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

Abstract

The DNA polymerase variant and kits containing the same have improved thermostable function and activity for template-independent nucleic acid synthesis using standard nucleotides and non-standard nucleotide analogs.

Description

[Technical field]

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

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

[0003] Sequence Listing This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 98kb file entitled 211019US-sequence listing.XML, created on July 12, 2022. The information in 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 decades-old toxic chemical phosphoramidite-based nucleic acid synthesis.

[0005] All living organisms depend 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 and incorporation of nucleotides into a growing nucleic acid chain. The template-dependent method of nucleic acid synthesis requires the association of the nucleic acid polymerase with the primer-template DNA before the nucleotide is added to the 3′ end of the primer by the polymerase. To ensure high-fidelity DNA synthesis, nucleic acid polymerases have evolved a robust nucleotide selection mechanism to precisely select and incorporate the correct nucleotide corresponding to its complementary template base during nucleic acid synthesis. The active site pocket of nucleic acid polymerases is pre-configured with the appropriate shape to accommodate a correctly matched canonical nucleotide with a normal 3′-hydroxyl (3′-OH) group. Thus, removal of the 3'-OH group or replacement with bulky chemical groups on nucleotides such as 2',3'-dideoxynucleotides (ddNTPs) and 3'-O-azidomethyl-dNTPs, respectively, can significantly alter the nucleotide organization within the active site pocket of a nucleic acid polymerase, reducing the nucleotide binding affinity and overall DNA synthesis efficiency of the nucleic acid polymerase. Similarly, modifications on the nucleobase or 5'-triphosphate group of the nucleotide disrupt the interactions between the nucleotide and the active site residues of the nucleic acid polymerase, rendering these modified nucleotides less usable for nucleic acid synthesis by the polymerase.

[0006] Unlike most nucleic acid polymerases, X-family terminal deoxynucleotidyl transferases (Tdts) are a unique class of mesophilic enzymes that are template-independent for adding nucleotides during nucleic acid synthesis. Tdt only requires a short initiator DNA or primer to direct the synthesis and incorporation of nucleotides into the growing initiator DNA or primer. Tdt can perform template-independent DNA synthesis, and the active site pocket of Tdt is also pre-configured with the proper shape to accommodate standard nucleotides with normal 3′-OH groups. As with other nucleic acid polymerases, replacement of the 3′-OH group on the nucleotide with bulky chemical groups causes steric hindrance of the nucleotide when entering the nucleotide-binding pocket of Tdt, resulting in a decrease in the nucleotide-binding affinity and overall DNA synthesis activity of Tdt. Not surprisingly, the template-independent DNA synthesis function of Tdt makes it a prime choice for application in de novo enzymatic DNA synthesis. However, several inherent properties of Tdt, such as its limited thermostability (mesophilic enzyme), preference for incorporation of certain nucleotides, intolerance to larger substitutions of the 3′-OH group on nucleotides, and synthetic inefficiency, pose barriers to the practical application of enzymatic DNA synthesis.

[0007] To expand the application of enzymatic DNA synthesis, there remains an unmet need for alternative nucleic acid polymerases and their derivatives that are thermostable and can accommodate a variety of non-canonical nucleotide analogs, such as reversible terminators and dye-terminator nucleotides. Summary of the Invention [Problem to be solved by the invention]

[0008] Due to the diverse structure-function relationships described above, naturally occurring nucleic acid polymerases cannot readily utilize standard nucleotides or nucleotide analogues as substrates for de novo nucleic acid synthesis, and tailor-made modified nucleic acid polymerases are therefore a prerequisite for their utility in a variety of nucleic acid synthesis applications. [Means for solving the problem]

[0009] The present inventors have discovered novel positions / regions in the amino acid sequences of B family DNA polymerase variants that play an important role in conferring template independence to the polymerase and increasing the nucleotide substrate binding affinity of the polymerase to both standard and modified nucleotides, thereby improving the efficiency of nucleic acid synthesis in template-independent nucleic acid synthesis methods.

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

[0011] In one embodiment, the family B DNA polymerase variant is modified from a wild-type family B 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 polymerase is selected from the group consisting of Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase (Methoxy), Methoxy, ... acetivorans DNA polymerase (Mac), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), 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).

[0013] In one embodiment, the family B DNA polymerase variants provided herein are deficient in 3' to 5' exonuclease activity.

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

[0015] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2, in which the amino acid L at position 408 of SEQ ID NO:2 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO:2 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 410 of SEQ ID NO:2 is unsubstituted or substituted with A, G, S, or T.

[0016] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2, in which the amino acid L at position 408 of SEQ ID NO:2 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO:2 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO:2 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3, in which the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or is substituted with A, G, S, or T.

[0018] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3, in which the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4, in which the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or is substituted with A, G, S, or T.

[0020] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4, in which the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO:5, in which the amino acid L at position 409 of SEQ ID NO:5 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 410 of SEQ ID NO:5 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 411 of SEQ ID NO:5 is not substituted or is substituted with A, G, S, or T.

[0022] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO:5, in which the amino acid L at position 409 of SEQ ID NO:5 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 410 of SEQ ID NO:5 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 411 of SEQ ID NO:5 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6, in which the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or is substituted with A, G, S, or T.

[0024] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6, in which the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7, in which the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or is substituted with A, G, S, or T.

[0026] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7, in which the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8, in which the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or is substituted with A, G, S, or T.

[0028] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8, in which the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9, in which the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or is substituted with A, G, S, or T.

[0030] In one embodiment, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9, in which the amino acid L at position 518 of SEQ ID NO: 9 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 519 of SEQ ID NO: 9 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 520 of SEQ ID NO: 9 is not substituted or is substituted with A, G, S, or T, and the 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 family B DNA polymerase variants provided herein exhibit the activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an extendible initiator.

[0032] In some embodiments, the extendible initiator comprises a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof.

[0033] In some embodiments, the extendible initiator is a nucleic acid in free form, as opposed to an immobilized nucleic acid, so as to react in a liquid phase, such as liquid medium or other aqueous solution.

[0034] In some embodiments, the extendible initiator is immobilized on a solid support, the solid support comprising a particle, a bead, a slide, an array surface, a membrane, a flow cell, a well, a microwell, a nanowell, a chamber, a microfluidic chamber, a channel, a microfluidic channel, or any other surface.

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

[0036] In some embodiments, the family B DNA polymerase variant exhibits nucleotide incorporating activity at reaction temperatures ranging from 10°C to 100°C.

[0037] In another embodiment, the disclosure further provides a kit for performing de novo enzymatic nucleic acid synthesis comprising a B family DNA polymerase variant derived 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. The B family DNA polymerase variant exhibits an activity to synthesize nucleic acid in a template-independent manner by adding at least one nucleotide selected from the group of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an extendible initiator, thereby synthesizing a desired nucleic acid sequence.

[0038] Thus, the present invention relates to a particular 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 carried out by the thermostable B family DNA polymerase variant with a wide range of nucleotides and nucleotide analogs.

