Polynucleotide kinase mutant and use thereof

Abstract

The present invention relates to the technical field of biology, and in particular to a polynucleotide kinase mutant and a use thereof. The polynucleotide kinase mutant comprises, compared with a wild type polynucleotide kinase, at least one amino acid mutation in the following sites or functional equivalent sites: the 47th site and the 129th site; and the wild type polynucleotide kinase has an amino acid sequence as shown in SEQ ID NO: 1.

Description

Polynucleotide kinase mutants and their applications Technical Field

[0001] The present application relates to the field of biotechnology, and in particular, to polynucleotide kinase mutants and applications thereof. Background Art

[0002] Polynucleotide kinase (PNK) is an enzyme that catalyzes the reversible phosphorylation of the 5' end of DNA or RNA. The PNK isolated from T4 bacteriophage (abbreviated as T4PNK) can transfer the gamma phosphate of 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.

[0003] 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.

[0004] The library construction process of nanopore sequencing technology (Figure 1) 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. In addition to conventional nucleic acid molecules, 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.

[0005] In addition to iSp18, there are other modified nucleotide bases that can play the role of spacer sequences, such as deoxyribonucleotides with alkyl substitutions on α-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 whether the sequencing library construction method can be successful. However, current polynucleotide kinases still have some shortcomings, such as low phosphorylation efficiency when using modified phosphate ATP as a phosphate donor.

[0006] Therefore, polynucleotide kinases still need to be improved.

[0007] Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, 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.

[0009] Another object of the present application is to provide a method for modifying and phosphorylating the termini of target nucleic acid molecules, thereby obtaining nucleic acid molecules containing modified nucleotides capable of blocking motor proteins at the termini.

[0010] Furthermore, another object of the present application is to provide a method for blocking motor proteins by causing the target sequencing nucleic acid molecule to contain modified nucleotides capable of blocking motor proteins at the end, thereby preventing the motor protein from further unwinding before sequencing.

[0011] 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.

[0012] Another purpose of this application is to avoid detecting a linker sequence after the spacer during sequencing, thereby further simplifying the bioinformatics analysis and processing process. Another purpose of this application is to effectively improve the blocking efficiency of motor proteins and enhance the linker recognition signal.

[0013] To this end, in the first aspect of the present application, the present application proposes a polynucleotide kinase mutant. According to an embodiment of the present application, the polynucleotide kinase mutant includes an amino acid mutation in at least one of the following sites or functionally equivalent sites compared to the wild-type polynucleotide kinase: position 47 and position 129; the wild-type polynucleotide kinase (PNK WT) has an amino acid sequence as shown in SEQ ID NO: 1 (Table 1). In some examples of the present application, the polynucleotide kinase mutant can be used to catalyze phosphorylation reactions with modified sites, and the phosphorylation reaction efficiency is higher. In some sequencing application scenarios, 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.

[0014] In a second aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the polynucleotide kinase mutant described in the first aspect of the present application. In some examples 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.

[0015] In a third aspect of the present application, an expression vector is provided. According to embodiments of the present application, the expression vector comprises the nucleic acid molecule described in the second aspect of the present application. In some examples of the present application, the expression vector can be used to express the polynucleotide kinase mutant in large quantities in vitro.

[0016] In a fourth aspect, the present application provides a recombinant cell. According to embodiments of the present application, 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. In some examples of the present application, the recombinant cell is used to produce large quantities of the polynucleotide kinase mutant in vitro.

[0017] In a fifth aspect of the present application, a recombinant strain is provided. According to embodiments of the present application, 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. In some examples of the present application, the recombinant strain is used to produce large quantities of the polynucleotide kinase mutant in vitro.

[0018] In a sixth aspect of this application, a method for obtaining a polynucleotide kinase mutant is provided. According to embodiments of this application, the method 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. In some examples of this application, the above method can be used to produce large quantities of polynucleotide kinase mutants in vitro.

[0019] In the seventh aspect of the present application, the present application proposes a substrate phosphorylation method. According to an embodiment of the present application, the method comprises: subjecting the nucleic acid substrate to be phosphorylated 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 substrate with a phosphorylation modification at the 5' end. In some examples of the present application, 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 modification phosphorylation of the nucleotide.

[0020] In the eighth aspect of the present application, the present application proposes a method for library construction. According to an embodiment of the present application, 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 having a phosphorylation modification at the 5' end; connecting the nucleic acid to be tested having 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. In some examples of the present application, 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.

[0021] In the ninth aspect of this application, a nucleic acid sequencing method is provided. According to embodiments of this application, the 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. In some examples of this application, nucleic acid sequencing using this method can effectively increase sequencing depth and reduce sequencing costs.

