WO2025166644A1 - 多核苷酸激酶突变体及其应用 - Google Patents

多核苷酸激酶突变体及其应用

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Publication number
WO2025166644A1
WO2025166644A1 PCT/CN2024/076748 CN2024076748W WO2025166644A1 WO 2025166644 A1 WO2025166644 A1 WO 2025166644A1 CN 2024076748 W CN2024076748 W CN 2024076748W WO 2025166644 A1 WO2025166644 A1 WO 2025166644A1
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Prior art keywords
nucleic acid
polynucleotide kinase
amino acid
present application
mutant
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English (en)
French (fr)
Inventor
师虓
郭斐
王欧
曾涛
董宇亮
黎宇翔
章文蔚
徐讯
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to PCT/CN2024/076748 priority Critical patent/WO2025166644A1/zh
Publication of WO2025166644A1 publication Critical patent/WO2025166644A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the present application relates to the field of biotechnology, and in particular, to polynucleotide kinase mutants and applications thereof.
  • Polynucleotide kinase is an enzyme that catalyzes the reversible phosphorylation of the 5' end of DNA or RNA.
  • the PNK isolated from T4 bacteriophage can transfer the ⁇ -phosphate on ATP to the 5' end of DNA or RNA. It is widely used in molecular biology experiments, such as specific labeling of the 5' end of polynucleotides and end preparation for polynucleotide ligation reactions. It has a wide range of applications in polynucleotide modification, molecular cloning, and sequencing library preparation.
  • Nanopore sequencing is a third-generation sequencing technology that has emerged in recent years. Due to its advantages such as long read length, high throughput, low cost and portability, it has brought disruptive changes to the gene sequencing industry. Nanopore sequencing technology has a wide range of applications in basic theoretical research in life sciences and clinical practice in biomedicine. Nanopore sequencing is a sequencing technology based on electrical signals. A nanopore (protein or solid) inserted in a membrane separates two electrolyte chambers filled with electrolyte. When voltage is applied between the two electrolyte chambers, a stable perforation current is generated. Different molecules entering the nanopore will hinder the flow of ions, which is called the current signal. When ssDNA passes through the nanopore, the magnitude of the current obstruction will vary due to the different bases. By detecting the current fluctuation signal of the nanopore and analyzing the current signal through computer deep learning model, the sequence of the perforated DNA can be determined.
  • the library construction process of nanopore sequencing technology starts with genomic DNA extraction, with genomic fragmentation as an optional operation, followed by end repair and 3' end dATP tailing, and then linker connection through ligase-mediated TA connection, followed by tether binding (optional), and the library construction is completed.
  • its linker sequence mainly contains three special functional regions ( Figure 2), one of which is the motor protein binding site, which is a single-stranded DNA sequence, usually a polythymine nucleotide, with a number of 6-10. It is followed by a spacer sequence, which is 4 iSp18 polymers in the specific embodiment of the prior art, and its main function is to block the motor protein and prevent it from unwinding forward before sequencing.
  • the third functional region is the binding site of the constraint sequence, which mainly functions to perform complementary pairing with the constraint sequence, so that the constraint sequence can bind to the sequence to be sequenced in the form of a non-covalent bond, thereby pulling the sequence to be sequenced to the vicinity of the membrane where the sequencing hole is located, increasing the probability of the library being tested.
  • modified nucleotide bases that can play the role of spacer sequences, such as deoxyribonucleotides with alkyl substitutions on ⁇ -phosphates ( Figure 3).
  • This modified nucleotide can be introduced into the 3' or 5' end of the DNA fragment by polymerase or polynucleotide kinase. Then, such modified DNA fragments are connected by DNA ligase to form a DNA library that can be sequenced. In this step, whether the polymerase or polynucleotide kinase can complete the reaction using the modified nucleic acid as a phosphate donor is a key part in determining the success of the sequencing library construction method.
  • current polynucleotide kinases still have some shortcomings, such as low phosphorylation efficiency when using modified phosphate ATP as a phosphate donor.
  • the present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent.
  • one object of the present application is to provide a polynucleotide kinase that can effectively improve the reaction efficiency when using modified ATP as a phosphate donor.
  • Another object of the present application is to use adapters without spacers during nanopore sequencing to reduce the difficulty of synthesizing spacers during adapter synthesis.
  • the polynucleotide kinase mutant comprises at least one amino acid residue in the following sites or functionally equivalent sites compared to the wild-type polynucleotide kinase: Amino acid mutations: positions 47 and 129; the wild-type polynucleotide kinase (PNK WT) has an amino acid sequence as shown in SEQ ID NO: 1 (Table 1).
  • the polynucleotide kinase mutant can be used to catalyze phosphorylation reactions with modified sites, and the phosphorylation reaction efficiency is higher.
  • the polynucleotide kinase mutant is used to introduce modified phosphates that can hinder motor proteins, replacing the currently commonly used ethylene glycol inter-arm modifications, which can significantly reduce raw material costs and preparation complexity.
  • a nucleic acid molecule is provided.
  • the nucleic acid molecule encodes the polynucleotide kinase mutant described in the first aspect of the present application.
  • the polynucleotide kinase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.
  • an expression vector comprises the nucleic acid molecule described in the second aspect of the present application.
  • the expression vector can be used to express the polynucleotide kinase mutant in large quantities in vitro.
  • the present application provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the second aspect of the present application, the expression vector described in the third aspect of the present application, or expresses the polynucleotide kinase mutant described in the first aspect of the present application.
  • the recombinant cell is used to produce large quantities of the polynucleotide kinase mutant in vitro.
  • a recombinant strain in a fifth aspect of the present application, a recombinant strain is provided.
  • the recombinant strain carries the nucleic acid molecule described in the second aspect of the present application, the expression vector described in the third aspect of the present application, or expresses the polynucleotide kinase mutant described in the first aspect of the present application.
  • the recombinant strain is used to produce large quantities of the polynucleotide kinase mutant in vitro.
  • a method for obtaining a polynucleotide kinase mutant comprises culturing the recombinant cell described in the fourth aspect of this application or the recombinant strain described in the fifth aspect of this application under conditions suitable for protein expression, thereby obtaining the polynucleotide kinase mutant.
  • the above method can be used to produce large quantities of polynucleotide kinase mutants in vitro.
  • a method for substrate phosphorylation comprises: subjecting a nucleic acid substrate to be phosphorylated to 5' end phosphorylation under the catalysis of the polynucleotide kinase mutant described in the first aspect of the present application and in the presence of a phosphate group, to obtain a nucleic acid substrate with a phosphorylation modification at the 5' end.
  • the use of this method can significantly improve the reaction efficiency when the modified phosphate is used as a donor, thereby improving the efficiency of the 5' end phosphorylation of nucleotides.
  • the present application proposes a method for library construction.
  • the method comprises: subjecting the nucleic acid to be tested to 5' end phosphorylation treatment under the catalysis of the polynucleotide kinase mutant described in the first aspect of the present application and in the presence of a phosphate group to obtain a nucleic acid to be tested with a phosphorylation modification at the 5' end; connecting the nucleic acid to be tested with a phosphorylation modification at the 5' end and a connector to obtain a connector connection product; and combining the connector connection product with a motor protein to obtain the sequencing library.
  • the use of this method for library construction can significantly reduce the complexity of library construction and effectively improve the success rate of library construction.
  • a nucleic acid sequencing method comprises: constructing a sequencing library based on the nucleic acid sample to be tested according to the method described in the eighth aspect of this application; and sequencing the sequencing library to determine the nucleic acid sequence of the nucleic acid to be tested.
  • nucleic acid sequencing using this method can effectively increase sequencing depth and reduce sequencing costs.
  • the present application proposes a use of the polynucleotide kinase 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, or the recombinant strain described in the fifth aspect in the preparation of products related to substrate phosphorylation or nucleic acid sequencing.
  • the aforementioned polynucleotide kinase mutant, nucleic acid molecule, expression vector, recombinant cell, or recombinant strain can be used to prepare products related to substrate phosphorylation or sequencing, such as substrate phosphorylation kits and sequencing kits.
  • the present application proposes a method for blocking a motor protein.
  • the method comprises: subjecting the nucleic acid molecule to 5'-terminal phosphorylation treatment under the catalysis of the polynucleotide kinase mutant described in the first aspect of the present application and the presence of a modified phosphate group to obtain a nucleic acid molecule with a modified phosphate at the end, and allowing the nucleic acid molecule to bind to the motor protein to block the motor protein.
  • this method can prevent the motor protein from further unwinding before sequencing.
  • FIG1 is a schematic diagram of the process of constructing a nanopore sequencing library in the prior art
  • FIG2 is a schematic diagram of a nanopore sequencing adapter in the prior art
  • FIG3 is a schematic diagram of the structure of an ⁇ -alkyl-substituted deoxyribonucleotide ( ⁇ -alkyl-dNTP) according to an embodiment of the present application; wherein R represents a modifying group; Base represents any base (i.e., A, T, C, G or other bases);
  • FIG4 is a schematic diagram of the structure of a wild-type T4PNK according to an embodiment of the present application.
  • FIG5 is a schematic diagram of the results of PNK protein purification according to an embodiment of the present application.
  • FIG6 is a schematic diagram of the mass spectrometry detection results of the phosphorylation reaction according to one embodiment of the present application.
  • FIG7 is a schematic diagram of the quality detection results of the sequencing adapter Ad1 according to one embodiment of the present application.
  • FIG8 is a schematic diagram of the purity test results of the helicase Dda described in one embodiment of the present application.
  • FIG9 is a schematic diagram showing the detection results of the sequencing adapter Ad1 ligation product according to one embodiment of the present application.
  • FIG10 is a schematic diagram of a typical nanopore sequencing signal of a PNK wild-type library constructed in one embodiment of the present application.
