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WO2025118178A1 - Mutant d'adn polymérase et son utilisation - Google Patents

Mutant d'adn polymérase et son utilisation Download PDF

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Publication number
WO2025118178A1
WO2025118178A1 PCT/CN2023/136740 CN2023136740W WO2025118178A1 WO 2025118178 A1 WO2025118178 A1 WO 2025118178A1 CN 2023136740 W CN2023136740 W CN 2023136740W WO 2025118178 A1 WO2025118178 A1 WO 2025118178A1
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mutated
dna polymerase
amino acid
mutant
mutation
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杨梦�
杨理想
张慧君
王艺
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present application relates to the field of biotechnology, and in particular, to DNA polymerase mutants and applications thereof.
  • DNA polymerase is widely used in PCR technology and plays an important role in life science research and related fields.
  • One of its main functions is to use single-stranded DNA as a template and deoxynucleotides (dNTPs) as substrates to efficiently and accurately synthesize a DNA sequence complementary to the template from the 5' end to the 3' end.
  • dNTPs deoxynucleotides
  • DNA polymerases can be divided into seven categories based on family classification, including A, B, C, D, X, Y and RT. There are some differences in the structure and function of DNA polymerases in different families. Among them, DNA polymerases in the B family usually have higher replication and elongation, which means that they can continuously polymerize a large number of nucleotides before dissociating from the DNA template.
  • the DNA polymerases of the B family also have DNA repair functions, including 3' ⁇ 5' exonuclease activity, which can detect mismatched bases during PCR amplification and remove them, and then reinsert the correct bases and continue DNA replication.
  • KOD is a representative of the B family DNA polymerase, derived from the thermophilic archaeon Thermococcuskodakarensis. Due to its high temperature resistance, KOD DNA polymerase has been widely used in PCR amplification, gene sequencing and other fields in recent years. However, the existing KOD DNA polymerase has a low polymerization ability in sequencing, which greatly limits the efficiency of sequencing.
  • the inventors obtained a new DNA polymerase mutant by enzyme engineering modification of the active sites related to the thermostable B family polymerase of thermophilic archaea, and improved the polymerization ability of KOD DNA polymerase in sequencing, thereby improving the sequencing speed and sequencing quality of the sequencing by synthesis (SBS) method.
  • a DNA polymerase mutant which, compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 24 positions and functionally equivalent positions: position 141, position 143, position 147, position 382, position 383, position 384, position 389, position 485, position 584, position 589, position 390, position 491, position 592, position 603, position 604, position 605, position 606, position 607, position 610, position 611, position 612, position 613, position 614, position 615 97, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; the amino acid sequence of the mutant other than the amino acid mutation site has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 23 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589, position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; the amino acid sequence of the mutant other than the amino acid mutation site has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the amino acid sequence of SEQ ID The amino acid sequence shown in NO:2.
  • amino acid positions in the amino acid sequence of the DNA polymerase mutant described in the present application are referenced to the amino acid positions in SEQ ID NO: 2.
  • the DNA polymerase mutant has a 408th (point) mutation, it means that the 408th amino acid in SEQ ID NO: 2 is mutated.
  • the "functionally equivalent sites" used in the present invention include sites where, under specific circumstances, amino acids or nucleotides mutate but the function or properties of the DNA polymerase do not change.
  • identity includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • amino acid sequence other than the amino acid mutation site of the mutant has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase" described in the present application refers to the amino acid sequence in the wild-type KOD DNA polymerase corresponding to the amino acid sequence other than the amino acid mutation site on the mutant.
  • the above-mentioned DNA polymerase mutant may further include at least one of the following technical features:
  • the amino acid sequence of the mutant other than the amino acid mutation site is the same as the corresponding amino acid sequence of the wild-type KOD DNA polymerase.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 9 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589.
  • the relative activity of the sequencing enzymes produced by different mutation sites is different, which can provide more options for actual production needs.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one of the following two positions and functionally equivalent positions: position 147, position 584.
  • the inventors first discovered after a large number of screenings that mutations to amino acids at positions 147 and 584 can increase the activity of DNA polymerase.
