A modified MPK2 helicase and its application

Abstract

The present invention provides a modified MPK2 helicase and its application, relating to the field of gene sequencing technology. The MPK2 helicase provided by the present invention includes a covalent linker introduced into a specific region of a parent MPK2 helicase; wherein the modified MPK2 helicase retains its ability to control the movement of polynucleotides. This invention solves the problem of polynucleotides easily falling off the helicase.

Description

Technical Field

[0001] The present invention relates to the technical fields of gene sequencing, molecular detection and clinical detection, and in particular to a modified MPK2 helicase and applications thereof. Background Art

[0002] Motor proteins are essential components in nanopore sequencing. The most commonly used motor protein is the helicase, which not only controls the translocation of single-stranded nucleic acids through the nanopore but also enables double-stranded DNA or RNA-DNA to unwind into single-stranded molecules for passage through the nanopore. The DNA-dependent ATPase (MPK2) helicase from Candida tropicalis MYA-3404 is useful for controlling polynucleotide translocation during strand sequencing, but it still suffers from weak binding to single-stranded nucleic acids, resulting in nucleic acids passing through the nanopore too quickly.

[0003] It is known in the prior art to mutate or modify helicase to improve its performance, see CN116334030A and CN107109380A. Summary of the Invention

[0004] In order to solve the existing problems, the present invention proposes the following technical solutions:

[0005] The present invention first provides a modified MPK2 helicase, wherein the parent sequence of the modified MPK2 helicase is SEQ ID NO: 1 or a sequence having 80% homology with SEQ ID NO: 1; the modified MPK2 helicase is covalently linked between at least one amino acid residue at positions 80 to 90 and at least one amino acid residue at positions 350 to 360, wherein the amino acid positions are based on SEQ ID NO: 1.

[0006] Preferably, the wild-type sequence of the modified MPK2 helicase is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% homologous to SEQ ID NO:1.

[0007] Preferably, the parent MPK2 helicase is a DNA-dependent ATPase (MPK2) helicase from Candida tropicalis MYA-3404, whose amino acid sequence is shown in SEQ ID NO: 1, with a length of 439 aa, obtained by truncation of the polypeptide with NCBI number XP_002548501.1.

[0008] In a specific embodiment of the present invention, the covalently linked amino acid residues are preferably at least one of positions 80 to 90 and at least one of positions 350 to 360; more preferably at least one of positions 85 to 90 and at least one of positions 354 to 360; more preferably at least one of positions 85, 86, 87, 88, 89 and 90 and at least one of positions 354, 355, 356, 357, 358, 359 or 360.

[0009] In a specific embodiment of the present invention, the covalent linkage is achieved by replacing or inserting cysteine ​​or an unnatural amino acid.

[0010] In a specific embodiment of the present invention, it is preferred to introduce at least one cysteine ​​each at positions 80-90 and 350-360; it is preferred to introduce at least one cysteine ​​each at positions 85-90 and 354-360; specifically: at least one of K85C, K86C, I87C, I88C, K89C, N90C and at least one of E354C, D355C, G356C, K357C, G35C8, N359C or P360C; more preferably N90C and K357C.

[0011] In a specific embodiment of the present invention, the covalent linkage between the two cysteines is achieved through a disulfide bond or linker molecules; preferably, the linker molecules include: BMOE (bismaleimide ethane), BMB (1,4-bismaleimide butane), BMH (bismaleimide hexane), DTME (dithiomaleimide ethane), TMEA (tris(2-bismaleimidoethyl)amine), Bis(PEG)2 (1,8-bismaleimide-diethylene glycol) and Bis(PEG)3 (1,11-bismaleimide-triethylene glycol).

[0012] In another embodiment of the present invention, at least one unnatural amino acid is introduced into positions 80-90 and 350-360, preferably at least one unnatural amino acid is introduced into positions 85-90 and 354-360 to achieve covalent linkage.

[0013] Preferably, the covalent linkage is achieved through a bioorthogonal reaction involving unnatural amino acids; more preferably, the covalent linkage is achieved through a bioorthogonal reaction involving linker molecules.