  • Figure 11 is a schematic diagram of a typical nanopore sequencing signal for PNK Mut3 library construction described in one embodiment of the present application.
  • first and second are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as “first” or “second” may explicitly or implicitly include at least one of the features.
  • plurality means at least two, for example, two, three, etc., unless otherwise specified.
  • 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).
  • nucleotide includes: phosphate, ribose or deoxyribose glycosides and purine or pyrimidine bases. Synthetic and/or naturally occurring nucleotides are included in the definition.
  • the term “identity” has the conventional meaning in the art and refers to the "homology" between two nucleic acid or amino acid sequences, where the percentage represents the statistically significant percentage of identical nucleotide or amino acid residues between the two sequences being compared after best alignment, with the differences between the two sequences being randomly distributed over their entire lengths.
  • the mutants are described according to their mutations at specific residues, the positions of which are located with reference to the positions of the amino acids in the wild-type polynucleotide kinase amino acid sequence.
  • nucleic acid sequencing refers to determining the order of base arrangement in the primary structure of a nucleic acid molecule.
  • T4PNK is the most widely used polynucleotide kinase in molecular biology experiments. It catalyzes the transfer of a phosphate at the gamma position of ATP to the hydroxyl group at the 5' end of single-stranded or double-stranded DNA or RNA, a process known as phosphorylation. This reaction is reversible, and in the presence of ATP or ADP, a 5'-terminal phosphate exchange reaction can also occur.
  • existing commercial T4PNK can only efficiently utilize natural ATP (as shown in Formula 1) as a substrate for polynucleotide 5' phosphorylation reactions, but cannot meet the needs of some specialized applications.
  • ATP modified with an alkylated gamma phosphate as shown in Formula 2, the electrical properties and steric hindrance caused by the alkyl group prevent the phosphate from entering the catalytic site, resulting in low efficiency in catalyzing the phosphorylation of the modified phosphate to the 5' end of DNA or RNA.
  • a polynucleotide kinase mutant is proposed. Compared with the wild-type polynucleotide kinase, the polynucleotide kinase mutant has an amino acid mutation at at least one of the following five sites or functionally equivalent sites: position 11, position 20, position 47, position 122 and position 129; the wild-type polynucleotide kinase has an amino acid sequence as shown in SEQ ID NO: 1 (Table 1).
  • the polynucleotide kinase mutant can be used to catalyze the phosphorylation reaction of a phosphate donor with a modified site, and the phosphorylation reaction is more efficient.
  • the polynucleotide kinase mutant is used to introduce a modified phosphate that hinders the motor protein, replacing the currently commonly used ethylene glycol spacer modification, which can significantly reduce the cost of raw materials and the complexity of preparation.
  • amino acid positions in the amino acid sequence of the polynucleotide kinase mutant described in the present application are located with reference to the amino acid positions in the amino acid sequence of the wild-type polynucleotide kinase.
  • the mutant is at least 90% identical to the wild-type polynucleotide kinase except for at least one of amino acids 11, 20, 47, 122, and 129.
  • the identity is optionally 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the mutant sequence is at least 95% identical.
  • the mutation type of the mutation site of the mutant is: (1) the amino acid P at position 11 mutates to G; (2) the amino acid R at position 20 mutates to G; (3) the amino acid R at position 47 mutates to G; (4) the amino acid R at position 122 mutates to G; or (5) the amino acid K at position 129 mutates to G.
  • the mutant has at least one mutation from (1) to (5).
  • the polynucleotide kinase activity produced by the free combination of different mutation sites has certain differences, and the combination of mutation types can provide more options for actual production needs.
  • the mutant has any one of the following mutations (1)-(14): (1) amino acid P at position 11 mutates to G; (2) amino acid R at position 20 mutates to G; (3) amino acid R at position 47 mutates to G; (4) amino acid R at position 122 mutates to G; (5) amino acid K at position 129 mutates to G; (6) amino acid R at position 47 mutates to G, and amino acid K at position 129 mutates to G; (7) amino acid R at position 20 mutates to G, and amino acid K at position 129 mutates to G; (8) amino acid R at position 122 mutates to G; (1) The amino acid at position 47 mutated to G, and the amino acid K at position 129 mutated to G; (2) The amino acid R at position 47 mutated to G, and the amino acid P at position 11 mutated to G; (3) The amino acid R at position 47 mutated to G, and the amino acid R at position 20 mutated to G; (4) The amino acid R at position 122 mutates to
  • the mutant has any one of the following mutations (1)-(8): (1) amino acid P at position 11 mutates to G; (2) amino acid R at position 20 mutates to G; (3) amino acid R at position 47 mutates to G; (4) amino acid R at position 122 mutates to G; (5) amino acid K at position 129 mutates to G; (6) amino acid R at position 47 mutates to G, and amino acid K at position 129 mutates to G; (7) amino acid R at position 20 mutates to G, and amino acid K at position 129 mutates to G; (8) amino acid R at position 122 mutates to G, and amino acid K at position 129 mutates to G.
  • the mutant can significantly improve the phosphorylation efficiency when modified or unmodified phosphate is used as a substrate.
  • the mutant has any one of the mutations (3) and (6): (3) the amino acid R at position 47 mutates to G; (6) the amino acid R at position 47 mutates to G, and the amino acid K at position 129 mutates to G. In some examples of the present application, the mutant can significantly improve the phosphorylation efficiency when the modified phosphate is used as a substrate.
  • a nucleic acid molecule in another aspect of the present application, encodes a polynucleotide kinase mutant as described above.
  • the polynucleotide kinase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.
  • the nucleic acid molecule is selected from DNA or RNA.
  • nucleic acid molecules mentioned in the specification and claims of this application those skilled in the art will understand that they actually include either or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is actually also disclosed.
  • nucleic acid sequences in this application include either DNA or RNA forms, and disclosure of one implies disclosure of the other.
  • an expression vector comprises the nucleic acid molecule described above.
  • the expression vector can be used to express the polynucleotide kinase mutant in large quantities in vitro.
  • nucleic acid molecule and the control elements on the vector can be directly or indirectly connected, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
  • nucleic acid molecule and the control elements can be operably connected.
  • "operably connected" means that the exogenous gene is connected to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can play their intended function of regulating the transcription and translation of the exogenous gene.
  • Commonly used vectors can be, for example, plasmids, bacteriophages, etc.
  • the expression vector may further include a promoter, which is operably linked 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.
  • a recombinant cell in another aspect of the present application, a recombinant cell is provided.
  • the recombinant cell carries a nucleic acid molecule as described above, an expression vector as described above, or expresses a polynucleotide kinase mutant as described above.
  • the recombinant cell can be used to produce large quantities of the polynucleotide kinase mutant in vitro under suitable conditions.
  • suitable conditions refers to conditions suitable for the expression of the polynucleotide kinase mutants described herein.
  • suitable conditions for the expression of the polynucleotide kinase mutants include, but are not limited to, a suitable transformation method, suitable transformation conditions, healthy host cells, suitable host cell density, a suitable cell culture environment, and a suitable cell culture time.
  • suitable conditions are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the polynucleotide kinase mutants based on the specific laboratory environment.
  • the recombinant cell is obtained by transfecting or transforming the expression vector.
  • the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.
  • the recombinant cells described herein are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages.
  • the prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, among others.
  • the eukaryotic cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, insect cells such as fall armyworms, plant cells such as tobacco, or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells.
  • the recombinant cells described herein are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.
  • a recombinant strain in another aspect of the present application, carries a nucleic acid molecule as described above, an expression vector as described above, or expresses a polynucleotide kinase mutant as described above. In some examples of the present application, the recombinant strain is used to produce a large amount of the polynucleotide kinase mutant in vitro.
  • a method for obtaining a polynucleotide kinase mutant comprises culturing the aforementioned recombinant cell or recombinant strain under conditions suitable for protein expression to obtain the polynucleotide kinase mutant.
  • the aforementioned method can be used to produce large quantities of polynucleotide kinase mutants in vitro.
  • condition suitable for protein expression refers to conditions suitable for the expression of the polynucleotide kinase mutants described herein.
  • conditions suitable for the expression of polynucleotide kinase mutants include, but are not limited to, appropriate transformation or transfection methods and conditions, healthy host cells, appropriate host cell density, a suitable cell culture environment, and an appropriate cell culture time.
  • Cons suitable for protein expression are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the polynucleotide kinase mutants based on the specific laboratory environment.
  • a substrate phosphorylation method comprises: subjecting a nucleic acid substrate to be phosphorylated to 5'-terminus phosphorylation under the catalysis of the aforementioned polynucleotide kinase mutant and in the presence of a phosphate group to obtain a nucleic acid substrate with a phosphorylated 5' terminus.
  • the use of this method can significantly improve the reaction efficiency when the modified phosphate serves as the phosphate donor, thereby increasing the efficiency of 5'-terminal phosphorylation of nucleotide modifications.
  • the phosphate group is provided in the form of at least one of dNTPs, NTPs, nucleoside-modified ribonucleotides, and deoxyribonucleotides.
  • dNTPs include dATP, dTTP, dCTP, dGTP, and dUTP; and NTPs include ATP, TTP, CTP, GTP, and UTP.
  • the nucleoside-modified ribonucleotides and deoxyribonucleotides can be base-modified ribonucleotides and deoxyribonucleotides, such as 3-methyladenine nucleotides, 7-methylguanine nucleotides, 1, N6 -vinylidene adenine inosinic acid, inosine nucleotides, uracil nucleotides, etc., or can be ribonucleotides and deoxyribonucleotides modified at the sugar ring, such as locked nucleotides, peptide nucleotides or threose nucleotides, etc.
