Recombinant b7 DNA polymerase

Abstract

Provided is a recombinant B7 polymerase. The recombinant B7 polymerase has a sequence having at least 85% identity to the sequence of a wild-type B7 polymerase. The recombinant B7 polymerase comprises mutations corresponding to one or more positions of the sequence of the wild-type B7 polymerase, selected from the following: positions 169, 171, 441, 442, and 443. The recombinant B7 polymerase has DNA polymerase activity. The sequence of the wild B7 polymerase is as shown in SEQ ID NO: 1.

Description

Recombinant B7 DNA polymerase Technical Field

[0001] This application relates to the field of molecular cloning technology, specifically to a recombinant B7 DNA polymerase and its applications. Background Technology

[0002] DNA polymerases, as key mediators of DNA amplification, play a crucial role in molecular cloning and nucleic acid sequencing. Commercially used DNA polymerases are mostly modified from B-family DNA polymerases derived from archaea, such as KOD (Thermococcus kodakarensis), 9°N (Thermococcus sp. 9oN-7), Tgo (Thermococcus gorgonarius), DTok (Desulfurococcus sp. Tok), Pab (Pyrococcus abyssi), and Deep Vent (Deep Vent DNA Polymerase|Pyrococcus). These modified DNA polymerases possess the ability to polymerize modified nucleotides (i.e., modified nucleotides with reversible blocking groups at the 3' end), and can be used in next-generation sequencing (NGS) technology, such as sequencing-by-synthesis (SBS).

[0003] In related technologies, the modification of the aforementioned B-family DNA polymerases has been ongoing for over 20 years. However, due to the high sequence similarity among these enzymes, modifications to these highly similar enzymes have limited effect on improving enzyme activity.

[0004] Therefore, there is an urgent need to develop novel B-family DNA polymerases with high catalytic activity.

[0005] Summary of the Invention

[0006] The embodiments of this application provide a recombinant B7 polymerase and its application.

[0007] The first aspect of this application provides a recombinant B7 polymerase having a sequence that is at least 85% identical to that of a wild-type B7 polymerase, wherein the recombinant B7 polymerase comprises one or more mutations corresponding to the wild-type B7 polymerase sequence selected from the following sites: positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443, wherein the recombinant B7 polymerase has DNA polymerase activity, and wherein the wild-type B7 polymerase sequence is shown in SEQ ID NO: 1.

[0008] In some embodiments, the recombinant B7 polymerase contains at least two, at least three, at least four, or at least five mutations corresponding to the wild-type B7 polymerase sequence selected from the following sites: positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443.

[0009] In some embodiments, the mutation is an amino acid substitution, deletion, and / or insertion.

[0010] In some embodiments, the mutation is an amino acid substitution, wherein the amino acid substitution at the site is independently selected from the following: amino acids at positions 169, 171, 241, 245, 246, 343, and 347 are replaced with non-negatively charged amino acids selected from: A, G, V, L, I, M, F, W, Y, N, C, Q, S, T, R, H, and K; amino acid at position 441 is replaced with A, C, E, F, G, H, I, M, Q, S, T, V, Y, or W; amino acid at position 442 is replaced with A, G, S, T, or V; and amino acid at position 443 is replaced with I, V, A, C, N, T, L, M, S, or G.

[0011] In some embodiments, the recombinant B7 polymerase comprises the following mutations corresponding to the wild-type B7 polymerase sequence: a. one or more mutations selected from the following sites: positions 169, 171, 241, 245, 246, 343, and 347; and / or b. one or more mutations selected from the following sites: positions 441, 442, and 443, wherein the recombinant B7 polymerase has DNA polymerase activity.

[0012] In some embodiments, the recombinant B7 polymerase comprises mutations at positions 169 and 171 corresponding to the wild-type B7 polymerase sequence and at least one, at least two, or at least three mutations selected from positions 441, 442, and 443.

[0013] In some embodiments, the recombinant B7 polymerase comprises mutations at positions 169 and 171 corresponding to the wild-type B7 polymerase sequence and two mutations selected from positions 441, 442, and 443, wherein the amino acid substitutions at these positions are independently selected from: amino acid substitution A at position 169; amino acid substitution A at position 171; amino acid substitution A, F, S, or Q at position 441; amino acid substitution A, G, S, T, or V at position 442; and amino acid substitution A, C, G, I, S, or V at position 443.

[0014] In some embodiments, the recombinant B7 polymerase comprises mutations at positions 169, 171, 442, and 441 or 443 corresponding to the wild-type B7 polymerase sequence. In some embodiments, the recombinant B7 polymerase has the following mutations: D169A+E171A+L441A+Y442A, D169A+E171A+L441F+Y442T, D169A+E171A+L441S+Y442A, D169A+E171A+L441Q+Y442T, D169A+E171A+Y442A+P443A, D169A+E171A+Y442A+P443C. D169A+E171A+Y442A+P443G, D169A+E171A+Y442A+P443I, D169A+E171A+Y442A+P443S, D169A+E171A+Y442A+P443V, D169A+E171A+Y442G+P443V, D169A+E171A+Y442S+P443S or D169A+E171A+Y442V+P443G.

[0015] In some embodiments, the recombinant B7 polymerase comprises mutations at the following sites corresponding to the wild-type B7 polymerase sequence: positions 169, 171, 441, 442, and 443, wherein the amino acid substitutions at these sites are independently selected from the following: amino acid substitution at position 169 is A; amino acid substitution at position 171 is A; amino acid substitution at position 441 is A, C, E, F, G, H, I, M, Q, S, T, V, or Y; amino acid substitution at position 442 is A, G, S, T, or V; and amino acid substitution at position 443 is I, V, A, C, N, T, L, M, S, or G.

[0016] In some embodiments, the amino acid substitutions at the site are independently selected from the following: the 169th amino acid is substituted with A; the 171st amino acid is substituted with A; the 441st amino acid is substituted with F, G, I, M, Q, S, V or Y; the 442nd amino acid is substituted with A, G or S; and the 443rd amino acid is substituted with V, A, C, T, L, M or S.

[0017] In some embodiments, the recombinant B7 polymerase has the following mutations:

[0018] D169A+E171A+L441S+Y442A+P443V, D169A+E171A+L441M+Y442A+P443A,

[0019] D169A+E171A+L441Q+Y442A+P443A, D169A+E171A+L441F+Y442A+P443C,

[0020] D169A+E171A+L441S+Y442A+P443M、D169A+E171A+L441Y+Y442S+P443S、

[0021] D169A+E171A+L441F+Y442A+P443T、D169A+E171A+L441Y+Y442A+P443T、

[0022] D169A+E171A+L441Q+Y442G+P443S、D169A+E171A+L441Y+Y442G+P443A、

[0023] D169A+E171A+L441F+Y442S+P443T、D169A+E171A+L441S+Y442A+P443C、

[0024] D169A+E171A+L441G+Y442A+P443V、D169A+E171A+L441I+Y442G+P443S、

[0025] D169A+E171A+L441Y+Y442G+P443L、D169A+E171A+L441S+Y442T+P443A、

[0026] D169A+E171A+L441I+Y442A+P443C、D169A+E171A+L441A+Y442A+P443V、

[0027] D169A+E171A+L441M+Y442S+P443C、D169A+E171A+L441F+Y442A+P443N、

[0028] D169A+E171A+L441V+Y442A+P443V、D169A+E171A+L441V+Y442A+P443A、

[0029] D169A+E171A+L441Y+Y442S+P443V、D169A+E171A+L441Q+Y442A+P443V、

[0030] D169A+E171A+L441Y+Y442A+P443V、D169A+E171A+L441Y+Y442T+P443T、

[0031] D169A+E171A+L441A+Y442A+P443I、D169A+E171A+L441T+Y442A+P443A、

[0032] D169A+E171A+L441E+Y442A+P443A、D169A+E171A+L441F+Y442T+P443M、

[0033] D169A+E171A+L441M+Y442A+P443I、D169A+E171A+L441S+Y442S+P443S、

[0034] D169A+E171A+L441Y+Y442A+P443S、D169A+E171A+L441F+Y442S+P443L、

[0035] D169A+E171A+L441V+Y442A+P443S、D169A+E171A+L441M+Y442G+P443V、

[0036] D169A+E171A+L441Y+Y442T+P443S、D169A+E171A+L441M+Y442S+P443T、

[0037] D169A+E171A+L441Q+Y442A+P443T、D169A+E171A+L441C+Y442S+P443A、

[0038] D169A+E171A+L441F+Y442G+P443V、D169A+E171A+L441M+Y442S+P443A、

[0039] D169A+E171A+L441Y+Y442T+P443V、D169A+E171A+L441H+Y442A+P443V、

[0040] D169A+E171A+L441S+Y442S+P443T、D169A+E171A+L441F+Y442S+P443V、

[0041] D169A+E171A+L441S+Y442S+P443V、D169A+E171A+L441Q+Y442A+P443G、

[0042] D169A+E171A+L441M+Y442S+P443V, D169A+E171A+L441V+Y442T+P443C,

[0043] D169A+E171A+L441Q+Y442V+P443V or D169A+E171A+L441G+Y442S+P443L

[0044] In some embodiments, the recombinant B7 polymerase has the following mutations:

[0045] D169A+E171A+L441S+Y442A+P443V, D169A+E171A+L441M+Y442A+P443A,

[0046] D169A+E171A+L441Q+Y442A+P443A, D169A+E171A+L441F+Y442A+P443C,

[0047] D169A+E171A+L441S+Y442A+P443M, D169A+E171A+L441Y+Y442S+P443S,

[0048] D169A+E171A+L441F+Y442A+P443T, D169A+E171A+L441Y+Y442A+P443T or

[0049] D169A+E171A+L441Q+Y442G+P443S.

[0050] The second aspect of this application provides a polynucleotide that encodes a recombinant B7 polymerase or its complementary sequence as described in any one of the first aspects of this application.

[0051] A third aspect of this application provides a vector comprising a polynucleotide as described in any one of the second aspects of this application.

[0052] The fourth aspect of this application provides a cell comprising a polynucleotide as described in any one of the second aspect of this application or a vector as described in any one of the third aspect of this application, or expressing a recombinant B7 polymerase as described in any one of the first aspect of this application.

[0053] The fifth aspect of this application provides a kit comprising a recombinant B7 polymerase as described in any one of the first aspect of this application, a polynucleotide as described in any one of the second aspect of this application, a vector as described in any one of the third aspect of this application, and / or cells as described in any one of the fifth aspect of this application.

[0054] The sixth aspect of this application provides for the use of the recombinant B7 polymerase as described in any one of the first aspects of this application in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing.

[0055] The seventh aspect of this application provides for the use of the recombinant B7 polymerase as described in any one of the first aspects of this application in incorporating nucleotides and / or nucleotide analogs into molecular sequences or in preparing polymers of nucleotides and / or nucleotide analogs, wherein the nucleotide analogs are selected from one or two of the following: modified nucleotides and non-natural nucleotides.

[0056] In some embodiments, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base.

[0057] In some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0058] An eighth aspect of this application provides a method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or preparing a polymer of nucleotides and / or nucleotide analogs, comprising: contacting a template nucleic acid sequence, nucleotides and / or nucleotide analogs with a recombinant B7 polymerase as described in any one of the first aspects of this application; and, under the catalysis of the recombinant B7 polymerase, attaching the nucleotides and / or nucleotide analogs to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or prepare the polymer of nucleotides and / or nucleotide analogs.

[0059] In some embodiments, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base. In some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0060] A ninth aspect of this application provides a nucleic acid sequencing method, comprising: contacting a nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with a recombinant B7 polymerase as described in any one of the first aspects of this application; and, under the catalysis of the recombinant B7 polymerase, adding the nucleotides and / or nucleotide analogs to the 3' end of the sequencing primers that are bound to the nucleic acid to be tested, and emitting a detectable signal to achieve the nucleic acid sequencing.

[0061] In some embodiments, the nucleotide analogue comprises a reversible blocking group modification.

[0062] The technical solution of this application achieves the following technical effects:

[0063] The recombinant B-family B7 DNA polymerase proposed in this application has a stronger nucleotide polymerization ability than its wild type, especially exhibiting higher incorporation efficiency and stronger catalytic activity for modified nucleotides (e.g., nucleotides containing blocking groups). Therefore, it can be used for molecular cloning and sequencing (especially NGS) and can effectively improve sequencing efficiency and sequencing accuracy. Attached Figure Description

[0064] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0065] Figure 1 shows the sequence alignment of B7 DNA polymerase and B family DNA polymerase according to embodiments of this application;

[0066] Figure 2 shows a structural comparison of B7 DNA polymerase and KOD DNA polymerase according to embodiments of this application;

[0067] Figure 3 shows a gel electrophoresis diagram of the B7 DNA polymerase mutant according to an embodiment of this application. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] This application is based on the inventor's following understanding:

[0070] Next-generation sequencing (NGS), based on the sequence-by-synthesis (SBS) principle, uses modified nucleotides with reversible blocking groups at the 3' end as substrates. During sequencing, DNA polymerase uses one DNA strand as a template and adds this type of modified nucleotide to the 3' end of another DNA primer strand. The presence of the 3' blocking group prevents the addition of subsequent nucleotides, thus stopping the reaction at this step. At this point, an optical imaging system detects the unique fluorescent signal on each nucleotide to determine the base type of the incorporated nucleotide. Then, AT and CG pairing is used to determine the base type of the nucleotide at the corresponding position on the DNA template strand, thereby achieving DNA sequencing. After reading a nucleotide sequence, a chemical cleavage reaction removes the reversible blocking group at the 3' end of the DNA primer strand, restoring the naturally free 3' hydroxyl group, allowing the next blocking nucleotide to polymerize. Thus, the cyclical process of "polymerization-optical imaging-chemical cleavage" enables the gene sequencing process from the DNA template strand.

[0071] Natural DNA polymerases lack the ability to polymerize nucleotides with 3' blocking modifications. It is typically necessary to modify the substrate specificity of natural DNA polymerases to enable them to polymerize modified nucleotides. These modified DNA polymerases can then be used in gene sequencing technologies. Currently, DNA polymerases used in NGS are usually derived from archaea-derived B-family DNA polymerases, including, for example, KOD (Thermococcus kodakarensis), 9°N (Thermococcus sp. 9oN-7), Tgo (Thermococcus gorgonarius), DTok (Desulfurococcus sp. Tok), Pab (Pyrococcus abyssi), and Deep Vent (Deep Vent DNA Polymerase|Pyrococcus). These enzymes have high sequence homogeneity, therefore, the improvement in enzyme activity resulting from continuous modification is relatively limited.