[0014] Preferably, the bioorthogonal reaction comprises a condensation reaction between a ketocarbonyl compound and hydroxylamine / hydrazide, a 1,3-dipolar cycloaddition reaction, a Staudinger ligation reaction, an inverse electron demand Diels-Alder reaction (IEDDA) reaction, or a photoinduced ligation reaction.

[0015] Preferably, the unnatural amino acid comprises a ketone group, an oxime / hydroxylamine group, an azide group, an alkyne group, a ring-strained olefin group, a tetrazine group, or a diazine group.

[0016] Preferably, the modified MPK2 helicase further comprises amino acid replacement of one or more cysteines in the parent sequence; preferably, all cysteines in SEQ ID NO: 1 are replaced; and the total number of cysteines in SEQ ID NO: 1 is 5.

[0017] Preferably, the original cysteines include: C60, C141, C171, C203 and C286.

[0018] Preferably, the modified MPK2 helicase further includes replacing the original cysteine ​​with another natural amino acid or a non-natural amino acid; the other natural amino acid or non-natural amino acid does not contain a sulfhydryl group; preferably, the other 19 natural amino acids include glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine; more preferably, threonine and / or serine.

[0019] Preferably, the original cysteine ​​is replaced by threonine and / or serine; preferably, the original cysteine ​​is replaced by the following amino acids: C60S, C141S, C171T, C203T and C286S.

[0020] In a specific embodiment of the present invention, the modified MPK2 helicase comprises the amino acid sequence shown in SEQ ID NO:2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% homology to the amino acid sequence shown in SEQ ID NO:2, and there is a covalent connection between N90C and K357C.

[0021] Preferably, the covalent linkage is achieved through bismaleimidoethane, Bis(PEG)2 or Bis(PEG)3; more preferably, the covalent linkage is achieved through bismaleimidoethane.

[0022] The present invention also provides a sequencing element and a sequencing device comprising the modified MPK2 helicase.

[0023] The present invention also provides sequencing applications of the modified MPK2 helicase.

[0024] On the other hand, the present invention provides a mutant MPK2 helicase, wherein the sequence of the MPK2 helicase before modification is SEQ ID NO: 1 or a sequence having 90% identity with SEQ ID NO: 1; and at least one amino acid residue at positions 80 to 90 and at least one amino acid residue at positions 350 to 360 are replaced with cysteine ​​or a non-natural amino acid.

[0025] In a specific embodiment of the present invention, it is preferred to introduce at least one cysteine ​​or unnatural amino acid at positions 80-90 and 350-360; it is preferred to introduce at least one cysteine ​​at positions 85-90 and 354-360; specifically, at least one of K85C, K86C, I87C, I88C, K89C, N90C and at least one of E354C, D355C, G356C, K357C, G35C8, N359C or P360C; more preferably, N90C and K357C.

[0026] Preferably, in the present invention, the mutated MPK2 helicase further comprises amino acid replacement of one or more original cysteines; preferably, all cysteines in SEQ ID NO: 1 are further replaced; and the total number of cysteines in SEQ ID NO: 1 is 5.

[0027] Preferably, the original cysteines include: C60, C141, C171, C203 and C286 (based on the amino acid positions of SEQ ID NO: 1).

[0028] Preferably, the mutated MPK2 helicase further comprises replacing the original cysteine ​​with another natural amino acid or a non-natural amino acid; the other natural amino acid or non-natural amino acid does not contain a sulfhydryl group; preferably, the other 19 natural amino acids include glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine; more preferably, threonine and / or serine.

[0029] Preferably, the original cysteine ​​is replaced by threonine and / or serine; preferably, the original cysteine ​​is replaced by the following amino acids: C60S, C141S, C171T, C203T and C286S.

[0030] Preferably, the mutated MPK2 helicase comprises the amino acid sequence shown in SEQ ID NO:2, or has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% homology to the amino acid sequence shown in SEQ ID NO:2.

[0031] The present invention also provides a polynucleotide encoding the above-mentioned mutated MPK2 helicase.

[0032] The present invention also provides biological materials such as vectors or recombinant microorganisms comprising the polynucleotide or expressing the mutated MPK2 helicase.

[0033] The present invention also provides a method for preparing the modified MPK2 helicase, comprising the following steps:

[0034] 1) Providing the above-mentioned mutant MPK2 helicase;

[0035] 2) reacting under covalent bonding conditions to obtain the modified MPK2 helicase.

