed back into E. coli cells. Plasmid DNA is then extracted from E. coli cells using the Qiagen Plasmid Mini-Kit (Whatman, MA). Mutation sequences in one or more desired motif regions of the polymerase variant are identified by DNA sequencing.

[0111] In step 3, E. coli Acella cells containing plasmid DNA with a specific polymerase variant gene are cultured at 37°C in 2 L of LB medium supplemented with 0.5% glucose and 50 μg / ml carbenicillin. When the cell density reaches approximately 0.6-0.8 as an absorbance at OD600 nm, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to induce protein expression. After culturing the cells at 37°C for a further 4 hours, the cells are harvested by centrifugation at 7,000 × g at 4°C for 10 minutes. The cell pellet is resuspended in buffer A containing 1 mM benzamidine hydrochloride [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v / v) glycerol]. After incubation on ice for 1 hour with 50 mg of lysozyme, the cells are sonicated to lyse the cells. The cell lysate is clarified by centrifugation at 18,000 × g for 25 minutes at 4°C. The clarified crude cell extract is incubated at 70°C for 30 minutes, then cooled to 4°C. The heat-treated cell extract is further clarified by centrifugation at 18,000 × g for 25 minutes at 4°C. After centrifugation, the supernatant is diluted with NaCl-free buffer A and loaded onto a HiTrap heparin column (Cytiva Life Sciences, Marlborough, MA, USA) pre-equilibriumized with buffer A in an AeKTA pure chromatography system (Cytiva Life Sciences, Marlborough, MA, USA). Proteins are eluted using buffer B [50 mM Tris-HCl (pH 8.0), 1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v / v) glycerol] with a linear gradient from 100 mM to 1 M NaCl. The column fraction is analyzed by 10% SDS-PAGE. The fraction containing the target protein is pooled and dialyzed overnight at 4°C in storage buffer [50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v / v) glycerol]. The dialyzed protein fraction pool containing the target protein is concentrated using an Amicon filter unit (MW cutoff 50,000).The concentrated protein pool was aliquoted and stored at -20°C. Each mutant polymerase variant was purified using the same procedure as described above. The final protein concentration was determined by the Bradford reaction (Bradford, 1976) using the Bio-Rad protein assay (Hercules, CA) with bovine serum albumin as the standard.

[0112] Example 2: Template-independent DNA synthesis assay The PolB variants provided herein are tested against template-independent DNA synthesis approaches. To further evaluate the activity of the PolB variants (their ability to incorporate naturally occurring nucleotides and nucleotide analogs), this specification uses ordinary dNTPs or modified nucleotides, and single-stranded DNA initiators or blunt-ended double-stranded DNA initiators.

[0113] In this example, the template-independent DNA synthesis activity of the PolB variant is determined using the following synthetic oligonucleotides.

[0114] FAM-45-merDNA initiator:5′-CTCGGCCTGGCACAGGTCCGTTCAGTGCTGCGGCGACCACCGAGG-3′ (SEQ ID NO: 18). This single-stranded oligonucleotide is labeled with a fluorescent fluoroceine amidite (FAM) dye at its 5′ end.

[0115] Blunt-end double-stranded DNA initiator: A double-stranded DNA consisting of a 38-mer primer (Cy5-38-mer primer) labeled with fluorescent cyanine 5 (Cy5) dye at its 5′ end, and its complementary 38-mer oligonucleotide (complementary 38-mer DNA). The sequence is as follows: Cy5-38-mer primer: 5′-GCTTGCACAAGTTCGTTCAATGATACGGCGACCACCGA-3′ (SEQ ID NO: 19) Complementary 38-mer DNA: 5′-TCGGTGGTCGCCGTATCATTGAACGAACTTGTGCAAGC-3′ (SEQ ID NO: 20)

[0116] The blunt-ended double-stranded DNA initiator is formed by annealing a 38-mer DNA complementary to a Cy5-38-mer primer in a 1:1.5 molar ratio in 1x TE buffer [10mM Tris-HCl (pH 8.0) and 1mM EDTA] containing 100mM NaCl. The DNA annealing reaction is carried out using a Bio-Rad Thermal Cycler (Hercules, CA) by first heating the sample mixture to 98°C for 3 minutes, and then gradually cooling it to 4°C (5°C / 30 seconds). The annealed product without overhangs is used as the blunt-ended double-stranded DNA initiator.

