WO2024056038A1 - Modified capif1 helicase and use thereof - Google Patents

Abstract

The present invention belongs to the field of genetic engineering and provides a modified CaPif1 helicase, a complex comprising the helicase, a construct comprising the helicase, a method for using the helicase or the complex or the construct to represent a target polynucleotide, and use. The provided modified CaPif1 helicase and the complex and the construct thereof all can control the movement of a polynucleotide when passing through a biological nanopore. Especially when the length of a polynucleotide chain is increased, the modified CaPif1 helicase and the complex and the construct thereof can still stably control the polynucleotide to move without falling off from the polynucleotide. Further provided is a specific CaPif1 helicase mutant, which improves the capability thereof in controlling the displacement of the polynucleotide when passing through the nanopore.

Description

A modified CaPif1 helicase and its application

This application claims the priority of the earlier application submitted to the State Intellectual Property Office of China on September 16, 2022, with patent application number 202211132258.8, titled "A modified CaPif1 helicase and its application". The entirety of this prior application is incorporated by reference into this application.

Technical field

The present invention relates to a modified CaPif1 helicase, in particular to a helicase that can control the movement of target polynucleotides through biological nanopores, and is particularly helpful in sequencing polynucleotides, and belongs to genetic engineering. and the field of genetic engineering.

Background technique

Nanopore sequencing technology developed in recent years is a new type of single-molecule sequencing technology that uses electric field force to drive single-stranded polynucleotides through nanoscale biopore proteins embedded in insulating phospholipid membranes. Due to different bases size, each nucleotide molecule will generate a characteristic obstruction current when passing through the biological nanopore, and these characteristic current signals recorded correspond to the sequence of the target nucleic acid.

In the "strand sequencing" method, a single polynucleotide strand is passed through the biological nanopore to enable identification of the nucleotide sequence. Compared with other sequencing technologies, the advantages of this sequencing technology are low cost, no need for PCR amplification, fast and real-time convenience, long sequencing reads (more than 150kb), and the ability to directly sequence RNA and epigenetic modifications. It is considered a revolutionary technology in the field of sequencing and has immeasurable application value.

However, nanopore sequencing technology also has serious limitations. Under the influence of an electric field, single-stranded polynucleotides pass through the nanopore so quickly that it is difficult to distinguish the current-blocking signal of a single nucleotide from the system noise. Therefore, to achieve identification of nucleotides, strand sequencing uses polynucleotide-bound molecular motors to control the movement of polynucleotides through the biological nanopore. However, the combination of molecular motors and polynucleotides is not static. When controlling the movement of polynucleotides, especially when facing very long nucleotide sequences, the molecular motors may fall off from the polynucleotides. In addition, in single-molecule level sequencing technology such as strand sequencing, molecular motors have many unexpected single-molecule dynamic behaviors. For example, some motor proteins may stagnate due to poor activity, causing channel obstruction and reducing Sequencing throughput. Secondly, the displacement of motor proteins on polynucleotides will slide forward, backward, and have uneven step sizes. These irregular single-molecule behaviors result in the inability of motor proteins to perfectly control single-stranded polynucleotides in a ratcheting manner. pass through the nanopore, thereby reducing the accuracy of base calling. Therefore, there is an urgent need for a technology that can stably and efficiently control polynucleotides to pass through biological nanopores.

Contents of the invention

In view of the above technical problems, the present invention overcomes the problem of too fast translocation speed of polynucleotides through nanopores in the prior art, and provides a DNA-dependent ATPase (CaPif1) helicase, which can Controlling polynucleotide punch movement is very useful.

The core of CaPif1 helicase contains five domains: 1A (RecA-like motor) domain, 2A (RecA-like motor) domain (RecA-like motor) domain, 1B (wedge domain) domain, 2B (SH3-like) domain and 2C domain (an additional structure unique to yeast). The structural information of the corresponding CaPif1 (PDB:7OTJ) protein can be obtained from the Protein Data Bank (PDB).