  • the mutation type of the mutation site is:
  • the inventors found that a DNA polymerase having one of the above mutation types (or any combination) has higher enzyme activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 amino acid mutation at the position or functionally equivalent position; and amino acid mutation at at least one position selected from the following 15 positions and functionally equivalent positions: position 382, position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, and position 725.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at at least one of the following 14 positions and functionally equivalent positions: 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725.
  • a polymerase mutant (SEQ ID NO: 1) having an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or a functionally equivalent position has a higher enzyme activity.
  • the inventors found that based on the amino acid sequence shown in SEQ ID NO: 1, mutations at one or more positions selected from position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711 and 725 can obtain mutants with different enzyme activities.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at position 408, position 409, position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589 or functionally equivalent positions; and amino acid mutations at at least one of the following 4 positions and functionally equivalent positions: position 397, position 424, position 481, position 553.
  • mutations at one or more positions selected from positions 397, 424, 481 and 553 can obtain mutants with higher enzyme activity.
  • the mutation types of the mutation sites are: (1) D at position 141 mutates to A, (2) E at position 143 mutates to A, (3) H at position 147 mutates to E, (4) S at position 383 mutates to T, (5) Y at position 384 mutates to F, (6) V at position 389 mutates to I, (7) A at position 485 mutates to E, (8) K at position 584 mutates to E, (9) V at position 589 mutated to H, (10) L at position 408 mutated to I, (11) Y at position 409 mutated to A, (12) W at position 397 mutated to Y or H, (13) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S, (14) D at position 432 mutated to R, E, H or M, (15) F mutates to R, (16) M at position 523 mutates to R
  • the mutation types of the mutation sites are: (1) D at position 141 mutates to A, (2) E at position 143 mutates to A, (3) H at position 147 mutates to E, (4) S at position 383 mutates to T, (5) Y at position 384 mutates to F, (6) V at position 389 mutates to I, (7) A at position 485 mutates to E, (8) K at position 584 mutates to E, (9) V at position 589 mutates to H, (10) L at position 408 mutates to I, (11) Y at position 409 mutates to A, (12) W at position 397 mutates to Y or H, (13) 3) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S (14) D at position 432 mutated to R, E, H or M, (15) F at position 445 mutated to R, (16) M at position 523 mutates
  • DNA polymerase mutants with the above mutation types still have different degrees of enzyme activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at one of the following 15 positions and functionally equivalent positions: positions 382, Position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; provided that the Q at position 382 cannot mutate to R, H, L and Y; or the N at position 424 cannot mutate to R and L; or the D at position 432 cannot mutate to M; or the H at position 663 cannot mutate to T, V, P and E.
  • Q at position 382 cannot mutate to R, H, L and Y means that Q at position 382 cannot mutate to R, or Q at position 382 cannot mutate to H, or Q at position 382 cannot mutate to L, or Q at position 382 cannot mutate to Y.
  • N at position 424 cannot mutate to R and L means that N at position 424 cannot mutate to R, or N at position 424 cannot mutate to L.
  • this expression method is also applicable to any embodiment. It will not be repeated in the following embodiments or implementation modes.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and one position selected from the following 14 positions and functionally equivalent positions Amino acid mutations: 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; provided that the N at position 424 cannot mutate to R and L; or the D at position 432 cannot mutate to M; or the H at position 663 cannot mutate to T, V, P and E.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at two positions selected from the following 14 positions and functionally equivalent positions.
  • Acid mutations position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; provided that, when the H at position 663 mutates to R, the A at position 553 cannot mutate to P or E; or when the H at position 663 mutates to Q, the Q at position 461 cannot mutate to Y.
  • the enzyme activity when the H at position 663 of the mutant mutates to R, and the A at position 553 mutates to P or E; or, when the H at position 663 mutates to Q, and the Q at position 461 mutates to Y, the enzyme activity is low.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has mutations (1)-(11), and any one mutation selected from (12)-(25).
  • amino acid sequence of a DNA polymerase mutant having mutations (1)-(11) is shown as SEQ ID NO:1.
  • a polymerase mutant with the amino acid sequence shown in SEQ ID NO:1 is used as a control group for polymerization activity detection.