[0022] In the tenth aspect of the present application, 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. In some examples of the present application, 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.

[0023] In the eleventh aspect of the present application, the present application proposes a method for blocking a motor protein. According to an embodiment of the present application, 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 binding the nucleic acid molecule to the motor protein to block the motor protein. In some sequencing scenarios (such as single-molecule nanopore sequencing), this method can prevent the motor protein from further unwinding before sequencing.

[0024] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0026] FIG1 is a schematic diagram of the process of constructing a nanopore sequencing library in the prior art;

[0027] FIG2 is a schematic diagram of a nanopore sequencing adapter in the prior art;

[0028] FIG3 is a schematic diagram of the structure of an α-alkyl-substituted deoxyribonucleotide (α-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);

[0029] FIG4 is a schematic diagram of the structure of a wild-type T4PNK according to an embodiment of the present application;

[0030] FIG5 is a schematic diagram of the results of PNK protein purification according to an embodiment of the present application;

[0031] FIG6 is a schematic diagram of the mass spectrometry detection results of the phosphorylation reaction according to one embodiment of the present application;

[0032] FIG7 is a schematic diagram of the quality detection results of the sequencing adapter Ad1 according to one embodiment of the present application;

[0033] FIG8 is a schematic diagram of the purity test results of the helicase Dda described in one embodiment of the present application;

[0034] FIG9 is a schematic diagram showing the detection results of the sequencing adapter Ad1 ligation product according to one embodiment of the present application;

[0035] 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;

[0036] FIG11 is a schematic diagram of typical nanopore sequencing signals for constructing a PNK Mut3 library according to one embodiment of the present application. DETAILED DESCRIPTION

[0037] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present application, but should not be understood as limiting the present application.

[0038] In this application, unless otherwise specified, the terms "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. In the description of this application, "plurality" means at least two, for example, two, three, etc., unless otherwise specified.

[0039] In this application, unless otherwise indicated, the term "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).

[0040] In this application, unless otherwise indicated, the term "nucleotide" includes: phosphate, ribose or deoxyribose glycosides and purine or pyrimidine bases. Synthetic and / or naturally occurring nucleotides are included in the definition.

[0041] In this application, unless otherwise indicated, 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 optimal alignment, with the differences between the two sequences being randomly distributed over their entire lengths. In this application, mutants are described based on their mutations at specific residues, which are positioned with reference to the positions of the amino acids in the wild-type polynucleotide kinase amino acid sequence.

[0042] In this application, unless otherwise specified, the term "nucleic acid sequencing" refers to determining the order of base arrangement in the primary structure of a nucleic acid molecule.

[0043] 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. However, 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. For example, when using ATP modified with an alkylated gamma phosphate (as shown in Formula 2) as a phosphate donor, 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.

[0044] In one aspect of the present application, the present application proposes a polynucleotide kinase mutant. 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: the 11th, 20th, 47th, 122nd and 129th positions; the wild-type polynucleotide kinase has an amino acid sequence as shown in SEQ ID NO: 1 (Table 1). In some examples of the present application, 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. In some sequencing application scenarios, 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.

[0045] It should be noted that the 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.

[0046] It should be noted that the so-called functionally equivalent sites refer to sites that have the same function as the designated sites in wild polynucleotide kinases, including positional equivalent sites or homologous sites.

[0047] In some examples herein, 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. In some examples herein, the identity is optionally 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Preferably, the mutant sequence is at least 95% identical.

[0048] In some examples of the present application, 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.

[0049] In some examples of the present application, the mutant has at least one mutation from (1) to (5). In some examples of the present application, 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.

[0050] In some examples of the present application, 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 47 mutated to G, and the amino acid R at position 122 mutated to G; (5) The amino acid R at position 47 mutated to G, and the amino acid P at position 11 mutated to G, and the amino acid R at position 20 mutated to G; (6) The amino acid R at position 47 mutated to G, and the amino acid P at position 11 mutated to G, and the amino acid R at position 122 mutated to G. mutated to G; (14) the 47th amino acid R mutated to G, the 11th amino acid P mutated to G, and the 129th amino acid K mutated to G; (15) the 47th amino acid R mutated to G, the 20th amino acid R mutated to G, and the 122nd amino acid R mutated to G; (16) the 47th amino acid R mutated to G, the 20th amino acid R mutated to G, and the 129th amino acid K mutated to G; (17) the 47th amino acid R mutated to G, the 122nd amino acid R mutated to G, and the 129th amino acid K mutated to G; (18) the 47th amino acid R mutated to G, the 11th amino acid P mutated to G, The amino acid R at position 20 mutated to G, and the amino acid R at position 122 mutated to G; (19) the amino acid R at position 47 mutated to G, the amino acid P at position 11 mutated to G, the amino acid R at position 20 mutated to G, and the amino acid K at position 129 mutated to G; (20) the amino acid R at position 47 mutated to G, the amino acid R at position 20 mutated to G, the amino acid R at position 122 mutated to G, and the amino acid K at position 129 mutated to G; (21) the amino acid P at position 11 mutated to G, the amino acid R at position 20 mutated to G, the amino acid R at position 47 mutated to G, the amino acid R at position 122 mutated to G, and the amino acid K at position 129 mutated to G.

