WO2024138472A1 - Porin monomer, porin, mutant thereof and use of same - Google Patents

Abstract

Provided in the present invention are a porin monomer, a porin, a mutant thereof and the use of same. The porin monomer comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein mutant the amino acid sequence of which is obtained by means of substitution, deletion and/or addition of one or several amino acids at at least one of the following sites in SEQ ID NO: 1: the 91st site, the 98th site, the 99th site, etc., the protein mutant having the function of forming pore structures by means of polymerization; or (c) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the protein of (a) or (b), and having the function of forming pore structures by means of polymerization. Provided is a novel nanopore sensor capable of being used for nanopore sequencing, which is applicable to the field of single molecule sequencing.

Description

Porin monomer, porin and its mutant and its application Technical Field

The present invention relates to the field of single molecule sequencing, and in particular to a porin monomer, a porin and a mutant thereof, and applications thereof.

Background technique

At present, some commercial nanopore sequencers have appeared on the market, such as MinION, GridION and PromethION of Oxford Nanopore Technologies in the UK, and QNome-3841 of Qi Carbon. However, they still have major deficiencies in sequencing accuracy, throughput, chip stability and applicable scenarios, and cannot meet the ultimate needs of molecular biology research. Therefore, it is urgent to develop a single-molecule sequencer with high accuracy, high integration and high stability. The nanopore-based single-molecule sequencer is a highly integrated detection system with multiple disciplines and technologies. The development of such instruments requires deep cross-disciplinary and collaborative innovation in multiple disciplines such as physics, biology, chemistry, semiconductors, and computers, and builds a high-precision single-molecule nanopore sequencing system from the underlying core modules.

Nanopore sequencing requires that the sensing region inside the pore protein is sharp enough to have high spatial resolution in both the horizontal and vertical directions. So far, only a few natural proteins such as Mycobacterium smegmatis porin A (MspA) and curli-specific transporter (CsgG) can meet the requirements of becoming pore proteins for single-molecule detectors in the industry. Finding more excellent pore proteins that can be used for single-molecule sequencing through gene mining methods is still an unresolved problem.

Summary of the invention

The main purpose of the present invention is to provide a porin monomer, a porin and a mutant thereof and applications thereof, so as to provide a new porin and a nanopore sensor that can be used for nanopore sequencing.

To achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided a poron monomer, which comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein mutant, the amino acid sequence of the protein mutant undergoes substitution, deletion and/or addition of one or more amino acids at at least one of the following positions of SEQ ID NO: 1: 91, 98, 99, 125, 126, 127, 131, 134, 136, 140, 152, 155, 174, 177, 183, 185, 188, 219, 222, 223, 230, 232, 238, 239, and the protein mutant has the function of forming a pore structure by polymerization; or (c) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in (a) or (b), and has the function of forming a pore structure by polymerization.

Further, in b), the types of substituted amino acids are independently selected from the following: P91 is mutated to P91G, P91A or P91T; S98 is mutated to S98G, S98A, S98T, S98N or S98Q; N99 is mutated to N99G, N99A, N99S, N99T or N99Q; E125 is mutated to E125K, E125R, E125 G, E125A, E125S, E125T, E125N or E125Q; R126 is mutated to R126K, R126G, R126A, R126S, R126T, R126N or R126Q; K127 is mutated to K127R, K127G, K127A, K127S, K127T, K127N or K127Q; D131 Mutation is D131K, D131R, D131G, D131A, D131S, D131T, D131N or D131Q; K134 mutation is K134R, K134G, K134A, K134S, K134T, K134N or K134Q; R136 mutation is R136K, R136G, R136A, R136S, R136T, R136N or R136Q; R140 mutation is R140K, R140G, R140A, R140S, R140T, R140N or R140Q; K152 mutation is K152R, K152G, K152A , K152S, K152T, K152N or K152Q; D155 is mutated to D155R, D155K, D155G, D155A, D155S, D155T, D155N or D155Q; T174 is mutated to T174A, T174G, T174V, T174L, T174I, T174Y, T174F or T174W; E177 is mutated to E177A, E177G, E177S, E177T, E177N or E177Q; H183 is mutated to H183A, H183G, H183V, H183L, H183I, H183Y, H183 83F or H183W; K185 mutated to K185A, K185G, K185V, K185L, K185I, K185Y, K185F or K185W; R188 mutated to R188A, R188G, R188S, R188T, R188N or R188Q; K219 mutated to K219A, K219G, K219V, K219L, K219I, K219Y, K219F or K219W; R222 mutated to R222A, R222G, R222S, R222T, R222N or R222Q; S223 mutated to S223A, S223G, R223S, R223T, R223N or R223Q; 3G, S223V, S223L, S223I, S223Y, S223F or S223W; R230 mutates to R230A, R230G, R230S, R230T, R230N or R230Q; K232 mutates to K232A, K232G, K232S, K232T, K232N or K232Q; E238 mutates to E238A, E238G, E238S, E238T, E238N or E238Q; R239 mutates to R239A, R239G, R239V, R239L, R239I, R239Y, R239F or R239W.

Furthermore, the porin monomer includes a protein having any amino acid sequence of SEQ ID NO: 2-SEQ ID NO: 4.

In order to achieve the above object, according to the second aspect of the present invention, a protein construct is provided. The protein construct is composed of two or more porin monomers mentioned above, connected by covalent or non-covalent bonding.

In order to achieve the above object, according to the third aspect of the present invention, a porin is provided, wherein the porin is composed of 7 to 11 porin monomers mentioned above, preferably 9, connected by covalent or non-covalent bonding.

Furthermore, the porin protein is composed of 9 porin monomers connected non-covalently, and the porin monomers include proteins having any amino acid sequence in SEQ ID NO: 1-SEQ ID NO: 4.

Furthermore, the pore diameter of the porin is 0.5 to 3 nm.