[0039] The present disclosure will be more readily understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 shows an amino acid sequence alignment of wild-type B family DNA polymerases (PolB) relevant to the present invention and their consensus sequences. [Figure 2A] FIG. 2A shows the results of the reaction described in Example 3. [Figure 2B] FIG. 2B shows the results of the reaction described in Example 3. [Figure 3A] FIG. 3A shows the results of the reaction described in Example 4. [Figure 3B] FIG. 3B shows the results of the reaction described in Example 4. [Figure 4] FIG. 4 shows the results of the reaction described in Example 5.1. [Figure 5A] FIG. 5A shows the results of the reaction described in Example 5.2. [Figure 5B] FIG. 5B shows the results of the reaction described in Example 5.2. [Figure 5C] FIG. 5C shows the results of the reaction described in Example 5.2. [Figure 5D] FIG. 5D shows the results of the reaction described in Example 5.2. [Figure 6] FIG. 6 shows the results of the reaction described in Example 5.3. [Figure 7A] FIG. 7A shows the results of the reaction described in Example 5.4. [Figure 7B]FIG. 7B shows the results of the reaction described in Example 5.4. [Figure 8] FIG. 8 shows the results of the reaction described in Example 5.5. [Figure 9] FIG. 9 shows the results of the reactions described in Example 6. [Figure 10] FIG. 10 shows the results of the reaction described in Example 7.1. [Figure 11] FIG. 11 shows the results of the reaction described in Example 7.2. [Figure 12] FIG. 12 shows the results of the reactions described in Example 8. Detailed Description of the Invention

[0041] ■Definition All terms used in this specification, including descriptive terms or technical terms, should be interpreted as having a meaning that can be understood by a person skilled in the art. However, the meaning of a term may differ depending on the intention of the explainer, precedents, the emergence of new technology, etc. In addition, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected term will be explained in detail in the description of this disclosure. Therefore, the terms used in this specification are defined based on their meaning together with the description throughout this specification. Furthermore, titles and subtitles may be given to the contents of the description to facilitate understanding, but these titles do not affect the scope of the present invention.

[0042] As used herein, the terms "a," "an," or "the" include plural reference terms unless expressly and unambiguously limited to one reference term. The term "or" is used interchangeably with the term "and / or" unless the context clearly indicates otherwise.

[0043] Furthermore, when a part or method "includes" or "comprises" an element or step, unless there is a specific statement to the contrary, the part or method may further include other elements or steps, and does not exclude other elements 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 twenty naturally occurring 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" residues are undefined, they shall be defined as "any amino acid."

[0045] The term "functionally equivalent" or "equivalent" is used to describe a particular B family DNA polymerase (PolB) variant that has a substitution or mutation that is believed to occur at an amino acid position in another PolB, PolB variant according to a sequence alignment or reference sequence, that has 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, and therefore equivalent amino acid substitution mutations between different PolBs generally occur at homologous amino acid positions. The term "functionally equivalent" or "equivalent" as used herein also encompasses 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. In fact, "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 FIG. 1, interspecies alignments were performed for multiple wild-type PolBs, and a consensus sequence (SEQ ID NO: 1) was used as a positional reference sequence.

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

[0048] The term "conserved" refers to a segment of a polymerase that has the same amino acid residues at homologous or equivalent positions in different PolBs from various sources. The term "semi-conserved" as used herein refers to a segment of a polymerase that has similar properties of amino acid residues 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" refer to a deoxyribonucleotide or ribonucleotide sequence in single- or double-stranded form, the source and length of which are not limited herein, and generally include naturally occurring nucleotides or artificial chemical mimetics. As used herein, the term "nucleic acid" is used interchangeably with terms including natural or non-natural "oligonucleotides", "polynucleotides", "DNA", "RNA", "gene", "complementary DNA" (cDNA), and "messenger RNA" (mRNA).

[0050] As used herein, "nucleic acid," "oligonucleotide," or "polynucleotide" refers to a polymer comparable to a ribose nucleic acid (RNA) or deoxyribose nucleic acid (DNA) polymer, or an analog thereof. 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, replacement of one or more naturally occurring nucleotides with an analog, modifications between nucleotides such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), pendant molecules (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Additionally, synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding or other chemical interactions are included. Typically, the nucleotide monomers are linked via phosphodiester bonds, although synthetic forms of nucleic acids can include other linkages (e.g., peptide nucleic acids as described in Nielsen et al. (Science 254:1497-1500, 1991)). Nucleic acids can be or include, for example, chromosomes or chromosome segments, vectors (e.g., expression vectors), expression cassettes, naked DNA or RNA polymers, products of polymerase chain reaction (PCR), oligonucleotides, probes, and primers. Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, and are not limited to a particular length. Unless otherwise noted, a particular nucleic acid sequence optionally includes or encodes complementary sequences in addition to the sequence explicitly indicated.

[0051] Nucleic acids as used herein also include nucleic acid analogs. The term nucleic acid analog is known to mean 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 the nucleotide (phosphate backbone, pentose, nucleobase) may be modified. These modifications of the nucleotide change the structure and shape of the nucleic acid and change the interaction with nucleic acid polymerase. Nucleic acid analogs also include a new category of artificial nucleic acids designed to have a new sugar backbone shape that does not exist in nature, such as xenonucleic acids (XNA).

[0052] Examples of nucleic acid analogs include universal bases that can base pair with all four standard bases, such as inosine, 3-nitropyrrole, and 5-nitroindole; phosphate-sugar backbone analogs, such as peptide nucleic acids (PNAs), that affect the backbone properties of the nucleic acid; analogs with chemical linkers or fluorophores attached, such as amine-reactive aminoallyl nucleotides, thiol-containing nucleotides, biotin-conjugated nucleotides, rhodamine-conjugated nucleotides, and cyanine-conjugated 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); fluorescent reporter dyes (ALEXA, FAM, TET, TAMRA, CY3, CY5, VIC, JOE, HEX, NED, PET, ROX, Texas These include, but are not limited to, nucleic acid probes for various genetic applications, such as oligonucleotides conjugated with fluorescent probes (e.g., Fluorescent Red) and / or fluorescent quenchers (BHQ); molecular beacons (MBs), which are single-stranded nucleic acid probes containing a stem-loop structure and dual fluorophore-quencher labels; and nucleic acid aptamers.

[0053] Generally, as used herein, a "template" is a polynucleotide or polynucleotide mimic that contains a desired or unknown target nucleotide sequence. In some instances, the terms "target sequence", "template polynucleotide", "target nucleic acid", "target polynucleotide", "nucleic acid template", "template sequence, and variations thereof" are used interchangeably. Specifically, the term "template" refers to a nucleic acid strand from which a complementary copy is synthesized from nucleotides or nucleotide analogs through template-dependent or template-directed replication by a nucleic acid polymerase. Within a nucleic acid duplex, the template strand is conventionally depicted and described as the "bottom" strand. Similarly, the non-template strand is often depicted and described as the "top" strand. The "template" strand may also be referred to as the "sense" or "plus" strand, and the non-template strand as the "antisense" 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 a nucleic acid polymerase. The term "initiator" may also refer to xenonucleic acids (XNA) or peptide nucleic acids (PNA) that have a 3'-hydroxyl group.

[0055] The terms "nucleotide incorporation", "analog incorporation", "incorporated nucleotide" and "incorporation analog" are known to those skilled in the art and are used to describe the process or reaction of nucleic acid synthesis. Thus, as used herein, the term "incorporation" is known to flexibly mean the addition of one or more nucleotides, or any designated nucleic acid precursor, to the 3'-hydroxyl end 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 elongated 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 express that "one dGTP is incorporated into DNA."

[0056] The term "nucleotide analog" is known to those skilled in the art and is known to refer to chemically modified or artificial nucleotides that are structural mimics of standard nucleotides. These nucleotide analogs can be substrates for nucleic acid polymerases to synthesize nucleic acids. In nucleotide analogs, one or more of the components of the nucleotide (e.g., phosphate backbone, pentose, nucleobase) can be changed, which changes the structure and arrangement of the nucleotide and affects its interaction with other nucleobases and nucleic acid polymerases. For example, nucleotide analogs with altered nucleobases can impart alternative base pairing and base stacking properties in DNA or RNA. Furthermore, by way of example, modifications in the bases can generate various nucleosides such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, diamino-2,6-purine or bromo-5-deoxyuridine, and any other analogs that allow hybridization. In other exemplary embodiments, modifications can occur at the level of the sugar moiety (e.g., replacement of deoxyribose with an analog) and / or at the level of the phosphate group (e.g., boronate, alkylphosphonate, or phosphorothioate derivatives). Nucleotide analog monomers can have a phosphate group selected from monophosphate, diphosphate, triphosphate, tetraphosphate, pentaphosphate, and hexaphosphate.