  • At least one of the aforementioned dNTPs and NTPs has a modified phosphate group. In some preferred examples of the present application, at least one of the aforementioned dNTPs and NTPs has a modified ⁇ -phosphate group. In some examples of the present application, nucleotides containing modified groups can inhibit motor proteins.
  • the modifying group is selected from at least one of an alkyl group, a thiol group, a seleno group, a fluorophore, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and/or anti-digoxigenin, and a diphenylmethylcyclooctyne group.
  • the alkyl group is selected from at least one of methyl, ethyl, propyl, butyl, isopropyl, silylmethyl or boryl. For example, as shown in Formula 3 and Formula 4.
  • R represents a modifying group
  • the R group can be selected from an alkyl group, a thiol group, a selenoyl group, a fluorophore, a streptavidin
  • the R group is selected from an alkyl group, and specifically, the R group is selected from a methyl group.
  • R represents a modifying group
  • the R group can be selected from at least one of an alkyl group, a sulfhydryl group, a selenoyl group, a fluorophore, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and/or antidigoxigenin, and a benzhydrylcyclooctynyl group.
  • the R group is selected from an alkyl group, specifically, a methyl group.
  • the present application proposes a method for library construction.
  • the method comprises: subjecting the nucleic acid to be tested to 5' end phosphorylation treatment under the catalysis of the polynucleotide kinase mutant described in the first aspect of the present application and the presence of a phosphate group to obtain a nucleic acid to be tested with a phosphorylation modification at the 5' end; connecting the nucleic acid to be tested with a phosphorylation modification at the 5' end and a connector to obtain a connector connection product; and combining the connector connection product with a motor protein to obtain the sequencing library.
  • the use of this method for library construction can significantly reduce the complexity of library construction and effectively improve the success rate of library construction.
  • a nucleic acid molecule containing a modified nucleotide at its terminus that blocks a motor protein is used for single-molecule sequencing.
  • a motor protein that moves from the 5' to the 3' direction of the nucleic acid molecule can be used, or a motor protein that moves from the 3' to the 5' direction of the nucleic acid molecule can be used.
  • Those skilled in the art will be able to select an appropriate motor protein based on actual needs.
  • a nucleic acid to be tested is subjected to 5'-terminal phosphorylation treatment under the catalysis of a polynucleotide kinase mutant and in the presence of a phosphate group to obtain a nucleic acid to be tested having a phosphorylated modification at the 5' end, wherein the polynucleotide kinase mutant has any one of the mutations (1)-(8), the phosphate group is provided in the form of a substrate to be phosphorylated selected from at least one of dNTPs, NTPs, nucleoside-modified ribonucleotides and deoxyribonucleotides, the ⁇ -position phosphate of at least one of the dNTPs and NTPs is modified, and the linker has or does not have an ethylene glycol inter-arm modification.
  • nucleotides containing modified groups can hinder motor proteins, thereby preventing the motor proteins from moving forward on the nucleic acid to be tested before sequencing.
  • the nucleic acid to be tested is subjected to 5'-terminal phosphorylation treatment under the catalysis of a polynucleotide kinase mutant and in the presence of a phosphate group to obtain a nucleic acid to be tested having a phosphorylated modification at the 5' end, wherein the polynucleotide kinase mutant has any one of mutations (3) or (6), and the phosphate group is provided in the form of a substrate to be phosphorylated selected from at least one of dNTPs, NTPs, nucleoside-modified ribonucleotides and deoxyribonucleotides, the ⁇ -position phosphate of at least one of the dNTPs and NTPs is modified, and the linker does not have an ethylene glycol inter-arm modification.
  • a nucleic acid sequencing method comprises: constructing a sequencing library based on the nucleic acid sample to be tested according to the method described in the eighth aspect of this application; and sequencing the sequencing library to determine the nucleic acid sequence of the nucleic acid to be tested.
  • nucleic acid sequencing using this method can effectively increase sequencing depth and reduce sequencing costs.
  • the nucleic acid sequencing is selected from nanopore sequencing.
  • the nanopore sequencing method comprises:
  • the termini of the target nucleic acid molecule contain modified nucleotides capable of blocking the motor protein to obtain a modified nucleic acid molecule to be tested;
  • step 2) Adding the product of step 2) to an electrophysiological detection system containing a nanopore to perform single-molecule nanopore sequencing.
  • the present application provides a use of the aforementioned polynucleotide kinase mutant, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned recombinant strain in the preparation of products related to substrate phosphorylation or nucleic acid sequencing.
  • the aforementioned polynucleotide kinase mutant, nucleic acid molecule, expression vector, recombinant cell, or recombinant strain can be used to prepare products related to substrate phosphorylation or sequencing, such as substrate phosphorylation kits and sequencing kits.
  • a method for blocking a motor protein comprises:
  • the nucleic acid molecule is subjected to 5'-terminal phosphorylation treatment under the catalysis of the polynucleotide kinase mutant described in the first aspect of the present application and in the presence of a modified phosphate group to obtain a nucleic acid product having a modified phosphate at the 5' end;
  • the nucleic acid product is allowed to bind to the motor protein so as to block the motor protein.
  • the library product with a modified phosphate at the 5' end is incubated with a motor protein to prevent the motor protein from further unwinding before sequencing.
  • the T4PNK mutants in Table 2 were gene synthesized and transferred into the PET.28a(+) plasmid using double restriction enzyme sites NdeI and XhoI.
  • the expressed T4DNA ligase mutant protein had a 6*His tag and a thrombin restriction enzyme site at the N-terminus.
  • Buffer A 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM Imidazole;
  • Buffer B 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;
  • Buffer C 20 mM Tris-HCl pH 7.5, 50 mM NaCl;
  • Buffer D 20 mM Tris-HCl pH 7.5, 100 mM NaCl;
  • Collect the expressed T4PNK Mut1-8 bacteria resuspend the bacteria with Buffer A, disrupt the bacteria with a cell disruptor, and then centrifuge to obtain the supernatant. Mix the supernatant with the Ni-NTA filler that has been equilibrated with Buffer A in advance and combine for 1 hour. Collect the filler and wash the filler with Buffer A in large quantities until no foreign proteins are washed out. Then add Buffer B to the filler to elute the target protein. Pass the eluted target protein through a desalting column equilibrated with Buffer C to replace the buffer. Then add thrombin and digest at 4°C overnight. After protein concentration, apply it to molecular sieve Superdex 200, and the molecular sieve B buffer used is Buffer D. Collect the target protein peak, concentrate it, and freeze it.
  • Example 2 Phosphorylation reaction and mass spectrometry detection
  • the mutant was prepared in Example 1. Place in a thermal cycler and incubate at 37°C for 30 minutes and 72°C for 30 minutes to perform the phosphorylation reaction, adding a phosphate or methylated phosphate to the 5' end of the target double-stranded DNA.
  • the reaction product was purified using a nucleic acid purification kit ( After purification using the PCR & DNA Cleanup Kit (New England Biolabs, T1030L), the reaction products were detected using electrospray ionization mass spectrometry (ESI-MS).
  • a nucleic acid purification kit After purification using the PCR & DNA Cleanup Kit (New England Biolabs, T1030L), the reaction products were detected using electrospray ionization mass spectrometry (ESI-MS).
  • sequencing linker Ad1 was prepared by annealing chemically synthesized SEQ ID NO: 21 and SEQ ID NO: 22.
  • helicase Dda (SEQ ID NO: 23) was prepared by recombinant expression in Escherichia coli, and the helicase was used as a motor protein.
  • Buffer A 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;
  • Buffer B 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;
  • Buffer C 20 mM Tris-HCl pH 7.5, 50 mM NaCl;
  • Buffer D 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;
  • Buffer E 20 mM Tris-HCl pH 7.5, 100 mM NaCl;
  • the ssDNA cellulose (Sigma, D8273-10G) filler was weighed and digested at 4°C overnight. The ssDNA cellulose filler was collected, washed 3-4 times with buffer C, and then eluted with buffer D. The protein purified from the ssDNA cellulose was concentrated and applied to a molecular sieve Superdex 200 (Sigma, GE28-9909-44) using buffer E. The target protein peak was collected, concentrated, and frozen. The concentration of the purified protein was quantified using Nanodrop. The protein purity was tested by HPLC and SDS-PAGE electrophoresis at the same time, and the results are shown in Figure 8.
  • Example 5 Generating a 5'-terminal methylphosphorylated sequencing library using ⁇ -methylphosphoATP
  • a fragment of the pUC57 plasmid (SEQ ID NO: 25) was obtained by enzyme digestion and used as the target nucleic acid sequence for sequencing.
  • ⁇ -methylphosphate ATP (shown in Formula 2) was used as the reaction substrate, and a phosphorylation reaction catalyzed by polynucleotide kinase was used to introduce a methylphosphate group to the 5' terminal nucleotide of the target nucleic acid sequence (i.e., the 5' terminal nucleoside is shown in Formula 5).
  • the resulting phosphorylated end-repair product ( Figure 3, methylphosphate-modified 5' terminal nucleotide) was then ligated with Ad1 prepared in Example 3, and then with the helicase Dda prepared in Example 4.
  • nanopore detection platform based on a patch clamp platform was constructed, and nanopore sequencing was performed on the target sequencing library (5' end methyl phosphorylated) prepared in Example 5 to verify the advantages of the sequencing library capable of blocking motor proteins constructed in this application in nanopore sequencing.
  • Example 6 The sequencing library obtained in Example 6 was added to the single-channel system, and the current amplitude change was detected and recorded using a patch clamp system.
  • Typical sequencing current plots for library construction using the wild-type PNK are shown in Figure 10, and typical sequencing current plots for library construction using the PNK Mut 3 mutant are shown in Figure 11.