In order to achieve the above object, according to the fourth aspect of the present invention, a kit is provided, which comprises the above porin monomer, or the above protein construct, or the above porin.

Furthermore, the kit also includes a membrane layer, and the membrane layer includes a lipid layer or an artificial polymer membrane.

Furthermore, the kit also includes at least one of the following: a sequencing buffer, a nuclease, a polymerase, a topoisomerase, a ligase, a helicase, and a single-stranded DNA linked to cholesterol.

To achieve the above object, according to the fifth aspect of the present invention, an isolated DNA molecule is provided, which has: a nucleotide sequence encoding the above protein monomer; or a nucleotide sequence encoding the above protein construct, or a nucleotide sequence encoding the above porin.

Furthermore, a DNA molecule that has more than 70%, preferably more than 80%, more preferably more than 90%, further preferably more than 99%, and most preferably more than 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 and encodes a protein with the same function.

In order to achieve the above object, according to the sixth aspect of the present invention, a recombinant vector is provided, which comprises the above DNA molecule.

In order to achieve the above object, according to the seventh aspect of the present invention, a host cell is provided, wherein the host cell is transformed with the above recombinant vector.

In order to achieve the above-mentioned purpose, according to the eighth aspect of the present invention, a nanopore sensor is provided, which comprises: a membrane layer; and a pore protein inserted into the membrane layer and forming a pore, and when a voltage is applied across the membrane layer, the pore generates current; wherein the pore protein comprises the above-mentioned pore protein.

Further, the membrane layer includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids contain a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; preferably, the liposome includes a multilayer liposome or a unilamellar liposome; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.

Furthermore, when a voltage is applied across the membrane layer, the biological molecules to be detected pass through the pores in the nanopore sensor and shift, and the pores generate a changing current; preferably, the biological molecules to be detected include DNA, RNA or polypeptides; preferably, the DNA and/or RNA include any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine, pseudouracil.

In order to achieve the above objective, according to a ninth aspect of the present invention, a nanopore sequencing device is provided, which comprises the above nanopore sensor.

Furthermore, the nanopore sequencing device includes: an electrolytic cell, the electrolytic cell contains a sequencing buffer; a nanopore sensor, the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrolyte area and a negative electrolyte area; a first electrode and a second electrode, the first electrode and the second electrode are respectively arranged in the positive electrolyte area and the negative electrolyte area, and the first electrode and the second electrode are connected to the signal processing chip; preferably, the first electrode and the second electrode include metal or composite electrode materials; preferably, the first electrode and the second electrode are different, silver and silver chloride, respectively; or the first electrode and the second electrode are the same, including gold, platinum, graphene or titanium nitride.

In order to achieve the above-mentioned purpose, according to the tenth aspect of the present invention, a sequencing method is provided, which utilizes the above-mentioned pore protein, or the above-mentioned nanopore sensor, or the above-mentioned nanopore sequencing device to determine the sequence of the biological molecule to be tested by detecting and analyzing the electrical signal generated when the biological molecule to be tested passes through the pore of the pore protein.

Furthermore, the biomolecules to be detected include modified or unmodified DNA, RNA or polypeptide; preferably, the electrical signal includes electric current.

Furthermore, the biological molecule to be detected is a target nucleic acid sequence, and the sequencing method includes: (a) contacting the nucleic acid sequence with the above-mentioned pore protein and nucleic acid binding protein, so that the nucleic acid binding protein controls the movement speed of the target nucleic acid sequence through the pore of the pore protein, wherein the nucleic acid binding protein is selected from any one or more of nucleases, polymerases, topoisomerases, ligases, helicases or single-stranded binding proteins; (b) when a voltage is applied across the pore, when the nucleic acid sequence moves through the pore, measuring the electrical signal passing through the pore, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the nucleic acid based on the electrical signal.

In order to achieve the above-mentioned purpose, according to the eleventh aspect of the present invention, there is provided the above-mentioned porin monomer, or the above-mentioned porin, or the above-mentioned kit, or the above-mentioned DNA molecule, or the above-mentioned recombinant vector, or the above-mentioned host cell, or the above-mentioned nanopore sensor, or the above-mentioned nanopore sequencing device, or the above-mentioned sequencing method for use in biological small molecule detection, nucleic acid sequencing or polypeptide sequencing.

The present invention provides a new pore protein and its mutants that can be used for nanopore sequencing. The nanopore sensor composed of the protein or its mutants has good stability, can meet the needs of single-molecule nanosequencing, and can realize the detection of biological small molecules such as nucleotides, amino acids, sugars, vitamins, etc. It can also be used for sequencing modified or unmodified DNA, RNA or polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

FIG1 shows a side view of the three-dimensional structure of BCP35 predicted according to Example 1 of the present invention.

FIG. 2 shows a top view of the three-dimensional structure of BCP35 predicted according to Example 1 of the present invention.

FIG3 shows a schematic diagram of the predicted structure of key amino acids in the gating region of BCP35 according to Example 1 of the present invention and the distances between key amino acids.

FIG. 4 shows an enlarged view of the structure of key amino acids in the gating region of BCP35 according to Example 1 of the present invention.

FIG5 shows a schematic diagram of key amino acids at the entrance of the predicted structure of BCP35 according to Example 1 of the present invention.

FIG6 shows a schematic diagram of key amino acids at the inner wall and the exit of the pore in the predicted structure of BCP35 according to Example 1 of the present invention.

FIG. 7 shows a schematic diagram of key amino acids in the transmembrane region of the predicted structure of BCP35 according to Example 1 of the present invention.

FIG. 8 shows an SDS-PAGE image obtained by purification of BCP35 protein according to Example 4 of the present invention.

Figure 9 shows a schematic diagram of the sequencing library structure according to Example 5 of the present invention, wherein A is a schematic diagram of the sequencing library structure, B is a schematic diagram of the sequencing library structure combined with single-stranded DNA containing cholesterol, a: positive chain; b: antisense chain; c: double-stranded target fragment to be tested; d: helicase BCH105; e: single-stranded DNA containing cholesterol.