  • a DNA polymerase mutant having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 has higher enzymatic activity. In some preferred examples, it has at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity, and in some most preferred examples, it has at least 100% identity.
  • the DNA polymerase mutant has (1)-(11) mutation; and any one mutation selected from the following mutations: W at position 397 mutates to H, N at position 424 mutates to Q, A at position 553 mutates to R, A at position 553 mutates to N, A at position 553 mutates to E, A at position 553 mutates to G, A at position 553 mutates to H, A at position 553 mutates to P, A at position 553 mutates to S, Q at position 461 mutates to W, Q at position 461 mutates to Y, Y at position 481 mutates to M, Y at position 481 mutates to F, G at position 711 mutates to R, and G at position 711 mutates to S.
  • the inventors have experimentally verified that further amino acid single-point mutation based on the amino acid sequence shown in SEQ ID NO:1 can further increase the enzyme polymerization activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has mutations (1)-(11); and any one mutation combination selected from the following mutation combinations:
  • some mutants may have base bias in sequencing applications and can be adaptively selected based on actual experimental needs.
  • the DNA polymerase mutant has mutations (1)-(11), and any one of the following mutation combinations: Y at position 481 mutates to F, and A at position 553 mutates to P; Y at position 481 mutates to F, and A at position 553 mutates to S; W at position 397 mutates to H, and A at position 553 mutates to P; Q at position 461 mutates to Y, and A at position 553 mutates to P; N at position 424 mutates to Q, and A at position 553 mutates to P; W at position 397 mutates to H, and Y at position 481 mutates to F; A at position 553 mutates to P, and G at position 711 mutates to R; W at position 397 mutates to H, and A at position 553 mutates to P; A mutated to S; Y mutated to F, and G mutated to
  • the inventors have experimentally verified that double-site amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 1 can further increase the enzyme polymerization activity.
  • the present application provides a nucleic acid molecule, which encodes the DNA polymerase mutant described in the first aspect of the present application.
  • the DNA polymerase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.
  • nucleic acids mentioned in the specification and claims of the present invention those skilled in the art should understand that they actually include any one or both of the complementary double strands.
  • the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the present application proposes an expression vector, which includes or carries the nucleic acid molecule described in the second aspect of the present application.
  • the type of expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in the host cell.
  • the expression vector may further include a promoter, and the promoter is operably connected to the nucleic acid molecule.
  • the expression vector is a non-pathogenic viral vector
  • the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.
  • the expression vector is a non-viral vector
  • the non-viral vector includes but is not limited to a plasmid vector.
  • the present application provides a recombinant cell, the recombinant cell carrying the nucleic acid molecule described in the second aspect of the present application and the expression vector described in the third aspect of the present application.
  • the recombinant cell is used to express or secrete the DNA polymerase mutant described in the first aspect of the present application.
  • the recombinant cell is selected from Escherichia coli, yeast and mammalian cells.
  • the recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express the above DNA polymerase mutant under appropriate conditions.
  • the present application provides a recombinant strain, wherein the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the present application.
  • the DNA polymerase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.
  • the present application proposes a method for obtaining a DNA polymerase mutant, the method comprising: The recombinant cell described in the first aspect or the recombinant strain described in the fifth aspect is cultured under conditions suitable for protein expression to obtain the DNA polymerase mutant.
  • the present application proposes a complex, which includes the DNA polymerase mutant described in the first aspect of the present application and a small molecule compound or a macromolecule, and the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled by a chemical bond.
  • the small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody, etc.
  • DNA polymerase mutants and complexes proposed in this application include but are not limited to nucleic acid synthesis and nucleic acid sequencing, etc. They can also be used to screen drugs against viruses or cell division, and have great development prospects in forensic medicine and criminology.
  • the present application proposes a method for nucleic acid synthesis, the method comprising: subjecting a nucleic acid template, an amplification primer, dNTPs and a mixed product of the DNA polymerase mutant described in the first aspect of the present application to an amplification treatment under conditions suitable for nucleic acid amplification, so as to obtain the nucleic acid.