  • the vertical axis in the figure represents the current value (unit: pA). Comparison of the two typical signals reveals that the sequencing signal for the complete SEQ ID NO: 25 using the PNK Mut 3 mutant is significantly higher per unit time (20 seconds) than when the phosphorylation reaction is performed using the wild-type PNK, indicating that higher methyl phosphorylation efficiency can yield more effective sequencing libraries.
  • the reference terms “one embodiment”, “some embodiments”, “example”, “specific example”, or “some examples” mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.
  • the schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
  • those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

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Abstract

涉及生物技术领域，具体地涉及多核苷酸激酶突变体及其应用。其中，该多核苷酸激酶突变体包括：与野生型多核苷酸激酶相比，包括在以下位点或功能等同位点中的至少一个具有氨基酸突变：第47位和第129位；所述野生型多核苷酸激酶具有如SEQ ID NO:1所示的氨基酸序列。

Description

多核苷酸激酶突变体及其应用 技术领域
本申请涉及生物技术领域，具体的，本申请涉及多核苷酸激酶突变体及其应用。
背景技术
多核苷酸激酶(polynucleotide kinase)是一种可以催化DNA或RNA 5’端可逆性磷酸化的酶。其中T4噬菌体中分离出的多核苷酸激酶(简称为T4PNK)，能够转移ATP上γ位磷酸至DNA或RNA的5’端，多用于分子生物实验中，如多核苷酸5’端的特异性标记，多核苷酸连接反应时的末端准备等，在多核苷酸修饰，分子克隆和测序文库制备中有广泛的应用。
纳米孔测序为近些年新兴的第三代测序技术，由于其长读长，高通量，低成本和便携性等优势，给基因测序行业带来了颠覆性的改变。纳米孔测序技术在生命科学基础理论研究以及生物医学临床实践中具有广泛的应用。纳米孔测序是基于电信号的测序技术。由一个插在膜上的(蛋白或固态)纳米孔将两个装有电解液的电解室分开。当电压施加给两个电解室之间时，会产生稳定的穿孔电流。进入纳米孔的不同分子会对离子的流动造成阻碍，这被称为电流信号。当ssDNA穿过纳米孔时，由于碱基的不同，会造成电流阻碍的大小不同。通过检测纳米孔的电流波动信号，并通过计算机深度学习建立模型分析该电流信号，从而测定得到穿孔的DNA的序列。
纳米孔测序技术的建库流程(图1)以基因组提取DNA为起始，基因组片段化为可选操作，随后进行末端修复和3’端加dATP尾，然后再通过连接酶介导的TA连接进行接头连接，随后再进行拘束序列(tether)结合(可选)，至此完成文库构建。其接头序列除了常规核酸分子以外，主要包含三个特殊功能区域(图2)，其一为马达蛋白结合位点，为一段单链DNA序列，通常为多聚胸腺嘧啶核苷酸，数量为6-10个。随后为间隔器序列，在现有技术的具体实施例中为4个iSp18聚合物，其作用主要为阻断马达蛋白，防止它在测序之前向前解旋。第三个功能区域为拘束序列的结合位点，主要作用是与拘束序列进行互补配对，使得拘束序列能够以非共价键形式与待测序序列进行结合，从而将待测序序列拉至测序孔所在的膜附近，增加文库被测概率。
除了iSp18以外，还有其他的一些修饰核苷酸碱基可以行使间隔器序列的作用，如在α磷酸上进行烷基取代的脱氧核糖核苷酸(图3)。这种修饰核苷酸可以通过聚合酶或者多核苷酸激酶(polynucleotide kinase)引入DNA片段的3’端或5’端。然后，通过DNA连接酶将这样的修饰DNA片段连接成为可以进行测序的DNA文库。在这一步骤中，聚合酶或者多核苷酸激酶能否以修饰核酸为磷酸供体完成反应，是决定测序建库方法能否成功的关键部分。然而，目前的多核苷酸激酶仍存在一些不足，如以修饰磷酸ATP为磷酸供体时磷酸化效率低等。
因此，多核苷酸激酶仍有待改进。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本申请的一个目的在于提供一种以修饰磷酸ATP为磷酸供体时，能够有效提升反应效率的多核苷酸激酶。
本申请的另一个目的是提供一种在目标核酸分子的末端进行修饰磷酸化的方法，从而获得在末端包含能够阻断马达蛋白的修饰核苷酸的核酸分子。
进一步地，本申请的另一个目的是提供一种阻断马达蛋白的方法，通过使目标测序核酸分子在末端包含能够阻断马达蛋白的修饰核苷酸，从而防止马达蛋白在测序之前进一步向前解旋。
本申请的另一个目的是在纳米孔测序时使用不带有间隔器的接头，减少接头合成时间隔器的合成难度。
本申请的另一个目的是避免在测序时检测到间隔器后的一段接头序列，从而进一步简化生物信息分析处理流程。本申请的另一个目的是有效提升马达蛋白阻断效率，增强接头识别信号。
为此，在本申请的第一方面，本申请提出了一种多核苷酸激酶突变体。根据本申请的实施例，所述多核苷酸激酶突变体与野生型多核苷酸激酶相比，包括在以下位点或功能等同位点中的至少一个具有氨 基酸突变：第47位和第129位；所述野生型多核苷酸激酶(PNK WT)具有如SEQ ID NO:1所示的氨基酸序列(表1)。在本申请的一些示例中，所述多核苷酸激酶突变体能够用于催化进行具有修饰位点的磷酸化反应，且磷酸化反应效率更高。在一些测序应用场景中，利用该多核苷酸激酶突变体引入可以阻碍马达蛋白的修饰磷酸，取代目前常用的乙二醇间臂类修饰，能够显著降低原料成本和制备复杂度。
在本申请的第二方面，本申请提出了一种核酸分子。根据本申请的实施例，所述核酸分子编码本申请第一方面所述的多核苷酸激酶突变体。在本申请的一些示例中，所述核酸分子编码的多核苷酸激酶突变体可在体内或体外进行大量获得。
在本申请的第三方面，本申请提出了一种表达载体。根据本申请的实施例，所述表达载体包括本申请第二方面所述的核酸分子。在本申请的一些示例中，所述表达载体可用于体外大量表达该多核苷酸激酶突变体。
在本申请的第四方面，本申请提出了一种重组细胞。根据本申请的实施例，所述重组细胞携带本申请第二方面所述的核酸分子、本申请第三方面所述的表达载体或表达本申请第一方面所述的多核苷酸激酶突变体。在本申请的一些示例中，所述重组细胞用于体外大量制备多核苷酸激酶突变体。
在本申请的第五方面，本申请提出了一种重组菌株。根据本申请的实施例，所述重组菌株携带本申请第二方面所述的核酸分子、本申请第三方面所述的表达载体或表达本申请第一方面所述的多核苷酸激酶突变体。在本申请的一些示例中，所述重组菌株用于体外大量制备多核苷酸激酶突变体。
在本申请的第六方面，本申请提出了一种获得多核苷酸激酶突变体的方法。根据本申请的实施例，所述方法包括：将本申请第四方面所述的重组细胞或第五方面所述的重组菌株在适于蛋白表达的条件下进行培养处理，以便获得所述多核苷酸激酶突变体。在本申请的一些示例中，利用上述方法能够体外大量制备多核苷酸激酶突变体。
在本申请的第七方面，本申请提出了一种底物磷酸化方法。根据本申请的实施例，所述方法包括：将待磷酸化核酸底物在本申请第一方面所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的核酸底物。在本申请的一些示例中，利用本方法能够显著提升修饰磷酸做供体时的反应效率，提升核苷酸5’端修饰磷酸化效率。
在本申请的第八方面，本申请提出了一种建库方法。根据本申请的实施例，所述方法包括：将待测核酸在本申请第一方面所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸；将所述5’末端具有磷酸化修饰的待测核酸和接头进行连接处理，得到接头连接产物；使所述接头连接产物与马达蛋白结合，以便获得所述测序文库。在本申请的一些示例中，利用本方法进行建库，能够显著降低建库复杂度，有效提高建库成功率。
在本申请的第九方面，本申请提出了一种核酸测序方法。根据本申请的实施例，所述方法包括：基于所述待测核酸样本，根据本申请第八方面所述方法构建测序文库；对所述测序文库进行测序，以确定所述待测核酸的核酸序列。在本申请的一些示例中，利用本方法进行核酸测序，能够有效增加测序深度，降低测序成本。
在本申请的第十方面，本申请提出了一种第一方面所述的多核苷酸激酶突变体、第二方面所述的核酸分子、第三方面所述的表达载体、第四方面所述的重组细胞或第五方面所述的重组菌株在制备用于底物磷酸化或核酸测序相关产品中的用途。在本申请的一些示例中，前述多核苷酸激酶突变体、核酸分子、表达载体、重组细胞或重组菌株可用于制备底物磷酸化或测序相关产品，如底物磷酸化试剂盒以及测序试剂盒等。