FIG. 10 shows the specific structure of iSp18 according to Embodiment 5 of the present invention.

FIG. 11 shows a graph of the pore opening current of BCP35 according to Example 6 of the present invention at different voltages in a phospholipid bilayer.

FIG. 12 shows a graph showing the current changes when the DNA to be tested passes through the nanopore BCP35 according to Example 7 of the present invention.

Detailed ways

It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

As mentioned in the background technology, there are very few types of pore proteins that can be used for single-molecule nanopore sequencing, and the selectivity is greatly limited. Therefore, in the present application, the inventors have mined a new nanopore protein from the deep-sea metagenome (derived from samples at a depth of 11,000 meters in the Mariana Trench) through gene mining methods using computer-assisted structure prediction, named BCP35, which is composed of nine monomers of the same type. Because it has a pore structure, it can be used as a detection protein and applied to the detection of biological small molecules such as nucleotides, amino acids, sugars, vitamins, or in nanopore-based DNA, RNA or polypeptide sequencing. Therefore, a series of protection schemes for the present application are proposed.

In a first typical embodiment of the present application, a poron monomer is provided, which comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; or (b) a protein mutant, wherein the amino acid sequence of the protein mutant is substituted, deleted and/or added with one or more amino acids at at least one of the following positions of SEQ ID NO: 1: 91, 98, 99, 125, 126, 127, 131, 134, 136, 140, 152, 155, 174, 177, 183, 185, 188, 219, 222, 223, 230, 232, 238, 239, and the protein mutant has the function of forming a pore structure by polymerization; or (c) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein in (a) or (b), and has the function of forming a pore structure by polymerization.

SEQ ID NO: 1:

The porin monomer defined in (a) above can polymerize to form the porin BCP35 having a pore structure, and when applied to nanopore sequencing, it can allow the biomolecules to be tested to pass through the pore one by one, generating a current signal. Based on the sequence in (a), the protein is mutated, for example, at other positions such as the mutation sites disclosed in (b), after substitution and/or deletion and/or addition of one or several amino acids, the pore structure and function of the porin can still be obtained. Mutating the porin monomer may affect the stability of the protein and aggregates, the inner diameter of the pore, and the amino acid residues on the inner wall of the pore, thereby affecting its physicochemical properties and the passing performance of the biomolecules to be tested, but the conventional operation mode of mutation, and the method of screening and obtaining proteins with nanopore structure and functional activity are well known to those skilled in the art.

Identity in this specification refers to the "identity" between amino acid sequences or nucleic acid sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function have a high probability of having the same active site, active pocket, active mechanism, protein structure, etc. as the protein provided by the sequence a).

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

Generally speaking, according to the rules of substitution and replacement, the effects of replacing amino acids with similar properties are similar. For example, in the above protein, conservative amino acid replacements may occur. "Conservative amino acid replacements" include but are not limited to:

Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

The hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;

Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

A person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

The "AlphaFold2-Multimer" used in this application is a public artificial intelligence model that can predict the conformation of protein complexes. The prediction of protein three-dimensional structure can be very close to the level observed by cryo-electron microscopes and other equipment in real experiments. It can obtain a more realistic protein structure, thereby guiding the exploration of protein structure and protein activity.

In a preferred embodiment, in b), the types of substituted amino acids are each independently selected from the following: P91 is mutated into P91G, P91A or P91T; S98 is mutated into S98G, S98A, S98T, S98N or S98Q; N99 is mutated into N99G, N99A, N99S, N99T or N99Q; E125 is mutated into E125K, E125R, E125G, E125A, E125S, E125T, E125N or E125Q; R126 is mutated into R126K, R126G, R126A, R126S, R126T, R126N or R126Q. 26N or R126Q; K127 mutated to K127R, K127G, K127A, K127S, K127T, K127N or K127Q; D131 mutated to D131K, D131R, D131G, D131A, D131S, D131T, D131N or D131Q; K134 mutated to K134R, K134G, K134A, K134S, K134T, K134N or K134Q; R136 mutated to R136K, R136G, R136A, R136S, R136T, R136N or R136Q; R140 mutates to R140K, R140G, R140A, R140S, R140T, R140N or R140Q; K152 mutates to K152R, K152G, K152A, K152S, K152T, K152N or K152Q; D155 mutates to D155R, D155K, D155G, D155A, D155S, D155T, D155N or D155Q; T174 mutates to T174A, T174G, T174V, T174L, T174I, T174Y, T174F or T174W; E17 7 mutated to E177A, E177G, E177S, E177T, E177N or E177Q; H183 mutated to H183A, H183G, H183V, H183L, H183I, H183Y, H183F or H183W; K185 mutated to K185A, K185G, K185V, K185L, K185I, K185Y, K185F or K185W; R188 mutated to R188A, R188G, R188S, R188T, R188N or R188Q; K219 mutated to K219A, K219G, K219V, K219L, K219I, K219Y, K219F or K219W; R222 mutated to R222A, R222G, R222S, R222T, R222N or R222Q; S223 mutated to S223A, S223G, S223V, S223L, S223I, S223Y, S223F or S223W; R230 mutated to R230A, R230G, R230S, R230T, R230N or R230Q; K232 mutates to K232A, K232G, K232S, K232T, K232N or K232Q; E238 mutates to E238A, E238G, E238S, E238T, E238N or E238Q; R239 mutates to R239A, R239G, R239V, R239L, R239I, R239Y, R239F or R239W.

Most of the above amino acid sites are located at the entrance, inner wall and exit of the porin, which is of great significance for the capture of the library and the smooth passage of the library DNA through the porin. Because nucleic acids are negatively charged, increasing or decreasing the amount of positive charge at the entrance can adjust the library capture ability of the porin.