[0051] In some examples of the present application, 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. In some examples of the present application, the mutant can significantly improve the phosphorylation efficiency when modified or unmodified phosphate is used as a substrate.

[0052] In some examples of the present application, 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.

[0053] In another aspect of the present application, a nucleic acid molecule is provided. The nucleic acid molecule encodes a polynucleotide kinase mutant as described above. In some examples 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.

[0054] In some examples of the present application, the nucleic acid molecule is selected from DNA or RNA.

[0055] It should be noted that, for 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. In addition, the nucleic acid sequences in this application include either DNA or RNA forms, and disclosure of one implies disclosure of the other.

[0056] In another aspect of the present application, an expression vector is provided. The expression vector comprises the nucleic acid molecule described above. In some examples of the present application, the expression vector can be used to express the polynucleotide kinase mutant in large quantities in vitro.

[0057] It should be noted that when the above-mentioned nucleic acid molecule is connected to a vector, the 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. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably connected. In this article, "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.

[0058] In some examples of the present application, the expression vector may further include a promoter, which is operably linked to the nucleic acid molecule.

[0059] In some examples of the present application, the expression vector is a non-pathogenic viral vector. In some examples of the present application, the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

[0060] 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. In some examples of the present application, the recombinant cell can be used to produce large quantities of the polynucleotide kinase mutant in vitro under suitable conditions.

[0061] The term "suitable conditions" refers to conditions suitable for the expression of the polynucleotide kinase mutants described herein. Those skilled in the art will readily appreciate that 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.

[0062] In some examples of the present application, the recombinant cell is obtained by transfecting or transforming the expression vector.

[0063] In some examples of the present application, the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

[0064] It should be noted that 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. In some embodiments, 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.

[0065] In another aspect of the present application, a recombinant strain is provided. The recombinant strain 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.

[0066] In another aspect of the present application, a method for obtaining a polynucleotide kinase mutant is provided. The method comprises culturing the aforementioned recombinant cell or recombinant strain under conditions suitable for protein expression to obtain the polynucleotide kinase mutant. In some examples of the present application, the aforementioned method can be used to produce large quantities of polynucleotide kinase mutants in vitro.

[0067] It should be noted that the "conditions suitable for protein expression" referred to in this specification refer to conditions suitable for the expression of the polynucleotide kinase mutants described herein. Those skilled in the art will readily understand that 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. "Conditions 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.

[0068] In another aspect of the present application, a substrate phosphorylation method is provided. The method comprises: subjecting a nucleic acid substrate to be phosphorylated to 5'-terminus phosphorylation under the catalysis of the polynucleotide kinase mutant described above and in the presence of a phosphate group to obtain a nucleic acid substrate having a phosphorylated 5' terminus. In some examples of the present application, 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.

[0069] In some examples of the present application, the phosphate group is provided in the form of at least one of dNTPs, NTPs, nucleoside-modified ribonucleotides, and deoxyribonucleotides.

[0070] In some examples of the present application, dNTPs include dATP, dTTP, dCTP, dGTP, and dUTP; and NTPs include ATP, TTP, CTP, GTP, and UTP.

[0071] In some examples of the present application, the nucleoside modified ribonucleotides and deoxyribonucleotides can be base modified ribonucleotides and deoxyribonucleotides, such as 3-methyladenine nucleotides, 7-methylguanine nucleotides, 1, N 6 -vinylidene adenine inosinic acid, inosine nucleotide, uracil nucleotide, etc., can also be ribonucleotides and deoxyribonucleotides modified at the sugar ring, such as locked nucleotides, peptide nucleotides or threose nucleotides.

[0072] In some examples of the present application, 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 γ-phosphate group. In some examples of the present application, nucleotides containing modified groups can inhibit motor proteins.

[0073] In some examples of the present application, 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.

[0074] The alkyl group is selected from at least one of methyl, ethyl, propyl, butyl, isopropyl, silylmethyl, and boryl groups.

[0075] Wherein, R represents a modifying group, and 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. In some preferred examples of the present application, the R group is selected from an alkyl group, specifically, a methyl group.

[0076] Wherein, R represents a modifying group, and 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. In some preferred examples of the present application, the R group is selected from an alkyl group, specifically, a methyl group.