[0117] Template-independent DNA synthesis is carried out in a reaction mixture (10 μl) containing 100 nM FAM-45-mer DNA initiator or blunt-ended double-strand DNA initiator, 0.25 mM manganese chloride (MnCl2), and 200 nM selective PolB variant. De novo enzymatic DNA synthesis is initiated by the addition of 200 μM standard nucleotide mixture (dNTPs) or nucleotide analogs (such as 3′-O-azidomethyl-dNTPs or dye nucleotides). The reaction is allowed to proceed for a set time (e.g., 5 minutes in the implementation of the examples in the following context), and then terminated by adding 10 μl of 2x quench solution (95% deionized formamide and 25 mM EDTA) at a predetermined reaction temperature. The sample mixture is denatured at 95°C for 10 minutes and analyzed by 20% polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea. Subsequently, the gel is imaged using an Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA) to visualize the de novo enzymatic DNA synthesis reaction products.

[0118] Alternatively, template-independent DNA synthesis assays are performed in a reaction mixture (10 μl) containing 50 nM FAM-45-mer DNA initiator and 200 nM terminal deoxynucleotidyltransferase (Tdt), obtained from New England BioLabs (Ipswich, MA, USA), in Tdt reaction buffer [50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate (pH 7.9), and 0.25 mM CoCl2]. De novo enzymatic DNA synthesis reactions are initiated by adding 200 μM dNTP mixture at various temperatures ranging from 10°C, 20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 70°C, and 80°C to 90°C. After allowing each reaction to proceed for 30 minutes (or a fixed time such as 5 or 10 minutes), 10 μl of 2x quench solution (95% deionized formamide and 25 mM EDTA) is added to stop the reaction. The sample mixture is denatured at 95°C for 10 minutes and analyzed by 20% polyacrylamide gel electrophoresis (Urea-PAGE) containing 8 M urea. Subsequently, the de novo enzymatic DNA synthesis reaction products are visualized by imaging the gel with an Amersham Typhoon Laser Scanner (Cytiva Life Sciences, Marlborough, MA, USA).

[0119] Example 3: Template-independent DNA synthesis activity of exonuclease-deficient PolB variant In this example, Tgo exo- , Kod1 exo- , 9°N exo- Pfu exo- , Vent exo- Mac exo- Pis exo- , and Sso exo- We selected Kod1 as the exonuclease-deficient enzyme constructed as described in Example 1. For brevity, we used Kod1. exo- , Vent exo- , and Pfu exo-This was used as an exemplary exonuclease-deficient PolB variant to demonstrate baseline template-independent enzymatic DNA synthesis activity. The template-independent enzymatic DNA synthesis activity of commercially available Tdt was also evaluated. The evaluation of template-independent enzymatic DNA synthesis activity was performed using the same procedure as in Example 2.

[0120] The assay results are shown in Figures 2A and 2B. In these figures, "S" indicates that the substrate (FAM-45-mer DNA initiator) was used as a blank (enzyme-free) control. As a result, Kod1 exo- , Vent exo- Pfu exo- Both enzymes exhibit template-independent enzymatic DNA synthesis activity when the reaction temperature is gradually increased using single-stranded DNA as an initiator (Figure 2B), but Tdt rapidly loses its activity when the reaction temperature is raised to around 45°C (Figure 2A). This result demonstrates that the exonuclease-deficient PolB variant has superior heat resistance compared to the conventional Tdt enzyme in template-independent nucleic acid synthesis.

[0121] Example 4: Template-independent DNA synthesis activity of exonuclease-deficient PolB B variant In this example, Kod1 described in Example 3 exo- and Pfu exo-This variant was further used as an exemplary exonuclease-deficient PolB variant to demonstrate the catalytic efficiency of the PolB variant. Catalytic efficiency was evaluated using the same activity assay as described above, and DNA synthesis activity was monitored over a predetermined time (e.g., 60 minutes). The results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control. "C" represents the reaction without the addition of dNTPs as a negative control. After a 5-minute reaction, newly synthesized DNA was clearly observed, indicating that the enzymatic DNA synthesis reaction was efficient. Furthermore, it was observed that the amount of FAM-45-mer DNA initiator decreased significantly over time, while the amount of newly synthesized DNA product increased, indicating that the enzymatic DNA synthesis reaction was completed within approximately 30 minutes. From these results, it can be concluded that the exonuclease-deficient PolB variant can effectively and efficiently perform template-independent enzymatic DNA synthesis.