[0072] Based on this, the inventors, through numerous experiments and tests, developed several novel DNA polymerases, namely recombinant B7 polymerases, which exhibit high sequence differences from traditional B-family polymerases (as shown in Figure 1, the sequence identity among B-family DNA polymerases is approximately 80%, while B7 differs significantly from these enzymes). The recombinant B7 polymerases proposed in this application have mutations at one or more sites compared to their wild-type (sequence shown in SEQ ID NO: 1). These recombinant B7 polymerases possess DNA polymerase activity and are capable of incorporating modified nucleotides (e.g., nucleotides modified with a 3'-O-blocking group containing a benzene ring structure) or non-natural nucleotides into the molecular sequence, exhibiting enhanced catalytic activity and incorporation rate.

[0073] In the embodiments of this application, "DNA polymerase activity" generally refers to the activity of DNA polymerase in catalyzing template-guided synthesis of polynucleotides. The enzyme activity of polymerase can be measured using various techniques and methods known in the art. For example, serial dilutions of the polymerase can be prepared in a buffer solution, the polymerase reaction mixture can be incubated at an appropriate temperature (e.g., 37°C, 74°C, etc.), and then the amount of product synthesized by the polymerase can be determined. Methods for measuring polymerase activity and other properties such as thermal stability, 3'-5" exonuclease activity, etc., are known in the art (see Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, etc.).

[0074] In this embodiment, "incorporation" refers to linking the nucleotide to the free 3' hydroxyl group of a second nucleotide by forming a phosphodiester bond with the 5' phosphate group of the nucleotide. The second nucleotide to which this nucleotide is linked typically appears at the 3' end of the polynucleotide chain, and this nucleotide can be a natural nucleotide dNTP, a modified nucleotide, or a non-natural nucleotide.

[0075] In this application, “improved / enhanced / enhanced incorporation” refers to an increase in the incorporation efficiency and / or observed incorporation rate of the recombinant B7 polymerase for at least one nucleotide, particularly a modified nucleotide, compared to the control polymerase. However, this application is not limited to an improvement in the absolute incorporation rate of modified nucleotides. As shown below, the recombinant B7 polymerase can also incorporate other types of modified nucleotides and so-called dark nucleotides or non-natural nucleotides (such as non-natural nucleotide aptamers, etc.). Therefore, “improved / enhanced / enhanced incorporation” should be interpreted accordingly to also cover any improvement in these other properties, regardless of whether the incorporation rate is increased. The “improvement” need not be constant across all cycles. Therefore, “improvement” can be the ability to incorporate the nucleotide and / or nucleic acid analog at low temperatures and / or over a wider temperature range than the control enzyme. In this document, “improvement” can be the ability to incorporate nucleotides and / or nucleic acid analogs when using lower concentrations of modified nucleotides as substrates.

[0076] In this application, "nucleotide" can refer to naturally occurring nucleotides, modified nucleotides, and xeno-nucleic acid (XNA), such as those disclosed in WO2022 / 083686A1. In some embodiments, the modified nucleotide can be a nucleotide modified with a reversible blocking group, wherein the reversible blocking group modification can occur at the 3' sugar ring hydroxyl group and / or base, and the reversible blocking group can be selected from one or more of the following: alkyl, aralkyl, alkenyl, alkynyl, allyl (e.g., 3'-O-allyl), aryl, heteroaryl, heterocyclic, benzyl, azide group, azide group (e.g., 3'-O-azidomethyl), amino, ketone, isocyanate group, phosphate ester group, carbonate group, thio, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amide group, etc., which dissociate under aqueous conditions to produce a molecule with a free 3'-OH. In this application, the xeno-nucleic acid can be a xeno-nucleic acid aptamer. In some embodiments, the substrates for the action of the B7 recombinant polymerase may also be the nucleotide types listed in WO2022083686A1, the entire contents of which are incorporated herein by reference.

[0077] In this embodiment, the nucleotides may also bear markers to facilitate their detection. Preferably, the markers are fluorescent markers. Each type of nucleotide may bear different fluorescent markers. However, the detectable markers are not necessarily fluorescent markers. Any marker that allows the detection of nucleotide incorporation in the DNA sequence can be used, and this application does not limit the specific type of marker.

[0078] In this application embodiment, recombinant B7 polymerase refers to polymerase having at least one amino acid mutation compared to wild-type B7 polymerase, where "mutation" can refer to substitution. In some cases, these mutations are conserved mutations to maintain the overall charge distribution of the protein. In this application embodiment, "conserved amino acid substitution" means that an amino acid residue is substituted by an amino acid residue having a similar side chain. Amino acid residues with similar side chains have been defined in the art, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, amino acids, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with B-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). However, this application is not limited to conserved substitutions. The recombinant B7 polymerase proposed in the embodiments of this application may also contain non-conserved substitutions. Furthermore, the mutation in the recombinant B7 polymerase proposed in the embodiments of this application can also be the deletion of the protein or the addition of one or more amino acids, as long as the recombinant B7 polymerase still has DNA polymerase activity and has improved incorporation efficiency / rate compared with the control polymerase.

[0079] In this application embodiment, the term "percentage of identity" for nucleic acid or polypeptide sequences is defined as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to a known polypeptide after arranging the sequence to obtain the maximum percentage of identity and introducing gaps (if necessary) to achieve the maximum percentage of homology. N-terminal or C-terminal insertions or deletions should not be interpreted as affecting homology. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using algorithms employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402 and Karlin (1990), Proc. Natl. Acad. Sci. USA. 87, 2264-2268), programs tailored for sequence similarity searches.

[0080] In the embodiments of this application, the recombinant B7 polymerase has a sequence (including endpoint values) that is at least 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to the wild-type B7 polymerase sequence (SEQ ID NO: 1), wherein the percentage of identity generally describes the degree to which the two sequences are the same, i.e., it generally describes the percentage of nucleotides that correspond to the same nucleotides in the reference sequence at their sequence positions. In some embodiments, the recombinant B7 polymerase has at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, or at least 99%, at least 99.1%, at least 99.2% compared to SEQ ID NO: 1. An amino acid sequence with at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99%, or any value between these values ​​(e.g., sequence identity represented by an infinite number of decimals between two adjacent integers), but less than 100% identical, wherein the recombinant B7 polymerase has one or more amino acid mutations compared to SEQ ID NO: 1.

[0081] In this embodiment, the recombinant B7 polymerase has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acid mutations compared to SEQ ID NO: 1. In some embodiments, unless otherwise specified, the remaining amino acids of the recombinant B7 polymerase are the same as those in SEQ ID NO: 1 or have conserved substitutions corresponding to the amino acids in SEQ ID NO: 1, except for the indicated mutations.

[0082] In some embodiments, the recombinant B7 polymerase comprises one or more mutations corresponding to the wild-type B7 polymerase sequence (SEQ ID NO: 1) selected from the following sites: positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443, and the recombinant B7 polymerase has DNA polymerase activity. The recombinant B7 polymerase with the above-mentioned site mutations proposed in this application exhibits improved catalytic activity compared to the wild-type B7 polymerase and demonstrates superior efficiency in incorporating modified nucleotides, thereby enabling its use in NGS and showing great application potential in improving NGS accuracy and throughput.

[0083] In some embodiments, the recombinant B7 polymerase has an amino acid mutation at position 169 of SEQ ID NO: 1. In some embodiments, the mutation at position 169 of the recombinant B7 polymerase can be a non-negatively charged amino acid.

[0084] In some embodiments, the recombinant B7 polymerase has an amino acid mutation at position 171 of SEQ ID NO: 1. In some embodiments, the mutation at position 171 of the recombinant B7 polymerase may be a non-negatively charged amino acid.