[0077] In another aspect of the present application, 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 having a phosphorylation modification at the 5' end; connecting the nucleic acid to be tested having 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. In some examples of the present application, 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.

[0078] Those skilled in the art will appreciate that examples of motor proteins are known in the art, and those skilled in the art will be able to select an appropriate motor protein based on actual needs. In a specific example of the present application, a nucleic acid molecule containing a modified nucleotide at its terminus that blocks a motor protein is used for single-molecule sequencing. For example, when used for nanopore 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.

[0079] In some examples of the present application, 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 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 γ-position phosphate of at least one of the dNTPs and NTPs has a modification, and the linker has or does not have an ethylene glycol inter-arm modification.

[0080] In some examples of the present application, 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.

[0081] In some specific examples of the present application, 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), 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 γ-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.

[0082] In another aspect of this application, a nucleic acid sequencing method is provided. The 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. In some examples of this application, nucleic acid sequencing using this method can effectively increase sequencing depth and reduce sequencing costs.

[0083] In some examples of the present application, the nucleic acid sequencing is selected from nanopore sequencing.

[0084] Exemplarily, the nanopore sequencing method comprises:

[0085] 1) 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;

[0086] 2) ligating the nucleic acid molecule to be tested to a sequencing adapter and then incubating with a motor protein, or incubating the sequencing adapter with a motor protein and then ligating with a sequencing nucleic acid molecule or a sequencing library;

[0087] 3) Adding the product of step 2) to an electrophysiological detection system containing a nanopore to perform single-molecule nanopore sequencing.

[0088] It should be noted that those skilled in the art can make appropriate selections of the motor protein and / or sequencing adapter used in the single-molecule sequencing method, as well as a suitable single-molecule sequencing device, according to actual needs.

[0089] In yet another aspect of the present application, 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. In some examples of the present application, 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.

[0090] In another aspect of the present application, a method for blocking a motor protein is provided. The method comprises:

[0091] Phosphorylating the nucleic acid molecule at its 5' end 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 its 5' end;

[0092] The nucleic acid product is allowed to bind to the motor protein so as to block the motor protein.

[0093] For example, during the nanopore sequencing process, the library product with a modified phosphate at the 5' end is incubated with the motor protein to prevent the motor protein from further unwinding before sequencing.

[0094] Table 1 Note: * indicates termination.

[0095] The present invention will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the examples, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not indicate the manufacturer are conventional products that can be obtained commercially.

[0096] Example 1: Design, cloning and expression of T4PNK mutants Mut 1-8

[0097] 1. T4 PNK mutant design

[0098] The phosphorylation reaction mechanism of T4 PNK is based on the catalytic transfer of γ-phosphate from ATP to the 5'-OH group of single-stranded or double-stranded DNA, RNA, oligonucleotides or nucleoside 3'-monophosphates.

[0099] Based on the protein structure (Figure 4), the inventors discovered that R47 and K129 act as a lid on the active site of T4PNK, controlling the entry (NTP) and exit (NDP) of the reaction substrate. In particular, when γ-alkyl-substituted ribonucleotides (γ-alkyl-NTPs) are used as phosphate donors, the volume and electrical properties of the amino acid residues at these two sites may hinder the substrate from entering the active site, affecting the normal progress of the reaction. Therefore, the inventors believe that amino acid residues with smaller volume and no significant positive charge may have a promoting effect on this reaction. Based on this, the inventors designed a series of T4PNK mutants, as shown in Table 2, and the mutant sequences are shown in Table 1.

[0100] Table 2

[0101] 2. T4 PNK mutant cloning

[0102] The T4PNK mutants in Table 2 were gene synthesized and transferred into the PET.28a(+) plasmid using double restriction enzyme sites NdeI and XhoI. Thus, the expressed T4DNA ligase mutant protein had a 6*His tag and a thrombin restriction enzyme site at the N-terminus.

[0103] 3. Expression and purification of T4 PNK mutants

[0104] Transform the cloned PET.28a(+)-T4PNK Mut 1-8 plasmid into E. coli BL21(DE3) or its derivatives. Pick a single colony and inoculate it into 5 mL of LB medium containing kanamycin resistance. Cultivate with shaking at 37°C overnight. Then, transfer the colony to 1 L of LB medium and incubate with shaking at 37°C until the OD600 reaches 0.6-0.8. Cool the culture to 16°C and induce expression overnight with IPTG at a final concentration of 500 μM.