Among the above-mentioned mutation sites, three types of sites are mainly included: mutation sites in the gate region (sensor region), mutation sites at the entrance of the porin, and mutation sites in the transmembrane region of the porin. Among them, the mutation sites in the sensor region mainly determine the opening of the porin, thereby directly determining the opening current. Because the nucleic acid to be tested is negatively charged, by adjusting the amino acids at the mutation sites at the entrance of the porin, including but not limited to mutating uncharged or negatively charged amino acids to positively charged amino acids, or mutating positively charged amino acids to other types of amino acids, this mutation can adjust the capture rate of the library. Mutations in the transmembrane region of the porin can enhance the insertion stability of the porin on lipid or polymer membranes. In addition, charged amino acids located at the inner wall or exit of the pore structure of the porin can also affect the perforation of the sample to be tested.

Among the above mutation sites, P91, S98 and N99 are located in the sensor region. E125, R126, K127, D131, K134, R136, R140, K152 and D155 are located at the entrance of the porin protein. Mutations in their charged properties can play an important role in regulating the capture rate of the library and sequencing noise. T174, H183, K185, K219, S223 and R239 are located on the outer wall of the transmembrane region of the porin protein. Mutations to hydrophobic amino acids can enhance the stability of the porin pore. E177, R188, R222 and E238 are located on the inner wall of the pore structure. They are charged amino acids on the inner wall of the porin barrel. Mutations in their charges can promote the smooth passage of the DNA molecules to be tested through the porin. R230 and K232 are located in the exit loop region of the pore structure. Mutations in them can promote the nucleic acid chain to leave the porin protein after sequencing.

In a preferred embodiment, the porin monomer includes a protein having any amino acid sequence of SEQ ID NO: 2-SEQ ID NO: 4.

SEQ ID NO: 2:

SEQ ID NO: 3:

SEQ ID NO: 4:

In a second typical embodiment of the present application, a protein construct is provided. The protein construct is composed of two or more of the above-mentioned porin monomers linked covalently or non-covalently.

In a third typical embodiment of the present application, a porin is provided, wherein the porin is composed of 7 to 11 porin monomers mentioned above, preferably 9, connected by covalent or non-covalent bonding.

Porin monomers can spontaneously aggregate together through hydrogen bonds, ionic bonds, hydrophobic interactions, etc. to form porins. Therefore, the porin monomers obtained by expression and purification exist in the form of polymers, especially nonamers, under non-denaturing conditions, while porin monomers exist after protein denaturation.

In a preferred embodiment, the porin protein is composed of 9 porin monomers connected non-covalently, and the porin monomers include proteins having any amino acid sequence of SEQ ID NO: 1-SEQ ID NO: 4.

In a preferred embodiment, the pore diameter of the porin is 0.5 to 3 nm.

Within a certain range, the smaller the pore diameter of the nanopore protein, the higher its accuracy when used for sequencing. If the pore diameter of the nanopore protein is too large (more than one molecule may pass through the pore at a time), it is difficult to meet the needs of single-molecule sequencing. When the biological molecule to be tested passes through the overly large pore, the generated current signal may be missed or erroneous, resulting in low sequencing accuracy. In single-molecule sequencing, accurate sequencing results are obtained by sequencing the same molecule multiple times. Therefore, the higher the accuracy of sequencing, the shorter the number and time of sequencing required. The sequencing accuracy of pore proteins with pore diameters within the above range is relatively high. Using this nanopore protein with high sequencing accuracy for sequencing can greatly reduce the sequencing time and reduce costs. This advantage is particularly evident in high-throughput sequencing.

In a fourth typical embodiment of the present application, a kit is provided, which includes the above-mentioned porin monomer, or the above-mentioned protein construct, or the above-mentioned porin.

To further improve the convenience of operation, in a preferred embodiment, the kit further comprises a membrane layer, and the membrane layer comprises a lipid layer or an artificial polymer membrane.

Preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipids comprise a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilayer liposome or a unilamellar liposome; preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine.

Artificial polymer membranes include, but are not limited to, polysiloxanes, polyolefins, perfluoropolyethers, perfluoroalkyl polyethers, polystyrenes, polyoxypropylenes, polyvinyl acetates, polyoxybutylenes, polyisoprene, polybutadiene, polyvinyl chlorides, polyalkyl acrylates, polyalkyl methacrylates, polyacrylonitrile, polypropylene, PTHF, polymethacrylates, polyacrylates, polysulfones, polyethylene ethers, poly(propylene oxide) and copolymers thereof, alkyl-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxy acrylates and methacrylates, polyethylene glycol monomethacrylates and polyethylene glycol monomethyl ether methacrylates, hydroxy-substituted (C1-C6 alkyl) acrylamides and methacrylamides, hydroxy-substituted C1-C6 alkyl vinyl ethers, sodium vinyl sulfonates, sodium styrene sulfonates, 2-acrylamido-2-methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl- One or more of 2-pyrrolidone, 2-vinyloxazoline, 2-vinyl-4,4′-bisalkyloxazolinyl-5-one, 2,4-vinylpyridine, ethylenically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, amino(C1-C6 alkyl)-, mono(C1-C6 alkylamino)(C1-C6 alkyl)- and bis(C1-C6 alkylamino)(C1-C6 alkyl)-acrylates and methacrylates, allyl alcohol, 3-trimethylammonium methacrylate 2-hydroxypropyl ester chloride, dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethyl methacrylamide, glycerol methacrylate, N-(1,1-dimethyl-3-oxobutyl)acrylamide, cyclic imino ethers, vinyl ethers, cyclic ethers containing epoxy derivatives, cyclic unsaturated ethers, N-substituted ethylenimines, β-lactones and β-lactams, vinyl ketone acetals, vinyl acetals or phosphoranes.

In a preferred embodiment, the kit further comprises at least one of the following: a sequencing buffer, a nuclease, a polymerase, a topoisomerase, a ligase, a helicase, and a single-stranded DNA linked to cholesterol.