  • the aforementioned nucleic acid synthesis method can be used to efficiently and quickly amplify the nucleic acid template.
  • DNA polymerase mutant described in the present application has polymerization activity for all dNTPs (including dNTPs with or without fluorescent labels).
  • the present application proposes a method for nucleic acid sequencing, the method comprising: subjecting a nucleic acid template to be tested and a mixed product of the DNA polymerase mutant and modified dNTP described in the first aspect of the present application to an amplification treatment and a fluorescent signal detection treatment under conditions suitable for nucleic acid amplification; and determining the nucleic acid sequence of the nucleic acid to be tested based on the fluorescent signal obtained by detection.
  • the nucleic acid sequencing method includes mixing the nucleic acid template to be tested with the DNA polymerase mutant and the non-natural dNTP with a fluorescently labeled 3'O-reversible terminator, the DNA polymerase mutant is responsible for matching the non-natural dNTP with the fluorescently labeled 3'O-reversible terminator with the nucleic acid template, and finally detecting multiple fluorescent labeling signals, and obtaining the nucleic acid sequence based on the obtained fluorescent labeling signals to obtain the nucleic acid sequence of the nucleic acid to be tested.
  • the above polymerization reaction and fluorescent labeling reaction can be performed for multiple cycles according to the length of the sequencing template.
  • the present application proposes a nucleic acid sequencing kit, which comprises the DNA polymerase mutant described in the first aspect or the complex described in the seventh aspect.
  • the kit described in the present application is used for efficient, accurate and rapid nucleic acid sequencing.
  • the present application proposes a use of the nucleic acid sequencing kit of the tenth aspect in sequencing.
  • the kit can be used for sequencing, including but not limited to sequencing by synthesis (SBS).
  • the present application proposes a use of the DNA polymerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the recombinant strain described in the fifth aspect, or the complex described in the seventh aspect in the preparation of products related to catalytic DNA amplification or nucleic acid sequencing.
  • the DNA polymerase mutant, nucleic acid molecule, expression vector, recombinant cell, recombinant strain or complex can be prepared alone or in combination for catalytic DNA amplification or nucleic acid sequencing related products.
  • Figure 1 is a schematic diagram of the recombinant KOD DNA polymerase expression plasmid pET22b-KOD plasmid map described in one embodiment of the present application;
  • Figure 2 is a schematic diagram of the expression of wild-type and some mutant KOD DNA polymerases and bacterial lysis results described in an embodiment of the present application;
  • Figure 3 is a schematic diagram of the results of detecting the activity of wild-type and some mutant KOD DNA polymerases using the FRET method described in an embodiment of the present application.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as “first” and “second” may explicitly or implicitly include up to In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • amino acid is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).
  • the term “identity” has the conventional meaning in the art and refers to the "homology” between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared after the best alignment, and the differences between the two sequences are randomly distributed over their entire length.
  • the mutants are described according to their mutations at specific residues, and their positions are located with reference to the positions of amino acids in the amino acid sequence shown in SEQ ID NO:2 of the wild-type KOD polymerase.
  • the KOD DNA polymerase mutant (SEQ ID NO: 1) was obtained by mutation of the wild-type KOD DNA polymerase (SEQ ID NO: 2).
  • the mutation sites include: D at position 141 mutated to A, E at position 143 mutated to A, H at position 147 mutated to E, S at position 383 mutated to T, Y at position 384 mutated to F, V at position 389 mutated to I, L at position 408 mutated to I, Y at position 409 mutated to A, A at position 485 mutated to E, K at position 584 mutated to E, and V at position 589 mutated to H.
  • the term "relative polymerization activity" refers to the polymerization activity of each experimental group relative to the reference group calculated when the polymerization activity of the reference group is assumed to be 100%.
  • the reference group DNA polymerase referred to in this application is DP01, and its amino acid sequence is shown in SEQ ID NO: 1.
  • transformation refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of foreign DNA into cells.
  • amino acid sequences are displayed from 5' end to 3' end.
  • non-natural dNTPs or dNTPs with modifications have the same meaning, including but not limited to dNTPs with labels (e.g., fluorescent labels) and/or dNTPs with O-reversible terminators at the 3' end.