在本申请的第十一方面，本申请提出了一种阻滞马达蛋白的方法。根据本申请的实施例，所述方法包括：将所述核酸分子在本申请第一方面所述多核苷酸激酶突变体催化下以及修饰磷酸基团存在的条件下进行5’端磷酸化处理获得末端具有修饰磷酸的核酸分子，使所述核酸分子与所述马达蛋白结合，以便阻滞所述马达蛋白。在一些测序场景中(如单分子纳米孔测序)，通过本方法能够防止马达蛋白在测序之前进一步向前解旋。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施方案)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1为现有技术中纳米孔测序文库构建流程示意图；
图2为现有技术中纳米孔测序接头示意图；
图3为本申请一个实施例所述α-烷基取代-脱氧核糖核苷酸(α-alkyl-dNTP)结构示意图；其中，R表示修饰基团；Base表示任意碱基(即A、T、C、G或其他碱基)；
图4为本申请一个实施例所述野生型T4PNK结构示意图；
图5为本申请一个实施例所述PNK蛋白纯化结果示意图；
图6为本申请一个实施例所述磷酸化反应质谱检测结果示意图；
图7为本申请一个实施例所述测序接头Ad1质量检测结果示意图；
图8为本申请一个实施例所述解旋酶Dda纯度检测结果示意图；
图9为本申请一个实施例所述测序接头Ad1连接产物检测结果示意图；
图10为本申请一个实施例所述PNK野生型建库典型纳米孔测序信号示意图；
图11为本申请一个实施例所述PNK Mut3建库典型纳米孔测序信号示意图。
具体实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制。
在本申请中，除非另有说明，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本申请的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本申请中，除非另有说明，术语“氨基酸”由单字母或三字母代码表示，具有如下含义：A：Ala(丙氨酸)；R：Arg(精氨酸)；N：Asn(天冬酰胺)；D：Asp(天冬氨酸)；C：Cys(半胱氨酸)；Q：Gln(谷氨酰胺)；E：Glu(谷氨酸)；G：Gly(甘氨酸)；H：His(组氨酸)；I：Ile(异亮氨酸)；L：Leu(亮氨酸)；K：Lys(赖氨酸)；M：Met(甲硫氨酸)；F：Phe(苯丙氨酸)；P：Pro(脯氨酸)；S：Ser(丝氨酸)；T：Thr(苏氨酸)；W：Trp(色氨酸)；Y：Tyr(酪氨酸)；V：Val(缬氨酸)。
本申请中，除非另有说明，术语“核苷酸”包括：磷酸、核糖或脱氧核糖糖苷以及嘌呤或嘧啶碱基。合成和/或天然存在的核苷酸，均包含在定义内。
本申请中，除非另有说明，术语“同一性”具有本领域常规的含义，是指两个核酸或氨基酸序列之间的“同源性”，其百分比表示在最佳比对(best alignment)后获得的待比较的两个序列之间的相同核苷酸或氨基酸残基的统计学意义的百分比，两个序列之间的差异随机地分布在其整个长度上。本申请中，所述突变体根据它们在特定残基上的突变来描述，其位置参照野生型多核苷酸激酶氨基酸序列中氨基酸的位置进行定位。
本申请中，除非另有说明，术语“核酸测序”是指测定核酸分子一级结构碱基排列次序。
T4PNK是分子生物学实验中应用最广泛的多核苷酸激酶，能催化ATP的γ位磷酸根转移到单链或双链DNA或RNA的5’末端羟基的反应，即磷酸化。该反应是可逆的，在ATP或ADP的存在下，也可进行5’末端磷酸交换反应。然而，现有商用T4PNK只能高效利用天然ATP(如式1所示)做底物进行多核苷酸5’的磷酸化反应，但不能满足一些特殊应用需求，如在利用γ位磷酸烷基化修饰的ATP(如式2所示)做磷酸供体时，由于烷基带来的电性改变和空间位阻，阻拦磷酸进入催化位点，进而导致催化修饰的磷酸至DNA或RNA的5’端的磷酸化反应效率较低。
在本申请的一方面，本申请提出了一种多核苷酸激酶突变体。该多核苷酸激酶突变体与野生型多核苷酸激酶相比，在以下五个位点或功能等同位点中的至少一个具有氨基酸突变：第11位、第20位、第47位、第122位和第129位；所述野生型多核苷酸激酶具有如SEQ ID NO:1所示的氨基酸序列(表1)。在本申请的一些示例中，所述多核苷酸激酶突变体能够用于催化具有修饰位点的磷酸供体的磷酸化反应，且磷酸化反应效率更高。在一些测序应用场景中，利用该多核苷酸激酶突变体引入阻碍马达蛋白的修饰磷酸，取代目前常用的乙二醇间臂类修饰(spacer)，能够显著降低原料成本和制备复杂度。
需要说明的是，本申请所述多核苷酸激酶突变体的氨基酸序列中的氨基酸位置参照野生型多核苷酸激酶氨基酸序列中氨基酸的位置进行定位。
需要说明的是，所称的功能等同位点是指与野生多核苷酸激酶中的指定位点具有相同功能的位点，包括位置等价位点或者同源位点。
在本申请的一些示例中，除第11位、第20位、第47位、第122位和第129位至少之一的氨基酸以外，所述突变体与所述野生型多核苷酸激酶具有至少90％的同一性。在本申请的一些示例中，同一性可选地为91％、92％、93％、94％、95％、96％、97％、98％或99％。优选具有至少95％同一性的突变体序列。
在本申请的一些示例中，所述突变体的突变位点的突变类型为：(1)第11位氨基酸P突变为G；(2)第20位氨基酸R突变为G；(3)第47位氨基酸R突变为G；(4)第122位氨基酸R突变为G；或(5)第129位氨基酸K突变为G。
在本申请的一些示例中，所述突变体具有(1)～(5)中的至少一个突变。在本申请的一些示例中，不同突变位点自由组合产生的多核苷酸激酶活性具有一定差异，其突变类型的组合可以为实际生产需要提供更多的选择。
在本申请的一些示例中，所述突变体具有以下(1)-(14)任意一种突变：(1)第11位氨基酸P突变为G；(2)第20位氨基酸R突变为G；(3)第47位氨基酸R突变为G；(4)第122位氨基酸R突变为G；(5)第129位氨基酸K突变为G；(6)第47位氨基酸R突变为G，第129位氨基酸K突变为G；(7)第20位氨基酸R突变为G，第129位氨基酸K突变为G；(8)第122位氨基酸R突变为G，第129位氨基酸K突变为G；(9)第47位氨基酸R突变为G，第11位氨基酸P突变为G；(10)第47位氨基酸R突变为G，第20位氨基酸R突变为G；(11)第47位氨基酸R突变为G，第122位氨基酸R突变为G；(12)第47位氨基酸R突变为G，第11位氨基酸P突变为G，第20位氨基酸R突变为G；(13)第47位氨基酸R突变为G，第11位氨基酸P突变为G，第122位氨基酸R 突变为G；(14)第47位氨基酸R突变为G，第11位氨基酸P突变为G，第129位氨基酸K突变为G；(15)第47位氨基酸R突变为G，第20位氨基酸R突变为G，第122位氨基酸R突变为G；(16)第47位氨基酸R突变为G，第20位氨基酸R突变为G，第129位氨基酸K突变为G；(17)第47位氨基酸R突变为G，第122位氨基酸R突变为G，第129位氨基酸K突变为G；(18)第47位氨基酸R突变为G，第11位氨基酸P突变为G，第20位氨基酸R突变为G，第122位氨基酸R突变为G；(19)第47位氨基酸R突变为G，第11位氨基酸P突变为G，第20位氨基酸R突变为G，第129位氨基酸K突变为G；(20)第47位氨基酸R突变为G，第20位氨基酸R突变为G，第122位氨基酸R突变为G，第129位氨基酸K突变为G；(21)第11位氨基酸P突变为G，第20位氨基酸R突变为G，第47位氨基酸R突变为G，第122位氨基酸R突变为G，第129位氨基酸K突变为G。
在本申请的一些示例中，所述突变体具有以下(1)-(8)任意一种突变：(1)第11位氨基酸P突变为G；(2)第20位氨基酸R突变为G；(3)第47位氨基酸R突变为G；(4)第122位氨基酸R突变为G；(5)第129位氨基酸K突变为G；(6)第47位氨基酸R突变为G，第129位氨基酸K突变为G；(7)第20位氨基酸R突变为G，第129位氨基酸K突变为G；(8)第122位氨基酸R突变为G，第129位氨基酸K突变为G。在本申请的一些示例中，所述突变体能够显著提升修饰或不修饰磷酸作为底物时的磷酸化效率。
在本申请的一些示例中，所述突变体具有(3)、(6)任意一种突变：(3)第47位氨基酸R突变为G；(6)第47位氨基酸R突变为G，第129位氨基酸K突变为G。在本申请的一些示例中，所述突变体能够显著提升修饰磷酸作为底物时的磷酸化效率。
在本申请的另一方面，本申请提出了一种核酸分子。该核酸分子编码如前所述的多核苷酸激酶突变体。在本申请的一些示例中，所述核酸分子编码的多核苷酸激酶突变体可在体内或体外进行大量获得。
在本申请的一些示例中，所述核酸分子选自DNA或RNA。
需要说明的是，对于本申请说明书和权利要求书中所提及的核酸分子，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本申请的另一方面，本申请提出了一种表达载体。该表达载体包括如前所述的核酸分子。在本申请的一些示例中，所述表达载体可用于体外大量表达该多核苷酸激酶突变体。
需要说明的是，在将上述核酸分子连接到载体上时，所述核酸分子与载体上的控制元件可直接或者间接相连，只要这些控制元件能够控制所述核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即，并非来自于载体本身。当然，所述核酸分子与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。常用的载体例如可以为质粒、噬菌体等。
在本申请的一些示例中，所述表达载体也可进一步包括启动子，所述启动子与所述核酸分子可操作的连接。
在本申请的一些示例中，所述表达载体为非致病性病毒载体。在本申请的一些示例中，所述非致病性病毒载体包括腺病毒载体或逆转录病毒载体。