By using the nanopore protein, membrane layer and sequencing buffer in the above kit, one or more nanopore proteins can be inserted into the membrane layer in the sequencing buffer to form a nanopore sensor. The nanopore experiment buffer can provide a neutral environment to maintain the stability of the nanopore protein and the membrane layer, and the metal ions contained in the nanopore experiment buffer have good conductivity. There are many options for the selection of the membrane layer. The nanopore protein can be inserted into a planar membrane layer or a spherical liposome, and on a lipid layer formed by different components to form a nanopore sensor.

The cholesterol on the single-stranded DNA molecule to be tested can bind to the above-mentioned lipid layer or artificial polymer membrane, which helps the nanopore capture the sequencing library and reduces the amount of sequencing library loading. When actually used, the cholesterol in the above-mentioned kit can first bind to the molecule to be tested and then be added to the space where the nanopore sensor is located for sequencing.

In a fifth typical embodiment of the present application, an isolated DNA molecule is provided, which has: a nucleotide sequence encoding the above-mentioned porin monomer; or a nucleotide sequence encoding the above-mentioned protein construct, or a nucleotide sequence encoding the above-mentioned porin.

In a preferred embodiment, the invention relates to a DNA molecule which has more than 70%, preferably more than 80%, more preferably more than 90%, further preferably more than 99%, and most preferably more than 99% (for example, it can be 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity with the nucleotide sequence shown in SEQ ID NO:5 and encodes a protein with the same function.

SEQ ID NO: 5:

The above DNA molecules can encode nanopore protein monomers having the above structure and function of the present application. Based on the sequence of (a), the nucleotides are mutated, hybridized with the DNA molecule defined by (a) under strict conditions, and no frameshift mutation occurs. If the mutation occurs in the nucleotides encoding the pore of the nanopore protein, it may result in the encoding of a nanopore protein with a changed pore, affecting the pore size of the nanopore protein and the properties of the amino acid residues on the inner wall of the pore; if the mutation occurs in the nucleotides encoding the non-pore part of the protein, it may affect the folding mode, three-dimensional structure and other properties of the encoded protein, thereby affecting the physicochemical properties and stability of the protein. (b) The nucleotide sequence that hybridizes with the DNA molecule defined under strict conditions includes a nucleotide sequence that is 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or 100% complementary to the DNA molecule defined by (a).

"Isolated" in this application means changed "by the hand of man" from its natural state, i.e., if it occurs in nature, it is changed and/or separated from its original environment. For example, a polynucleotide or polypeptide naturally present in a living organism is not "isolated", however, the same polynucleotide or polypeptide separated from its coexisting state in its natural state is "isolated" (as the term is used in this article).

In a sixth typical embodiment of the present application, a recombinant vector is provided, which comprises the above-mentioned DNA molecule.

The above DNA molecule, i.e., the nanopore protein expression gene, is inserted into a recombinant vector, and the nanopore protein expression gene is replicated in large quantities by utilizing the ability of the recombinant vector to replicate itself in large quantities. "Recombinant" here refers to genetically engineered DNA prepared by transplanting or splicing a gene from one species into the cells of a host organism of a different species. This DNA becomes part of the host gene structure and is replicated.

In a seventh typical embodiment of the present application, a host cell is provided, wherein the host cell is transformed with the above-mentioned recombinant vector.

The above recombinant vector is transformed into a host cell, and the host cell is used to replicate, transcribe and translate the nanopore protein expression gene on the recombinant vector, so that a large amount of nanopore protein can be produced. The host cell includes common host cells such as Escherichia coli, yeast, mammalian cells, insect cells, etc. The host cell is used to fold the nanopore protein to form a correct three-dimensional structure, and a nanopore protein with normal structure and function is obtained.

In the eighth typical embodiment of the present application, a nanopore sensor is provided, which includes: a membrane layer; and a pore protein inserted into the membrane layer and forming a pore, and when a voltage is applied across the membrane layer, the pore generates an electric current; wherein the pore protein includes the above-mentioned pore protein.

The nanopore sensor in this application specifically refers to a membrane layer with nanopore protein inserted. This type of nanopore sensor inserts a nanopore protein with a pore in the membrane layer, which can fix the pore direction of the nanopore protein. When an electric field force is applied across the membrane layer, the pore diameter is perpendicular to the direction of the electric field force, and the ions on both sides of the membrane layer pass through the pore of the pore protein under the action of the electric field force, generating current.

In a preferred embodiment, the membrane layer comprises a lipid layer or an artificial polymer membrane; preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipids comprise a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilayer liposome or a unilamellar liposome; preferably, the lipid layer comprises a phospholipid bilayer composed of diphytanoylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine).

There are many options for the selection of lipid layers. Nanopore proteins can be inserted into planar membrane layers or spherical liposomes, or into lipid layers formed of different components to form nanopore sensors.

In a preferred embodiment, when a voltage is applied across the membrane layer, the biological molecules to be detected pass through the pores in the nanopore sensor and shift, and the pores generate a changing current; preferably, the biological molecules to be detected include DNA, RNA or polypeptides; preferably, the DNA and/or RNA include any one or more of the following modified bases: 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine (m7G), pseudouracil (Ψ).

When an electric field force is applied across the membrane layer, the biomolecule to be tested passes through the nanopore protein through the pore under the action of the electric field force. The biomolecule to be tested includes biomacromolecules carrying biological genetic information such as DNA, RNA, polypeptides or proteins. The biomolecule to be tested can carry a group molecule for modification. The group molecule includes but is not limited to cholesterol, polyethylene glycol with different polymerization degrees, biotin or fluorescent group molecules.

In a ninth typical embodiment of the present application, a nanopore sequencing device is provided, wherein the nanopore sequencing device comprises the above-mentioned nanopore sensor.