  • polymerase mutants used in the examples of the present application are all polymerase mutant fusion proteins with 6 His tags connected to the C-terminus. As known to those skilled in the art, they can all be replaced by untagged polymerase mutants.
  • thermostable B-family polymerases of thermophilic archaea perform well in incorporating natural nucleotides or their analogs in high-throughput sequencing.
  • thermostable B-family polymerases include KOD (Thermococcuskodakaraensis), 9°N (Thermococcussp.9°N), TGO (Thermococcusgorgonarius), TOK (Desulfurococcussp.Tok), VentDNA polymerase (Thermococcuslitoralis), JDF-3, and pfuDNA polymerase (Pyrococcusfuriosis).
  • KOD Thermococcuskodakaraensis
  • 9°N Thermococcussp.9°N
  • TGO Thermococcusgorgonarius
  • TOK Desulfurococcussp.Tok
  • VentDNA polymerase Thermococcuslitoralis
  • the inventors protected the functional domain sites of the wild-type polymerase to ensure that it can still perform its original basic functions. Through dynamic simulation and statistical inference of the palm, finger and thumb regions of KOD DNA polymerase, mutation sites that can be used for experimental screening were obtained. And through a large number of experimental verifications, DNA polymerases suitable for attachment to DNBs (DNA nanoballs) on the surface of the chip were finally obtained.
  • amino acid sequence of KOD DNA polymerase (DP01) is shown in SEQ ID NO:1.
  • the recombinant expression vector pET22b-WT is a vector in which the KOD DNA polymerase encoding gene fused with a His tag is recombined into the vector pET22b according to the instructions of the Seamless Cloning Kit (manufacturer: Novozymes; catalog number: C112-01).
  • the resulting vector is a KOD DNA polymerase encoding gene fused with a His tag ( Figure 1), and expression is induced by IPTG.
  • the nucleotide sequence of the KOD DNA polymerase encoding gene fused with a His tag is a sequence obtained by connecting six His tag codons to the 3' end of SEQ ID NO: 3;
  • the amino acid sequence of KOD DNA polymerase fusion protein is obtained by connecting 6 His tags to the C-terminus of the amino acid shown in SEQ ID NO:1.
  • the recombinant expression vector pET22b-WT was introduced into Escherichia coli BL21 competent cells (Beijing Solebold Technology Co., Ltd.), and the positive colonies were screened by smearing resistance plates (containing 50 ⁇ g/ml of ampicillin). 3-5 positive monoclonal colonies were selected, and the positive colonies were identified by bacterial liquid PCR using primers SQF (Table 1) and primers SQR (Table 1). The fragment of 2500 bp in size that was basically consistent with the expected theoretical value was obtained as a positive clone, and the positive clone was named BL21/pET22b-WT.
  • Lane 1 is protein Marker (Page Ruler Prestained Protein Ladder, 26616, Thermo Fisher Scientific)
  • lane 12 is 5 ⁇ l KOD DNA polymerase fusion protein (DP01) + 5 ⁇ l 2X loading buffer
  • lanes 2-11 are 5 ⁇ l KOD DNA polymerase mutant fusion protein (DP001, DP002, DP004, DP005, DP007, DP008, DP009, DP024, DP011, DP012) + 5 ⁇ l 2X loading buffer; it can be seen that the protein size in lanes 2-12 is about 91.5kDa, which is consistent with the molecular weight reported in the literature.
  • the target protein of about 91.5 kDa was not obtained from the uninduced BL21/pET22b-WT bacterial solution.
  • the empty vector pET22b was introduced into E. coli BL21 to obtain BL21/pET22b.
  • the target protein of about 91.5 kDa was not obtained by expression and lysis using the above method.
  • the KOD DNA polymerase mutant fusion protein is a protein obtained by performing amino acid mutation on at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or all fourteen amino acids in the 14 positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711 and 725 of the amino acid sequence shown in DP01 (SEQ ID NO: 1); if only one amino acid is mutated, a single-point mutant of KOD DNA polymerase is obtained; if two amino acid mutations are obtained, a two-point combination mutation of KOD DNA polymerase is obtained, and so on.