Among them, the 1A and 2A domains are mainly involved in the binding and hydrolysis of ATP. The 1B domain consists of a short helix and an extended loop structure, which is called the "wedge region". The 2B domain adopts a SH-3-like folding manner, and the orbital-like β hairpin structure on it is close to the 1B domain. The 2C domain has not yet been resolved in the crystal structure.

Therefore, when reducing the size of the opening through modified connection, the present invention focuses on the two domains 1B and 2B, and tends to reduce or close the opening through covalent connection.

There are two ways of connection. One is through its own naturally occurring amino acids and the insertion of new amino acids, such as cysteine and unnatural amino acids, to achieve connection. The other is through linker molecules, which tend to connect cysteines. Linker molecules include: BMOE, Bis(PEG)2, Bis(PEG)3, etc.

The present invention introduces cysteine into the two structural domains 1B and 2B respectively. The above-mentioned connecting molecules usually contain two functional terminals, which will be covalently connected to the cysteine to realize the connection of the two parts 1B and 2B, so that The bound polynucleotide will not dissociate from the helicase.

To this end, one aspect of the present invention provides a modified CaPif1 helicase, which modifies five natural cysteines in the amino acid sequence of wild-type CaPif1 (368-883). Cystine is C426, C507, C584, C592 and C662 respectively.

In a preferred embodiment of the invention, the five natural cysteines are replaced with alanine (A) or serine (S).

Another aspect of the present invention provides a modified CaPif1 helicase, which introduces new cysteine for connection, and the introduction sites include Q443, N448, K452, R455, I633, L634, P635, Q638, Q641, V642, D795, E796, D797 and T799.

In preferred embodiments of the invention, preferred mutation combinations include R455C, E796C, C426A, C507A, C584A, C592A and C662A.

In a further preferred embodiment of the present invention, the amino acid sequence of the helicase is shown in SEQ ID NO.1.

In preferred embodiments of the invention, preferred mutation combinations include Q443C, L634C, C426A, C507A, C584A, C592A and C662A.

In a further preferred embodiment of the present invention, the amino acid sequence of the helicase is shown in SEQ ID NO. 3.

In preferred embodiments of the invention, the helicase binds to internal nucleotides of a single-stranded polynucleotide or a double-stranded polynucleotide.

Another aspect of the invention provides a nucleotide sequence encoding a helicase of the invention.

In a preferred embodiment of the present invention, the nucleotide sequence is shown in SEQ ID NO.2 or SEQ ID NO.4.

Another aspect of the present invention provides a complex formed between the helicase of the present invention and bismaleimidoethane or bismaleimide PEG3.

Another aspect of the invention provides a construct comprising a helicase according to the invention and a binding moiety for binding to a polynucleotide.

In a preferred embodiment of the invention, the binding moiety is selected from eukaryotic single-chain binding proteins, bacterial single-chain binding proteins, archaeal single-chain binding proteins, viral single-chain binding proteins, or double-chain binding proteins.

Another aspect of the present invention provides the helicase of the present invention or the nucleotide sequence encoding the helicase or the complex comprising the helicase or the construct comprising the helicase in characterizing the target polynucleoside. Application of acids or controlled passage of target polynucleotides through nanopores.

Another aspect of the present invention provides a kit for characterizing a target polynucleotide or controlling the passage of a target polynucleotide through a nanopore, the kit comprising the helicase of the present invention or a nucleoside encoding the helicase. acid sequence or a complex comprising the helicase or a construct comprising the helicase.

Another aspect of the invention provides a device for characterizing a target polynucleotide or controlling the passage of a target polynucleotide through a nanopore, the device comprising the helicase of the invention or a nucleotide sequence encoding the helicase. or a complex containing the helicase or Constructs comprising said helicase.

Another aspect of the invention provides a method for characterizing a target polynucleotide or controlling the passage of a target polynucleotide through a nanopore, comprising the following steps:

1. Contact the target polynucleotide with the nanopore, and the helicase of the present invention or the complex containing the helicase or the construct containing the helicase, so that the helicase, complex or construct The construct controls passage of the target polynucleotide through the nanopore; and

2. Obtain one or more characteristics of the interaction between the nucleotides in the target polynucleotide and the nanopore to characterize the target polynucleotide, and the construct includes a helicase and a helicase for binding the polynucleotide the combined part.