In a preferred embodiment, the nanopore sequencing device includes: an electrolytic cell, the electrolytic cell contains a sequencing buffer; a nanopore sensor, the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrolyte region and a negative electrolyte region; a first electrode and a second electrode, the first electrode and the second electrode are respectively arranged in the positive electrolyte region and the negative electrolyte region, and the first electrode and the second electrode are connected to a signal processing chip; preferably, the first electrode and the second electrode include metal or composite electrode materials; preferably, the first electrode and the second electrode are different, silver and silver chloride, respectively; or the first electrode and the second electrode are the same, including gold, platinum, graphene or titanium nitride.

In the nanopore sequencing device, there are an electrolytic cell containing an electrolyte, a nanopore sensor, a first electrode, and a second electrode. The nanopore sensor is placed in the center of the electrolytic cell containing an electrolyte, and the electrolytic cell is decomposed into a positive electrolyte area and a negative electrolyte area. The two areas are respectively provided with electrodes, and the electrodes are used to form an electric field applied to the nanopore sensor. The biological molecules to be tested pass through the nanopore protein on the membrane, generating a current amplitude. By receiving this current amplitude and transmitting the current amplitude to a signal processing chip connected to the electrode. According to the difference in current amplitude, the signal processing chip, that is, the nanopore sequencing device including the signal processing chip, can perform data analysis and determination on the sequence of the biological molecules to be tested.

In the tenth typical embodiment of the present application, a sequencing method is provided, which utilizes the above-mentioned pore protein, or the above-mentioned nanopore sensor, or the above-mentioned nanopore sequencing device to determine the sequence of the biological molecule to be tested by detecting and analyzing the electrical signal generated when the biological molecule to be tested passes through the pore of the pore protein.

In a preferred embodiment, the biomolecule to be detected includes modified or unmodified DNA, RNA or polypeptide; preferably, the electrical signal includes electric current.

In a preferred embodiment, the biological molecule to be detected is a target nucleic acid sequence, and the sequencing method comprises: (a) contacting the nucleic acid sequence with the above-mentioned pore protein and nucleic acid binding protein, so that the nucleic acid binding protein controls the movement speed of the target nucleic acid sequence through the pore of the pore protein, wherein the nucleic acid binding protein is selected from any one or more of nucleases, polymerases, topoisomerases, ligases, helicases or single-stranded binding proteins; (b) when a voltage is applied across the pore, when the nucleic acid sequence moves through the pore, measuring the electrical signal passing through the pore, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the nucleic acid based on the electrical signal.

In the eleventh typical embodiment of the present application, a porin monomer, or the porin, or the kit, or the DNA molecule, or the recombinant vector, or the host cell, or the nanopore sensor, or the nanopore sequencing device, or the sequencing method for use in biological small molecule detection, nucleic acid sequencing or polypeptide sequencing is provided.

"Small molecules" in this application include but are not limited to small molecules such as nucleotides, amino acids, polysaccharides or vitamins.

Based on the major advantage of nanopore sequencing over traditional sequencing: it will not affect accuracy due to accumulated errors, so it can achieve extremely long read lengths. It can make up for the gap problem that cannot be avoided when assembling short sequencing fragments in traditional sequencing, determine whether long fragments are missing, repeated, inverted, or translocated in chromosomes, and cover the full length of the transcriptome with a typical length of several kb, thus providing a new solution for scientific research such as genome assembly, structural variation, and variable splicing.

Since nanopore sequencing does not require PCR amplification, the original base modification information on the nucleic acid molecule to be tested can be retained, and the type, site and abundance of the modified base can be directly sequenced at one time. Therefore, the nanopore protein of the present application can also detect several nucleic acid molecules with DNA/RNA modified bases: including 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine (m7G), pseudouracil (pseudouridine, Ψ), etc. Through specific model training and algorithm development for various modified bases, nanopore sequencing can complete the identification and positioning of more modified bases, thereby constructing a more complete genome/transcriptome modification map.

In addition, from the perspective of clinical application, nanopore sequencing has the characteristics of long read length, high portability, fast sequencing speed and real-time readout, so it is suitable for major epidemic monitoring and rapid detection of pathogens (for example, large-scale epidemic operations such as Zika virus, Ebola virus, Dengue virus and Coronavirus), which is very timely. In addition to viruses, nanopore sequencing can also be used for rapid detection of other pathogens such as bacteria and fungi.

Based on the common composition of proteins and nucleic acid molecules, the nanopore sequencing platform also has great application potential in the field of protein sequencing. For example, according to the exploration that has been carried out so far, by using protein unfolding enzymes as rate control tools, the characteristic signals of proteins have been successfully observed, and the initial identification of protein types and modification states has been achieved, verifying the possibility of nanopore protein sequencing. In future development, by further optimizing the rate control system, developing suitable nanopore proteins and signal analysis algorithms, it will eventually be possible to achieve fingerprint recognition and even sequence identification of proteins at the single-molecule level.

In addition to being used in the field of sequencing, the nanopore platform can also be used as a basic detection platform, combined with sensing methods, to complete the metabolomics detection of various small and large molecules. Combining genomics, proteomics, and metabolomics, the nanopore platform can eventually develop into a universal measurement platform that meets the needs of full-omics analysis, providing a powerful research tool for a deeper understanding of the laws of life and the mechanisms of disease occurrence.

The beneficial effects of the present application will be further explained in detail below in conjunction with specific examples. However, those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. The reagents or instruments used without indicating the manufacturer are conventional products that can be obtained on the market. The experimental methods used are conventional methods unless otherwise specified.

Example 1 Predicted structure of the Alphafold2-Multimer of wild-type BCP35

Nine porin monomers (SEQ ID NO: 1) are non-covalently polymerized into nonamers to obtain porin BCP35, and the structure of BCP35 is predicted using Alphafold2-Multimer. The prediction results are shown in Figures 1, 2, 3, 4, 5, 6 and 7. Figure 1 is a side view of the predicted structure of BCP35, and Figure 2 is a top view of the predicted structure of BCP35.