[0105] Purification of T4PNK Mut 1-8

[0106] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0107] Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0108] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0109] Buffer D: 20 mM Tris-HCl pH 7.5, 100 mM NaCl;

[0110] Collect the expressed T4PNK Mut1-8 cells, resuspend them in Buffer A, disrupt them with a cell disruptor, and centrifuge to collect the supernatant. Mix the supernatant with Ni-NTA filler previously equilibrated with Buffer A and allow to bind for 1 hour. Collect the filler and wash it extensively with Buffer A until all contaminants 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 and perform a buffer exchange. Then, add thrombin and digest overnight at 4°C. After protein concentration, apply it to Superdex 200 using Buffer D as the buffer for the sieve B buffer. Collect the target protein peak, concentrate it, and freeze it.

[0111] The results are shown in FIG5 , and the purification results of the mutant were close to those of the wild-type T4PNK (1000007870, BGI).

[0112] Example 2: Phosphorylation reaction and mass spectrometry detection

[0113] Sequences SEQ ID NO: 19 and SEQ ID NO: 20 were synthesized and dissolved in TE buffer (pH = 8) to a final concentration of 100 μM. Subsequently, 10 μL of each stock solution was diluted to a final concentration of 20 μM by adding 40 μL of TE buffer (pH = 8).

[0114] 30 μL of the working solution of SEQ ID NO: 19 and 30 μL of the working solution of SEQ ID NO: 20, obtained by dilution in the previous step, were mixed together and thoroughly vortexed. The solution was heated to 70°C in a thermal cycler and incubated for 10 minutes. The temperature was then cooled to 25°C at a rate of 0.1°C / s and incubated for another half hour. This yielded a 10 μM annealed double-stranded DNA solution, designated short-1, which served as the double-stranded DNA substrate for the phosphorylation reaction. SEQ ID NO: 19 served as the reaction substrate strand for verifying T4PNK activity, while SEQ ID NO: 20 served as the reference strand, phosphorylated at the 5' end and not participating in the reaction.

[0115] Prepare the phosphorylation reaction mixture according to Table 3. 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.

[0116] Table 3 Phosphorylation reaction solution formula

[0117] 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).

[0118] The test results showed that only the T4PNK Mut 3 and Mut 6 mutants could transfer the methylated phosphate on γ-methyl-ATP to the 5' end of double-stranded DNA (as shown in Figures 6A to D). When using standard commercial ATP as a phosphate donor, a clear reaction product was observed, indicating that all phosphokinases were capable of phosphorylation. When using γ-methyl-ATP as a phosphate donor, no clear reaction product was observed with either wild-type or commercial phosphokinases. However, when using the phosphokinase mutants, a clear reaction product was observed, indicating that phosphorylation could proceed (Table 4).

[0119] Table 4

[0120] Example 3: Preparation of sequencing adapter Ad1

[0121] In this example, sequencing adapter Ad1 was prepared by annealing chemically synthesized SEQ ID NO: 21 and SEQ ID NO: 22.

[0122] 1. Order SEQ ID NO: 21 and SEQ ID NO: 22 from Sangon Biotechnology and dissolve them in TE buffer (pH 8) to a final concentration of 100 μM stock solution according to the manufacturer's instructions. Then, take 10 μL of each stock solution and add 40 μL of TE buffer (pH 8) to dilute the solution to a final concentration of 20 μM working solution.

[0123] 2. 30 μL of the working solution of SEQ ID NO: 21 and 30 μL of the working solution of SEQ ID NO: 22 obtained by dilution in the previous step were mixed together and thoroughly vortexed. The mixture was heated to 70°C in a thermal cycler and incubated for 10 minutes. The temperature was then cooled to 25°C at a rate of 0.1°C / s and incubated for another half hour. This yielded a 10 μM annealed linker solution. The linker product was designated Ad1. The Ad1 product was quality tested using a 15% native PAGE gel, as shown in Figure 7.

[0124] Example 4: Cloning, expression and purification of helicase Dda

[0125] In this example, helicase Dda (SEQ ID NO: 23) was prepared by recombinant expression in Escherichia coli, and the helicase was used as a motor protein.

[0126] 1. Order the full-length Dda cDNA sequence (SEQ ID NO: 24) from Sangon Biotechnology and ligate it into the PET.28a(+) plasmid. Use the double restriction sites Nde1 and Xho1 so that the expressed Dda protein has a 6*His tag and a thrombin restriction site at the N-terminus.

[0127] 2. Transform the cloned PET.28a(+)-Dda plasmid into ArcticExpress (DE3) competent bacteria (Tolo Biotech., 96183-02) or its derivatives. Pick a single colony and inoculate it into 5 mL of LB medium containing kanamycin. Cultivate with shaking at 37°C overnight. Then, inoculate the culture into 1 L of LB medium (containing kanamycin) and incubate with shaking at 37°C until the OD600 reaches 0.6-0.8. Cool the culture to 16°C and induce Dda expression overnight by adding IPTG at a final concentration of 500 μM.