[0085] In some embodiments, the non-negatively charged recombinant B7 polymerase may also have amino acid mutations at positions 241, 245, 246, 343, and 347 of SEQ ID NO: 1, for example, mutations to non-negatively charged amino acids.

[0086] In some embodiments, the non-negatively charged amino acids include: A, G, V, L, I, M, F, W, Y, N, C, Q, S, T, R, H, and K.

[0087] In some embodiments, the recombinant B7 polymerase has an amino acid mutation at position 441 of SEQ ID NO: 1. In some embodiments, the mutation at position 441 of the recombinant B7 polymerase can be L441A, L441C, L441E, L441F, L441G, L441H, L441I, L441M, L441Q, L441S, L441T, L441V, L441Y, or L441W.

[0088] In some embodiments, the recombinant B7 polymerase has an amino acid mutation at position 442 of SEQ ID NO: 1. In some embodiments, the mutation at position 442 of the recombinant B7 polymerase can be Y442A, Y442G, Y442S, Y442T, or Y442V.

[0089] In some embodiments, the recombinant B7 polymerase has an amino acid mutation at position 443 of SEQ ID NO: 1. In some embodiments, the mutation at position 443 of the recombinant B7 polymerase can be P443I, P443V, P443A, P443C, P443N, P443T, P443L, P443M, P443S, or P443G.

[0090] The recombinant B7 polymerase proposed in this application embodiment may also have any combination of the above-mentioned mutation sites, for example, including the following mutations: a. one or more mutations selected from the following sites: positions 169, 171, 241, 245, 246, 343, and 347; and / or b. one or more mutations selected from the following sites: positions 441, 442, and 443, wherein the recombinant B7 polymerase has DNA polymerase activity. The inventors have found that these sites in a are conserved amino acid sites located in the 3'-5' exonuclease region of the polymerase, and their individual mutations or / and combined mutations (e.g., double mutations) can weaken the 3'-5' exonuclease activity of the polymerase, preventing the non-natural nucleotides polymerized to the 3' end of the primer chain from being cleaved by the exonuclease activity, thereby improving the polymerase's incorporation efficiency for non-natural amino acids / modified amino acids. Furthermore, the inventors discovered that sites 441, 442, and 443 in b are located within the conserved motif A of the 5'-3' polymerase region of the polymerase. Mutations at these three sites enable the polymerase to efficiently polymerize non-natural nucleotides. This application aims to protect each site in a and b and its various combinations, as well as the various mutation forms listed in the above examples, such as mutations at any site at positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443; mutations only in sites a and their combinations, such as two-site mutation combinations, three-site mutation combinations, four-site mutation combinations, five-site mutation combinations, six-site mutation combinations, or seven-site mutation combinations in sites a; mutations only in sites b and their combinations, such as two-site mutation combinations or three-site mutation combinations in sites b; and arbitrary combinations of one, two, three, four, five, six, or seven sites in a with one, two, or three sites in b.

[0091] In some embodiments, the recombinant B7 polymerase may contain mutations at at least two, at least three, at least four, or at least five sites selected from positions 169, 171, 441, 442, and 443, wherein the mutation at each site may optionally be a substitution as described in the above embodiments.

[0092] In some embodiments, the recombinant B7 polymerase contains mutations at positions 169 and 171 corresponding to the wild-type B7 polymerase sequence, and at least one, at least two, or at least three mutations selected from positions 441, 442, and 443, corresponding to three-mutation combinations, four-mutation combinations, and five-mutation combinations.

[0093] In some embodiments, the recombinant B7 polymerase comprises mutations at positions 169 and 171 and at positions 441, 442 or 443 (i.e., a combination of three mutations) corresponding to the wild-type B7 polymerase sequence, wherein the mutation at each position may optionally be a substitution form as described in the above embodiments.

[0094] In some embodiments, the recombinant B7 polymerase comprises mutations at positions 169 and 171 corresponding to the wild-type B7 polymerase sequence and two mutations selected from positions 441, 442, and 443 (i.e., a quadruple mutation combination), wherein the mutation at each position may optionally be a substitution as described in the above embodiments. In some specific embodiments, the amino acid substitutions at each position of the quadruple mutation combination are independently selected from the following:

[0095] The amino acid at position 169 is replaced with A;

[0096] The amino acid at position 171 is replaced with A;

[0097] The amino acid at position 441 is replaced with A, F, S, or Q;

[0098] The 442nd amino acid is replaced with A, G, S, T, or V;

[0099] The 443rd amino acid is replaced with A, C, G, I, S, or V.

[0100] In some embodiments, the four mutation combinations of the recombinant B7 polymerase can occur at positions 169, 171, 441, and 442. In some embodiments, the mutations at these four sites can be: amino acid position 169 replaced with A; amino acid position 171 replaced with A; amino acid position 441 replaced with A, F, S, or Q; and amino acid position 442 replaced with A, G, S, T, or V, preferably A or T. In some embodiments, the recombinant B7 polymerase has the following mutations:

[0101] D169A+E171A+L441A+Y442A、

[0102] D169A+E171A+L441F+Y442T、

[0103] D169A+E171A+L441S+Y442A or

[0104] D169A+E171A+L441Q+Y442T.

[0105] In some embodiments, the four mutation combinations of the recombinant B7 polymerase can occur at positions 169, 171, 442, and 443. In some embodiments, the mutations at these four sites can be: amino acid position 169 replaced with A; amino acid position 171 replaced with A; amino acid position 442 replaced with A, G, S, T, or V, preferably A, G, S, or V; and amino acid position 443 replaced with A, C, G, I, S, or V. In some embodiments, the recombinant B7 polymerase has the following mutations:

[0106] D169A+E171A+Y442A+P443A、

[0107] D169A+E171A+Y442A+P443C、

[0108] D169A+E171A+Y442A+P443G、

[0109] D169A+E171A+Y442A+P443I、

[0110] D169A+E171A+Y442A+P443S

[0111] D169A+E171A+Y442A+P443V

[0112] D169A+E171A+Y442G+P443V

[0113] D169A+E171A+Y442S+P443S or

[0114] D169A+E171A+Y442V+P443G.

[0115] In some embodiments, the recombinant B7 polymerase comprises mutations at the following sites corresponding to the wild-type B7 polymerase sequence: positions 169, 171, 441, 442, and 443 (i.e., a five-mutation combination), wherein the amino acid substitutions at each site are independently selected from the following:

[0116] The amino acid at position 169 is replaced with A;

[0117] The amino acid at position 171 is replaced with A;

[0118] The 441st amino acid is replaced with A, C, E, F, G, H, I, M, Q, S, T, V, or Y;

[0119] The 442nd amino acid is replaced with A, G, S, T, or V;

[0120] The 443rd amino acid is replaced with I, V, A, C, N, T, L, M, S, or G.

[0121] In some embodiments, the amino acid substitutions at each site of the five mutant combinations of recombinant B7 polymerase can be independently selected from the following:

[0122] The amino acid at position 169 is replaced with A;

[0123] The amino acid at position 171 is replaced with A;

[0124] The 441st amino acid is replaced with F, G, I, M, Q, S, V, or Y;

[0125] The amino acid at position 442 is replaced with A, G, or S;

[0126] The 443rd amino acid is replaced with V, A, C, T, L, M, or S.