Since the composition of the amino acids in the sensor region (gating region) plays a decisive role in the generation of current signals, the first mutants of BCP35 that were tried were mutations in the sensor region. Figures 3 and 4 show the side chain structures of the important amino acids in the sensor region of the predicted structure of BCP35, showing that the three amino acids in the amino acid side chains are P91, S98 and N99.

Since nucleic acids are negatively charged, if the amino acids at the entrance of the porin are positively charged, the library capture efficiency of nanopore sequencing can be increased, otherwise, the library capture rate will be reduced. At the same time, the strength of the binding between the library and the porin will also have a certain impact on the sequencing speed and sequencing current signal. Figure 5 shows some important amino acids in the entrance region of BCP35, which are E125, R126, K127, D131, K134, R136, R140, K152, and D155.

At the same time, some amino acids on the inner wall and outlet of the barrel have an impact on the smooth passage of the analyte through and out of the nanopore. Figure 6 shows some amino acids on the inner wall and outlet of the barrel of BCP35 that may affect the smooth passage of the analyte through the pore, especially charged amino acids, which are E177, R188, R222, R230, K232, and E238.

Since the amino acids in the membrane-facing direction of the porin transmembrane region prefer hydrophobic amino acids, charged amino acids and polar amino acids may affect the efficiency of pore insertion. Figure 7 shows multiple charged and polar amino acids facing the membrane in the transmembrane region of BCP35, which are T174, H183, K185, K219, S223, and R239.

Example 2 Construction of expression vectors for porin BCP35 and its mutants

By the In-fusion method, after digestion with NdeI and XhoI, the DNA sequence encoding the porin monomer (SEQ ID NO: 5) was inserted into the multi-cloning region of the vector pET24a. StrepII amino acid was added to the C-terminus of the amino acid sequence of the porin monomer (SEQ ID NO: 1) as a purification tag, wherein the screening tag was kanamycin, and the constructed vector was named pET24a-BCP35. By the site-directed mutagenesis method, the Agilent site-directed mutagenesis kit was used, and the expression vector of porin BCP35 was used as a template to construct the corresponding mutants. This embodiment constructs three mutants of P91A, S98N, and N99S. The constructed mutant vectors were named pET24a-BCP35-P91A, pET24a-BCP35-S98N, and pET24a-BCP35-N99S, respectively.

Example 3 Cultivation and induction of porin monomer strains

The constructed porin monomer or its mutant expression plasmids were independently transformed into the E. coli expression strain E. coli BL21 (DE3), and the bacterial solution was evenly spread on a plate containing 50μg/mL kanamycin and cultured at 37°C overnight. The next day, a single colony was picked and cultured in 5mL LB medium containing 50μg/mL kanamycin at 37°C, 200rpm, overnight. The above-obtained bacterial solution was inoculated into 50mL LB containing 50μg/mL kanamycin at a ratio of 1:100, and cultured at 37°C, 200rpm for 4h. The expanded cultured bacterial solution was inoculated into 2L LB containing 50μg/mL kanamycin at a ratio of 1:100 and cultured at 37°C, 200rpm. When the OD600 value reached about 0.6-0.8, IPTG was added at a final concentration of 0.5mM, and cultured at 16°C, 200rpm for about 16-18h. The bacterial solution was collected by centrifugation at 8000 rpm and the bacteria were frozen at -20°C until use.

Example 4 Extraction and purification of recombinant porin monomers

(1) Buffer preparation:

Buffer A: 20 mM Tris-HCl, 250 mM NaCl, 1% DDM, pH 8.0.

Buffer B: 20 mM Tris-HCl, 250 mM NaCl, 0.05% DDM, pH 8.0.

Buffer C: 20 mM Tris-HCl, 250 mM NaCl, 0.05% DDM, 5 mM desthiobiotin, pH 8.0.

(2) Purification step:

Resuspend the cells thoroughly at a ratio of 10 mL Buffer A per 1 g of cells, and ultrasonically disrupt the cells until the cell solution is clear. Then, place the cells on a rotator and rotate them at 4°C overnight. Centrifuge at 18,000 rpm at 4°C for 1 hour the next day, take the supernatant, filter with a 0.22 μm filter membrane, and store at 4°C for later use.

On an AKTA pure chromatograph, the Strep-Tactin beads (IBA Lifesciences) column was equilibrated with Buffer A for 5 column volumes (CV) and then loaded at 2 mL/min. After loading, it was rinsed with Buffer B for 20 CV and eluted with Buffer C to collect the target protein.

The obtained protein was concentrated to 1 mL, passed through a Superdex 6increase 10/300GL (Cytiva) column equilibrated with buffer B, and the target protein was collected and then stored at -80°C. The target protein obtained after purification was subjected to SDS-PAGE electrophoresis, and the results of the wild-type protein monomer are shown in Figure 8. Lanes 2 and 3 and lanes 4 and 5 are the electrophoresis bands of uncooked (porin nonamer BCP35) and boiled at 95°C (porin monomer after denaturation). The results showed that the target protein was in a polymer state before boiling and in a monomer state after boiling. The SDS-PAGE results of the mutant were consistent with those of the wild-type protein, and will not be specifically displayed here.

Example 5 Library construction

The sense strand and antisense strand of two partially complementary DNA strands (SEQ ID NO: 8) were connected to the double-stranded target fragment pUC57 (SEQ ID NO: 9) to be tested using T4 DNA ligase at room temperature and purified to prepare a sequencing library. The sequencing library was then incubated with helicase BCH105 (SEQ ID NO: 10) at 25°C for 1 hour (molar concentration ratio 1:8) to form a sequencing library containing the BCH105 motor protein with a structure as shown in Figure 9A. During sequencing, the sequencing library can further complementarily pair with single-stranded DNA with cholesterol (SEQ ID NO: 11, cholesterol is connected to the 5′ end of DNA) to form a structure as shown in Figure 9B.