[0036] Preferably, the reaction in step 2) is a reaction involving thiol groups.

[0037] Preferably, the reaction in step 2) is a bioorthogonal reaction.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention has demonstrated that a modified MPK2 helicase can control the movement of a polynucleotide through a biological nanopore, particularly under the influence of an electric field. The helicase can enable the movement of a target polynucleotide through the nanopore in a controlled and stepwise manner.

[0040] (2) The present invention also demonstrates that the stability of the binding between the MPK2 helicase of the present invention and the polynucleotide is improved by modifying the linker molecule. In particular, when the length of the polynucleotide chain increases, the helicase of the present invention can still stably control the movement of the polynucleotide without falling off the polynucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the SDS-PAGE gel electrophoresis image of the purified MPK2 helicase;

[0042] Figure 2 Schematic diagram of fluorescence analysis for detecting helicase activity;

[0043] Figure 34-10% SDS-PAGE gel electrophoresis image of MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S;

[0044] Figure 4 Gel migration diagram of MPK2 helicase binding to DNA before and after modification.

[0045] Band 4 represents the pure DNA substrate without added protein, bands 1, 2, and 3 represent the results of the binding of parent MPK2 to the DNA substrate at a molar ratio of 1:1, 3:1, and 5:1, respectively, and bands 5, 6, and 7 represent the results of the binding of the modified MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S to the substrate DNA at a molar ratio of 1:1, 3:1, and 5:1;

[0046] Figure 5 is a schematic diagram of DNA construct X;

[0047] Figure 6 To show that MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S is able to control the movement of the intact DNA construct X through the nanopore;

[0048] Figure 7 To show Figure 6 The enlarged image of the area where MPK2 helicase controls the movement of DNA construct X through the pore is shown in FIG. The X-axis represents time (s) and the Y-axis represents current (nA);

[0049] Figure 8 Figure 3. Transwell signal plot of the no-helicase control. DETAILED DESCRIPTION

[0050] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0051] Technical terms

[0052] As used in the present invention, "covalent linkage" refers to the connection between two or more amino acid residues at specific positions of MPK2 helicase formed by any chemical group, including direct connection through the functional groups of the amino acid residues, or connection through a bifunctional linker molecule or a multifunctional linker molecule.

[0053] For direct connection, for example, two cysteines can form a disulfide bond, or two amino acid residues carrying bioorthogonal functional groups such as azide or alkyne can form a triazole connection under Cu ion catalysis.

[0054] When a bifunctional linker molecule is used to form a covalent linkage, the "covalent linkage" has a typical structure of AL-A1, wherein A and A1 are structures formed by coupling the functional group of the linker with the functional group of the amino acid residue, such as thioether (for example, formed by the reaction of the sulfhydryl group of cysteine ​​with maleimide) or triazole (formed by the reaction of azide and alkyne), wherein L is the spacer of the bifunctional linker, which can be any divalent chemical group, preferably a C1-10 (including C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10) straight or branched alkylene or 1, 2 or 3 repeating units of polyethylene glycol subunits.

[0055] Bifunctional linkers are well known to those skilled in the art and can be obtained commercially or customized. In certain embodiments of the present invention, homofunctional bifunctional linkers such as BMOE (bismaleimidoethane) or BMB (1,4-bismaleimidobutane) are used; in other embodiments of the present invention, heterofunctional bifunctional linkers such as alkyne-hydrazide bifunctional linkers are used. The type and length of the spacer arm of the bifunctional linker can be selected based on, for example, the distance between the amino acid residues to be connected.

[0056] The linking molecule in the present invention has its broadest meaning, and also includes what are called disulfide bridging or disulfide re-bridging reagents or disulfides tapling reagents in the art, such as the thioester bireactive reagents reported in the prior art (Chem. Sci., 2022, 13, 11533–1153); another example is 3Br–5MP reagent (Zhang, Y., Zang, C., An, G. et al. Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylenepyrrolones. Nat Commun 11, 1015 (2020). https: / / doi.org / 10.1038 / s41467-020-14757-4) and the like.