在本申请的另一方面，本申请提出了一种重组细胞。该重组细胞携带如前所述的核酸分子、如前所述的表达载体或表达如前所述的多核苷酸激酶突变体。在本申请的一些示例中，该重组细胞在适合条件下，可用于体外大量制备多核苷酸激酶突变体。
所称的“适合条件”，是指适合本申请所述多核苷酸激酶突变体表达的条件。本领域技术人员容易理解的是，适合所述多核苷酸激酶突变体表达的条件包括但不限于合适的转化方式、合适的转化条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述多核苷酸激酶突变体表达的条件。
在本申请的一些示例中，所述重组细胞是通过转染或者转化所述表达载体获得的。
在本申请的一些示例中，所述重组细胞选自大肠杆菌、酵母或哺乳动物细胞。
需要注意的是，本申请所述重组细胞不受特别限制，可以为原核细胞、真核细胞或噬菌体。所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核细胞可以为包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中，本申请所述重组细胞优选为哺乳动物细胞，包括BHK细胞、CHO细胞、NSO细胞或COS细胞，且不包括动物生殖细胞、受精卵或胚胎干细胞。
在本申请的另一方面，本申请提出了一种重组菌株。该重组菌株携带如前所述的核酸分子、如前所述的表达载体或表达如前所述的多核苷酸激酶突变体。在本申请的一些示例中，所述重组菌株用于体外大量制备多核苷酸激酶突变体。
在本申请的另一方面，本申请提出了一种获得多核苷酸激酶突变体的方法。该方法包括：将如前所述的重组细胞或如前所述的重组菌株在适于蛋白表达的条件下进行培养处理，以便获得所述多核苷酸激酶突变体。在本申请的一些示例中，利用上述方法能够体外大量制备多核苷酸激酶突变体。
需要说明的是，本申请说明书中所述的“适于蛋白表达的条件”，是指适合本申请所述多核苷酸激酶突变体表达的条件。本领域技术人员容易理解的是，适合多核苷酸激酶突变体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适于蛋白表达的条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述多核苷酸激酶突变体表达的条件。
在本申请的又一方面，本申请提出了一种底物磷酸化方法。该方法包括：将待磷酸化核酸底物在如前所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的核酸底物。在本申请的一些示例中，利用本方法能够显著提升修饰磷酸做磷酸供体时的反应效率，提升核苷酸5’端修饰磷酸化效率。
在本申请的一些示例中，所述磷酸基团是以dNTPs、NTPs、核苷修饰核糖核苷酸和脱氧核糖核苷酸中的至少之一的形式提供的。
在本申请的一些示例中，dNTPs包括dATP、dTTP、dCTP和dGTP、dUTP；NTPs包括ATP、TTP、CTP、GTP和UTP。
在本申请的一些示例中，所述核苷修饰核糖核苷酸和脱氧核糖核苷酸可以是碱基修饰的核糖核苷酸和脱氧核糖核苷酸，如3-甲基腺嘌呤核苷酸、7-甲基鸟嘌呤核苷酸、1，N6-亚乙烯基腺嘌呤肌苷酸、次黄嘌呤核苷酸、尿嘧啶核苷酸等，也可以是在糖环处进行修饰的核糖核苷酸和脱氧核糖核苷酸中，如锁核苷酸、肽核苷酸或苏糖核苷酸等。
在本申请的一些示例中，前述dNTPs和NTPs中的至少之一的磷酸根具有修饰。在本申请的一些优选示例中，所述dNTPs和NTPs中的至少之一的γ位磷酸具有修饰。在本申请的一些示例中，含有修饰基团的核苷酸能够阻碍马达蛋白。
在本申请的一些示例中，修饰基团选自烷基基团、硫基基团、硒基基团、荧光团、链霉亲和素和/或生物素、胆固醇、亚甲蓝、二硝基苯酚(DNP)、地高辛配基和/或抗地高辛配基和二苯甲基环辛炔基中的至少之一。
其中，所述烷基基团选自甲基、乙基、丙基、丁基、异丙基、硅烷甲基或硼烷基中的至少之一。示例性地，如式3和式4所示。
其中，R表示修饰基团，所述R基团可以选自烷基基团、硫基基团、硒基基团、荧光团、链霉亲和 素和/或生物素、胆固醇、亚甲蓝、二硝基苯酚(DNP)、地高辛配基和/或抗地高辛配基和二苯甲基环辛炔基中的至少之一。在本申请的一些优选示例中，所述R基团选自烷基，具体地，所述R基团选自甲基。
其中，R表示修饰基团，所述R基团可以选自烷基基团、硫基基团、硒基基团、荧光团、链霉亲和素和/或生物素、胆固醇、亚甲蓝、二硝基苯酚(DNP)、地高辛配基和/或抗地高辛配基和二苯甲基环辛炔基中的至少之一。在本申请的一些优选示例中，所述R基团选自烷基，具体地，所述R基团选自甲基。
在本申请的又一方面，本申请提出了一种建库方法。该方法包括：将待测核酸在本申请第一方面所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸；将所述5’末端具有磷酸化修饰的待测核酸和接头进行连接处理，得到接头连接产物；使所述接头连接产物与马达蛋白结合，以便获得所述测序文库。在本申请的一些示例中，利用本方法进行建库，能够显著降低建库复杂度，有效提高建库成功率。
本领域技术人员应该理解，马达蛋白的实例在本领域中是已知的，本领域技术人员能够根据实际需要选择合适的马达蛋白。在本申请的一个具体示例中，末端包含能够阻断马达蛋白的修饰核苷酸的核酸分子用于单分子测序，例如，用于纳米孔测序时，可以使用移动方向为从核酸分子的5’至3’方向的马达蛋白，也可以使用移动方向为从核酸分子的3’至5’方向的马达蛋白。本领域技术人员可以根据实际需要选择适当的马达蛋白。
在本申请的一些示例中，将待测核酸在多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸，所述多核苷酸激酶突变体具有(1)-(8)任意一种突变，所述磷酸基团是以待磷酸化底物选自dNTPs、NTPs、核苷修饰核糖核苷酸和脱氧核糖核苷酸中的至少之一的形式提供的，所述dNTPs和NTPs中的至少之一的γ位磷酸具有修饰，所述接头具有或不具有乙二醇间臂类修饰。
在本申请的一些示例中，含有修饰基团的核苷酸能够阻碍马达蛋白，进而阻止马达蛋白在测序之前在待测核酸上向前移动。
在本申请的一些具体示例中，将待测核酸在多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸，所述多核苷酸激酶突变体具有(3)或(6)任意一种突变，所述磷酸基团是以待磷酸化底物选自dNTPs、NTPs、核苷修饰核糖核苷酸和脱氧核糖核苷酸中的至少之一的形式提供的，所述dNTPs和NTPs中的至少之一的γ位磷酸具有修饰，所述接头不具有乙二醇间臂类修饰。
在本申请的再一方面，本申请提出了一种核酸测序方法。该方法包括：基于所述待测核酸样本，根据本申请第八方面所述方法构建测序文库；对所述测序文库进行测序，以确定所述待测核酸的核酸序列。在本申请的一些示例中，利用本方法进行核酸测序，能够有效增加测序深度，降低测序成本。
在本申请的一些示例中，所述核酸测序选自纳米孔测序。
示例性地，所述纳米孔测序方法包括：
1)使目标核酸分子的末端包含能够阻断马达蛋白的修饰核苷酸，得到修饰的待测核酸分子；
2)使待测核酸分子与测序接头连接然后与马达蛋白孵育，或使测序接头与马达蛋白孵育然后与测序核酸分子或测序文库连接；
3)将步骤2)的产物加入到含有纳米孔的电生理检测系统中，进行单分子纳米孔测序。
需要说明的是，对于单分子测序方法中所用到的马达蛋白和/或测序接头、以及合适的单分子测序装置，本领域技术人员根据实际需要能够进行适当的选择。
在本申请的再一方面，本申请提出了一种如前所述的多核苷酸激酶突变体、如前所述的核酸分子、如前所述的表达载体、如前所述的重组细胞或如前所述的重组菌株在制备用于底物磷酸化或核酸测序相关产品中的用途。在本申请的一些示例中，前述多核苷酸激酶突变体、核酸分子、表达载体、重组细胞或重组菌株可用于制备底物磷酸化或测序相关产品，如底物磷酸化试剂盒以及测序试剂盒等。
在本申请的再一方面，本申请提出了一种阻滞马达蛋白的方法。该方法包括：
将核酸分子在本申请第一方面所述多核苷酸激酶突变体催化下以及修饰磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有修饰磷酸的核酸产物；
使核酸产物与马达蛋白结合，以便阻滞所述马达蛋白。
示例性地，在纳米孔测序过程中，将5’末端具有修饰磷酸的建库产物与马达蛋白进行孵育处理，防止马达蛋白在测序之前进一步向前解旋。
表1








注：*表示终止。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：T4PNK突变体Mut 1-8的设计、克隆和表达
1、T4 PNK突变体设计
T4 PNK的磷酸化反应机理基于催化γ-磷酸盐从ATP转移至单链或双链DNA、RNA、寡核苷酸或核苷3’-单磷酸盐的5’-OH基团。
根据蛋白结构(图4)，发明人发现R47和K129对T4PNK的酶活位点起到盖子作用，控制反应底物的进(NTP)出(NDP)。特别地，对于γ-烷基取代-核糖核苷酸(γ-alkyl-NTP)作为磷酸供体时，这两个位点的氨基酸残基的体积和电性可能会阻碍底物进入酶活位点，影响反应的正常进行。因此，发明人认为，更小体积且没有显著正电荷的氨基酸残基可能会对这个反应有提升作用。基于此，发明人设计了一系列T4PNK突变体，如表2所示，突变体序列见表1。
表2

2、T4 PNK突变体克隆
对表2中的T4PNK突变体进行基因合成，转入PET.28a(+)质粒中，使用双酶切位点为NdeI和XhoI，因此，表达出来的T4DNA连接酶突变体蛋白N端具有6*His标签和thrombin酶切位点。