In a preferred embodiment of the invention, said one or more characteristics are selected from the group consisting of origin, length, identity, sequence, secondary structure of the target polynucleotide or whether the target polynucleotide is modified.

In a preferred embodiment of the invention, said one or more characteristics are carried out by electrical measurements and/or optical measurements.

In a preferred embodiment of the present invention, the target polynucleotide is single-stranded, double-stranded, or at least part of it is double-stranded.

In a preferred embodiment of the present invention, the nanopores are transmembrane pores, and the transmembrane pores are biological pores, solid pores or hybrid pores between biology and solid.

Another aspect of the invention provides a vector comprising the nucleotide sequence of the helicase of the invention.

Yet another aspect of the present invention provides a host cell comprising a nucleotide sequence of the helicase of the present invention or a vector comprising the nucleotide sequence.

beneficial effects

The present invention has demonstrated that the modified CaPif1 helicase can control the movement of polynucleotides through biological nanopores, especially under the action of electric field force. The helicase enables target polynucleotides to move through the nanopore in a controlled and stepwise manner.

The specific CaPif1 helicase mutants provided by the present invention have improved ability to control the translocation of polynucleotides through nanopores. These mutants usually have one or more modifications on the 1B or 2B domain. . Therefore, the modified CaPif1 helicase provided by the present invention has at least one cysteine or unnatural amino acid inserted, and still maintains its ability to control the movement of polynucleotides.

The present invention also provides a modified CaPif1 helicase that covalently connects 1B and 2B domains through a linker molecule, which improves the stability of the CaPif1 helicase binding to polynucleotides, especially when polynucleotides are used. When the length of the acid chain increases, the helicase of the present invention can still stably control the movement of the polynucleotide without falling off from the polynucleotide.

Description of drawings

Figure 1 is a diagram showing the results of SDS-PAGE gel electrophoresis purification of the CaPif1 helicase of the present invention;

M: Marker;

1: Electrophoresis results of CaPif1 helicase.

Figure 2 is a schematic diagram of fluorescence analysis for detecting helicase enzyme activity.

Figure 3 is a graph showing the changes in fluorescence value generated by CaPif1 helicase unwinding the fluorescent substrate over time.

Figure 4 is an SDS-PAGE gel electrophoresis picture of the cross-linking results of the CaPif1 mutant.

Figure 5 is a gel shift image of CaPif1 binding to DNA before and after modification.

1: Pure DNA substrate without added protein;

2: Results of wild-type CaPif1 binding to DNA;

3: Modified CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A) The result of combining DNA.

Figure 6 is a schematic diagram of DNA construct X used in the present invention.

A: 50 T;

B: Helicase used in the present invention;

C: 1 iSpC18 spacer;

D: SEQ ID NO: 11;

E: 2 iSpC18 spacers;

F: SEQ ID NO: 12;

G: SEQ ID NO: 13;

H: SEQ ID NO: 14 with a cholesterol tag at the 3’ end.

Figure 7 is a schematic diagram of DNA construct Y used in the present invention.

A: 50 T;

B: Helicase used in the present invention;

C: 1 iSpC18 spacer;

D: SEQ ID NO: 11;

E: 2 iSpC18 spacers;

F: SEQ ID NO: 12;

K: SEQ ID NO: 15;

I: 30 T;

J: SEQ ID NO: 16.

Figure 8 is a schematic diagram of the current trajectories of mutants CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane when controlling the movement of the complete DNA construct X through the nanopore.

X-axis: time (s);

Y-axis: current (nA).

Figure 9 is an enlarged view of an area showing the movement of the DNA construct X via the helicase shown in Figure 8.

X-axis: time (s);

Y-axis: current (nA).

Figure 10 is a schematic diagram of the current trajectory when CaPif1 helicase controls the displacement of DNA construct X through the nanopore.

Figure 11 is an enlarged view of an area showing the movement of the via hole of DNA construct X controlled by the helicase shown in Figure 10.

X-axis: time (s);

Y-axis: current (nA).