[0057] Structure of the MPK2 helicase

[0058] Through protein structure modeling, the present invention discovered that positions 80-90 and 350-360 of the amino acid sequence of the wild-type MPK2 protein (shown in SEQ ID NO: 1) (NCBI Reference Sequence: XP_002548501.1) are spatially close to each other, forming the opening of the helicase. If the amino acid residues in the above two segments are covalently linked, it helps to narrow the opening of the helicase, thereby enabling the target polynucleotide to move through the nanopore in a controllable and stepwise manner.

[0059] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0060] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The equipment and reagents used in each example are conventionally available on the market.

[0061] Example 1. Preparation of MPK2 helicase protein

[0062] Based on the amino acid sequence of the wild-type MPK2 protein (shown in SEQ ID NO: 1) (NCBI Reference Sequence: XP_002548501.1), its nucleic acid sequence was obtained through in vitro gene synthesis. The relevant codons were then optimized using E. coli as the host, replacing them with codons commonly used in E. coli. This optimized nucleic acid sequence of the MPK2 protein was obtained, as shown in SEQ ID NO: 3. This protein was then inserted into the pET28 expression vector via the Nde I and Xho I restriction enzyme sites. After sequencing verification of the correct sequence, a recombinant expression plasmid for the MPK2 helicase was obtained.

[0063] The recombinant plasmid was transformed into the BL21 (DE3) Escherichia coli expression host by heat shock method. During the induction expression process, the host bacteria containing the expression plasmid were first cultured at 37°C overnight with LB medium containing kanamycin resistance, and then amplified at 37°C at a ratio of 1:100. When the OD (600) value reached 0.4-0.6, the culture was stopped and the cells were placed at 4°C for 1 hour for cooling. Subsequently, isopropyl β-D-Thiogalactoside (IPTG) was added at a final concentration of 0.5mM to induce expression at 16°C for 12-16 hours. Then, the bacteria were collected by centrifugation at 4°C, 15000rpm, and the cells were crushed by high pressure at 4°C. The supernatant was then collected by centrifugation at 4°C. The target protein was then separated and purified step by step through nickel column, heparin column, Q column and molecular sieve, and a large amount of high-purity wild-type helicase protein was finally obtained.

[0064] Figure 1 The SDS-PAGE gel electrophoresis image of the purified MPK2 helicase is shown, showing only a single band, indicating that the protein is highly pure.

[0065] Example 2. Fluorescence analysis of the unwinding activity of MPK2 helicase

[0066] like Figure 2 As shown in (1), the fluorescent substrate chain (final concentration 100 nM, d, SEQ ID NO: 5) has a 20-base single-stranded DNA portion at the 5' end and an 18-base hybridized double-stranded DNA portion, and its 3' end has a fluorescent group (Cy3, f). The upper short chain (c, SEQ ID NO: 6) that is complementary to it has a fluorescence quenching group (BHQ-1, e) at the 5' end. When the two chains are complementary, the fluorescence of Cy3 (f) is quenched by BHQ-1 (e), and the substrate is essentially non-fluorescent.

[0067] During the experiment, Figure 2 As shown in (2), MPK2 helicase binds to the 5' end single-stranded DNA portion of the fluorescent substrate, and after adding ATP (2 mM) and MgCl2 (2 mM), it shifts along the 5'-3' direction and unwinds the double-stranded portion.

[0068] Then, if Figure 2 As shown in (3), the excess capture chain (b, SEQ ID NO: 7) preferentially pairs with the short chain (c) to prevent re-annealing between the initial substrates, and the released substrate main chain (d) emits fluorescence.

[0069] The results showed that the substrate changed from being essentially non-fluorescent to emitting fluorescence.

[0070] Example 3. Preparation of MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S

[0071] MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S is SEQ ID NO: 2 having the mutation combination N90C / K357C / C60S / C141S / C171T / C203T / C286S reacted with a bismaleimidoethane linker to form a covalent linkage between cysteines at positions 90 and 357 in the modified MPK2 helicase sequence.