[0057] Other examples of nucleotide analogs include nucleotides with 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-azido (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, O-acyl. Examples of base blocking moieties include reversible dye terminators. Examples of reversible dye terminators include, but are not limited to, the Illumina MiSeq reversible dye terminator, the Illumina HiSeq reversible dye terminator, the Illumina Genome Analyzer IIX reversible dye terminator, the Helicos Biosciences Heliscope reversible dye terminator, and the LaserGen Lightning Terminators reversible dye terminator. As used herein, "family B DNA polymerase (PolB)" refers to the most common template-dependent nucleic acid polymerase or replicase in all domains of life and many DNA viruses. Like most nucleic acid polymerases, native PolB requires a duplex primer-template DNA with a free 3′-hydroxyl (3′-OH) group at the primer terminus, all four nucleoside triphosphates (dATP, dTTP, dCTP, dGTP), and a catalytic divalent cation (Mg 2+ or Mn 2+PolB enzymes, like bacterial PolII and archaeal B-family DNA polymerases, are replication and repair polymerases that naturally contain a catalytic polymerase domain and a 3′ to 5′ exonuclease or proofreading domain to remove misincorporated nucleotides from the growing primer strand during nucleic acid replication. The term “3′ to 5′ exonuclease domain (Exo domain)” refers to the region of the amino acid sequence of a polymerase that exerts nucleolytic activity from the 3′ end of a primer or polynucleotide strand. Cooperatively, the term “catalytic polymerase domain” (Pol domain) refers to the region of the amino acid sequence of a polymerase that exerts catalytic DNA / RNA polymerase activity to add nucleotides to the 3′ end of a primer or polynucleotide strand.

[0058] All known structures of PolB catalytic polymerase domains resemble the shape of a human right hand, with the major functional regions characterized as finger, palm, and thumb subdomains. The most conserved region is the palm subdomain, which contains residues essential for catalysis. Protein sequence alignments between various B family DNA polymerases from different kingdoms of life and DNA viruses revealed that PolB polymerases generally possess six semi-conserved 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] As used herein, the term "mutant" in the context of a DNA polymerase of the invention refers to a polypeptide, typically recombinant, that contains one or more amino acid substitutions relative to the corresponding functional DNA polymerase.

[0060] As used herein, the expression "corresponding to another sequence" (e.g., region, fragment, nucleotide position or amino acid position, etc.) in the context of DNA polymerase variants is based on the convention of numbering according to the nucleotide or amino acid position number and aligning sequences to maximize the probability of sequence identity. An amino acid "corresponding to position X of a particular sequence" refers to an amino acid in a polypeptide of interest that matches the equivalent amino acid in the particular sequence. In general, as described herein, amino acids corresponding to positions in a polymerase can be determined using alignment algorithms such as BLAST and other currently available tools for performing amino acid sequence alignment. Since not all positions in a given "corresponding region" need be identical, non-matching positions in a corresponding region can be considered or defined as "corresponding positions". Thus, as used herein, "amino acid position corresponding to amino acid position X of a particular DNA polymerase" refers to the equivalent position based on alignment in other DNA polymerases and structural homologs and families.

[0061] The term "consensus sequence of SEQ ID NO:1" herein refers to a reference sequence that comprises conserved amino acids of family B DNA polymerases across species. The consensus sequence of SEQ ID NO:1 is a hypothetical sequence, and the following 16 wild-type B family DNA polymerases were used to obtain the conserved amino acids: Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase (McA), Methanococcus acetivorans DNA polymerase (Mm ... acetivorans DNA polymerase (Mac), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta 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 DNA polymerase (T4), or Bacillus phage Phi29 DNA polymerase (Phi29).These PolB sequences are aligned to obtain an aligned sequence as a reference for functionally equivalent positions.

[0062] It should be noted that the positions of motifs ExoI, ExoII, ExoIII, A, B and C have been defined by the inventors using the consensus sequence of SEQ ID NO:1 of the present invention, and therefore the positions of these motifs defined in the present invention are not entirely identical to those described in the literature or prior art.

[0063] the purpose The present inventors have discovered PolB variants with improved function and activity for utilizing standard nucleotides, nucleotide analogs, and initiators in template-independent polynucleotide synthesis, which efficiently add the standard nucleotides or nucleotide analogs to the initiators in the absence of a replication template, allowing 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 natural nucleotides and nucleotide analogs to the 3′-OH terminus of single-stranded or blunt-ended double-stranded nucleic acid initiators in the absence of a replication template to generate polynucleotides with desired nucleic acid sequences. Furthermore, the PolB variants provided herein generally have broader substrate specificity. This means that the PolB variants can utilize not only naturally occurring nucleotides, but also various modified nucleotides and nucleic acid analogs for de novo nucleic acid synthesis. Thus, modified nucleotides can also be further designed to be incorporated into initiators to generate specific functional polynucleotides. Thus, these PolB variants expand the scope and utility of template-free enzymatic nucleic acid synthesis applications to synthesize polynucleotides with desired sequences and characteristics.

[0065] Protein sequence alignment of B family DNA polymerasesFIG. 1 shows an amino acid sequence alignment of 16 wild-type B family DNA polymerases (PolB) used by the present inventors, and the resulting aligned consensus sequence is listed at the bottom (SEQ ID NO: 1).The 16 aligned wild-type PolBs were Thermococcus gorgonarius DNA polymerase (Tgo, SEQ ID NO: 2), Thermococcus kodakarensis DNA polymerase (Kod1, SEQ ID NO: 3), Thermococcus sp. (strain 9°N-7) 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), Pyrobaculum israndicum DNA polymerase (Pyrobaculum spp., SEQ ID NO: 8), and Thermococcus spp. (strain 9°N-7) DNA polymerase (9°N, SEQ ID NO: 9). 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 delta catalytic p125 subunit (hPOLD, SEQ ID NO: 11), Saccharomyces cerevisiae DNA polymerase delta 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 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 FIG. 1, various sequence regions among these exemplary wild-type PolBs are highly conserved, while other regions are more variable. One of skill in the art will readily recognize and understand that the variable regions of wild-type PolB can be further mutated in addition to those specifically identified and discussed herein without altering or substantially altering the polymerase activity of the mutated enzyme. Similarly, conservative mutations at conserved residues / positions of any 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 that allows the inventors to reasonably predict the effect of a given mutation on the catalytic activity of an enzyme. Based on the present disclosure, one of skill in the art can use the sequence, structural data, and known physical properties of amino acids to mutate enzymes such as DNA polymerases encompassed by the present invention without altering or substantially altering the essential intrinsic properties of the enzyme.

[0067] In addition, the present disclosure focuses on motifs ExoI, ExoII, ExoIII, A, B, and C, which correspond 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 of the present invention are based on substitution mutations at one or more residues present in each of the above motifs.

[0068] B family DNA polymerase variants In view of the above, there is provided herein a modified polymerase described based on the amino acid sequence of the consensus sequence of SEQ ID NO:1. The modified polymerase comprises a substitution mutation at one or more residues compared to the consensus sequence of SEQ ID NO:1. The substitution mutation can be at the same or homologous position, or at a functionally equivalent position, compared to the consensus sequence of SEQ ID NO:1. Those skilled in the art can easily understand that the modified polymerase described herein is not naturally occurring. Thus, the modified polymerase described herein is based on the consensus sequence of SEQ ID NO:1 and further comprises a substitution mutation at one or more residues of the corresponding wild-type polymerase (parent polymerase). In one embodiment, at least one substitution mutation is at a functionally equivalent position to the amino acid of the consensus sequence of SEQ ID NO:1. By "functionally equivalent" it is meant that the modified polymerase has an amino acid substitution at an amino acid position according to the consensus sequence of SEQ ID NO:1 that has 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 amino acid sequences of the polymerases. Thus, "functionally equivalent" also 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. Regions of functionally equivalent and positionally equivalent amino acid residues in the amino acid sequences of two or more different polymerases can be identified based on sequence alignment and / or molecular modeling. For example, an amino acid sequence alignment of 16 exemplary wild-type B family DNA polymerases from different domains of life is used to identify positionally equivalent and / or functionally equivalent residues. Figure 1 shows the results of a protein sequence alignment between these PolBs. That is, residue 171 of Pis, residue 231 of Sso, and residue 198 of Mac polymerase are functionally equivalent and positionally equivalent to exemplary residue 141 of Tgo, Kod1, 9°N, Pfu, and Vent polymerases. Similarly, residue 173 of Pis, residue 233 of Sso, and residue 200 of Mac polymerase are functionally and positionally equivalent to exemplary residue 143 of Tgo, Kod1, 9°N, Pfu, and Vent polymerases. Those of skill in the art can readily identify functionally equivalent residues in DNA polymerases.