  • the KOD DNA polymerase mutant encoding gene is a nucleic acid obtained by mutating the nucleotide sequence of the KOD DNA polymerase mutant DP01 encoding gene (SEQ ID NO:3) according to the corresponding amino acid sequence (SEQ ID NO:1) of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or all fourteen amino acid codons at positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725.
  • the N at position 424 is mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S;
  • the D at position 432 mutated to R, E, H, or M;
  • the A at position 553 is mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I or C;
  • the T at position 605 mutated to I or V;
  • H at position 663 is mutated to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E, or K;
  • the H at position 725 was mutated to R.
  • amino acids at two positions selected from the 14 positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 in the amino acid sequence of the KOD DNA polymerase SEQ ID NO: 1 are mutated, and the other amino acid sequences remain unchanged to obtain a protein having DNA polymerase activity:
  • the amino acid sequence of DP80 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP81 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to S;
  • the amino acid sequence of DP82 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 553rd A is mutated to P;
  • the amino acid sequence of DP83 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the A at position 553 is mutated to P;
  • the amino acid sequence of DP84 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to G;
  • the amino acid sequence of DP85 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 553rd A is mutated to P;
  • the amino acid sequence of DP86 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 481st Y is mutated to F;
  • the amino acid sequence of DP87 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 711th position G is mutated to R;
  • the amino acid sequence of DP88 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 553rd A is mutated to S;
  • the amino acid sequence of DP89 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to P;
  • the amino acid sequence of DP90 is that the Q at position 461 of SEQ ID NO: 1 is mutated to W, and the A at position 553 is mutated to P;
  • the amino acid sequence of DP91 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 711th position G is mutated to S;
  • the amino acid sequence of DP92 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the A at position 553 is mutated to S;
  • the amino acid sequence of DP93 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 561st position M is mutated to W;
  • the amino acid sequence of DP94 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 561st position M is mutated to R;
  • the amino acid sequence of DP96 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 711th G is mutated to R;
  • the amino acid sequence of DP97 is that the 461st Q in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP98 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 553rd A is mutated to S;
  • the amino acid sequence of DP99 is that the 432nd position D of SEQ ID NO:1 is mutated to H, and the 553rd position A is mutated to P;
  • the amino acid sequence of DP100 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 605th position T is mutated to V;
  • the amino acid sequence of DP101 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 561st M is mutated to W;
  • the amino acid sequence of DP102 is that the 424th N of SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP103 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 605th T is mutated to I;
  • the amino acid sequence of DP104 is that the 461st Q of SEQ ID NO:1 is mutated to F, and the 481st Y is mutated to F;
  • the amino acid sequence of DP105 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to R;
  • the amino acid sequence of DP106 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to S;
  • the amino acid sequence of DP107 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 564th position L is mutated to M;
  • the amino acid sequence of DP108 is that the 432nd position D of SEQ ID NO:1 is mutated to E, and the 553rd position A is mutated to P;
  • the amino acid sequence of DP109 is that the 461st Q of SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to S;
  • the amino acid sequence of DP110 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to G;
  • the amino acid sequence of DP111 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 564th L is mutated to M;
  • the amino acid sequence of DP112 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to N;
  • the amino acid sequence of DP113 is that the 432nd position D of SEQ ID NO:1 is mutated to H, and the 553rd position A is mutated to S;
  • the amino acid sequence of DP114 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to F;
  • the amino acid sequence of DP115 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 564th position L is mutated to R;
  • the amino acid sequence of DP116 is that the 424th N of SEQ ID NO:1 is mutated to S, and the 553rd A is mutated to P;
  • the amino acid sequence of DP117 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to F;
  • the amino acid sequence of DP118 is that the 553rd position A of SEQ ID NO:1 is mutated to E, and the 663rd position H is mutated to R;
  • the amino acid sequence of DP119 is that the 481st Y in SEQ ID NO:1 is mutated to L, and the 711th G is mutated to R;
  • the amino acid sequence of DP120 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 725th H is mutated to R;
  • the amino acid sequence of DP121 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 663rd H is mutated to R;
  • the amino acid sequence of DP122 is that the 461st Q in SEQ ID NO:1 is mutated to W, and the 561st M is mutated to R;
  • the amino acid sequence of DP123 is that the D at position 432 of SEQ ID NO: 1 is mutated to R, and the A at position 553 is mutated to S;
  • the amino acid sequence of DP124 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 663rd H is mutated to Y;
  • the amino acid sequence of DP126 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the H at position 663 is mutated to Q;
  • the amino acid sequence of DP127 is that the 461st Q of SEQ ID NO:1 is mutated to I, and the 481st Y is mutated to M;
  • the amino acid sequence of DP128 is that the 553rd position A of SEQ ID NO:1 is mutated to Y, and the 605th position T is mutated to V;
  • the amino acid sequence of DP130 is that the 424th N of SEQ ID NO:1 is mutated to K, and the 663rd H is mutated to L;
  • the amino acid sequence of DP131 is that the 461st Q in SEQ ID NO:1 is mutated to F, and the 481st Y is mutated to R.