[0072] Based on the amino acid sequence of the wild-type MPK2 protein (NCBI Reference Sequence: XP_002548501.1), its nucleic acid sequence was obtained by in vitro gene synthesis. The relevant codons were then optimized using E. coli as the host, replacing the codons with those commonly used in E. coli to obtain the optimized nucleic acid sequence of the MPK2 protein. This was then inserted into the pET28 expression vector via the Nde I and Xho I restriction enzyme sites. After sequencing verified the correct sequence, a recombinant expression plasmid for the MPK2 helicase was obtained. Next, site-directed mutagenesis was performed by overlapping PCR to obtain the nucleic acid sequence encoding the mutant combination N90C / K357C / C60S / C141S / C171T / C203T / C286S (SEQ ID NO: 4).

[0073] The mutated recombinant plasmid was transformed into the BL21 (DE3) Escherichia coli expression host by heat shock method. During the induction expression process, the host bacteria containing the expression plasmid were first cultured overnight at 37°C using LB medium with kanamycin resistance, and then amplified at 37°C at a ratio of 1:100. When the OD (600) value reached 0.4-0.6, the culture was stopped and the cells were placed at 4°C for 1 hour for cooling. Subsequently, isopropyl β-D-Th iogalactoside (IPTG) was added at a final concentration of 0.5 mM to induce expression at 16°C for 12-16 hours. Then, the bacteria were collected by centrifugation at 4°C, 15000 rpm, and the cells were crushed by high pressure at 4°C using a high pressure crusher. The supernatant was then collected by centrifugation at 4°C, and the target protein was separated and purified step by step through nickel column, heparin column, Q column and molecular sieve, and finally a large amount of high-purity helicase mutant protein was obtained, the sequence of which is shown in SEQ ID NO: 2.

[0074] Specifically, 1 μL of 1 M DTT was added to 100 μL of MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S (SEQ ID NO: 2 with mutations N90C / K357C / C60S / C141S / C171T / C203T / C286S, stored in 25 mM Tris-HCl pH 7.5, 500 mM NaCl, 10% glycerol) and incubated at room temperature for 30 minutes.

[0075] The buffer was exchanged with PBS buffer (pH 7.0) using a 0.5 ml Zeba desalting column (7k MWCO) to obtain 100 μl of sample. 0.5 μl of 10 mM bismaleimidoethane was added and the mixture was incubated at room temperature for 1 hour with rotation at 20 rpm. The cross-linking results were then analyzed using a 4-10% polyacrylamide gel.

[0076] Figure 3 Shown is a 4-10% SDS-PAGE gel electrophoresis image of MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S (SEQ ID NO: 2 having the mutations N90C / K357C / C60S / C141S / C171T / C203T / C286S).

[0077] It can be clearly seen from the gel that the cross-linking reaction of MPK2 mutants achieved almost 100% yield.

[0078] Example 4. Gel shift assay to measure the ability of modified MPK2 helicase to bind DNA

[0079] DNA substrates for gel shift assays were prepared by annealing (SEQ ID NO: 8 was annealed with SEQ ID NO: 9 and SEQ NO. 10 at a molar ratio of 1:1.1:1.1, with a final concentration of 10 μM). These substrates were then incubated with wild-type MPK2 (NCBI Reference Sequence: XP_002548501.1) and MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S (SEQ ID NO: 2 with the mutation combination N90C / K357C / C60S / C141S / C171T / C203T / C286S linked to bismaleimidoethane) at molar ratios of 1:1, 1:3, and 1:5, respectively, in a buffer solution (25 mM Tris-HCl pH 7.0, 100 mM NaCl, 10% glycerol, 1 mM The cells were incubated in EDTA at room temperature for one hour to obtain a final DNA concentration of 500 nM in the reaction solution. The total reaction volume was 20 μL.

[0080] Bismaleimidoethane was added to the corresponding helicase mutant samples to a final concentration of 5 μM and incubated at room temperature for 1 hour. The samples were then examined using a 4%-10% TBE gel, run at 120V for 1.5 hours, and the DNA bands were visualized under UV light using Gel Red staining.

[0081] like Figure 4As shown, lane 4 represents a simple DNA substrate without protein binding. When wild-type MPK2 was mixed with the DNA substrate at different molar ratios of 1:1, 3:1 and 5:1, protein-bound bands appeared on the gel image, i.e., new bands appeared above lanes 1, 2 and 3 relative to lane 4. After the MPK2 helicase was modified, the simple DNA substrate bands at the bottom of lanes 5, 6 and 7 decreased compared to lanes 1, 2 and 3, while the protein-bound bands at the top increased, indicating that the binding ability of the modified MPK2 to polynucleotides was significantly enhanced.