[0122] Example 5: Catalytic activity of PolB variant in the incorporation of standard nucleotides into FAM-45-mer DNA initiator Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolB (e.g., Tgo) are selected to contain more amino acid substitutions and to include different amino acids in various conserved regions or motifs of each protein. exo- , Kod1 exo- , 9°N exo- Pfu exo- , Vent exo- Mac exo- Pis exo- , Sso exo- Further modifications were made to )

[0123] Example 5.1: Template-independent DNA synthesis activity of the Sso variant In this example, a PolB variant derived from Sso (SEQ ID NO: 9) is used exemplarily to evaluate the template-independent DNA synthesis activity of PolB variants having combined substitutions in motifs ExoI, A, and B. Furthermore, U.S. Patent No. US11136564B2 discloses an AAI motif for substituting conserved motifs in several archaeal DNA polymerases to improve the uptake of nucleotide analogs for template-dependent DNA synthesis reactions (i.e., DNA sequencing). The conserved motif is functionally and positionally equivalent to L715, Y716, and P717 present in motif A of the consensus sequence (SEQ ID NO: 1) as defined herein. Thus, this conserved motif is also functionally and positionally equivalent to L518, Y519, and P520 present in motif A of wild-type Sso (SEQ ID NO: 9). In this example, AAI motif substitutions are also included and compared to take into account the effect of the AAI motif on template-directed nucleotide uptake.

[0124] In this example, the exemplified Sso exo-(S01) Variants modified from the skeleton were numbered, and a list of these is shown in Table 5.1. The template-independent enzymatic DNA synthesis activity of these Sso variants was evaluated using the same activity assay as above. The results are shown in Figure 4. In this figure, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control. As shown in Figure 4, variant S02, which has amino acid substitutions in motif ExoI (D231A+E233A) and motif A (L518Y+Y519A+P520G), and variant S03, which has amino acid substitutions in motif ExoI (D231A+E233A) and motif B (A601L), both exhibited significant catalytic activity for template-independent enzymatic DNA synthesis. Furthermore, almost all of the initiator (>95%) substrate reacted, and a large amount of newly synthesized DNA product was obtained. Furthermore, variant S05, which has combined amino acid substitutions in motifs ExoI (D231A+E233A), A (L518Y+Y519A+P520G), and B (A601L), exhibited further enhanced catalytic activity and longer lengths of newly synthesized DNA products compared to variants S02 and S03. However, variant S04, which has combined amino acid substitutions in motifs ExoI (D231A+E233A), A (L518A+Y519A+P520I), and B (A601L), showed only slight activity.

[0125] [Table 5.1]

[0126] Example 5.2: Template-independent DNA synthesis activity of Vent variants In this example, a PolB variant derived from Vent (SEQ ID NO: 6) is used to illustrate the evaluation of template-independent DNA synthesis activity of PolB variants having combined substitutions in motifs ExoI, A, and B.

[0127] In this example, the Vent exo-(S01) Variants modified from the skeleton were numbered, and a list of these is shown in Table 5.2. The template-independent enzymatic DNA synthesis activity of these Vent variants was evaluated using the same activity assay as above. The results are shown in Figures 5A, 5B, 5C, and 5D. In these figures, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control. As shown in Figure 5A, variant V01, which has an amino acid substitution in motif ExoI (D141A+E143A), exhibited baseline catalytic activity for template-independent enzymatic DNA synthesis at a reaction temperature of 55°C, while variant V03, which has amino acid substitutions in motif ExoI (D141A+E143A) and motif B (A488L), showed improved DNA synthesis activity compared to variant V01. Furthermore, as shown in Figure 5B, variants V02, V05, and V06, which have amino acid substitutions (detailed substitutions are shown in Table 5.2) in motifs ExoI and motif A, also showed robust DNA synthesis activity at a high reaction temperature of 70°C. Furthermore, as shown in Figure 5C, variants V06 and V07, which have combined amino acid substitutions (detailed amino acid substitutions are shown in Table 5.2) in motifs ExoI, motif A, and motif B, also showed significant DNA synthesis activity at a high reaction temperature of 70°C. Additionally, variants V08, V09, and V10, which have combined amino acid substitutions (detailed amino acid substitutions are shown in Table 5.2) in motifs ExoI, motif A, and motif B, were also used exemplary in this example to demonstrate the functional substitutions of motifs ExoI, motif A, and motif B in comparison to the control enzyme (Tdt). As shown in Figure 5C, variants with combined substitutions in both motif A and motif B, such as V04, V07, V08, V09, and V10, exhibit excellent catalytic activity over a wide range of high-temperature reaction temperatures (i.e., 60°C to 90°C).