Detailed ways

In order to better explain the purpose and advantages of this method, the specific implementation content of the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments.

Example 1: Preparation of CaPif1 helicase protein

According to the amino acid sequence of wild-type CaPif1 protein (PDB ID: 7OTJ, related website: RCSB PDB-7OTJ: Crystal structure of Pif1 helicase from Candida albicans), its nucleic acid sequence was obtained through in vitro gene synthesis, and then E. coli was used as the host. The relevant codons were optimized and replaced with codons commonly used in E. coli to obtain the optimized nucleic acid sequence of CaPif1 protein, whose sequence is shown in SEQ ID NO.5. Then it was ligated and inserted into the pET28 expression vector through two restriction enzyme sites of NdeⅠ and XhoⅠ. After sequencing to verify the correct sequence, the recombinant expression plasmid of CaPif1 helicase was finally obtained.

The recombinant plasmid was transformed into the BL21(DE3) E. coli expression host by heat shock method. In the process of inducing expression, the host bacteria containing the expression plasmid were first cultured overnight at 37°C in LB medium with kana resistance, and then amplified and cultured at 37°C at a ratio of 1:100, until the OD (600 ) value reaches 0.4-0.6, stop culturing and place it at 4°C for 1 hour of cooling treatment, then add isopropylβ-D-Thiogalactoside (IPTG) at a final concentration of 0.5mM to induce expression at 16°C12 -16h. Then, collect the bacteria by centrifugation at 4°C and 15000rpm, crush the bacteria at 4°C with a high-pressure crusher, and then centrifuge at 4°C to collect the supernatant, and then pass through the nickel column, heparin column, Q column and molecular sieve to achieve the goal step by step. The protein is separated and purified, and a large amount of highly pure wild-type helicase protein is finally obtained.

Figure 1 shows the SDS-PAGE gel electrophoresis pattern of purified CaPif1 helicase.

Example 2: Fluorescence experiment to analyze the unwinding activity of CaPif1 helicase

Figure 2 is a schematic diagram of fluorescence analysis for detecting helicase enzyme activity.

As shown in (1) of Figure 2, the fluorescent substrate chain (final concentration 100 nM, d, SEQ ID NO: 6) has a single-stranded DNA portion of 20 bases at the 5' end and a double-stranded DNA portion of 18 bases hybridized, and its 3' end has a fluorescent group (Cy3, f). The upper short chain (c, SEQ ID NO: 7) that is complementary to it has a fluorescent quenching group (BHQ-1, e) at the 5' end. When the two chains are complementary, the fluorescence of Cy3 (f) will be quenched by BHQ-1 (e), and the substrate is basically non-fluorescent.

During the experiment, as shown in (2) in Figure 2, CaPif1 helicase will bind to the 5'-end single-stranded DNA portion of the fluorescent substrate, and after adding ATP (2mM) and MgCl ₂ (2mM), Shift in the 5'-3' direction and unwind the double-stranded portion.

Subsequently, as shown in (3) in Figure 2, the excess capture chain (b, SEQ ID NO: 8) is preferentially complementary to the short chain (c) to prevent re-annealing between the initial substrates, and the released substrates The substance backbone (d) emits fluorescence.

Figure 3 shows the change in fluorescence value generated by the unwinding of the fluorescent substrate by CaPif1 helicase over time in a 300mM NaCl buffer (25mM Tris-HCl pH 7.5, 2mM ATP, 2mM MgCl ₂ , 300mM NaCl). The results show that the substrate ranges from essentially non-fluorescent to fluorescent.

Example 3: Preparation of CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A mutation combinations and CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A and C662A mutation combinations

Among them, CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A are SEQ ID NO: 1 and bismaleimidoethane with mutation combinations R455C, E796C, C426A, C507A, C584A, C592A and 662A. Alkane connection;

CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A and C662A is SEQ ID NO: 3 with the mutation combination Q443C, L634C, C426A, C507A, C584A, C592A and C662A linked to bismaleimide PEG3.

In this case, by reacting with bismaleimidoethane or bismaleimide PEG3 linker molecules, codons were formed between cysteines 455 and 796 or 443 and 634 in the initial sequence of CaPif1. Valence connection.