[0070] According to some embodiments, there is provided a B-family DNA polymerase variant comprising motif ExoI, motif ExoII, motif ExoIII, motif A, motif B, and motif 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 positions present in motif ExoI, motif ExoII, and motif ExoIII; and at least one amino acid substitution (one or more amino acid substitutions, or a combination of amino acid substitutions) at positions present in motif A, motif B, and motif C.

[0071] In accordance with the objectives of the present invention, there is provided a PolB variant having an amino acid sequence set forth in SEQ ID NO: 1 and a functionally or positionally equivalent sequence to any of the amino acid substitutions or combinations thereof set forth in Table 1. In Table 1, "essential substitutions" include substitution motifs that, by themselves, confer de novo nucleic acid synthesis activity to the above-mentioned PolB variant, and "enhanceable substitutions" include replacement residues that, in a minor capacity, may confer the above-mentioned activity by themselves. Thus, preferably, "essential substitutions" can be used alone or in combination with other mutations, and "enhanceable substitutions" can be used optionally in combination with "essential substitutions" or other mutations.

[0072] [Table 1] TIFF2024533804000003.tif196143 TIFF2024533804000004.tif196143TIFF2024533804000005.tif196143TIFF2024533804000006.tif196143TIFF2024533804000007.tif196143TIFF2024533804000008.tif196143TIFF2024533804000009.tif196143TIFF2024533804000010.tif196143TIFF2024533804000011.tif196143TIFF2024533804000012.tif196143TIFF2024533804000013.tif196143TIFF2024533804000014.tif196143TIFF2024533804000015.tif196143 TIFF2024533804000016.tif196143TIFF2024533804000017.tif196143TIFF2024533804000018.tif196143TIFF2024533804000019.tif196143TIFF2024533804000020.tif196143TIFF2024533804000021.tif196143TIFF2024533804000022.tif196143 TIFF2024533804000023.tif196143TIFF2024533804000024.tif196143TIFF2024533804000025.tif134143

[0073] In some embodiments, the B family DNA polymerase variant is selected from the group consisting of Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (Vent), Methanosarcina acetivorans DNA polymerase (Mt), ... acetivorans DNA polymerase (Mac), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus DNA polymerase (Sso), Methanococcus maripaludis DNA polymerase (Mma), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (SecPOLD), Pseudomonas aeruginosa DNA polymerase II (Pae), Escherichia coli DNA polymerase II (Eco), Escherichia phage RB69 DNA polymerase (RB69), which is modified from wild-type B family DNA polymerases derived from Escherichia phage T4 DNA polymerase (T4), and Bacillus phage Phi29 DNA polymerase (Phi29).

[0074] In some embodiments, the polymerase is substantially devoid of 3' exonuclease or other editing activity, such that the PolB variants provided herein have a deficiency in 3' to 5' exonuclease activity. The deficiency in 3' to 5' exonuclease activity may be generated by any means. For example, the 3' to 5' exonuclease domain of the polymerase may be modified to generate a polymerase that is deficient or lacks 3' to 5' exonuclease activity, thereby actually reducing, attenuating, eliminating or inactivating the 3' to 5' exonuclease activity. Preferably, the amino acid substitution means is adapted to modify the 3' to 5' exonuclease domain. For example, a PolB variant may have a functionally equivalent or positionally equivalent substitution in the motif ExoI of SEQ ID NO:1 with an A in the native D at position 354 (D354A) and an A in the native E at position 356 (E356A), resulting in an exonuclease deficiency in the 3' to 5' direction.

[0075] According to a particular embodiment, the 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, the amino acid Y corresponding to position 716 of SEQ ID NO:1 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N or Q, and the amino acid P corresponding to position 717 of SEQ ID NO:1 is not substituted or is substituted with A, G, S or T.

[0076] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Hermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2, in which the amino acid L at position 408 of SEQ ID NO:2 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO:2 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 410 of SEQ ID NO:2 is unsubstituted or substituted with A, G, S, or T.

[0077] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Hermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO:2, in which the amino acid L at position 408 of SEQ ID NO:2 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO:2 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO:2 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3, in which the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y, the 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, and the amino acid P at position 410 of SEQ ID NO: 3 is unsubstituted or substituted with A, G, S, or T.

[0079] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3, in which the amino acid L at position 408 of SEQ ID NO: 3 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 3 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO: 3 is not substituted or is substituted with A, G, S, or T, and the 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 some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4, in which the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or is substituted with A, G, S, or T.

[0081] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N) having a wild-type amino acid sequence of SEQ ID NO: 4, in which the amino acid L at position 408 of SEQ ID NO: 4 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 409 of SEQ ID NO: 4 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 410 of SEQ ID NO: 4 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO:5, in which the amino acid L at position 409 of SEQ ID NO:5 is substituted with A, F, H, I, Q, S, W, or Y, the 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, and the amino acid P at position 411 of SEQ ID NO:5 is unsubstituted or substituted with A, G, S, or T.

[0083] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO:5, in which the amino acid L at position 409 of SEQ ID NO:5 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 410 of SEQ ID NO:5 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 411 of SEQ ID NO:5 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6, in which the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y, the 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, and the amino acid P at position 413 of SEQ ID NO: 6 is unsubstituted or substituted with A, G, S, or T.

[0085] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6, in which the amino acid L at position 411 of SEQ ID NO: 6 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 412 of SEQ ID NO: 6 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 413 of SEQ ID NO: 6 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7, in which the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y, the 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, and the amino acid P at position 487 of SEQ ID NO: 7 is unsubstituted or substituted with A, G, S, or T.

[0087] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7, in which the amino acid L at position 485 of SEQ ID NO: 7 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 486 of SEQ ID NO: 7 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 487 of SEQ ID NO: 7 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8, in which the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, and the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or is substituted with A, G, S, or T.

[0089] According to some embodiments, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8, in which the amino acid M at position 426 of SEQ ID NO: 8 is substituted with A, F, H, I, Q, S, W, or Y, the amino acid Y at position 427 of SEQ ID NO: 8 is not substituted or is substituted with A, C, D, F, G, H, I, K, L, M, N, or Q, the amino acid P at position 428 of SEQ ID NO: 8 is not substituted or is substituted with A, G, S, or T, and the 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, the B-family DNA polymerase variant having a defective 3' to 5' exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO:9, in which the amino acid L at position 518 of SEQ ID NO:9 is substituted with A, F, H, I, Q, S, W, or Y, the 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 the 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, the B-family DNA polymerase variant with a 3' to 5' exonuclease activity deficiency is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO:9, in which the amino acid L at position 518 of SEQ ID NO:9 is substituted with A, F, H, I, Q, S, W, or Y, the 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, the amino acid P at position 520 of SEQ ID NO:9 is unsubstituted or substituted with A, G, S, or T, and the 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, the family B DNA polymerase variant exhibits the activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an extendible initiator.

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

[0094] In certain embodiments, the extendible initiator is immobilized on a solid support, the solid support comprising a particle, a bead, a slide, an array surface, a membrane, a flow cell, a well, a microwell, a nanowell, a chamber, a microfluidic chamber, a channel, a microfluidic channel, or any other surface.