3、T4 PNK突变体表达、纯化
将克隆好的PET.28a(+)-T4PNK Mut 1-8质粒转化入大肠杆菌表达菌BL21(DE3)或其衍生菌中。挑取单菌落，接入5mL含有卡纳抗性的LB培养基中，37℃震荡培养过夜。然后转接入1L的LB中，37℃震荡培养至OD600＝0.6-0.8,降温至16℃，加入终浓度500μM的IPTG诱导表达过夜。
T4PNK Mut 1-8的纯化
Buffer A(缓冲液A):20mM Tris-HCl pH 7.5，250mM NaCl,20mM咪唑(Imidazole)；
Buffer B(缓冲液B):20mM Tris-HCl pH 7.5，250mM NaCl,300mM咪唑；
Buffer C(缓冲液C):20mM Tris-HCl pH 7.5，50mM NaCl；
Buffer D(缓冲液D):20mM Tris-HCl pH 7.5，100mM NaCl；
收集表达的T4PNK Mut1-8菌体，使用Buffer A重悬菌体,用细胞破碎仪破碎菌体，然后离心取上清。将上清与事先用Buffer A平衡好的Ni-NTA填料混合，结合1h。收集填料，用Buffer A大量清洗填料，直至没有杂蛋白被洗出。然后在填料中加入Buffer B洗脱目的蛋白。将洗脱得到的目的蛋白过Buffer C平衡好的脱盐柱，进行buffer更换。然后加入适凝血酶(thrombin)，4℃酶切过夜。蛋白浓缩后上分子筛Superdex 200，所用分子筛Bbuffer为Buffer D。收集目的蛋白峰，浓缩，冻存。
结果如图5所示，突变体的纯化结果与野生型T4PNK(1000007870，BGI)接近。
实施例2：磷酸化反应与质谱检测
合成序列SEQ ID NO:19和SEQ ID NO:20，按生产商说明将SEQ ID NO:19和SEQ ID NO:20序列用TE缓冲液(pH＝8)溶解成终浓度100μM的储液。随后分别取10μL储液，加入40μL TE缓冲液(pH＝8)稀释为终浓度20μM的工作液。
分别取上一步稀释得到的30μL SEQ ID NO:19的工作液和30μL SEQ ID NO:20的工作液混合在一起，并使用涡旋振荡仪充分震荡混匀，使用热循环仪加热至70℃孵育10分钟，随后按照0.1℃/s的降温速度降至25℃后继续孵育半小时，至此得到退火后的10μM的双链DNA溶液，命名为short-1，即位进行磷酸化反应所用的双链DNA底物。其中SEQ ID NO:19作为验证T4PNK活性的反应底物链，SEQ ID NO:20作为5’端已磷酸化，不参与反应的参考链。
按照表3配制磷酸化反应混合溶液，突变体为实施例1中制备。置于热循环仪上，孵育37℃30分钟，72℃30分钟，进行磷酸化反应，在目标双链DNA的5’末端添加一个磷酸或者甲基化磷酸。
表3磷酸化反应液配方表
将反应产物使用核酸纯化试剂盒(PCR&DNA Cleanup Kit，New England Biolabs，T1030L)进行纯化后，使用电离子喷雾质谱(ESI-MS)对反应产物进行成分检测。
检测结果表明，只有使用T4PNK Mut 3和Mut 6突变体时，可以将γ-methyl-ATP上的甲基化磷酸转移至双链DNA的5’末端(如图6A至D所示)。在使用普通商业ATP作为磷酸供体时，有明显反应产物，即所有磷酸激酶均可进行磷酸化反应。在使用在γ-methyl-ATP作为磷酸供体时，使用野生型及商业磷酸激酶均无明显反应产物，在使用磷酸激酶突变体时，发现有明显反应产物，即可进行磷酸化反应(表4)。
表4
实施例三：制备测序接头Ad1
本实施例中，通过使化学合成的SEQ ID NO:21和SEQ ID NO:22退火，制得测序接头Ad1。
1、从生工生物订购SEQ ID NO:21和SEQ ID NO:22序列，按生产商说明将SEQ ID NO:21和SEQ ID NO:22序列用TE缓冲液(pH＝8)溶解成终浓度100μM的储液。随后分别取10μL储液，加入40μL TE缓冲液(pH＝8)稀释为终浓度20μM的工作液。
2、分别取上一步稀释得到的30μL SEQ ID NO:21的工作液和30μL SEQ ID NO:22的工作液混合在一起，并使用涡旋振荡仪充分震荡混匀，使用热循环仪加热至70℃孵育10分钟，随后按照0.1℃/s的降温速度降至25℃后继续孵育半小时，至此得到退火后的10μM的接头溶液，接头产物命名为Ad1。使用15％非变性PAGE胶对Ad1产物进行质量检测，结果如图7所示。
实施例四：解旋酶Dda的克隆、表达和纯化
本实施例通过在大肠杆菌中重组表达而制备解旋酶Dda(SEQ ID NO:23)，该解旋酶用作马达蛋白。
1、从生工生物订购全长Dda的cDNA全长序列(SEQ ID NO:24)，将其连接入PET.28a(+)质粒中，使用双酶切位点为Nde1和Xho1，因此表达出来的Dda蛋白N端具有6*His标签和凝血酶(thrombin)酶切位点。
2、将克隆好的PET.28a(+)-Dda质粒转化入ArcticExpress(DE3)感受态细菌(Tolo Biotech.,96183-02)或其衍生菌中。挑取单菌落，接入5mL含有卡那霉素的LB培养基中，37℃震荡培养过夜。然后转接入1L的LB(包含卡那霉素)中，37℃震荡培养至OD600＝0.6-0.8,降温至16℃，加入终浓度500μM的IPTG诱导Dda表达过夜。
3、按照下述配方配制五种缓冲液：
缓冲液A:20mM Tris-HCl pH 7.5，250mM NaCl，20mM咪唑；
缓冲液B:20mM Tris-HCl pH 7.5，250mM NaCl，300mM咪唑；
缓冲液C:20mM Tris-HCl pH 7.5，50mM NaCl；
缓冲液D:20mM Tris-HCl pH 7.5，1000mM NaCl；
缓冲液E:20mM Tris-HCl pH 7.5，100mM NaCl；
4、收集表达Dda的菌体，使用缓冲液A重悬菌体，用细胞破碎仪破碎菌体，然后离心取上清。将上清与事先用缓冲液A平衡好的Ni-NTA填料混合，结合1h。收集填料，用缓冲液A大量清洗填料，直至没有杂蛋白被洗出。然后在填料中加入缓冲液B洗脱Dda。将洗脱得到的Dda过缓冲液C平衡好的脱盐柱，进行缓冲液更换。然后加入适量凝血酶(thrombin)(翊圣生物，20402ES05)，然后加入到缓冲液C平 衡好的ssDNA纤维素(Sigma，D8273-10G)填料中，4℃酶切和结合过夜。收集ssDNA纤维素填料，用缓冲液C洗3-4次，然后用缓冲液D洗脱。将ssDNA纤维素纯化后的蛋白浓缩后上分子筛Superdex 200(Sigma，GE28-9909-44)，所用分子筛缓冲液为缓冲液E。收集目的蛋白峰，浓缩，冻存。采用Nanodrop对纯化后的蛋白进行浓度定量。同时使用HPLC和SDS-PAGE电泳对蛋白进行纯度检测，结果如图8所示。
实施例五：使用γ甲基磷酸ATP产生5’末端甲基磷酸化的测序文库
本实施例通过酶切得到pUC57质粒的片段(SEQ ID NO:25)，用作目标测序核酸序列，使用γ甲基磷酸ATP(式2所示)作为反应底物，通过多聚核苷酸激酶催化磷酸化反应，从而在目标测序核酸序列的5’末端核苷酸上引入甲基磷酸基团(即，5’末端核苷如式5所示)。然后，将得到的磷酸化末端修复产物(图3，甲基磷酸修饰后的5’末端核苷酸)与实施例三制备的Ad1连接，之后再与实施例四制备的解旋酶Dda连接。
1、将空载pUC57质粒转化进DH5α(Vazyme Biotech，C502-02)感受态细菌中，涂布后挑取单克隆送测序，将测序正确(即，包含序列正确的pUC57质粒序列)的克隆菌液补充无菌甘油至甘油终浓度为50％(v/v)，标记后保存于-80℃冰箱。
2、将测序正确的菌液进行大量培养并进行质粒大提(天根，DP117)，使用Qubit dsDNA BR试剂盒(Thermofisher，Q32853)对提取后的质粒进行浓度测定并标记。
3、按下表5配制双酶切体系，将体系放置于热循环仪上，37℃孵育一小时。
表5双酶切体系配制表
4、将Ampure XP磁珠(Beckman Coulter,A63882)提前从冰箱中取出，振荡混匀后置于室温平衡半小时。取50μL平衡后的磁珠加入上述双酶切体系中，振荡混匀并短暂离心后室温静置10分钟。
5、将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
6、使用200μL 75％v/v乙醇溶液将磁珠重悬并使用移液器吹打清洗，将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
7、重复上述乙醇溶液清洗步骤一次，移除上清后将离心管放置于磁力架上静置，待磁珠表面变干燥后，加入22μL TE缓冲液(pH＝8)将磁珠重悬，置于室温静置10分钟。
8、将离心管放置于磁力架上，待磁珠全部吸附到磁力架侧，将上清转移至新的离心管中，至此得到纯化后的酶切产物，其序列如SEQ ID NO:25所示。
9、合成γ甲基磷酸ATP，结构式如式2，并使用超纯水溶解配制100mM浓度储液。取10μL储液，加入90μL超纯水，稀释为10mM浓度工作液。
10、按照下表6在冰上配制5’磷酸化和末端加A反应液，配制好之后放置于热循环仪上，37℃孵育30分钟，72℃孵育30分钟。
表6磷酸化和末端加A反应液配方表

11、将Ampure XP(Beckman Coulter,A63882)磁珠提前从冰箱中取出，振荡混匀后置于室温平衡半小时。取50μL平衡后的磁珠加入上述磷酸化和末端加A反应体系中，振荡混匀并短暂离心后室温静置10分钟。
12、将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