[0127] In some specific embodiments, the five-mutation combination of recombinant B7 polymerase can have the following mutation forms:

[0128] D169A+E171A+L441S+Y442A+P443V, D169A+E171A+L441M+Y442A+P443A,

[0129] D169A+E171A+L441Q+Y442A+P443A, D169A+E171A+L441F+Y442A+P443C,

[0130] D169A+E171A+L441S+Y442A+P443M, D169A+E171A+L441Y+Y442S+P443S,

[0131] D169A+E171A+L441F+Y442A+P443T, D169A+E171A+L441Y+Y442A+P443T,

[0132] D169A+E171A+L441Q+Y442G+P443S, D169A+E171A+L441Y+Y442G+P443A,

[0133] D169A+E171A+L441F+Y442S+P443T, D169A+E171A+L441S+Y442A+P443C,

[0134] D169A+E171A+L441G+Y442A+P443V, D169A+E171A+L441I+Y442G+P443S,

[0135] D169A+E171A+L441Y+Y442G+P443L、D169A+E171A+L441S+Y442T+P443A、

[0136] D169A+E171A+L441I+Y442A+P443C、D169A+E171A+L441A+Y442A+P443V、

[0137] D169A+E171A+L441M+Y442S+P443C、D169A+E171A+L441F+Y442A+P443N、

[0138] D169A+E171A+L441V+Y442A+P443V、D169A+E171A+L441V+Y442A+P443A、

[0139] D169A+E171A+L441Y+Y442S+P443V、D169A+E171A+L441Q+Y442A+P443V、

[0140] D169A+E171A+L441Y+Y442A+P443V、D169A+E171A+L441Y+Y442T+P443T、

[0141] D169A+E171A+L441A+Y442A+P443I、D169A+E171A+L441T+Y442A+P443A、

[0142] D169A+E171A+L441E+Y442A+P443A、D169A+E171A+L441F+Y442T+P443M、

[0143] D169A+E171A+L441M+Y442A+P443I、D169A+E171A+L441S+Y442S+P443S、

[0144] D169A+E171A+L441Y+Y442A+P443S、D169A+E171A+L441F+Y442S+P443L、

[0145] D169A+E171A+L441V+Y442A+P443S、D169A+E171A+L441M+Y442G+P443V、

[0146] D169A+E171A+L441Y+Y442T+P443S, D169A+E171A+L441M+Y442S+P443T,

[0147] D169A+E171A+L441Q+Y442A+P443T, D169A+E171A+L441C+Y442S+P443A,

[0148] D169A+E171A+L441F+Y442G+P443V, D169A+E171A+L441M+Y442S+P443A,

[0149] D169A+E171A+L441Y+Y442T+P443V, D169A+E171A+L441H+Y442A+P443V,

[0150] D169A+E171A+L441S+Y442S+P443T, D169A+E171A+L441F+Y442S+P443V,

[0151] D169A+E171A+L441S+Y442S+P443V, D169A+E171A+L441Q+Y442A+P443G,

[0152] D169A+E171A+L441M+Y442S+P443V, D169A+E171A+L441V+Y442T+P443C,

[0153] D169A+E171A+L441Q+Y442V+P443V or D169A+E171A+L441G+Y442S+P443L.

[0154] The recombinant B7 polymerase proposed in this application has a stronger nucleotide polymerization ability than its wild type, especially for modified nucleotides (e.g., 3'-O-blocking modified nucleotides), exhibiting higher incorporation efficiency and stronger catalytic activity. Therefore, it can be used for molecular cloning and sequencing (especially NGS), and can effectively improve sequencing efficiency and sequencing accuracy.

[0155] It is understood that the recombinant B7 polymerase proposed in this application embodiment may also have other modifications as needed, such as surface modifications like macromolecular modifications, small molecule modifications, cross-linking modifications, and / or immobilization modifications; internal modifications like modifications targeting non-catalytic groups, catalytic groups, the main chain, cofactors, and peptide chain extension; and / or chemical modifications combined with site-directed mutagenesis. The recombinant B7 polymerase proposed in this application embodiment may also have its primary structure modified as needed, for example, optionally by attaching adapters or tags at positions such as the sequence ends, such as FLAG tags, MYC tags, polyhistidine tags, or any detectable tag. These modifications can be implemented using conventional methods in the art as needed, and they also fall within the protection scope of this application.

[0156] This application also provides a polynucleotide encoding the recombinant B7 polymerase described in any of the above embodiments, a vector containing the polynucleotide, a cell containing the polynucleotide, the vector and / or expressing the recombinant B7 polymerase, and a kit containing the recombinant B7 polymerase, the polynucleotide, the vector and / or the cell.

[0157] This application also proposes the use of the recombinant B7 polymerase described in any of the above embodiments in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing, wherein the sequencing may optionally be NGS.

[0158] This application also proposes the application of the recombinant B7 polymerase described in any of the above embodiments in incorporating nucleotides and / or nucleotide analogs into molecular sequences or in preparing nucleotide and / or nucleotide analog polymers, wherein the nucleotide analogs are selected from one or two of the following: modified nucleotides and non-natural nucleotides. In some embodiments, the modification is a reversible blocking group modification, preferably the reversible blocking group modification optionally occurring at the 3' sugar ring hydroxyl group and / or base; in some embodiments, the non-natural nucleotide is a non-natural nucleotide aptamer.

[0159] This application also proposes a method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or preparing a polymer of nucleotides and / or nucleotide analogs, comprising: contacting a template nucleic acid sequence, nucleotides and / or nucleotide analogs with a recombinant B7 polymerase as described in any of the above embodiments; and, under the catalysis of the recombinant B7 polymerase, attaching the nucleotides and / or nucleotide analogs to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or prepare the polymer of nucleotides and / or nucleotide analogs.

[0160] This application also proposes a nucleic acid sequencing method, comprising: contacting a nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with a recombinant B7 polymerase as described in any of the above embodiments; and, under the catalysis of the recombinant B7 polymerase, adding the nucleotides and / or nucleotide analogs to the 3' end of the sequencing primers that are bound to the nucleic acid to be tested, and emitting a detectable signal to achieve the nucleic acid sequencing. In some embodiments, the nucleotide analogs contain reversible blocking group modifications.

[0161] It should be noted that the above explanations and descriptions of the recombinant B7 polymerase examples are also applicable to the application of the polynucleotides, vectors, cells, kits, and recombinant B7 polymerases in the embodiments of this application, and will not be repeated here.

[0162] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0163] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0164] Example 1 - Sequence alignment between B7 DNA polymerase and B family DNA polymerases

[0165] The wild-type B7 DNA polymerase was sequence-aligned with several typical B-family DNA polymerases using the Clustal Omega online sequence alignment website (http: / / www.clustal.org / omega / ). The sequences of each enzyme used are shown in Table 1 below, and the alignment results are shown in Figure 1. As shown in Figure 1, the sequence identity between the B7 polymerase and the other enzymes was between 40.6% and 43.8%, suggesting that the wild-type B7 DNA polymerase has significant potential for modification.

[0166] Table 1

[0167] Example 2 - Structural comparison between B7 DNA polymerase and B family DNA polymerases

[0168] The wild-type sequence of B7 DNA polymerase shown in SEQ ID NO: 1 was predicted using an AlphaFold-based protein structure model. The predicted structure model of B7 DNA polymerase was then compared with that of the existing KOD DNA polymerase (PDB ID: 4k8z) using the TM-align online structure alignment website. The results showed a TM-score of 0.9464, indicating that although the sequence similarity between the two is only 43%, their three-dimensional structures are highly homologous and they are likely to have similar functional activities (Figure 2).

[0169] Figure 2 shows the structural comparison between B7 DNA polymerase and KOD DNA polymerase, with KOD DNA polymerase represented in black and B7 DNA polymerase in gray. This comparison indicates that their three-dimensional structures are highly homologous, demonstrating that B7 DNA polymerase potentially possesses the activity of B-family DNA polymerases, suggesting that B7 DNA polymerase has extremely high potential for modification and application.