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

[0096] According to some embodiments, the family B DNA polymerase variant exhibits template-independent nucleic acid synthesis activity at a reaction temperature in the range of 10° C. to 100° C. For example, the reaction temperature may be 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., or 95° C. to 100° C., or 15° C., 20° C., 25° C., 30° C., 35° C., 37° C., or 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., or 95° C. to 100° C. , 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 as upper limits, and 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 as lower limits.

[0097] Generation of polymerase variants In order to modify polymerase to generate variants of the present application, various types of mutagenesis techniques are optionally used in the present disclosure, for example, using random or semi-random mutation approaches. In general, any available mutagenesis technique can be used to generate polymerase mutants. Such mutagenesis techniques optionally include the selection of nucleic acids and polypeptides modified for one or more activities of interest. Techniques that can be used 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-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, constraint selection and constraint purification, deletion mutagenesis, mutagenesis by total 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 for performing a de novo enzymatic nucleic acid synthesis reaction, comprising a PolB variant as described herein, the kit comprising the above-mentioned B family DNA polymerase variant, wherein the PolB variant exhibits an activity to synthesize nucleic acid in a template-independent manner by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof, to an extendible initiator, thereby synthesizing a desired or predetermined nucleic acid sequence.

[0099] Optionally, other reagents such as buffers or solutions required for the PolB variant and nucleotide solutions are also included. Instructions for use for bundled or packaged components are also typically, but not necessarily, included.

[0100] Use of B family DNA polymerase variants In some embodiments, the PolB variants described herein can be used to add natural 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 a template-independent synthetic manner 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 termini of an array of isolated, clustered, single-stranded or blunt-ended, double-stranded nucleic acid initiators immobilized or physically confined on a solid support, preferably made of glass, and implemented in the form of a silicon wafer, as described above. In this way, multiplexed parallel de novo nucleic acid synthesis can be performed to synthesize large amounts of a variety of polynucleotides or nucleic acids with different sequences.

[0102] Massively parallel de novo enzymatic nucleic acid synthesis methods based on PolB variants can lower the overall cost of de novo nucleic acid synthesis and simultaneously reduce the time to manufacture 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, DNA-based digital data storage, etc. In certain embodiments, the PolB variants described herein can be used to add natural nucleotides or 3'-modified nucleotide analogs to the 3'-OH termini of extendable initiator or polynucleotide chains in a template-independent synthetic manner to produce polynucleotides having desired sequences.

[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 linked to a chemical moiety / group, such as an enzyme, an antibody, biotin, desthiobiotin, or a fluorophore on the base, phosphate moiety, or pentose of the nucleotide, at the 3' end of a nucleic acid initiator in a template-independent synthetic manner.

[0104] Incorporation of these nucleotide analogs into nucleic acids by PolB variants during nucleic acid synthesis simultaneously adds desired moieties, 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 moieties 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, thiol, azide, or amine groups, fluorophores, enzymes, and antibodies.

[0105] Alternatively, in other embodiments, post-synthetic modification of nucleic acids to label or generate nucleic acid probes can be accomplished by covalent or non-covalent attachment of enzymes, antibodies, chemical moieties / groups, or fluorophores via modified linkers on the base, phosphate moiety, or pentose sugar of the synthetic nucleotide, such that desired components can be covalently or non-covalently attached to specific bases or to newly synthesized nucleic acids.

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

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

[0108] In some embodiments, the procedure for obtaining a PolB variant generally includes the steps of: Step 1: cloning wild-type PolB and its 3′ to 5′ exonuclease deficient (Exo - 1) gene synthesis of the mutant, step 2: construction of a specific PolB variant with a given mutation in the desired region, and step 3: synthesis of wild-type PolB, Exo - The process is divided into three steps, including expression and purification of the PolB mutants and PolB variants. As detailed below, the techniques used in the above procedures are well known to those skilled in the art.

[0109] In step 1, a codon-optimized gene fragment encoding a wild-type, intein-free PolB polymerase was synthesized by Genomics BioSci&Tech Co. (New Taipei City, Taiwan). - The PolB polymerase (also referred to as "exo" in this specification) is also available from the same supplier. - " means that the wild-type PolB of interest has been modified to remove the inherent 3' to 5' exonuclease activity, which indicates that the PolB is a 3' to 5' exonuclease-deficient PolB. Preferably, in the embodiments of the present disclosure, Exo - refer to PolB mutants having a combination mutation at positions corresponding to D354 (D354A) of SEQ ID NO: 1, which is substituted with an alanine residue, and E356 (E356A) of SEQ ID NO: 1, which is also substituted with an alanine residue, respectively.

[0110] In step 2, the synthesized wild-type and Exo - The PolB gene is subcloned into the pET28b vector using the NdeI and NotI restriction sites, respectively. The sequence of the recombinant plasmid is confirmed by DNA sequencing. The polymerase variants are then cloned into the pET28b vector using the NdeI and NotI restriction sites, respectively. -Site-directed mutagenesis is performed to create the desired motif region of the protein scaffold. Briefly, site-directed mutagenesis PCR is performed using the Q5 Site-Directed Mutagenesis Kit from New England Biolabs (Ipswich, MA) with the recombinant plasmid to introduce amino acid substitutions. The resulting products are first analyzed on a 1% agarose gel to confirm the size of the amplicon, and the remainder of the PCR reaction mixture is treated with DpnI for 1 h at 37°C. The mixture is further incubated at 70°C for 10 min to inactivate the function of DpnI. The DpnI-treated PCR reaction mixture is then purified with Qiagen's QIAquick PCR Purification Kit (Whatman, MA). The purified DNA fragment is treated with a mixture of T4 PNK and T4 DNA ligase. The recircularized PCR-amplified DNA is again transformed into E. coli cells. Plasmid DNA was then extracted from the E. coli cells using Qiagen's Plasmid Mini Kit (Whatman, MA). Mutated sequences at one or more desired motif regions of the polymerase variants are confirmed by DNA sequencing.

[0111] In step 3, E. coli Acella cells carrying plasmid DNA with specific polymerase variant genes 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 an absorbance of 0.6-0.8 at OD600 nm, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to induce protein expression. The cells are cultured for another 4 h at 37 °C and then centrifuged at 7,000 × g for 10 min at 4 °C to harvest the cells. The cell pellet is resuspended in buffer A [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v / v) glycerol] containing 1 mM benzamidine hydrochloride. After incubation with 50 mg of lysozyme on ice for 1 h, the cells are lysed by sonication. The cell lysate is clarified by centrifugation at 18,000×g for 25 min at 4°C. The clarified crude cell extract is incubated at 70°C for 30 min and then cooled at 4°C. The heat-treated cell extract is further clarified by centrifugation at 18,000×g for 25 min 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-equilibrated with buffer A on an AeKTA Pure Chromatography System (Cytiva Life Sciences, Marlborough, MA, USA). Proteins are eluted with a linear gradient from 100 mM to 1 M NaCl using buffer B [50 mM Tris-HCl (pH 8.0), 1 M NaCl, 0.5 mM EDTA, 1 mM DTT, 5% (v / v) glycerol]. Column fractions are analyzed by 10% SDS-PAGE. Fractions containing the protein of interest are pooled and dialyzed overnight at 4°C against 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 protein of interest is concentrated using an Amicon filter unit (MW cutoff 50,000).The concentrated protein pool is aliquoted and stored at -20°C. Each mutant polymerase variant is purified using the same procedure as above. Final protein concentrations are determined by the Bradford reaction (Bradford, 1976) using a 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 for template-independent DNA synthesis approaches.To further evaluate the activity of PolB variants (the ability to incorporate naturally occurring nucleotides and nucleotide analogs), normal dNTPs or modified nucleotides, and single-stranded DNA initiators or blunt-ended double-stranded DNA initiators are used herein.