[0128] 3. Prepare five buffer solutions according to the following formula:

[0129] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0130] Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0131] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0132] Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

[0133] Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl;

[0134] 4. Collect Dda-expressing bacteria, resuspend them in buffer A, disrupt them with a cell disruptor, and centrifuge to collect the supernatant. Mix the supernatant with Ni-NTA filler previously equilibrated with buffer A and allow to bind for 1 hour. Collect the filler and wash it extensively with buffer A until no impurities are washed out. Then, add buffer B to the filler to elute Dda. Pass the eluted Dda through a desalting column equilibrated with buffer C to perform buffer exchange. Then, add an appropriate amount of thrombin (Yishen Bio, 20402ES05) to the ssDNA cellulose filler equilibrated with buffer C. Digest and bind overnight at 4°C. Collect the ssDNA cellulose filler, wash it 3-4 times with buffer C, and then elute it with buffer D. Concentrate the protein purified from the ssDNA cellulose and apply it to a Superdex 200 molecular sieve (Sigma, GE28-9909-44) using buffer E. The target protein peak was collected, concentrated, and frozen. The purified protein concentration was quantified using Nanodrop. Protein purity was also tested using HPLC and SDS-PAGE electrophoresis. The results are shown in Figure 8.

[0135] Example 5: Generating a 5'-terminal methylphosphorylated sequencing library using γ-methylphosphoATP

[0136] In this example, 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. γ-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.

[0137] 1. Transform the empty pUC57 plasmid into DH5α (Vazyme Biotech, C502-02) competent bacteria. After plating, pick a single clone for sequencing. Supplement the clone solution with correct sequencing (i.e., containing the correct pUC57 plasmid sequence) with sterile glycerol to a final glycerol concentration of 50% (v / v), label it, and store it in a -80°C refrigerator.

[0138] 2. The correctly sequenced bacterial suspension was cultured in large quantities and the plasmid was extracted (Tiangen, DP117). The concentration of the extracted plasmid was measured and labeled using the Qubit dsDNA BR kit (Thermofisher, Q32853).

[0139] 3. Prepare the double enzyme digestion system according to Table 5 below, place the system on a thermal cycler, and incubate at 37°C for one hour.

[0140] Table 5 Preparation of double enzyme digestion system

[0141] 4. Remove Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator in advance, vortex to mix, and then equilibrate at room temperature for half an hour. Add 50 μL of the equilibrated magnetic beads to the double enzyme digestion system, vortex to mix, centrifuge briefly, and let stand at room temperature for 10 minutes.

[0142] 5. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0143] 6. Use 200 μL 75% v / v ethanol solution to resuspend the magnetic beads and use a pipette to wash them. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0144] 7. Repeat the ethanol solution washing step once. After removing the supernatant, place the centrifuge tube on a magnetic stand and let it stand. After the surface of the magnetic beads becomes dry, add 22 μL TE buffer (pH = 8) to resuspend the magnetic beads and let it stand at room temperature for 10 minutes.

[0145] 8. Place the centrifuge tube on a magnetic stand. After all the magnetic beads are adsorbed to the side of the magnetic stand, transfer the supernatant to a new centrifuge tube. The purified enzyme digestion product is obtained, and its sequence is shown in SEQ ID NO: 25.

[0146] 9. Synthesize γ-methyl phosphate ATP, with the structural formula shown in Formula 2, and dissolve it in ultrapure water to prepare a 100 mM stock solution. Take 10 μL of the stock solution and add 90 μL of ultrapure water to dilute it to a 10 mM working solution.

[0147] 10. Prepare 5' phosphorylation and end addition A reaction solutions on ice according to Table 6 below. Place the solution in a thermal cycler and incubate at 37°C for 30 minutes and 72°C for 30 minutes.

[0148] Table 6 Phosphorylation and terminal addition A reaction solution formula

[0149] 11. Remove Ampure XP (Beckman Coulter, A63882) magnetic beads from the refrigerator in advance, vortex to mix, and then equilibrate at room temperature for half an hour. Add 50 μL of the equilibrated beads to the phosphorylation and terminal addition reaction system described above, vortex to mix, briefly centrifuge, and let stand at room temperature for 10 minutes.

[0150] 12. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0151] 13. Use 200 μL 75% ethanol solution to resuspend the magnetic beads and use a pipette to wash them. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0152] 14. Repeat the above ethanol solution washing step once, remove the supernatant and place the centrifuge tube on a magnetic stand. After the surface of the magnetic beads becomes dry, add 22 μL TE buffer (pH = 8) to resuspend the magnetic beads and let it stand at room temperature for 10 minutes.