[0170] Example 3 - Construction, protein expression, and purification of B7 DNA polymerase mutant B7-M

[0171] 3.1 Construction and transformation of plasmid for B7 DNA polymerase mutant B7-M

[0172] Based on the wild-type sequence of B7 DNA polymerase (SEQ ID NO: 1), a bimutation B7-M containing B7 DNA polymerase was artificially synthesized (its amino acid sequence is shown in SEQ ID NO: 2), which, compared to SEQ ID NO: 1, contains a D169A+E171A mutation. A recombinant plasmid pET-28a(+) / B7-M was constructed based on this sequence and transformed into BL21(DE3) competent cells (Tiangen Biotech (Beijing) Co., Ltd., catalog number: CB105-02). The specific transformation steps are as follows: Add 0.5 μL of 50 ng / μL pET-28a(+) / B7-M plasmid to 100 μL of BL21(DE3) competent cells, gently mix and place on ice for 5 minutes; heat shock at 42℃ for 1 minute and place on ice again for 5 minutes; add 400 μL of antibiotic-free LB liquid medium and shake at 220 rpm at 37℃ for 1 hour; take 100 μL of culture and spread it on kanamycin-resistant LB plates and incubate at 37℃ for 12-16 hours.

[0173] B7 DNA polymerase wild-type sequence SEQ ID NO: 1:

[0174] B7 DNA polymerase mutant B7-M sequence (SEQ ID NO: 2):

[0175] 3.2 Protein Expression and Purification of B7 DNA Polymerase Mutant B7-M

[0176] The affinity chromatography column and anion exchange column used for protein purification were HisTrap FF 5mL (brand: Cytiva, catalog number: 17528601) and HiTrap Q FF 5mL (brand: Cytiva, catalog number: 17515601), respectively. The specific purification steps are as follows.

[0177] 3.2.1 Pick 3-6 healthy single colonies from an LB agar plate and inoculate them into 50 mL of LB liquid medium. Incubate at 37°C for 5-7 h until the OD600 reaches 0.6-4.0. Then, inoculate 1% of the bacterial culture into 2 L of LB liquid medium and incubate at 37°C for 2-4 h until the OD600 reaches 0.8-1.0. Pre-cool the original shaker to 16°C. Add IPTG to the medium to a final concentration of 0.5 mM. Place the bacterial culture in a shaker at 16°C and induce expression at 220 rpm for 12-16 h.

[0178] 3.2.2 After induction of expression, all bacterial cultures were centrifuged at 8000g for 30 min to collect the bacterial cells. Subsequently, Ni column affinity solution was added at a ratio of 1:20 to resuspend the bacterial cells, and the cells were disrupted by sonication in an ice bath environment.

[0179] 3.2.3 Heating treatment: Preheat the water bath to 75-80℃, put the broken bacterial cells into the water bath, shake and mix well, use a thermometer to detect the internal temperature of the bacterial solution. When it reaches 75℃, start timing for 30 minutes. During this period, shake and mix 3 times every 10 minutes to ensure that it is heated evenly.

[0180] 3.2.4 The heat-treated ultrasonically disrupted liquid was centrifuged at 12000 rpm at 4℃ for 60 min. The supernatant was filtered through a 0.22 μM filter membrane and used as the sample for the purification column.

[0181] 3.2.5 The above sample was loaded into a pretreated nickel chromatography column (HisTrap FF) at a flow rate of 3 mL / min. After loading, the column was washed with Ni column affinity solution A for 20 column volumes (20 CV). Linear elution with Ni column affinity solution B (0-70%) at 10.5 CV was then performed. Eluted proteins were collected when the UV absorption peak reached 100 mAu, and collection was stopped when the UV absorption peak decreased to 200 mAu.

[0182] 3.2.6 Dilute the collected eluent 6 times with B7 diluent and load it onto the pretreated Q column (HiTrap Q FF 5mL). After loading, rinse with Q column A solution for 30 CV until the baseline is stable, at a flow rate of 5 min / min.

[0183] 3.2.7 Gradient elution (0-100% Q column B solution, 10 CV) was performed on the target protein using Q column B solution at a flow rate of 5 mL / min. The collected samples were analyzed by SDS-PAGE to determine protein purity, and the results are shown in Figure 3.

[0184] 3.2.8 The collected protein elution peak samples were dialyzed overnight at 4°C. After dialysis, the protein concentration was determined and stored in stock solution at -20°C for subsequent functional activity analysis. The enzyme stock concentration was 1 mg / mL.

[0185] The specific components of the buffer solution used in the purification process are shown below:

[0186] Ni column affinity solution A: 20 mM Tris-HCl, 300 mM NaCl, 20 mM Imidazole, 5% Glycerol, pH 8.5

[0187] Ni column affinity solution B: 20 ​​mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole, 5% Glycerol, pH 8.5

[0188] Q column-A Buffer: 20mM Tris-HCl, 100mM NaCl, 5% Glycerol, pH 8.5

[0189] Q column-B Buffer: 20mM Tris-HCl, 500mM NaCl, 5% Glycerol, pH 8.5

[0190] Diluent: 20 mM Tris-HCl, 5% Glycerol, pH 8.5

[0191] 2× Dialysis buffer: 40mM Tris-HCl, 200mM KCl, 0.2mM EDTA, 5% Glycerol, pH 8.0 @ 25℃

[0192] Stock solution: 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol, pH 8.0 @ 25℃

[0193] Example 4 - Construction, protein expression, and purification of B7 DNA polymerase mutant

[0194] 4.1 Construction of B7 DNA polymerase mutant vector

[0195] Using the B7 DNA polymerase mutant expression plasmid pET-28a(+) / B7-M from step 3.1 as a template, rapid PCR amplification was performed, and mutations were simultaneously introduced into sites 441, 442, and 443 of B7-M, resulting in a total of 65 mutants: B7-M1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18 / 19 / 20 / 21 / 22 / 23 / 24 / 25 / 26 / 27 / 28 / 29 / 30 / 31 / 32 / 33 / 34 / 35 / 36 / 37 / 38 / 39 / 40 / 41 / 42 / 43 / 44 / 45 / 46 / 47 / 48 / 49 / 50 / 51 / 52 / 53 / 54 / 55 / 56 / 57 / 58 / 59 / 60 / 61 / 62 / 63 / 64 / 65. Specific mutation information for each mutant is shown in Table 1 below.

[0196] The rapid PCR reaction system consisted of: 5 μL Pfu DNA Polymerase 10X Buffer with MgSO4, 1 μL dNTP Mix (10 mM each), 1.5 μL forward primer (10 μM), 1.5 μL reverse primer (10 μM), 50 ng template DNA, 0.5 μL Pfu DNA Polymerase (3 U / μL, Promega, catalog number: M7741), and nuclease-free water to a total volume of 50 μL. The forward and reverse primers for each mutant are shown in Table 2. The rapid PCR reaction program was set as follows: 95℃ pre-denaturation for 2 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 8 min, 16 cycles; final extension at 72℃ for 5 min, and storage at 4℃.