[0128] [Table 5.2]

[0129] Example 5.3: Template-independent DNA synthesis activity of the 9°N variant In this example, a PolB variant derived from 9°N (SEQ ID NO: 4) is used to illustrate the template-independent DNA synthesis activity of PolB variants having combined substitutions in motifs ExoI and A.

[0130] Similarly, the example 9°N exo- (S01) Variants modified from the skeleton were numbered, and a list of these is shown in Table 5.3. The template-independent DNA synthesis activity of these 9°N variants was evaluated using the same activity assay as above. The results are shown in Figure 6. In this figure, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control. As shown in Figure 6, variants N02, N03, and N04, which have amino acid substitutions in motif ExoI (D141A+E143A) and motif A (detailed amino acid substitutions are shown in Table 5.3), exhibited robust DNA synthesis activity at a high reaction temperature of 70°C.

[0131] [Table 5.3]

[0132] Example 5.4: Template-independent DNA synthesis activity of the Kod1 variant In this example, a PolB variant derived from Kod1 (SEQ ID NO: 3) is used to illustrate the evaluation of template-independent DNA synthesis activity of PolB variants having combined substitutions in motifs ExoI, A, and B. Furthermore, as stated in Example 5.1, the AAI motif substitution is functionally and positionally equivalent to the conserved motifs L715, Y716, and P717 present in motif A of the consensus sequence (SEQ ID NO: 1). Therefore, this conserved motif is functionally and positionally equivalent to L408, Y409, and P410 present in motif A of wild-type Kod1 (SEQ ID NO: 3). The AAI motif substitution is also included in this example for comparison.

[0133] Similarly, the Kod1 example exo-Variants modified from the (K01) skeleton were numbered, and a list of these is shown in Table 5.4. The template-independent enzymatic DNA synthesis activity of these Kod1 variants was evaluated using the same activity assay as described above. The results are shown in Figures 7A (synthesis reaction carried out at 55°C) and 7B (reaction carried out at 70°C). As shown in Figure 7A, variant K02, which has amino acid substitutions in motif ExoI (D141A+E143A) and motif B (A485L), and variant K05, which has amino acid substitutions in motif ExoI (D141A+E143A) and motif A (L408Y+Y409A+P410G), both exhibited template-independent DNA synthesis activity. Furthermore, variant K03, which has combined amino acid substitutions in motifs ExoI (D141A+E143A), A (L408Y+Y409A+P410G), and B (A485L), showed improved DNA synthesis activity compared to variants K02 and K05. Variant K04, which has combined amino acid substitutions in motifs ExoI (D141A+E143A), A (L408A+Y409A+P410I), and B (A485L), showed lower DNA synthesis activity compared to variants K02, K03, and K05. Moreover, as shown in Figure 7B, similar results were observed for these variants even at a high reaction temperature of 70°C. In the same figure, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control.

[0134] [Table 5.4]

[0135] Example 5.5: Template-independent DNA synthesis activity of Pfu variants In this example, a PolB variant derived from Pfu (SEQ ID NO: 5) is used exemplarily to evaluate the template-independent DNA synthesis activity of PolB variants having combined substitutions in motifs ExoI, A, and B. Similarly, as described above in Example 5.1, the AAI motif substitution is functionally and positionally equivalent to the conserved motifs L715, Y716, and P717 present in motif A of the consensus sequence (SEQ ID NO: 1). Therefore, this conserved motif is functionally and positionally equivalent to L409, Y410, and P411 present in motif A of wild-type Kod1 (SEQ ID NO: 5). The AAI motif substitution is also included in this example for comparison.