[0153] 15. Place the centrifuge tube on a magnetic rack. When all the magnetic beads are adsorbed to the side of the magnetic rack, transfer 21 μL of the supernatant to a new centrifuge tube. This will obtain the purified end-repair product in which the 5'-terminal nucleotide of SEQ ID NO. 25 is phosphorylated with a methylphosphate group. Take 1 μL of the purified product and quantify it using the Qubit dsDNA HS kit (Thermofisher, Q32854).

[0154] 16. Prepare 5X ligation buffer containing 330 mM Tris, 50 mM MgCl2, 5 mM DTT, 30% PEG6000, pH 7.6.

[0155] 17. Prepare the ligation system on ice according to Table 7. Gamma-S ATP is used only to power the ligase and is not consumed by the motor protein. Incubate the system in a thermal cycler at 25°C for 30 minutes.

[0156] Table 7 Connection system formula

[0157] 18. Remove Ampure XP magnetic beads (Beckman Coulter, A63882) from the refrigerator, vortex to mix, and allow to equilibrate at room temperature for at least half an hour. After equilibration, transfer 20 μL of the beads to a new centrifuge tube. Place the tube on a magnetic stand for 10 minutes. Once the beads are completely absorbed and the solution becomes clear, carefully remove the supernatant.

[0158] 19. Remove Ampure XP (Beckman Coulter, A63882) magnetic beads from the refrigerator, vortex to mix, and equilibrate at room temperature for half an hour. Add 20 μL of the equilibrated beads to the phosphorylation and terminal addition reaction system described above, vortex to mix, centrifuge briefly, and let stand at room temperature for 10 minutes.

[0159] 20. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0160] 21. Use 200 μL of 75% ethanol solution to resuspend the magnetic beads and use a pipette to wash them. Place the centrifuge tube on the magnetic stand for 10 minutes. After the magnetic beads are completely adsorbed to the side of the magnetic stand and the solution becomes completely clear, carefully remove the supernatant.

[0161] 22. Repeat the above ethanol solution washing step once, remove the supernatant and place the centrifuge tube on a magnetic stand. After the surface of the magnetic beads becomes dry, add 22 μL TE buffer (pH = 8) to resuspend the magnetic beads and let it stand at room temperature for 10 minutes.

[0162] 23. Place the centrifuge tube on the magnetic rack. After all the magnetic beads are adsorbed to the side of the magnetic rack, transfer 21 μL of the supernatant to a new centrifuge tube to obtain the purified ligation product. Take 1 μL of the purified product and quantify it using the Qubit dsDNA HS kit (Thermofisher, Q32854).

[0163] 24. Take 3 μL of the eluted product and perform native polyacrylamide gel electrophoresis, as shown in FIG9 , where SEQ25+Ad1 in lane 2 represents the ligation product of SEQ ID NO. 25 and Ad1 after repair of the methyl phosphate group phosphorylation end).

[0164] 25. Add 100 mL of 1 M Tris-HCl pH 7.5 buffer and 100 mL of 1 M KCl solution to a volumetric flask and dilute to 1 L with ultrapure water to prepare 2X binding buffer.

[0165] 26. Prepare a mixed solution of helicase Dda (prepared in Example 4) and the library on ice according to Table 8 below, and then incubate at 30°C for one hour.

[0166] Table 8 Motor protein and library binding system

[0167] 27. Use the Qubit DNA HS kit to quantify the library. After clearly marking the concentration, store the product in a 4°C refrigerator until use.

[0168] Example 6: Nanopore sequencing

[0169] In this example, a 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.

[0170] 1. With reference to the single-channel electrophysiological detection system in Geng Jia and Guo Peixuan (“Application of phage phi29 DNA packaging motor phospholipid membrane chimera in single molecule detection and nanomedicine”. Life Science, 2011, 23(11):1114-1129), a nanopore detection platform based on the patch clamp platform was constructed, and porin (Sigma-Aldrich, H9395-5mg) was inserted into the phospholipid bilayer membrane to form a single-channel nanopore.

[0171] 2. 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.

[0172] 3. A typical sequencing current plot for library construction using the wild-type PNK is shown in Figure 10, and a typical sequencing current plot for library construction using the PNK Mut 3 mutant is shown in Figure 11, with the ordinate representing the current value (in pA). Comparison of these two typical signals reveals that per unit time (20 seconds), the sequencing signal for the complete SEQ ID NO: 25 using the PNK Mut 3 mutant is significantly higher than that obtained using the wild-type PNK for phosphorylation, indicating that higher methyl phosphorylation efficiency can yield more effective sequencing libraries.

[0173] In the description of this specification, 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. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, 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.