[0082] Example 5. MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S-bismaleimidoethane has the ability to control the movement of the intact DNA construct X through the nanopore

[0083] Among them, MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S-bismaleimidoethane is SEQ ID NO: 2 having the mutation combination N90C / K357C / C60S / C141S / C171T / C203T / C286S connected to bismaleimidoethane.

[0084] Prepare as Figure 5 DNA construct X is shown.

[0085] First, primers containing sequences A, C, D, E, and F were designed and used to amplify a 1000-base sequence (G) on λ DNA. The resulting PCR product was purified and then annealed and hybridized with sequence H at a molar ratio of 1:1.1 to obtain the final DNA construct X.

[0086] A represents 50 Ts, B represents MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S, C represents an iSpC18 spacer, D represents SEQ ID NO: 11, E represents 2 iSpC18 spacers, F represents SEQ IN NO. 12, G represents SEQ ID NO: 13, and H represents SEQ ID NO: 14 with a cholesterol tag at the 3' end.

[0087] The prepared DNA construct X (final concentration 0.1 nM) was preincubated with MPK2-N90C / K357C / C60S / C141S / C171T / C203T / C286S (final concentration 10 nM) in a buffer solution (10 mM Hepes, pH 8.0, 100 mM KCl, 10% glycerol) at room temperature for 30 minutes. Bismaleimidoethane was added to the corresponding helicase mutant sample to a final concentration of 5 μM and incubated at room temperature for 1 hour.

[0088] Electrical signal measurements were obtained from CsgG nanopores embedded in DPhPC phospholipid bilayers in a buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0). The bilayers were formed using the Montal-Mueller technique using ~25 μm diameter pores in a PTFE membrane, separating two approximately 100 μL volumes of buffer solution. All experiments were performed in this buffer solution. Single-channel currents were measured using an amplifier equipped with a digitizer. Ag / AgCl electrodes were connected to the buffer solution such that the cis compartment was connected to the amplifier ground terminal and the trans compartment was connected to the active electrode. After achieving single-pore insertion into the phospholipid bilayer, 2 ml of buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM Hepes, pH 8.0) was flowed through the system to remove excess residual nanopores. The pre-incubated sample, ATP (final concentration 2 mM), and MgCl2 (final concentration 10 mM) were then flowed into the single nanopore experimental system (total volume 100 μL), and the signal was measured at a constant voltage of +180 mV for 6 h (including a potential 2 s voltage reversal at -180 mV).

[0089] Figure 6 showed that the mutants were able to control the movement of the complete DNA construct X through the nanopore, Figure 7 An enlarged view of a portion of the region (0 to 1 s) where the DNA is moved is shown. Figure 8 This is the case of a simple nucleic acid sample passing through the well without helicase control. The burr-like signal marked by the arrow represents the simple nucleic acid sample passing through the well. It can be seen that the speed of the simple nucleic acid passing through the well is very fast (<<1s), and the base information of the nucleic acid cannot be identified. However, after adding the modified MPK2 helicase, a clear nucleic acid passing through signal can be seen. When using the wild-type helicase for the experiment, due to its weak binding to the nucleic acid sequence to be tested, it slips from the spacer region, resulting in no signal detection.

Claims

1. A modified MPK2 helicase, characterized in that The sequence of the modified MPK2 helicase is SEQ ID NO: 2, and the modified MPK2 helicase is covalently linked between positions 90 and 357 via bismaleimidoethane.

2. A sequencing element and a sequencing device comprising the modified MPK2 helicase according to claim 1.

3. Sequencing application of the modified MPK2 helicase according to claim 1.

4. A method for preparing the modified MPK2 helicase according to claim 1, comprising the following steps: 1) Providing an MPK2 helicase having an amino acid sequence as set forth in SEQ ID NO: 2; 2) reacting under conditions for forming a covalent linkage to obtain the modified MPK2 helicase, wherein the covalent linkage is formed by reacting the sulfhydryl groups of the cysteines at positions 90 and 357 of the MPK2 helicase with bismaleimidoethane.

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