[0136] Similarly, the example Pfu exo- (S01) Variants modified from the skeleton were numbered, and a list of these is shown in Table 5.5. The template-independent enzymatic DNA synthesis activity of these Pfu variants was evaluated using the same activity assay as above, at a reaction temperature of 55°C. The results are shown in Figure 8. As shown in Figure 8, variant P02, which has amino acid substitutions in motif ExoI (D141A+E143A) and motif B (A486L), exhibited template-independent DNA synthesis activity. Furthermore, variant P03, which has combination amino acid substitutions in motif ExoI (D141A+E143A), motif A (L409Y+Y410A+P411G), and motif B (A486L), showed improved DNA synthesis activity compared to variants P02 and P04. Variant P04, which has combination amino acid substitutions in motif ExoI (D141A+E143A), motif A (L409A+Y410A+P411I; AAI motif), and motif B (A486L), exhibited lower DNA synthesis activity compared to variants P02 and P03.

[0137] [Table 5.5]

[0138] Example 5.6: Summary of template-independent DNA synthesis activity of PolB variants Considering the above examples and other comparable template-independent DNA synthesis activities of various PolB variants (data not shown), these results indicate that amino acid substitutions, such as those provided herein, are important in conferring or improving the template-independent DNA synthesis activity of PolB variants.

[0139] The functional or positional substitutions present in motifs A and B of the selected PolB variants are summarized and listed in Table 5.6.

[0140] [Table 5.6] JPEG2026102853000031.jpg148142

[0141] Example 6: Template-independent DNA synthesis activity of PolB variant against blunt-ended double-stranded DNA initiator in the presence of standard nucleotides. Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolB (e.g., Tgo) are selected to include additional amino acid substitutions in various regions of the protein. exo- , Kod1 exo- , 9°N exo- Pfu exo- , Vent exo- Mac exo- Pis exo- , Sso exo- Further modifications were made to )

[0142] For brevity, in this embodiment, Vent exo-(V01) Only variants modified from the skeleton are selected and illustrated. The template-independent DNA synthesis activity of these Vent variants for extending blunt-ended double-stranded DNA initiators was evaluated using the same activity assay as above. Only illustrative results for representative Vent variants are shown (Figure 9). Table 6.1 lists these variants and their corresponding amino acid substitutions. Figure 9 shows gel image results for variant V04 and variants V11-V15. As shown in Figure 9, "S" represents the substrate (blunt-ended double-stranded DNA initiator) and is used as a blank DNA control, as well as a baseline for scoring the relative DNA synthesis activity of each variant, as detailed below.

[0143] [Table 6.1]

[0144] The relative template-independent DNA synthesis activity of each variant is scored and represented by the number of "+" symbols. The overall activity score of each variant is divided into the following four levels:

[0145] 1) "+++" indicates that the initiator was completely converted into newly synthesized DNA of various lengths, compared to the band intensity and position of the substrate control. Therefore, this variant is considered to have 100% DNA synthesis activity.

[0146] 2) "++" indicates that the initiator was converted to newly synthesized DNA of various lengths by approximately 50% to 100% compared to the band intensity and position of the substrate control. Therefore, this variant is considered to have 50% to 100% DNA synthesis activity.

[0147] 3) A "+" indicates that the initiator was converted to newly synthesized DNA of various lengths by approximately 10% to 50% compared to the band intensity and position of the substrate control. Therefore, this variant is considered to have 10% to 50% DNA synthesis activity.

[0148] 4) "+ / -" indicates that the initiator was converted to newly synthesized DNA of various lengths with less than 10% efficiency, compared to the band intensity and position of the substrate control. Therefore, this variant is considered to have <10% DNA synthesis activity.

[0149] Based on the above criteria, the activity scoring results for each functionally selected Vent variant are shown in Table 6.2.

[0150] [Table 6.2] JPEG2026102853000034.jpg139143

[0151] Example 7: Template-independent DNA synthesis activity of PolB variant against FAM-45-mer DNA initiator in the presence of nucleotide analogue (3′-O-azidomethyl-dNTP) Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolB (e.g., Tgo) are selected to include further additional amino acid substitutions in various protein regions. exo- , Kod1 exo- , 9°N exo- Pfu exo- , Vent exo- Mac exo- Pis exo- , Sso exo-Further modifications were made to ). In this example, the exemplary nucleotide analog 3′-O-azidomethyl-dNTP (3′-O-AZ-dNTP) is a well-known reversible terminator nucleotide in synthetic DNA sequencing and is used to demonstrate the PolB variant's ability to utilize non-standard nucleotides for template-independent nucleic acid synthesis.