  • the recombinant vectors expressing different KOD DNA polymerase point mutants are taken as templates using the KOD DNA polymerase vector pET22b-WT and the operating steps in the instruction manual of the Mut Express II Fast Mutagenesis Kit V2 (manufacturer: Novazonics; item number: C214-02) to perform at least one site-directed mutagenesis to obtain each mutant, and the protein encoding genes of different KOD DNA polymerase point mutants fused with the His tag are recombined into the vector pET22b.
  • the obtained vectors, the protein encoding genes of different point mutants fused with the His tag are expressed by inducing IPTG.
  • each KOD DNA polymerase point mutant fusion protein is a point mutation based on SEQ ID NO: 1 and 6 His tags are connected to its C-terminus.
  • His tags are connected to its C-terminus.
  • a tag can be added to the N-terminus or C-terminus of the amino acid sequence to facilitate purification.
  • the tag is generally a sequence shown in Table 3; adding or not adding a tag has no effect on the performance of the enzyme.
  • the construction steps are the same as those in Example 1.
  • the recombinant vector expressing different KOD DNA polymerase point mutants prepared in step 3 is introduced into BL21 to obtain recombinant bacteria expressing different KOD DNA polymerase mutant fusion proteins.
  • the preparation steps are the same as those in Example 1, and the recombinant bacteria expressing different KOD DNA polymerase point mutant fusion proteins prepared in step 4 are expressed and lysed to obtain different KOD DNA polymerase point mutant fusion protein crude extracts.
  • the point mutant fusion proteins in this example were expressed and cleaved according to the method in Example 1.
  • dATP dATP-AF532
  • dTTP dTTP-AF532
  • DNA template template DNA-AF647 labeled with AF532 fluorescent dye
  • the single-stranded primers S1A (Table 1) and S2A (Table 1) (synthesized by Sangon Biotech Co., Ltd.) with 5'AF647 fluorescent label were mixed at 1:1 equimolar concentration, annealed at 80°C for 10 min, and the annealed product was stored at -20°C in the dark to obtain the template DNA-AF647 labeled with AF647 fluorescent dye.
  • Enzyme activity was detected using a TECAN microplate reader and the reaction was carried out in 96 plates (Corning black, clear bottom 96 plates). The total volume of the solution was 50 ⁇ l.
  • the reaction system was: 20 ⁇ l crude extract of KOD polymerase mutant fusion protein, 0.25 ⁇ M dATP-AF532, 0.25 ⁇ M dTTP, 0.25 ⁇ M dCTP, 0.25 ⁇ M dGTP, 0.1 ⁇ M template DNA-AF647 for the experiment, the enzyme reaction buffer was 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO4, 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton, pH 8.8; the reaction temperature was 40°C.
  • the enzyme reaction was carried out in kinetic detection mode, and data were recorded once every minute.
  • the detection conditions were as shown in Table 4.
  • the data table or enzyme activity curve can be directly exported, and the reaction rate of its relative fluorescence value can be approximately calculated.
  • the polymerization reaction was carried out using KOD DNA polymerase fusion protein (DP01) and KOD DNA polymerase point mutant fusion protein as examples.