[0197] Table 1

[0198] Table 2

[0199] 1 μL of DpnI (20 U / μL, NEB, catalog number: R0176) was added to the PCR product and incubated at 37°C for 2 hours to degrade the template. The reaction product was then transformed into *E. coli* DH5α competent cells (Tiangen, catalog number: CB101-02). The transformation procedure was as follows: 10 μL of the reaction product was added to 100 μL of DH5α competent cells, gently swirl to mix, and incubated on ice for 30 minutes; heat-shocked at 42°C for 1 minute, then incubated on ice again for 10 minutes; 400 μL of antibiotic-free LB agar was added, and the mixture was shaken at 37°C for 220 rpm / min for 1 hour. After centrifuging the culture at 3000g for 3 minutes, a portion of the supernatant was discarded, leaving approximately 100 μL. The culture was resuspended, mixed, and plated onto kanamycin-resistant LB agar plates, and incubated at 37°C for 12-16 hours. Several single colonies were picked and cultured overnight at 37°C for small-scale plasmid DNA extraction. The plasmid was sequenced to verify that the mutation site was correctly introduced. Subsequent protein expression and purification were performed using the verified vectors shown in Table 1.

[0200] Example 5 - Test of the ability of the B7 DNA mutant to inhibit the polymerization of 3'-blocking nucleotides

[0201] Using dATP labeled with Cy5 fluorescent dye and modified with a 3'-O-azidomethyl reversible blocking group as a substrate, and DNA double strands labeled with Cy3 fluorescent dye as templates / primers (PT-1: Cy3-CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG (SEQ ID NO: 133); PT-2: Cy3-CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC (SEQ ID NO: 134), wherein PT-1 and PT-2 with Cy3 dye modified at the 5' end were mixed in equal proportions, incubated at 80℃ for 10 min, and then cooled to room temperature to obtain the templates / primers), the incorporation of modified nucleotides containing reversible blocking groups during high-throughput sequencing was simulated. The polymerization activity of B7 wild-type (B7_WT), B7-M DNA polymerase and its mutants B7-M1 to B7-M65 for the incorporation of modified single nucleotides was detected by detecting the FRET-Cy5 fluorescence signal generated when the substrate polymerizes onto the template strand due to the interaction distance between Cy3 and Cy5.

[0202] The detection method is as follows:

[0203] Prepare a 50 μL reaction mixture as follows: 5 μL 10× reaction buffer, 40 μM dT / C / GTP, 0.1 mg / mL BSA, 1 μM Cy3-DNA double-stranded template, 4 μM Cy5-modified 3'-O-AzidoMethyl-dATP, 0.5 μg B7 DNA polymerase (SEQ ID NO: 1) or B7-M DNA polymerase (SEQ ID NO: 2) or its mutants (B7-M1 to B7-M65), and bring the nuclease-free water to a final volume of 50 μL. The 10× reaction buffer is formulated with 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, pH 8.5 at 25°C. The reaction mixture was prepared on ice and transferred to a 384-well plate for fluorescence detection using a BioTek Synergy H1 (Agilent) microplate reader. The reaction temperature was set at 40℃, and fluorescence signals at 530 / 568 nm and 630 / 676 nm (FRET Cy5) were collected every 60 seconds for a total reaction time of 2 hours. The negative control was achieved by replacing the B7-M DNA polymerase with an equal volume of enzyme stock solution. After the reaction, the raw data were exported and the maximum slope, i.e., the ΔFRET-Cy5 fluorescence value per unit time (ΔRFU), was calculated to characterize the polymerase's polymerization activity for the incorporation of modified single nucleotides.

[0204] The experimental results are shown in Table 3 below. As can be seen from Table 3, no FRET Cy5 fluorescence signal was detected in the negative control group. An increase in fluorescence signal was detected in the experimental groups of 65 DNA polymerase mutants from B7-M1 to B7-M65, while no signal was detected in the wild-type B7 DNA polymerase (SEQ ID NO: 1) and B7-M (SEQ ID NO: 2). This demonstrates that through the above-mentioned mutation modification, B7 DNA polymerase has improved polymerization activity for 3'-O-blocking modified nucleotides. This indicates that by performing one or more mutations at positions 169, 171, 441, 442, and 443 of B7 DNA polymerase, the catalytic activity of B7 DNA polymerase can be effectively improved, giving it a higher incorporation efficiency / rate of modified nucleotides. Therefore, it can be used for sequencing and the preparation of polymers of non-natural nucleotides.

[0205] Table 3

[0206] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0207] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0208] All embodiments of this application can be executed individually or in combination with other embodiments, and are all considered to be within the scope of protection claimed by this disclosure.

Claims

1. A recombinant B7 polymerase having a sequence with at least 85% identity to a wild-type B7 polymerase sequence, wherein the recombinant B7 polymerase comprises one or more mutations corresponding to the wild-type B7 polymerase sequence selected from positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443, the recombinant B7 polymerase having DNA polymerase activity. The wild-type B7 polymerase sequence is shown in SEQ ID NO:

1.

2. The recombinant B7 polymerase according to claim 1, wherein the recombinant B7 polymerase comprises at least two, at least three, at least four, or at least five mutations corresponding to the wild-type B7 polymerase sequence selected from the following sites: positions 169, 171, 241, 245, 246, 343, 347, 441, 442, and 443.

3. The recombinant B7 polymerase according to claim 1 or 2, wherein the mutation is an amino acid substitution, deletion, and / or insertion.

4. The recombinant B7 polymerase according to claim 3, wherein the mutation is an amino acid substitution, and the amino acid substitution at the site is independently selected from the following: The amino acids at positions 169, 171, 241, 245, 246, 343, and 347 are replaced with non-negatively charged amino acids, wherein the non-negatively charged amino acids are selected from: A, G, V, L, I, M, F, W, Y, N, C, Q, S, T, R, H, and K. The amino acid at position 241 is replaced with A; The amino acid at position 245 is replaced with A; The 246th amino acid is replaced with A; The amino acid at position 343 is replaced with A; The 347th amino acid is replaced with A; The 441st amino acid is replaced with A, C, E, F, G, H, I, M, Q, S, T, V, Y, or W; The 442nd amino acid is replaced with A, G, S, T, or V; The 443rd amino acid is replaced with I, V, A, C, N, T, L, M, S, or G.

5. The recombinant B7 polymerase according to any one of claims 1 to 4, wherein the recombinant B7 polymerase comprises the following mutation corresponding to the wild-type B7 polymerase sequence: a. One or more mutations selected from the following sites: positions 169, 171, 241, 245, 246, 343, and 347; and / or b. Selected from one or more mutations at the following sites: positions 441, 442, and 443. The recombinant B7 polymerase has DNA polymerase activity.

6. The recombinant B7 polymerase according to any one of claims 1 to 5, wherein the recombinant B7 polymerase comprises mutations at positions 169 and 171 corresponding to the wild-type B7 polymerase sequence and at least one, at least two, or at least three mutations selected from positions 441, 442, and 443.

7. The recombinant B7 polymerase according to any one of claims 1 to 6, wherein the recombinant B7 polymerase comprises mutations corresponding to positions 169 and 171 of the wild-type B7 polymerase sequence and two mutations selected from positions 441, 442, and 443. The amino acid substitutions at the site are independently selected from the following: The amino acid at position 169 is replaced with A; The amino acid at position 171 is replaced with A; The amino acid at position 441 is replaced with A, F, S, or Q; The 442nd amino acid is replaced with A, G, S, T, or V; The 443rd amino acid is replaced with A, C, G, I, S, or V.