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

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

[0115] Blunt-end double-stranded DNA initiator: A double-stranded DNA consisting of a 38-mer primer (Cy5-38-mer primer) labeled with a fluorescent Cyanine 5 (Cy5) dye at the 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-end duplex DNA initiator is formed by annealing the Cy5-38-mer primer with the complementary 38-mer DNA at a molar ratio of 1:1.5 in 1x TE buffer [10 mM Tris-HCl (pH 8.0) and 1 mM EDTA] containing 100 mM NaCl. The DNA annealing reaction is carried out by first heating the sample mixture to 98°C for 3 min and then gradually cooling to 4°C (5°C / 30 s) using a Bio-Rad Thermal Cycler (Hercules, CA). The annealed product without overhangs is used as the blunt-end duplex DNA initiator.

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

[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 deoxynucleotidyl transferase (Tdt) 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 mix 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, 80°C to 90°C, respectively. Each reaction is allowed to proceed for 30 min (or a fixed time such as 5 min, 10 min, etc.) and then stopped by adding 10 μl of 2x quench solution (95% deionized formamide and 25 mM EDTA). The sample mixture is denatured at 95°C for 10 min and analyzed by 20% polyacrylamide gel electrophoresis containing 8 M urea (Urea-PAGE). The de novo enzymatic DNA synthesis reaction products are then 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 variants In this example, Tgo exo- , Kod1 exo- , 9°N exo- , P.F.U. exo- , Vent exo- , Mac exo- , Pis exo- , and Sso exo- was selected as the exonuclease-deficient enzyme constructed as described in Example 1. For simplicity, Kod1 exo- , Vent exo- , and Pfu exo-was used as an exemplary exonuclease-deficient PolB variant to demonstrate the baseline of template-independent enzymatic DNA synthesis activity. The template-independent enzymatic DNA synthesis activity of commercially available Tdt was also evaluated. The template-independent enzymatic DNA synthesis activity was evaluated in the same manner as in Example 2.

[0120] The assay results are shown in Figures 2A and 2B. In the figures, "S" indicates the use of substrate (FAM-45-mer DNA initiator) as a blank (no enzyme) control. As a result, Kod1 exo- , Vent exo- , P.F.U. exo- Both of these showed template-independent enzymatic DNA synthesis activity when the reaction temperature was increased stepwise using single-stranded DNA as an initiator (Fig. 2B), but Tdt was found to rapidly lose activity when the reaction temperature was increased to around 45°C (Fig. 2A). This result demonstrated that the exonuclease-deficient PolB variant has superior thermostability in template-independent nucleic acid synthesis to the conventional Tdt enzyme.

[0121] Example 4: Template-independent DNA synthesis activity of exonuclease-deficient PolB B variants In this example, Kod1, as described in Example 3, exo- and Pfu exo-was further used as an exemplary exonuclease-deficient PolB variant to demonstrate the catalytic efficiency of the PolB variant. The catalytic efficiency was evaluated using the same activity assay described above, and the DNA synthesis activity was monitored over a set time period (e.g., 60 min). The results are shown in Figure 3A and Figure 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. And "C" represents the reaction without the addition of dNTPs as a negative control. After 5 min of reaction, newly synthesized DNA was clearly observed, indicating that the enzymatic DNA synthesis reaction was efficient. Furthermore, it was observed that the amount of newly synthesized DNA product increased while the amount of FAM-45-mer DNA initiator decreased significantly with time, indicating that the enzymatic DNA synthesis reaction was completed within about 30 min. 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 variants in the incorporation of standard nucleotides into FAM-45-mer DNA initiators Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolBs (e.g., Tgo) were used to contain more amino acid substitutions and different amino acids in various conserved regions or motifs of each protein. exo- , Kod1 exo- , 9°N exo- , P.F.U. exo- , Vent exo- , Mac exo- , Pis exo- , Sso exo- ) was further modified.

[0123] Example 5.1: Template-independent DNA synthesis activity of Sso variants In this example, the PolB variant from Sso (SEQ ID NO: 9) is illustratively used to evaluate the template-independent DNA synthesis activity of PolB variants with combination substitutions in motifs ExoI, motif A, and motif B. Furthermore, US Patent No. US11136564B2 discloses an AAI motif to replace conserved motifs in several archaeal DNA polymerases to improve the incorporation of nucleotide analogues 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) 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). Thus, in this example, the effect of the AAI motif on template-directed nucleotide incorporation was considered, and the AAI motif substitution was also included and compared equally.

[0124] In this example, the example Sso exo-The variants modified from the (S01) backbone were numbered and listed 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 the figure, "S" represents the substrate (FAM-45-mer DNA initiator), used as a blank DNA control. As shown in Figure 4, variant S02 with amino acid substitutions in motif ExoI (D231A+E233A) and motif A (L518Y+Y519A+P520G) and variant S03 with amino acid substitutions in motif ExoI (D231A+E233A) and motif B (A601L) both exhibited significant catalytic activity for template-independent enzymatic DNA synthesis. Moreover, most of the initiator (>95%) substrates reacted, resulting in large amounts of newly synthesized DNA products. Furthermore, variant S05, which has combined amino acid substitutions in motifs ExoI (D231A+E233A), motif A (L518Y+Y519A+P520G), and motif B (A601L), showed further enhanced catalytic activity and further extended the length of the newly synthesized DNA product compared to variants S02 and S03. However, variant S04, which has combined amino acid substitutions in motifs ExoI (D231A+E233A), motif A (L518A+Y519A+P520I), and motif 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 from Vent (SEQ ID NO: 6) is illustratively used to evaluate the template-independent DNA synthesis activity of PolB variants with combination substitutions in motif ExoI, motif A, and motif B.

[0127] In this example, the Vent exo-The variants modified from the (S01) backbone are numbered and listed 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 the 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 with amino acid substitutions in motif ExoI (D141A+E143A) exerted baseline catalytic activity for template-independent enzymatic DNA synthesis at a reaction temperature of 55 °C, and variant V03 with amino acid substitutions in motif ExoI (D141A+E143A) and motif B (A488L) had improved DNA synthesis activity compared to variant V01. Furthermore, as shown in FIG. 5B, variants V02, V05 and V06 with amino acid substitutions in motif ExoI and motif A (detailed substitutions are shown in Table 5.2) also showed robust DNA synthesis activity at a high reaction temperature of 70° C. Furthermore, as shown in FIG. 5C, variants V06 and V07 with combined amino acid substitutions in motif ExoI, motif A and motif B (detailed amino acid substitutions are shown in Table 5.2) also showed significant DNA synthesis activity at a high reaction temperature of 70° C. Furthermore, variants V08, V09 and V10 with combined amino acid substitutions in motif ExoI, motif A and motif B (detailed amino acid substitutions are shown in Table 5.2) were also exemplarily used in this example to demonstrate the functional substitutions of motif ExoI, motif A and motif B as a comparison with the control enzyme (Tdt). As shown in Figure 5C, variants with combinatorial 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 9°N variants In this example, the PolB variant from 9°N (SEQ ID NO: 4) is used exemplarily for the evaluation of the template-independent DNA synthesis activity of PolB variants with combined substitutions in motif ExoI and motif A.

[0130] Similarly, the example 9°N exo- The variants modified from the (S01) backbone are numbered and listed 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 the figure, "S" represents the substrate (FAM-45-mer DNA initiator), used as a blank DNA control. As shown in Figure 6, variants N02, N03 and N04 with 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 Kod1 variants In this example, the PolB variant from Kod1 (SEQ ID NO: 3) is used as an example to evaluate the template-independent DNA synthesis activity of the PolB variant with combination substitutions in motifs ExoI, motif A, and motif B. Furthermore, as previously explained in Example 5.1, the AAI motif substitution is also functionally and positionally equivalent to the conserved motifs L715, Y716, and P717 present in motif A of the consensus sequence (SEQ ID NO: 1). Thus, this conserved motif is also 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 equally in this example for comparison.