  • the C-terminus of the fusion protein contained 6 his tags.
  • KOD DNA polymerase point mutants For detailed information on KOD DNA polymerase point mutants, please refer to Table 2; for preparation methods, please refer to Examples 1 and 2.
  • DP01 fusion protein is a highly active KOD DNA polymerase, and its relative polymerization activity is 2-3 times higher than that of the polymerase with only mutations 408 and 409 (see patent number PCT/CN2019/102493). Based on the highly active variants, this application further optimizes and improves the polymerization activity of KOD DNA polymerase mutants.
  • the activity determination kinetic curves of some KOD DNA polymerase mutants are shown in Figure 3.
  • mutants DP001, DP002, DP004, DP005, DP006, DP007, DP008, DP009, DP010, DP015, DP016, DP017, DP180, etc. showed comparable polymerization activities.

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Abstract

L'invention concerne un mutant d'ADN polymérase et son utilisation. Par comparaison avec une ADN polymérase KOD de type sauvage, le mutant de l'ADN polymérase présente des mutations d'acides aminés au niveau des sites 408 et 409, ou de sites fonctionnellement équivalents, et des mutations d'acides aminés au niveau d'au moins l'un des 23 sites suivants ou de sites fonctionnellement équivalents : site 141, site 143, site 147, site 383, site 384, site 389, site 485, site 584, site 589, site 397, site 424, site 432, site 445, site 523, site 553, site 561, site 564, site 461, site 481, site 605, site 663, site 711 et site 725 ; une séquence d'acides aminés du mutant autre que les sites de mutation d'acides aminés présente une identité d'au moins 90 % avec une séquence d'acides aminés correspondante de l'ADN polymérase KOD de type sauvage ; et l'ADN polymérase KOD de type sauvage présente une séquence d'acides aminés représentée dans SEQ ID NO : 2.
PCT/CN2023/136740 2023-12-06 2023-12-06 Mutant d'adn polymérase et son utilisation Pending WO2025118178A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2004038007A2 (fr) * 2002-10-25 2004-05-06 Stratagene Adn polymerases a activite de detection analogique a base reduite
CN108018270A (zh) * 2016-11-01 2018-05-11 Pgi股份有限公司 用以提升核苷酸类似物并入的重组dna聚合酶
WO2018126470A1 (fr) * 2017-01-09 2018-07-12 深圳华大智造科技有限公司 Adn polymérase recombinante
WO2020047695A1 (fr) * 2018-09-03 2020-03-12 深圳华大生命科学研究院 Polymérase kod recombinante
WO2023056394A1 (fr) * 2021-09-29 2023-04-06 Chen cheng yao Variants de polymérase pour la synthèse d'acides nucléiques enzymatiques indépendante de la matrice et kit les comprenant

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WO2004038007A2 (fr) * 2002-10-25 2004-05-06 Stratagene Adn polymerases a activite de detection analogique a base reduite
CN108018270A (zh) * 2016-11-01 2018-05-11 Pgi股份有限公司 用以提升核苷酸类似物并入的重组dna聚合酶
WO2018126470A1 (fr) * 2017-01-09 2018-07-12 深圳华大智造科技有限公司 Adn polymérase recombinante
WO2020047695A1 (fr) * 2018-09-03 2020-03-12 深圳华大生命科学研究院 Polymérase kod recombinante
WO2020048329A1 (fr) * 2018-09-03 2020-03-12 深圳华大生命科学研究院 Kod polymérase recombinante
WO2023056394A1 (fr) * 2021-09-29 2023-04-06 Chen cheng yao Variants de polymérase pour la synthèse d'acides nucléiques enzymatiques indépendante de la matrice et kit les comprenant

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ZHAI LILI, WANG ZI, LIU FEN, XU CHONGJUN, WANG JINGJING, HAN HONGYAN, XIE QINGQING, ZHANG WENWEI, ZHENG YUE, BUELL ALEXANDER K., D: "Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 20, no. 2, 14 February 2025 (2025-02-14), US , pages e0316531, XP093322291, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0316531 *

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