According to the amino acid sequence of wild-type CaPif1 protein (PDB ID: 7OTJ, related website: RCSB PDB-7OTJ: Crystal structure of Pif1 helicase from Candida albicans), its nucleic acid sequence was obtained through in vitro gene synthesis, and then E. coli was used as the host. The relevant codons were optimized and replaced with codons commonly used in E. coli to obtain the optimized nucleic acid sequence of the CaPif1 protein, whose sequence is shown in SEQ ID NO.5. Then it was ligated and inserted into the pET28 expression vector through two restriction enzyme sites of NdeⅠ and XhoⅠ. After sequencing to verify the correct sequence, the recombinant expression plasmid of CaPif1 helicase was finally obtained. Then, site-directed mutagenesis was performed by overlapping PCR to obtain the nucleic acid sequences encoding mutation combinations R455C, E796C, C426A, C507A, C584A, C592A, and 662A (SEQ ID NO. 2). and the nucleic acid sequence encoding mutation combinations Q443C, L634C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO. 4).

The mutated recombinant plasmid was transformed into the BL21 (DE3) E. coli expression host by heat shock method. In the process of inducing expression, the host bacteria containing the expression plasmid were first cultured overnight at 37°C in LB medium with kana resistance, and then amplified and cultured at 37°C at a ratio of 1:100, until the OD (600 ) value reaches 0.4-0.6, stop culturing and place it at 4°C for 1 hour of cooling treatment, then add isopropylβ-D-Thiogalactoside (IPTG) at a final concentration of 0.5mM to induce expression at 16°C12 -16h. Then, collect the bacteria by centrifugation at 4°C and 15000rpm, crush the bacteria at 4°C with a high-pressure crusher, and then centrifuge at 4°C to collect the supernatant, and then pass through the nickel column, heparin column, Q column and molecular sieve to achieve the goal step by step. After protein separation and purification, a large number of highly pure helicase mutant proteins were finally obtained, with sequences such as SEQ ID NO.1 and SEQ ID NO.3.

Add 1μL of 1M DTT to 100μl of CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A, stored at 25 mM Tris-HCl pH 7.5, 500mM NaCl, 10% glycerol, 2mM EDTA) and CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A, and C662A (with mutation combinations Q443C, L634C, C426A, C507A, C584A, C592A, C66 2A's SEQ ID NO: 3, stored in 25mM Tris-HCl pH 7.5, 500mM NaCl, 10% glycerol, 2mM EDTA) and incubated at room temperature for 30 minutes.

The buffer was replaced with PBS buffer (pH7.5) through a 0.5ml Zeba desalting column (7k MWCO) to obtain a 100μl sample. Add 0.5 μl, 10 mM bismaleimidoethane and bismaleimide PEG3 respectively, and incubate with rotation at 20 rpm for 1 hour at room temperature. After that, add 1 μl, 1M DTT to stop the reaction. Cross-linking results were analyzed on a 4-10% polyacrylamide gel.

The results in Figure 4 show that the reaction of CaPif1 mutants CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A with the linker molecules reached almost 100% yield.

Example 4: Using gel shift assay to measure the ability of modified CaPif1 helicase to bind DNA

The DNA substrate required for gel shift experiments was prepared by annealing (SEQ ID NO: 9 was hybridized with SEQ ID NO: 10 containing a 5' end Cy3 label), and then it was mixed with SEQ ID NO: 10 in a molar ratio of 1:1. Wild-type CaPif1 (PDB ID: 7OTJ, related website: RCSB PDB-7OTJ: Crystal structure of Pif1 helicase from Candida albicans), CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (with mutation combinations R455C, E796C, SEQ ID NO: 1) for C426A, C507A, C584A, C592A and C662A and CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A and C662A (with mutation combination Q443C, L634C, C426A, C507A, C584A, C592 A and C662A SEQ ID NO: 3) Incubate in buffer (25mM Tris-HCl pH 7.5, 300mM NaCl) for one hour at room temperature to obtain a final concentration of CaPif1 helicase in the final reaction solution of 250nM and a final concentration of DNA of 25nM. The total reaction The volume is 20 μL.