[0133] Similarly, the example Kod1 exo-The variants modified from the (K01) backbone are numbered and listed in Table 5.4. The template-independent enzymatic DNA synthesis activity of these Kod1 variants was evaluated using the same activity assay as above. The results are shown in Figure 7A (synthesis reaction was performed at 55°C) and 7B (reaction was performed at 70°C). As shown in Figure 7A, variant K02 with amino acid substitutions in motif ExoI (D141A+E143A) and motif B (A485L) and variant K05 with amino acid substitutions in motif ExoI (D141A+E143A) and motif A (L408Y+Y409A+P410G) both exhibited template-independent DNA synthesis activity. In addition, variant K03, which has combination amino acid substitutions in motifs ExoI (D141A+E143A), motif A (L408Y+Y409A+P410G), and motif B (A485L), had improved DNA synthesis activity compared with variants K02 and K05. Variant K04, which has combination amino acid substitutions in motifs ExoI (D141A+E143A), motif A (L408A+Y409A+P410I), and motif B (A485L), had lower DNA synthesis activity compared with variants K02, K03, and K05. Furthermore, as shown in Figure 7B, comparable results were observed for these variants even at a high reaction temperature of 70 °C. In the figure, "S" represents the substrate (FAM-45-mer DNA initiator), which 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, the PolB variant from Pfu (SEQ ID NO: 5) is illustratively used to evaluate the template-independent DNA synthesis activity of PolB variants with combination substitutions in motifs ExoI, motif A, and motif B. Similarly, as described above in Example 5.1, the AAI motif substitution is also functionally and positionally equivalent to the conserved motifs L715, Y716, and P717 present in motif A of the consensus sequence (SEQ ID NO: 1). Thus, this conserved motif is also 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 equally in this example for comparison.

[0136] Similarly, the exemplified Pfu exo- The variants modified from the (S01) backbone are numbered and listed 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), exerted template-independent DNA synthesis activity. In addition, variant P03, which has combined amino acid substitutions in motif ExoI (D141A + E143A), motif A (L409Y + Y410A + P411G), and motif B (A486L), had improved DNA synthesis activity compared to variants P02 and P04. Variant P04, which has combined amino acid substitutions in motifs ExoI (D141A+E143A), motif A (L409A+Y410A+P411I; AAI motif), and motif B (A486L), had reduced DNA synthesis activity compared with 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 the amino acid substitutions as provided herein are important in conferring or improving template-independent DNA synthesis activity of PolB variants.

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

[0140] [Table 5.6] TIFF2024533804000032.tif149143

[0141] Example 6: Template-independent DNA synthesis activity of PolB variants toward blunt-ended duplex DNA initiators in the presence of standard nucleotides Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolBs (e.g., Tgo exo- , Kod1 exo- , 9°N exo- , P.F.U. exo- , Vent exo- , Mac exo- , Pis exo- , Sso exo- ) was further modified.

[0142] For simplicity, in this example, Vent exo-Only variants modified from the (V01) backbone are selected and exemplified. The template-independent DNA synthesis activity of these Vent variants to extend blunt-end duplex DNA initiators was evaluated using the same activity assay as above. Only exemplary results for representative Vent variants are shown (Figure 9). Table 6.1 shows a list of these variants and the corresponding amino acid substitutions. Figure 9 shows the gel image results for variant V04 and variants V11-V15. As shown in Figure 9, "S" represents the substrate (blunt-end duplex DNA initiator), which is used as a blank DNA control and also 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 expressed as 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, in comparison with 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% in comparison with the band intensity and position of the substrate control. Therefore, this variant is considered to have DNA synthesis activity of 50% to 100%.

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

[0148] 4) "+ / -" indicates that the initiator was converted to newly synthesized DNA of various lengths less than 10% in comparison with 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 function and activity scoring results for each selected Vent variant are shown in Table 6.2.

[0150] [Table 6.2] TIFF2024533804000035.tif139143

[0151] Example 7: Template-independent DNA synthesis activity of PolB variants toward FAM-45-mer DNA initiator in the presence of nucleotide analogues (3′-O-azidomethyl-dNTP) Based on the improved template-independent DNA synthesis properties of exonuclease-deficient PolB, selected exonuclease-deficient PolBs (e.g., Tgo exo- , Kod1 exo- , 9°N exo- , P.F.U. exo- , Vent exo- , Mac exo- , Pis exo- , Sso exo-In this example, the exemplary nucleotide analog 3′-O-azidomethyl-dNTP (3′-O-AZ-dNTP), a well-known reversible terminator nucleotide in DNA sequencing by synthetic chemistry, is used to demonstrate the ability of PolB variants to utilize non-standard nucleotides to perform template-independent nucleic acid synthesis.

[0152] Example 7.1: Template-independent DNA synthesis activity of 9°N variants For simplicity, in this example, 9° N exo- Variants modified from the (N01) backbone were selected and exemplified. The template-independent DNA synthesis activity of these 9°N variants to extend 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. In addition, Figure 10 also shows the scoring results of each 9°N variant in parallel according to the criteria for relative DNA synthesis activity scoring described in Example 6. Here, "S" stands for 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 motif ExoI and motif A, showed approximately 50% to 95% DNA synthesis activity in the presence of 3′-O-AZ-dCTP. Addition of 3′-O-AZ-dCMP to FAM-45-mer DNA produced 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-Variants modified from the (V01) backbone are selected and exemplified. The template-independent DNA synthesis activity of these Vent variants to extend 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. In addition, Figure 11 also illustrates the relative DNA synthesis activity scoring of each variant according to the criteria for relative DNA synthesis activity scoring 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 motif ExoI, motif A, and motif B, exhibited 100% DNA synthesis activity in the presence of 3′-O-AZ-dATP and generated a 46-mer DNA product.

[0157] [Table 7.2]

[0158] Considering the above results, 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, Vent variants are further selected to demonstrate their ability for template-independent DNA synthesis function. Table 7.3 shows a list of these variants. As mentioned above, Table 7.3 also shows the relative DNA synthesis activity score in the presence of 3'-O-AZ-dNTPs for each variant.

[0159] [Table 7.3] TIFF2024533804000039.tif121143

[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- Variants modified from the (S01) backbone were selected and exemplified. The template-independent DNA synthesis activity of these variants to extend 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 shows a list of the variants used in this example and their relative DNA synthesis activity.

[0161] This result indicates that Pfu exo- , Kod1 exo- and Sso exo- The variants were shown to exert 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), motif A (L408A+Y409A+P410I) and motif B (A485L), was only slightly higher than that of K03.

[0162] [Table 7.4]

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

[0164] From the above results, it was further demonstrated that the PolB variants and kits provided herein can effectively and efficiently incorporate various nucleotides for de novo enzymatic nucleic acid synthesis, and that they can reliably exert the template-independent DNA synthesis function provided thereto under a wider range of reaction temperatures, from ambient temperature to high temperature conditions, demonstrating high thermostability. Thus, the PolB variants and kits within the scope of the present disclosure can broaden the range of various applications of template-independent enzymatic nucleic acid synthesis under different reaction conditions.

[0165] Although the present disclosure has been described based on its embodiments, it will be understood that various modifications without departing from the scope of the present disclosure may also be made to the embodiments of the present disclosure. Therefore, the described embodiments are not intended to limit the present disclosure, but are also intended to include modifications within the scope of the present disclosure. Therefore, the claims should be accorded the broadest interpretation so as to encompass all such modifications.

Claims

1. A B family DNA polymerase variant 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, the amino acid L corresponding to position 408 of SEQ ID NO: 2, 3 or 4 is substituted with F, H, I, Q, S, W or Y; the amino acid L corresponding to position 409 of SEQ ID NO:5 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 411 of SEQ ID NO:6 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 485 of SEQ ID NO:7 is substituted with F, H, I, Q, S, W, or Y; the amino acid M corresponding to position 426 of SEQ ID NO:8 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 518 of SEQ ID NO:9 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 417 of SEQ ID NO: 10 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 606 of SEQ ID NO: 11 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 612 of SEQ ID NO: 12 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 425 of SEQ ID NO: 13 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 423 of SEQ ID NO: 14 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 415 of SEQ ID NO: 15 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 412 of SEQ ID NO: 16 is substituted with F, H, I, Q, S, W, or Y; A B family DNA polymerase variant in which the amino acid L corresponding to position 253 of SEQ ID NO: 17 is substituted with F, H, I, Q, S, W, or Y.