8. The recombinant B7 polymerase according to any one of claims 1 to 6, wherein the recombinant B7 polymerase comprises a mutation at positions 169, 171, 442, and 441 or 443 corresponding to the wild-type B7 polymerase sequence. Optionally, the recombinant B7 polymerase has the following mutations: D169A+E171A+L441A+Y442A、 D169A+E171A+L441F+Y442T、 D169A+E171A+L441S+Y442A、 D169A+E171A+L441Q+Y442T、 D169A+E171A+Y442A+P443A、 D169A+E171A+Y442A+P443C、 D169A+E171A+Y442A+P443G、 D169A+E171A+Y442A+P443I、 D169A+E171A+Y442A+P443S D169A+E171A+Y442A+P443V D169A+E171A+Y442G+P443V D169A+E171A+Y442S+P443S or D169A+E171A+Y442V+P443G.

9. The recombinant B7 polymerase according to any one of claims 1 to 7, wherein the recombinant B7 polymerase comprises a mutation at the following sites corresponding to the wild-type B7 polymerase sequence: positions 169, 171, 441, 442, and 443, wherein the amino acid substitution at said site is independently selected from the following: The amino acid at position 169 is replaced with A; The amino acid at position 171 is replaced with A; The 441st amino acid is replaced with A, C, E, F, G, H, I, M, Q, S, T, V, or Y; The 442nd amino acid is replaced with A, G, S, T, or V; The 443rd amino acid is replaced with I, V, A, C, N, T, L, M, S, or G. Preferably, the amino acid substitutions at the site are independently selected from the following: The amino acid at position 169 is replaced with A; The amino acid at position 171 is replaced with A; The 441st amino acid is replaced with F, G, I, M, Q, S, V, or Y; The amino acid at position 442 is replaced with A, G, or S; The 443rd amino acid is replaced with V, A, C, T, L, M, or S.

10. The recombinant B7 polymerase according to any one of claims 1 to 8, wherein the recombinant B7 polymerase has the following mutations: D169A+E171A+L441S+Y442A+P443V, D169A+E171A+L441M+Y442A+P443A, D169A+E171A+L441Q+Y442A+P443A, D169A+E171A+L441F+Y442A+P443C, D169A+E171A+L441S+Y442A+P443M, D169A+E171A+L441Y+Y442S+P443S, D169A+E171A+L441F+Y442A+P443T, D169A+E171A+L441Y+Y442A+P443T, D169A+E171A+L441Q+Y442G+P443S, D169A+E171A+L441Y+Y442G+P443A, D169A+E171A+L441F+Y442S+P443T, D169A+E171A+L441S+Y442A+P443C, D169A+E171A+L441G+Y442A+P443V, D169A+E171A+L441I+Y442G+P443S, D169A+E171A+L441Y+Y442G+P443L, D169A+E171A+L441S+Y442T+P443A, D169A+E171A+L441I+Y442A+P443C, D169A+E171A+L441A+Y442A+P443V, D169A+E171A+L441M+Y442S+P443C, D169A+E171A+L441F+Y442A+P443N, D169A+E171A+L441V+Y442A+P443V, D169A+E171A+L441V+Y442A+P443A, D169A+E171A+L441Y+Y442S+P443V, D169A+E171A+L441Q+Y442A+P443V, D169A+E171A+L441Y+Y442A+P443V, D169A+E171A+L441Y+Y442T+P443T, D169A+E171A+L441A+Y442A+P443I, D169A+E171A+L441T+Y442A+P443A, D169A+E171A+L441E+Y442A+P443A, D169A+E171A+L441F+Y442T+P443M,D169A+E171A+L441M+Y442A+P443I, D169A+E171A+L441S+Y442S+P443S, D169A+E171A+L441Y+Y442A+P443S, D169A+E171A+L441F+Y442S+P443L, D169A+E171A+L441V+Y442A+P443S, D169A+E171A+L441M+Y442G+P443V, D169A+E171A+L441Y+Y442T+P443S, D169A+E171A+L441M+Y442S+P443T, D169A+E171A+L441Q+Y442A+P443T, D169A+E171A+L441C+Y442S+P443A, D169A+E171A+L441F+Y442G+P443V, D169A+E171A+L441M+Y442S+P443A, D169A+E171A+L441Y+Y442T+P443V, D169A+E171A+L441H+Y442A+P443V, D169A+E171A+L441S+Y442S+P443T, D169A+E171A+L441F+Y442S+P443V, D169A+E171A+L441S+Y442S+P443V, D169A+E171A+L441Q+Y442A+P443G, D169A+E171A+L441M+Y442S+P443V, D169A+E171A+L441V+Y442T+P443C, D169A+E171A+L441Q+Y442V+P443V, or D169A+E171A+L441G+Y442S+P443L. Preferably, the recombinant B7 polymerase has the following mutations: D169A+E171A+L441S+Y442A+P443V, D169A+E171A+L441M+Y442A+P443A, D169A+E171A+L441Q+Y442A+P443A, D169A+E171A+L441F+Y442A+P443C, D169A+E171A+L441S+Y442A+P443M, D169A+E171A+L441Y+Y442S+P443S, D169A+E171A+L441F+Y442A+P443T, D169A+E171A+L441Y+Y442A+P443T or D169A+E171A+L441Q+Y442G+P443S.

11. A polynucleotide encoding a recombinant B7 polymerase or its complementary sequence as described in any one of claims 1 to 10.

12. A vector comprising the polynucleotide as described in claim 11.

13. A cell comprising the polynucleotide of claim 11 or the vector of claim 12, or expressing the recombinant B7 polymerase of any one of claims 1 to 10.

14. A kit comprising the recombinant B7 polymerase as described in any one of claims 1 to 10, the polynucleotide as described in claim 11, the vector as described in claim 12, and / or the cells as described in claim 13.

15. Use of the recombinant B7 polymerase as described in any one of claims 1 to 10 in molecular cloning, molecular quantitative analysis, molecular marker detection and / or sequencing.

16. The use of the recombinant B7 polymerase as described in any one of claims 1 to 10 in incorporating nucleotides and / or nucleotide analogs into molecular sequences or in preparing nucleotide and / or nucleotide analog polymers.

17. The application according to claim 16, wherein the nucleotide analogue is selected from one or two of the following: modified nucleotides and non-natural nucleotides. Optionally, the modification is a reversible blocking group modification, which may optionally occur at the 3' sugar ring hydroxyl group and / or base. Optionally, the non-natural nucleotide is a non-natural nucleotide aptamer.

18. A method for incorporating nucleotides and / or nucleotide analogs into a nucleic acid sequence or for preparing polymers of nucleotides and / or nucleotide analogs, comprising: Contact the template nucleic acid sequence, nucleotide and / or nucleotide analog with the recombinant B7 polymerase as described in any one of claims 1 to 10; and Catalyzed by the recombinant B7 polymerase, the nucleotides and / or nucleotide analogs are attached to the complementary strand of the template nucleic acid sequence to incorporate the nucleotides and / or nucleotide analogs into the nucleic acid sequence or to prepare the polymer of the nucleotides and / or nucleotide analogs.

19. The method of claim 18, wherein the modification is a reversible blocking group modification, the reversible blocking group modification optionally occurring at the 3' sugar ring hydroxyl group and / or base. Optionally, the non-natural nucleotide is a non-natural nucleotide aptamer.

20. A nucleic acid sequencing method, comprising: Contact the nucleic acid to be tested, sequencing primers, nucleotides and / or nucleotide analogs with the recombinant B7 polymerase as described in any one of claims 1 to 10; and Catalyzed by the recombinant B7 polymerase, the nucleotide and / or nucleotide analogue are added to the 3' end of the sequencing primer that binds to the nucleic acid to be tested, and a detectable signal is emitted to achieve the nucleic acid sequencing.

21. The method of claim 20, wherein the nucleotide analog comprises a reversible blocking group modification.

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