Add bismaleimidoethane and bismaleimide PEG3 to the corresponding helicase mutant samples to a final concentration of 5 μM, and incubate at room temperature for 1 hour. Subsequently, 4%-10% TBE gel was used for detection, and the gel was run at 160V for 1.5 hours, and then the DNA bands were observed under 550nM excitation light.

The results in Figure 5 show the effect of CaPif1 helicase on DNA binding ability before and after modification, that is, the interaction between modified CaPif1 (CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A mutants) and polynucleotides The binding ability is significantly enhanced.

Example 5: CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-Bismaleimidoethane and CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A and C662A-Bismaleimidoethane Imine PEG3 has the ability to control the passage of intact DNA constructs through nanopores ability to move

Among them, CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane are SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A. Linked to bismaleimidoethane; CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A, and C662A-Bismaleimide PEG3 is a protein with mutations Q443C, L634C, C426A, C507A, C584A, C592A, and SEQ ID NO: 3 of C662A is linked to bismaleimide PEG3.

DNA construct X as shown in Figure 6 was prepared.

First, design primers, which contain sequences A, C, D, E, and F, and then use them to amplify a 1000-base sequence (G) on lambda DNA. The obtained PCR product is purified and matched with sequence H at a ratio of 1:1.1 Annealing hybridization is performed at the molar ratio to obtain the final DNA construct X.

The prepared DNA construct C507A, C584A, C592A, and C662A-bismaleimide PEG3 (final concentration 10 nM) were preincubated in buffer (10 mM Hepes, pH 8.0, 100 Mm KCl, 10% glycerol) for 30 min at room temperature.

In buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0), phospholipids embedded in the DPhPC phospholipid bilayer were Electrical signal measurements were obtained from Csgg nanopores. After achieving single-hole insertion into the phospholipid bilayer, 2 ml buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0 ) is flowed through the system to remove residual excess nanopores. The pre-incubated sample, ATP (final concentration 2mM) and MgCl ₂ (final concentration 10mM) were then flowed together into a single nanopore experimental system (total volume 100μL), and the signal was measured at a constant voltage of +180mV for 6h (including potential 2s -180mV voltage inversion).

The results in Figure 8 show that CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A mutants are able to control the movement of intact DNA construct X through the nanopore.

Figure 9 shows an enlarged view of part of the region where CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A mutants control DNA movement.

Example 6: CaPif1 helicase controls movement of entire DNA construct X through a single Csgg nanopore

This example takes CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A) as an example to verify CaPif1 unwinding How the enzyme controls the movement of the entire DNA construct X through a single Csgg nanopore.

DNA construct X as shown in Figure 6 was prepared.

First, design primers, which contain sequences A, C, D, E, and F, and then use them to amplify a 1000-base sequence (G) on lambda DNA. The obtained PCR product is purified and matched with sequence H at a ratio of 1:1.1 Annealing hybridization is performed at the molar ratio to obtain the final DNA construct X.

Pre-incubate the prepared DNA construct .

In buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0), phospholipids embedded in the DPhPC phospholipid bilayer were Electrical signal measurements were obtained from Csgg nanopores. After achieving single-hole insertion into the phospholipid bilayer, 2 ml buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0 ) is flowed through the system to remove residual excess nanopores. The pre-incubated sample, ATP (final concentration 2mM) and MgCl ₂ (final concentration 10mM) were then flowed together into a single nanopore experimental system (total volume 100μL), and the signal was measured at a constant voltage of +180mV for 6h (including potential 2s -180mV voltage inversion).

As shown in Figure 10, CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A) was observed to unwind The enzyme controls the movement of DNA construct X through the nanopore. The duration of helicase-controlled DNA movement of 14 seconds corresponds to the movement of nearly 1000 bp of DNA construct through the Csgg nanopore.

Among them, Figure 11 shows CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A (SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A) helicase control A magnified view of part of the DNA movement.

Example 7: Ability of wild-type CaPifl and CaPifl-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane to control movement of intact DNA construct Y through the nanopore

Among them, CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane are SEQ ID NO: 1 with mutation combination R455C, E796C, C426A, C507A, C584A, C592A and C662A. Connected with bismaleimidoethane.