2. 2. The variant of claim 1, wherein the variant is a modified wild-type family B DNA polymerase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, and 9.

3. 2. The B family DNA polymerase variant according to claim 1, wherein the wild-type B family DNA polymerase is selected from the group consisting of Thermococcus gorgonarius DNA polymerase (Tgo), Thermococcus kodakarensis DNA polymerase (Kod1), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N), Pyrococcus furiosus DNA polymerase (Pfu), Thermococcus litoralis DNA polymerase (TfU), Thermococcus sp. (strain 9°N-7) DNA polymerase (9°N ... litoralis DNA polymerase (Vent), Methanococcus maripaludis DNA polymerase (Mma), Methanosarcina acetivorans DNA polymerase (Mac), human DNA polymerase delta catalytic p125 subunit (hPOLD), Saccharomyces cerevisiae DNA polymerase delta catalytic subunit (ScePOLD), Pyrobaculum islandicum DNA polymerase (Pis), Sulfolobus solfataricus 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. 4. The family B DNA polymerase variant of claim 3, wherein the family B DNA polymerase variant has a deficiency in 3' to 5' exonuclease activity.

5. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; i. the amino acid Y at position 409 of SEQ ID NO:2 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; ii. A B-family DNA polymerase variant, wherein the amino acid P at position 410 of SEQ ID NO:2 is unsubstituted or substituted with A, G, S, or T.

6. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus gorgonarius DNA polymerase (Tgo) having a wild-type amino acid sequence of SEQ ID NO: 2; i. the amino acid Y at position 409 of SEQ ID NO:2 is unsubstituted or substituted with A, C, D, F, G, H, I, K, L, M, N, or Q; ii. the amino acid P at position 410 of SEQ ID NO:2 is unsubstituted or substituted with A, G, S, or T; iii. A B-family DNA polymerase variant, wherein 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.

7. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; i. the 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; ii. A B-family DNA polymerase variant, wherein the amino acid P at position 410 of SEQ ID NO: 3 is unsubstituted or substituted with A, G, S, or T.

8. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus kodakarensis DNA polymerase (Kod1) having a wild-type amino acid sequence of SEQ ID NO: 3; i. the 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; ii. the amino acid P at position 410 of SEQ ID NO: 3 is unsubstituted or substituted with A, G, S, or T; iii. 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.

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

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

11. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; i. the 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; ii. A B-family DNA polymerase variant, wherein the amino acid P at position 411 of SEQ ID NO:5 is unsubstituted or substituted with A, G, S, or T.

12. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a 3' to 5' exonuclease activity deficiency is derived from Pyrococcus furiosus DNA polymerase (Pfu) having a wild-type amino acid sequence of SEQ ID NO: 5; i. the 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; ii. the amino acid P at position 411 of SEQ ID NO:5 is unsubstituted or substituted with A, G, S, or T; iii. 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.

13. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; i. the 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; ii. A B-family DNA polymerase variant, wherein the amino acid P at position 413 of SEQ ID NO: 6 is unsubstituted or substituted with A, G, S, or T.

14. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Thermococcus litoralis DNA polymerase (Vent) having a wild-type amino acid sequence of SEQ ID NO: 6; i. the 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; ii. the amino acid P at position 413 of SEQ ID NO:6 is unsubstituted or substituted with A, G, S, or T; iii. A B-family DNA polymerase variant, wherein 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.

15. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; i. the 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; ii. A B-family DNA polymerase variant, in which the amino acid P at position 487 of SEQ ID NO: 7 is unsubstituted or substituted with A, G, S, or T.

16. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Methanosarcina acetivorans DNA polymerase (Mac) having a wild-type amino acid sequence of SEQ ID NO: 7; i. the 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; ii. the amino acid P at position 487 of SEQ ID NO:7 is unsubstituted or substituted with A, G, S, or T; iii. 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.

17. 5. The family B DNA polymerase variant of claim 4, wherein the family B DNA polymerase variant having a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; i. the 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; ii. A B-family DNA polymerase variant, in which the amino acid P at position 428 of SEQ ID NO:8 is unsubstituted or substituted with A, G, S, or T.

18. 5. The family B DNA polymerase variant of claim 4, wherein the family B DNA polymerase variant having a 3' to 5' exonuclease activity deficiency is derived from Pyrobaculum islandicum DNA polymerase (Pis) having a wild-type amino acid sequence of SEQ ID NO: 8; i. the 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; ii. the amino acid P at position 428 of SEQ ID NO:8 is unsubstituted or substituted with A, G, S, or T; iii. A B-family DNA polymerase variant, wherein 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.

19. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; i. the 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; ii. A B-family DNA polymerase variant, in which the amino acid P at position 520 of SEQ ID NO:9 is unsubstituted or substituted with A, G, S, or T.

20. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant having a deficiency in 3' to 5' exonuclease activity is derived from Sulfolobus solfataricus DNA polymerase (Sso) having a wild-type amino acid sequence of SEQ ID NO: 9; i. the 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; ii. the amino acid P at position 520 of SEQ ID NO:9 is unsubstituted or substituted with A, G, S, or T; iii. 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.

21. 5. The family B DNA polymerase variant according to claim 4, wherein the family B DNA polymerase variant exhibits an activity of synthesizing nucleic acids in a template-independent manner by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an extendible initiator.

22. 22. The family B DNA polymerase variant of claim 21, wherein the extendible initiator comprises a single-stranded oligonucleotide initiator, a blunt-ended double-stranded oligonucleotide initiator, or a mixture thereof.

23. 22. The family B DNA polymerase variant of claim 21, wherein the extendible initiator is a nucleic acid in free form and the reaction is carried out in a liquid phase.

24. 22. The B-family DNA polymerase variant of claim 21 , wherein the extendible initiator is immobilized on a solid support, the solid support comprising a particle, a bead, a slide, an array surface, a membrane, a flow cell, a well, a microwell, a nanowell, a chamber, a microfluidic chamber, a channel, a microfluidic channel, or any other surface.

25. 22. The family B DNA polymerase variant of claim 21, wherein the at least one nucleotide is linked to a detectable label.

26. 22. The family B DNA polymerase variant of claim 21, wherein the family B DNA polymerase variant exhibits the activity at a reaction temperature ranging from 10°C to 100°C.

27. A kit for de novo enzymatic nucleic acid synthesis, comprising a B family DNA polymerase variant derived 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, wherein the B family DNA polymerase variant exhibits an activity to synthesize nucleic acid in a template-independent manner by adding at least one nucleotide selected from the group consisting of naturally occurring nucleotides, nucleotide analogs, or mixtures thereof to an extendible initiator, thereby synthesizing a desired nucleic acid sequence; the amino acid L corresponding to position 408 of SEQ ID NO: 2, 3 or 4 is substituted with F, H, I, Q, S, W or Y; the amino acid L corresponding to position 409 of SEQ ID NO:5 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 411 of SEQ ID NO:6 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 485 of SEQ ID NO:7 is substituted with F, H, I, Q, S, W, or Y; the amino acid M corresponding to position 426 of SEQ ID NO:8 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 518 of SEQ ID NO:9 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 417 of SEQ ID NO: 10 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 606 of SEQ ID NO: 11 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 612 of SEQ ID NO: 12 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 425 of SEQ ID NO: 13 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 423 of SEQ ID NO: 14 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 415 of SEQ ID NO: 15 is substituted with F, H, I, Q, S, W, or Y; the amino acid L corresponding to position 412 of SEQ ID NO: 16 is substituted with F, H, I, Q, S, W, or Y; A kit in which the amino acid L corresponding to position 253 of SEQ ID NO: 17 is substituted with F, H, I, Q, S, W, or Y.

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