[0152] Example 7.1: Template-independent DNA synthesis activity of the 9°N variant For simplicity, in this embodiment, 9°N exo- (N01) Variants modified from the skeleton are selected and illustrated. The template-independent DNA synthesis activity of these 9°N variants for extending the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dCTP was evaluated using the same activity assay as above. Table 7.1 shows a list of variants derived from 9°N. Furthermore, Figure 10 shows the scoring results for each 9°N variant in parallel, according to the relative DNA synthesis activity scoring criteria described in Example 6. Here, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control.

[0153] As shown in Figure 10, variants N02, N03, and N04, which have substitutions in motifs ExoI and A, showed approximately 50% to 95% DNA synthesis activity in the presence of 3′-O-AZ-dCTP. Adding 3′-O-AZ-dCMP to FAM-45-mer DNA generates a 46-mer DNA product.

[0154] [Table 7.1]

[0155] Example 7.2: Template-independent DNA synthesis activity of Vent variants In this embodiment, Vent exo-(V01) Variants modified from the skeleton are selected and illustrated. The template-independent DNA synthesis activity of these Vent variants for extending the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dATP was evaluated using the same activity assay as above. Table 7.2 shows a list of Vent-derived variants. Furthermore, Figure 11 also illustrates the relative DNA synthesis activity scoring of each variant according to the relative DNA synthesis activity scoring criteria described in Example 6. Here, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control.

[0156] As shown in Figure 11, variants V04, V17, and V18, which have combination substitutions in motifs ExoI, A, and B, showed 100% DNA synthesis activity in the presence of 3′-O-AZ-dATP and produced 46-mer DNA products.

[0157] [Table 7.2]

[0158] Considering the results above, robust template-independent DNA synthesis activity in the presence of 3′-O-AZ-dNTPs was commonly observed in various PolB variants derived from different sources of PolB provided by the present invention. Therefore, further Vent variants are selected to demonstrate their ability to perform template-independent DNA synthesis. A list of these variants is shown in Table 7.3. As mentioned above, Table 7.3 also shows the relative DNA synthesis activity score for each variant in the presence of 3′-O-AZ-dNTPs.

[0159] [Table 7.3] JPEG2026102853000038.jpg121142

[0160] Example 7.3: Template-independent DNA synthesis activity of Pfu, Kod1, and Sso variants In this embodiment, Pfu exo- (P01), Kod1 exo- (K01) and Sso exo- (S01) Variants modified from the skeleton are selected and illustrated. The template-independent DNA synthesis activity of these variants for extending the FAM-45-mer DNA initiator in the presence of 3′-O-AZ-dATP was evaluated using the same activity assay as above. Table 7.4 lists the variants used in this example and their relative DNA synthesis activity.

[0161] This result indicates that Pfu has combination substitutions. exo- , Kod1 exo- and Sso exo- The variants were shown to exhibit 100% DNA synthesis activity in the presence of 3′-O-AZ-dATP. However, the activity of variant K04, which has combination amino acid substitutions in motifs ExoI (D141A+E143A), A (L408A+Y409A+P410I), and B (A485L), was only slightly greater than that of K03.

[0162] [Table 7.4]

[0163] Example 8: Template-independent DNA synthesis activity of PolB variant against FAM-45-mer DNA initiator in the presence of dye-labeled nucleotide analogues In this example, the ability of PolB variants to utilize dye-labeled nucleotide analogs in template-independent DNA synthesis is further demonstrated by exemplary PolB variants. exo-(V01) Variants modified from the skeleton were selected and illustrated. The template-independent DNA synthesis activity of these Vent variants for extending the FAM-45-mer DNA initiator in the presence of Cy5-labeled dTTP (Cy5-dTTP) was evaluated using the same activity assay as above. The DNA synthesis activity assay was performed at a high reaction temperature of 70°C. Meanwhile, the Tdt enzyme was also used for direct comparison. The Tdt enzyme activity assay was performed at a reaction temperature of 37°C, which is the standard operating temperature of the Tdt enzyme. For brevity, variant V04 was selected to demonstrate template-independent DNA synthesis activity in the presence of Cy5-dTTP. The results are shown in Figure 12. In the same figure, "S" represents the substrate (FAM-45-mer DNA initiator) and is used as a blank DNA control. As shown in Figure 12, variant V04 exhibits robust DNA synthesis activity by incorporating Cy5-dTTP into the FAM-45-mer DNA initiator.