DNA construct Y as shown in Figure 7 was prepared.

First, design the upstream primer and downstream primer. The upstream primer contains A, C, D, E and J sequences. The J sequence is as shown in SEQ ID NO:16, and the downstream primer is as shown in SEQ ID NO:17. They were then used to amplify the 4115 base length sequence (K) on λ DNA, so that a poly T sequence (I) was added to the 3' end of the K sequence (shown as SEQ ID NO: 15), and the obtained After purification, the PCR product is annealed and hybridized with sequence H at a molar ratio of 1:1.1 to obtain the final DNA construct Y.

The prepared DNA construct Y (final concentration 0.1nM) was combined with wild-type CaPif1 (PDB ID: 7OTJ, related website: RCSB PDB-7OTJ: Crystal structure of Pif1 helicase from Candida albicans) and CaPif1-R455C, E796C, C426A respectively. , C507A, C584A, C592A, and C662A-bismaleimidoethane (final concentration 10 nM) were preincubated in buffer (10mM Hepes, pH 8.0, 100mM KCl, 10% glycerol) at room temperature for 30 minutes.

In buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0), phospholipids embedded in the DPhPC phospholipid bilayer were Electrical signal measurements were obtained from Csgg nanopores. After achieving single-hole insertion into the phospholipid bilayer, 2 ml buffer (600mM KCl, 75mM K ₃ [Fe(CN) ₆ , 25mM K ₄ [Fe(CN) ₆ ]·3H ₂ O, 100mM Hepes, pH 8.0 ) is flowed through the system to remove residual excess nanopores. The pre-incubated sample, ATP (final concentration 2mM) and MgCl ₂ (final concentration 10mM) were then flowed together into a single nanopore experimental system (total volume 100μL), and the signal was measured at a constant voltage of +180mV for 6h (including potential 2s -180mV voltage inversion).

The results show that adding the complex formed by CaPif1 helicase and DNA construct to the Csgg nanopore system can generate a typical nucleic acid through-hole current signal. The wild-type CaPif1 helicase monomer and CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane can control the pore movement of DNA construct Y.

Statistical comparison of the same number of DNA via trajectories controlled by the helicase found that for the wild-type CaPif1 helicase monomer, only a smaller proportion of electrical signal trajectories observed the typical poly-T (I region) Via current signal.

However, for CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A-bismaleimidoethane, the typical process of polyT (I region) was observed in most of the electrical signal traces. hole signal. This means that the helicase reaches the 3’ end of DNA construct Y.

The above results indicate that CaPif1-R455C, E796C, C426A, C507A, C584A, C592A, and C662A-bismaleimidoethane have better ability to control DNA perforation movement than wild-type CaPif1.

The details of all sequences in the present invention are summarized as follows:

1. SEQ ID NO: 1, which is the amino acid sequence of CaPif1-R455C, E796C, C426A, C507A, C584A, C592A and C662A mutants. The specific sequence is:

2. SEQ ID NO: 2, which is the coding sequence of SEQ ID NO: 1. The specific sequence is:

3. SEQ ID NO: 3, which is the amino acid sequence of CaPif1-Q443C, L634C, C426A, C507A, C584A, C592A and C662A mutants. The specific sequence is:

4. SEQ ID NO: 4, which is the coding sequence of SEQ ID NO: 3. The specific sequence is:

5. SEQ ID NO: 5, which is the optimized nucleic acid sequence of CaPif1 protein. The specific sequence is:

6. SEQ ID NO: 6, which is the sequence of the fluorescent substrate chain. The specific sequence is:

7. SEQ ID NO: 7, which is a short chain sequence that is complementary to the fluorescent substrate chain sequence. The specific sequence is:

8. SEQ ID NO: 8, which is the capture strand sequence. The specific sequence is:

9. SEQ ID NO: 9, which is the DNA substrate sequence. The specific sequence is:

10. SEQ ID NO: 10, which is a sequence that hybridizes with SEQ ID NO: 9. The specific sequence is:

11. SEQ ID NO: 11, which is the D sequence of DNA construct X and construct Y. The specific sequence is:

12. SEQ ID NO: 12, which is the F sequence of DNA construct X and construct Y. The specific sequence is:

13. SEQ ID NO: 13, which is the G sequence of DNA construct X. The specific sequence is:

14. SEQ ID NO: 14, which is the H sequence of DNA construct X. The specific sequence is:

15. SEQ ID NO: 15, which is the K sequence of DNA construct Y. The specific sequence is:

16. SEQ ID NO: 16, which is the J sequence of DNA construct Y. The specific sequence is:

17. SEQ ID NO: 17, which is the downstream primer sequence of DNA construct Y. The specific sequence is:

The above embodiments are only preferred embodiments of the present invention, and the present invention should not be limited to the contents disclosed in the embodiments and drawings. All equivalents or modifications made without departing from the spirit disclosed in the present invention fall within the scope of protection of the present invention.

Claims (11)

A modified CaPif1 helicase, characterized in that five natural cysteines in the amino acid sequence of wild-type CaPif1 (368-883) are modified, and the five natural cysteines are C426 respectively. , C507, C584, C592 and C662, it is preferred to replace the five natural cysteine with alanine (A) or serine (S). A modified CaPif1 helicase, characterized by introducing new cysteine for connection, and the introduction sites include Q443, N448, K452, R455, I633, L634, P635, Q638, Q641, V642, D795, E796, D797 and T799, wherein the mutation combination preferably includes R455C, E796C, C426A, C507A, C584A, C592A and C662A, and more preferably the amino acid sequence of the helicase is as shown in SEQ ID NO.1. The helicase according to claim 1 or 2, characterized in that the helicase binds to the internal nucleotides of single-stranded polynucleotides or double-stranded polynucleotides. The nucleotide sequence encoding the helicase described in any one of claims 1-3, the nucleotide sequence is preferably as shown in SEQ ID NO. 2. The complex formed by the helicase according to any one of claims 1 to 3 and bismaleimidoethane. A construct, characterized in that the construct comprises the helicase of any one of claims 1-3 or the complex of claim 5 and a binding portion for binding to a polynucleotide, wherein The binding moiety is preferably selected from eukaryotic single-chain binding proteins, bacterial single-chain binding proteins, archaeal single-chain binding proteins, viral single-chain binding proteins, or double-chain binding proteins. The helicase of any one of claims 1 to 3 or the nucleotide sequence of claim 4 or the complex of claim 5 or the construct of claim 6 is useful in characterizing the target polynucleotide. or applications in controlling the passage of target polynucleotides through nanopores. A kit or device for characterizing a target polynucleotide or controlling the passage of a target polynucleotide through a nanopore, characterized in that the kit or device comprises the helicase described in any one of claims 1 to 3, the nucleotide sequence described in claim 4, the complex described in claim 5, or the construct described in claim 6. A method for characterizing a target polynucleotide or controlling the passage of a target polynucleotide through a nanopore, characterized by comprising the following steps: (1) Combining the target polynucleotide with the nanopore, and the helicase according to any one of claims 1-3 or The complex of claim 5 or the construct of claim 6 is contacted such that the helicase, complex or construct controls the passage of the target polynucleotide through the nanopore; and (2) Obtain one or more characteristics of the interaction between the nucleotides in the target polynucleotide and the nanopore to characterize the target polynucleotide, and the construct includes a helicase and a polynucleoside for binding Acid binding part; wherein said one or more characteristics are preferably selected from the group consisting of origin, length, identity, sequence, secondary structure of the target polynucleotide or whether the target polynucleotide is modified; Said one or more characteristics are preferably carried out by electrical measurements and/or optical measurements; The target polynucleotide is preferably single-stranded, double-stranded, or at least part of it is double-stranded; The nanopores are preferably transmembrane pores, and the transmembrane pores are preferably biological pores, solid pores or hybrid pores between biology and solid. A vector comprising the nucleotide sequence of claim 4. A host cell comprising the nucleotide sequence of claim 4 or the vector of claim 10.