[0174] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A polynucleotide kinase mutant, characterized in that: Compared to the wild-type polynucleotide kinase, the polynucleotide kinase comprises an amino acid mutation in at least one of the following positions or functionally equivalent positions: 47th and 129th; The wild-type polynucleotide kinase has an amino acid sequence as shown in SEQ ID NO:

1.

2. The polynucleotide kinase mutant according to claim 1, characterized in that The mutant has at least 90% identity to the wild-type polynucleotide kinase, preferably 95%.

3. The polynucleotide kinase mutant according to claim 1, characterized in that The mutant has any one of the following mutations (1)-(3): (1) The amino acid R at position 47 mutated to G; (2) amino acid K at position 129 mutated to G; (3) The amino acid R at position 47 mutated to G, and the amino acid K at position 129 mutated to G.

4. The polynucleotide kinase mutant according to claim 3, characterized in that The polynucleotide kinase mutant comprises the sequence shown in SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO:

13.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polynucleotide kinase mutant according to any one of claims 1 to 4.

6. An expression vector, characterized in that Comprising the nucleic acid molecule according to claim 5.

7. The expression vector according to claim 6, characterized in that The expression vector is a non-pathogenic viral vector.

8. A recombinant cell, characterized in that Carrying the nucleic acid molecule according to claim 5, the expression vector according to any one of claims 6 to 7, or expressing the polynucleotide kinase mutant according to any one of claims 1 to 4.

9. The recombinant cell according to claim 8, characterized in that The recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

10. A recombinant strain, characterized in that The nucleic acid molecule according to claim 5, the expression vector according to any one of claims 6 to 7 or the recombinant strain expresses the polynucleotide kinase mutant according to any one of claims 1 to 4.

11. A method for obtaining a polynucleotide kinase mutant, characterized in that: include: The recombinant cell according to any one of claims 8 to 9 or the recombinant strain according to claim 10 is cultured under conditions suitable for protein expression to obtain the polynucleotide kinase mutant.

12. A substrate phosphorylation method, characterized in that: include: The nucleic acid substrate to be phosphorylated is subjected to 5'-end phosphorylation treatment under the catalysis of the polynucleotide kinase mutant according to any one of claims 1 to 4 and in the presence of a phosphate group to obtain a nucleic acid substrate with phosphorylation modification at the 5' end.

13. The method according to claim 12, characterized in that The phosphate groups are provided in the form of at least one of dNTPs, NTPs, nucleoside-modified ribonucleotides, and deoxyribonucleotides.

14. The method according to claim 13, characterized in that The phosphate group of at least one of the dNTPs and NTPs is modified; preferably the γ-position phosphate is modified.

15. The method according to claim 13, characterized in that The nucleoside modified ribonucleotides and deoxyribonucleotides are selected from 3-methyladenine nucleotides, 7-methylguanine nucleotides, 1, N 6 - at least one of vinylidene adenine inosinic acid, inosine nucleotide, uracil nucleotide, ribonucleotide modified at the sugar ring, and deoxyribonucleotide.

16. The method according to claim 14, characterized in that The modifying group is 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 diphenylmethylcyclooctyne group.

17. The method according to claim 16, characterized in that The alkyl group is selected from at least one of a methyl group, an ethyl group, a propyl group, a butyl group, an isopropyl group, a silylmethyl group, and a boryl group.

18. A library construction method, characterized in that: include: The nucleic acid to be tested is subjected to 5'-end phosphorylation treatment under the catalysis of the polynucleotide kinase mutant according to any one of claims 1 to 4 and in the presence of a phosphate group to obtain a nucleic acid to be tested having a phosphorylation modification at the 5' end; ligating the nucleic acid to be tested having a phosphorylation modification at the 5' end to a linker to obtain a linker ligation product; The adapter-ligated product is allowed to bind to the motor protein to obtain the sequencing library.

19. The method according to claim 18, characterized in that 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 the mutations (1) to (3), 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 γ-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.

20. A nucleic acid sequencing method, characterized in that: include: Based on the nucleic acid sample to be tested, constructing a sequencing library according to the method according to any one of claims 18 to 19; The sequencing library is sequenced to determine the nucleic acid sequence of the nucleic acid to be tested.

21. Use of the polynucleotide kinase mutant according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to any one of claims 6 to 7, the recombinant cell according to any one of claims 8 to 9, or the recombinant strain according to claim 10 in the preparation of products related to substrate phosphorylation or nucleic acid sequencing.

22. A method for blocking a motor protein, characterized in that: include: The nucleic acid molecule is subjected to 5'-end phosphorylation treatment under the catalysis of the polynucleotide kinase mutant according to any one of claims 1 to 4 and in the presence of a modified phosphate group to obtain a nucleic acid molecule with a modified phosphate at the end, and the nucleic acid molecule is mixed with a motor protein to block the motor protein.

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