13、使用200μL 75％乙醇溶液将磁珠重悬并使用移液器吹打清洗，将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
14、重复上述乙醇溶液清洗步骤一次，移除上清后将离心管放置于磁力架上静置，待磁珠表面变干燥后，加入22μL TE缓冲液(pH＝8)将磁珠重悬，置于室温静置10分钟。
15、将离心管放置于磁力架上，待磁珠全部吸附到磁力架侧，将21μL上清转移至新的离心管中，至此得到纯化后的SEQ ID NO.25的5’末端的核苷酸被甲基磷酸基团磷酸化的末端修复产物，取1μL纯化产物使用Qubit dsDNA HS试剂盒(Thermofisher,Q32854)试剂盒进行定量。
16、配制330mM Tris，50mM MgCl2，5mM DTT,30％PEG6000，pH 7.6的5X连接缓冲液。
17、按下表7在冰上配制连接体系，其中Gamma-S ATP仅给连接酶供能，本身不会被马达蛋白消耗。将体系置于热循环仪上，25℃孵育30分钟。
表7连接体系配方
18、将Ampure XP磁珠(Beckman Coulter,A63882)预先从冰箱中取出，振荡混匀，置于室温至少半小时进行平衡。平衡后取20μL的磁珠于一个新的离心管中。将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
19、将Ampure XP(Beckman Coulter,A63882)磁珠提前从冰箱中取出，振荡混匀后置于室温平衡半小时。取20μL平衡后的磁珠加入上述磷酸化和末端加A反应体系中，振荡混匀并短暂离心后室温静置10分钟。
20、将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
21、使用200μL 75％乙醇溶液将磁珠重悬并使用移液器吹打清洗，将离心管放置于磁力架上10分钟，待磁珠完全被吸附至磁力架侧，溶液完全变澄清后，小心移除上清。
22、重复上述乙醇溶液清洗步骤一次，移除上清后将离心管放置于磁力架上静置，待磁珠表面变干燥后，加入22μL TE缓冲液(pH＝8)将磁珠重悬，置于室温静置10分钟。
23、将离心管放置于磁力架上，待磁珠全部吸附到磁力架侧，将21μL上清转移至新的离心管中，至此得到纯化后的连接产物，取1μL纯化产物使用Qubit dsDNA HS试剂盒(Thermofisher,Q32854)试剂盒进行定量。
24、取3μL洗脱产物进行非变性聚丙烯酰胺凝胶电泳检测，如图9所示，其中泳道2的SEQ25+Ad1表示甲基磷酸基团磷酸化末端修复的SEQ ID NO.25与Ad1的连接产物)。
25、向容量瓶中100mL 1M Tris-HCL PH 7.5缓冲液及100mL 1M KCl溶液，加入超纯水定容至1L，配制成2X结合缓冲液。
26、按照下表8在冰上配制解旋酶Dda(由实施例四制备)及文库的混合溶液，随后30℃孵育一小时。
表8马达蛋白与文库结合体系
27、使用Qubit DNA HS试剂盒对文库进行定量，标记清楚浓度后，将产物置于4℃冰箱保存备用。
实施例六：纳米孔测序
本实施例构建基于膜片钳平台的纳米孔检测平台，对实施例五制备的目标测序文库(5’末端甲基磷酸化)进行纳米孔测序，以验证本申请构建的能够阻断马达蛋白的测序文库在纳米孔测序中的优点。
1、参考耿佳,郭培宣(“噬菌体phi29DNA包装马达磷脂膜嵌合体在单分子检测及纳米医学领域的应用”.生命科学,2011,23(11):1114-1129)中的单通道电生理检测系统，搭建基于膜片钳平台的纳米孔检测平台，将孔蛋白(Sigma-Aldrich,H9395-5mg)插入磷脂双分子层膜上，形成单通道纳米孔。
2、将实施例六中获得的测序文库加入到该单通道体系中，通过膜片钳体系检测并记录电流振幅变化。
3、使用PNK野生型建库时的典型测序电流图如图10所示，使用PNK Mut 3突变体建库时的典型测序电流图如图11所示，图中纵坐标为电流值(单位：pA)。通过两种典型信号的对比，发现单位时间内(20秒)，使用PNK Mut 3突变体的完整SEQ ID NO:25测序信号显著高于使用PNK野生型进行磷酸化反应时，表示更高的甲基磷酸化反应效率可以得到更多的有效测序文库。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (22)

  1. 一种多核苷酸激酶突变体，其特征在于，
    与野生型多核苷酸激酶相比，包括在以下位点或功能等同位点中的至少一个具有氨基酸突变：
    第47位和第129位；
    所述野生型多核苷酸激酶具有如SEQ ID NO:1所示的氨基酸序列。
  2. 根据权利要求1所述的多核苷酸激酶突变体，其特征在于，所述突变体与所述野生型多核苷酸激酶具有至少90％的同一性，优选95％。
  3. 根据权利要求1所述的多核苷酸激酶突变体，其特征在于，所述突变体具有以下(1)-(3)任意一种突变：
    (1)第47位氨基酸R突变为G；
    (2)第129位氨基酸K突变为G；
    (3)第47位氨基酸R突变为G，第129位氨基酸K突变为G。
  4. 根据权利要求3所述的多核苷酸激酶突变体，其特征在于，所述多核苷酸激酶突变体包含有SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:13所示的序列。
  5. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～4任一项所述的多核苷酸激酶突变体。
  6. 一种表达载体，其特征在于，包含权利要求5所述的核酸分子。
  7. 根据权利要求6所述的表达载体，其特征在于，所述表达载体为非致病性病毒载体。
  8. 一种重组细胞，其特征在于，携带权利要求5所述的核酸分子、权利要求6～7任一项所述的表达载体或表达权利要求1～4任一项所述的多核苷酸激酶突变体。
  9. 根据权利要求8所述的重组细胞，其特征在于，所述重组细胞选自大肠杆菌、酵母或哺乳动物细胞。
  10. 一种重组菌株，其特征在于，携带权利要求5所述的核酸分子、权利要求6～7任一项所述的表达载体或所述重组菌株表达权利要求1～4任一项所述的多核苷酸激酶突变体。
  11. 一种获得多核苷酸激酶突变体的方法，其特征在于，包括：将权利要求8～9任一项所述的重组细胞或权利要求10所述的重组菌株在适于蛋白表达的条件下进行培养处理，以便获得所述多核苷酸激酶突变体。
  12. 一种底物磷酸化方法，其特征在于，包括：
    将待磷酸化核酸底物在权利要求1～4任一项所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的核酸底物。
  13. 根据权利要求12所述的方法，其特征在于，所述磷酸基团是以dNTPs、NTPs、核苷修饰核糖核苷酸和脱氧核糖核苷酸中的至少之一的形式提供的。
  14. 根据权利要求13所述的方法，其特征在于，所述dNTPs和NTPs中的至少之一的磷酸根具有修饰；优选γ位磷酸具有修饰。
  15. 根据权利要求13所述的方法，其特征在于，所述核苷修饰核糖核苷酸和脱氧核糖核苷酸选自3-甲基腺嘌呤核苷酸、7-甲基鸟嘌呤核苷酸、1，N6-亚乙烯基腺嘌呤肌苷酸、次黄嘌呤核苷酸、尿嘧啶核苷酸、在糖环处进行修饰的核糖核苷酸和脱氧核糖核苷酸中的至少之一。
  16. 根据权利要求14所述的方法，其特征在于，修饰基团选自烷基基团、硫基基团、硒基基团、荧光团、链霉亲和素和/或生物素、胆固醇、亚甲蓝、二硝基苯酚(DNP)、地高辛配基和/或抗地高辛配基和二苯甲基环辛炔基中的至少之一。
  17. 根据权利要求16所述的方法，其特征在于，所述烷基基团选自甲基、乙基、丙基、丁基、异丙基、硅烷甲基或硼烷基中的至少之一。
  18. 一种建库方法，其特征在于，包括：
    将待测核酸在权利要求1～4任一项所述多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸；
    将所述5’末端具有磷酸化修饰的待测核酸和接头进行连接处理，得到接头连接产物；
    使所述接头连接产物与马达蛋白结合，以便获得所述测序文库。
  19. 根据权利要求18所述的方法，其特征在于，将待测核酸在多核苷酸激酶突变体催化下以及磷酸基团存在的条件下进行5’端磷酸化处理，以获得5’末端具有磷酸化修饰的待测核酸，所述多核苷酸激酶突变体具有(1)-(3)任意一种突变，所述磷酸基团是以待磷酸化底物选自dNTPs、NTPs、核苷修饰核糖核苷酸和脱氧核糖核苷酸中的至少之一的形式提供的，所述dNTPs和NTPs中的至少之一的γ位磷酸具有修饰，所述接头具有或不具有乙二醇间臂类修饰。
  20. 一种核酸测序方法，其特征在于，包括：
    基于所述待测核酸样本，根据权利要求18～19任一项所述方法构建测序文库；
    对所述测序文库进行测序，以确定所述待测核酸的核酸序列。
  21. 权利要求1～4任一项所述的多核苷酸激酶突变体、权利要求5所述的核酸分子、权利要求6～7任一项所述表达载体、权利要求8～9任一项所述的重组细胞或权利要求10所述的重组菌株在制备用于底物磷酸化或核酸测序相关产品中的用途。
  22. 一种阻滞马达蛋白的方法，其特征在于，包括：
    将所述核酸分子在权利要求1～4任一项所述多核苷酸激酶突变体催化下以及修饰磷酸基团存在的条件下进行5’端磷酸化处理获得末端具有修饰磷酸的核酸分子，将所述核酸分子与马达蛋白进行混合处理，以便阻滞所述马达蛋白。
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