[0164] The results above further demonstrate that the PolB variants and kits provided herein effectively and efficiently incorporate various nucleotides for de novo enzymatic nucleic acid synthesis, and that they reliably exhibit the conferred template-independent DNA synthesis function under a wider reaction temperature range from ambient temperature to high-temperature conditions, demonstrating high heat resistance. Therefore, the PolB variants and kits within the scope of this disclosure can broaden the range of applications for template-independent enzymatic nucleic acid synthesis under different reaction conditions.

[0165] While this disclosure has been described based on its embodiments, it will be understood that various modifications that do not deviate from the scope of this disclosure also conform to the embodiments of this disclosure. Accordingly, the embodiments described are not intended to limit this disclosure and are intended to include modifications that fall within the scope of this disclosure. Accordingly, the claims should be given the broadest possible interpretation to encompass all such modifications.

Claims

1. A B-family DNA polymerase variant having an amino acid sequence having positions and numbers corresponding to the consensus sequence (SEQ ID NO: 1), wherein the variant includes multiple amino acid substitution mutations at positions in motifs selected from motif ExoI, motif ExoII, motif ExoIII, motif A, motif B, motif C, or combinations thereof, and the motifs ExoI, ExoII, ExoIII, motif A, motif B, and motif C correspond to positions 349-364, 450-476, 590-608, 706-730, 843-855, and 940-956 of the consensus sequence, respectively.

2. A B-family DNA polymerase variant according to claim 1, which is modified from a wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17.

3. A B-family DNA polymerase variant according to claim 2, wherein the wild-type B-family DNA polymerase is Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litralis litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetylans DNA polymerase (Mac), Human DNA polymerase δ catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase δ catalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfatalicus B-family DNA polymerase variants include solfataricus DNA polymerase (Sso), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), Escherichia phage T4 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29).

4. A B-family DNA polymerase variant according to claim 3, wherein the B-family DNA polymerase variant has a deficiency in exonuclease activity in the 3' to 5' direction.

5. A B-family DNA polymerase variant according to claim 4, i. The amino acid L or M corresponding to position 715 of Sequence ID No. 1 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y, corresponding to position 716 of sequence number 1, is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P, corresponding to position 717 of SEQ ID NO: 1, is either unsubstituted or substituted with A, G, S, or T.

6. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, i. The amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 410 of SEQ ID NO: 2 is either unsubstituted or substituted with A, G, S, or T.

7. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having the wild-type amino acid sequence of SEQ ID NO: 2, i. The amino acid L at position 408 of SEQ ID NO: 2 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 2 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 410 of sequence number 2 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 485 of SEQ ID NO: 2 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

8. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having a deficiency in exonuclease activity in the 3' to 5' direction is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, i. The amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 3 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 410 of SEQ ID NO: 3 is either unsubstituted or substituted with A, G, S, or T.

9. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having a deficiency in exonuclease activity in the 3' to 5' direction is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having the wild-type amino acid sequence of SEQ ID NO: 3, i. The amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 3 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 410 of sequence number 3 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 485 of SEQ ID NO: 3 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

10. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having exonuclease activity deficiency in the 3' to 5' direction is derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, i. The amino acid L at position 408 of sequence number 4 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 410 of sequence number 4 is either unsubstituted or substituted with A, G, S, or T.

11. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having exonuclease activity deficiency in the 3' to 5' direction is derived from Thermococcus sp. (9°N-7 strain) DNA polymerase (9°N) having the wild-type amino acid sequence of SEQ ID NO: 4, i. The amino acid L at position 408 of sequence number 4 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 409 of sequence number 4 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 410 of sequence number 4 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 485 of SEQ ID NO: 4 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

12. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, i. The amino acid L at position 409 of sequence number 5 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 410 of sequence number 5 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 411 of sequence number 5 is either unsubstituted or substituted with A, G, S, or T.

13. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Pyrococcus furiosus DNA polymerase (Pfu) having the wild-type amino acid sequence of SEQ ID NO: 5, i. The amino acid L at position 409 of sequence number 5 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 410 of sequence number 5 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 411 of sequence number 5 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 486 of SEQ ID NO: 5 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

14. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the exonuclease activity deficiency in the 3' to 5' direction is derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, i. The amino acid L at position 411 of sequence number 6 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 412 of sequence number 6 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 413 of sequence number 6 is either unsubstituted or substituted with A, G, S, or T.

15. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the exonuclease activity deficiency in the 3' to 5' direction is derived from Thermococcus litoralis DNA polymerase (Vent) having the wild-type amino acid sequence of SEQ ID NO: 6, i. The amino acid L at position 411 of sequence number 6 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 412 of sequence number 6 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 413 of sequence number 6 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 488 of SEQ ID NO: 6 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

16. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the exonuclease activity deficiency in the 3' to 5' direction is derived from methanosarcina acetylvorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, i. The amino acid L at position 485 of sequence number 7 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 486 of sequence number 7 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 487 of sequence number 7 is either unsubstituted or substituted with A, G, S, or T.

17. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the exonuclease activity deficiency in the 3' to 5' direction is derived from methanosarcina acetylvorans DNA polymerase (Mac) having the wild-type amino acid sequence of SEQ ID NO: 7, i. The amino acid L at position 485 of sequence number 7 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 486 of sequence number 7 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 487 of sequence number 7 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 565 of SEQ ID NO: 7 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

18. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, i. The amino acid M at position 426 of sequence number 8 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 427 of sequence number 8 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 428 of sequence number 8 is either unsubstituted or substituted with A, G, S, or T.

19. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from Pyrobaculum islandicum DNA polymerase (Pis) having the wild-type amino acid sequence of SEQ ID NO: 8, i. The amino acid M at position 426 of sequence number 8 is substituted with A, F, H, I, Q, S, W, or Y. ii. Amino acid Y at position 427 of sequence number 8 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 428 of sequence number 8 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 508 of SEQ ID NO: 8 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

20. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, i. The amino acid L at position 518 of sequence number 9 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 519 of sequence number 9 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. A B-family DNA polymerase variant in which amino acid P at position 520 of sequence number 9 is either unsubstituted or substituted with A, G, S, or T.

21. The B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant having the deficiency of exonuclease activity in the 3' to 5' direction is derived from sulfolobus solfataricus DNA polymerase (Sso) having the wild-type amino acid sequence of SEQ ID NO: 9, i. The amino acid L at position 518 of sequence number 9 is substituted with A, F, H, I, Q, S, W, or Y. ii. The amino acid Y at position 519 of sequence number 9 is either unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q. iii. The amino acid P at position 520 of sequence number 9 is either unsubstituted or substituted with A, G, S, or T. iv. A B-family DNA polymerase variant in which amino acid A at position 601 of SEQ ID NO: 9 is substituted with C, D, E, F, G, H, K, L, R, T, or Y.

22. A B-family DNA polymerase variant according to claim 5, wherein the B-family DNA polymerase variant exhibits template-independent activity of synthesizing nucleic acids by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator.

23. A B-family DNA polymerase variant according to claim 22, wherein the extensible initiator comprises a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof.

24. A B-family DNA polymerase variant according to claim 22, wherein the extendable initiator is a nucleic acid in free form and is reacted in a liquid phase.

25. A B-family DNA polymerase variant according to claim 22, wherein the extensible initiator is immobilized on a solid support, the solid support comprising particles, beads, slides, array surfaces, membranes, flow cells, wells, microwells, nanowells, chambers, microfluidic chambers, channels, microfluidic channels, or any other surface.

26. A B-family DNA polymerase variant according to claim 22, wherein at least one nucleotide is linked to a detectable label.

27. A B-family DNA polymerase variant according to claim 22, wherein the B-family DNA polymerase variant exhibits the activity in the reaction temperature range of 10°C to 100°C.

28. A kit for de novo enzymatic nucleic acid synthesis comprising a B-family DNA polymerase variant derived from wild-type B-family DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17, wherein the B-family DNA polymerase variant exhibits template-independent activity in synthesizing nucleic acids by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an elongable initiator, thereby synthesizing a desired nucleic acid sequence.

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