Linker sequence positive chain: S1-(iSp18)4-S2.

The sequence of S1 is shown in SEQ ID NO: 6, the sequence of S2 is shown in SEQ ID NO: 7, and the structure of iSp18 is shown in Figure 10.

SEQ ID NO：6：ttttttttttttttttttttttttttttttttttttttttttttt.

SEQ ID NO:7:ggttgtttctgttggtgctgatattgct.

SEQ ID NO: 8 (antisense linker sequence):

SEQ ID NO: 9:

SEQ ID NO: 10:

Example 6 Construction of nanopore biosensor using porin BCP35 and its mutants

The current signal is collected using a patch clamp amplifier. The Ag/AgCl electrode is immersed in the sequencing buffer and the electrodes are respectively located in the cis region and the trans region of the electrolytic cell. After diluting the porin (i.e., the porin BCP35 purified and obtained in Example 4) by a certain multiple using 1xPBS buffer, a single nanoporin BCP35 is inserted into a phospholipid bilayer composed of diacylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) under the action of an external electric field force to form a nanopore biosensor. The dilution multiple is based on whether the porin is embedded in the membrane (i.e., embedded in the pore). Generally, a protein concentration of 0.1 mg/ml is used, and it is diluted 100 times, 50 times, or other times with PBS for an attempt. If a certain dilution concentration fails to embed the pore, it is necessary to reduce the dilution multiple and continue to try until the nanoporin is successfully embedded in the membrane layer. Apply an applied voltage to obtain the current amplitude value of a single porin. Figure 11 shows the biosensing current generated by the pore of nanoporin BCP35 when voltages of 0.02 V, 0.04 V, 0.10 V, 0.14 V and 0.18 V are applied. It can be seen that the pore current of porin BCP35 is stable at different voltages with little noise. Similarly, porin BCP35 mutants (P91A, S98N and N99S) also produce stable biosensing current.

Example 7 Use of porin BCP35 and its mutants for DNA sequencing

The sequencing library containing the pUC57 sequence prepared in Example 5 and the single-stranded DNA with cholesterol (SEQ ID NO: 11, cholesterol is connected to the 5′ end of the DNA) were mixed with the sequencing buffer and added to the nanopore biosensor; after applying an external voltage of 0.14V or 0.18V, it was observed that the DNA was captured by the nanopore, generating a characteristic blocking current amplitude value. And as the DNA moves through the nanopore, the current amplitude value changes. Different DNA sequences produce different blocking current amplitude values. Single-stranded DNA with cholesterol can bind to the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduce the amount of sequencing library loaded. Figure 12 shows the current changes generated when the library DNA passes through the nanopore protein BCP35 under the action of an applied voltage of 0.14V. It can be seen that the library DNA can pass through the wild-type BCP35 pore protein to generate current, and the current value fluctuates as different nucleotides penetrate the pore. Similarly, when the library DNA passed through the porin BCP35 mutants (P91A, S98N, and N99S), current changes were also generated, indicating that the porin BCP35 and its mutants can be used for nanopore sequencing.

Single-stranded DNA sequence with cholesterol (SEQ ID NO: 11):

cholestero-ttgaccgctcgcctc.

Sequencing buffer: 0.47 M KCl, 25 mM HEPES, 1 mM EDTA, 5 mM ATP, 25 mM MgCl ₂ , pH 7.6.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present invention has discovered a new nanopore protein monomer, which is polymerized into the porin BCP58. The porin and its mutants have good stability and can meet the needs of single-molecule nanopore sequencing. The above nanopore protein and its mutants can be used to form a nanopore sensor and a further nanopore sequencing device to realize the detection of small molecules such as nucleotides, amino acids, sugars, vitamins, etc., and can also be used for sequencing samples such as DNA, RNA and polypeptides.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (23)

A porin monomer, characterized in that the porin monomer comprises: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) a protein mutant, wherein the amino acid sequence of the protein mutant undergoes substitution, deletion and/or addition of one or more amino acids at at least one of the following positions of SEQ ID NO: 1: 91, 98, 99, 125, 126, 127, 131, 134, 136, 140, 152, 155, 174, 177, 183, 185, 188, 219, 222, 223, 230, 232, 238, 239, and the protein mutant has the function of forming a pore structure through polymerization; or (c) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the protein described in (a) or (b), and has the function of forming a pore structure through polymerization. The porin monomer according to claim 1, characterized in that in b), the types of substituted amino acids are each independently selected from the following: P91 mutated to P91G, P91A, or P91T; S98 mutation to S98G, S98A, S98T, S98N, or S98Q; N99 mutated to N99G, N99A, N99S, N99T, or N99Q; E125 mutation is E125K, E125R, E125G, E125A, E125S, E125T, E125N, or E125Q; R126 mutation to R126K, R126G, R126A, R126S, R126T, R126N, or R126Q; K127 mutation is K127R, K127G, K127A, K127S, K127T, K127N, or K127Q; D131 mutation is D131K, D131R, D131G, D131A, D131S, D131T, D131N or D131Q; K134 mutation is K134R, K134G, K134A, K134S, K134T, K134N, or K134Q; R136 mutation to R136K, R136G, R136A, R136S, R136T, R136N, or R136Q; R140 mutation to R140K, R140G, R140A, R140S, R140T, R140N, or R140Q; K152 mutation is K152R, K152G, K152A, K152S, K152T, K152N, or K152Q; D155 is mutated to D155R, D155K, D155G, D155A, D155S, D155T, D155N, or D155Q; T174 mutation is T174A, T174G, T174V, T174L, T174I, T174Y, T174F, or T174W; E177 mutation to E177A, E177G, E177S, E177T, E177N, or E177Q; H183 mutated to H183A, H183G, H183V, H183L, H183I, H183Y, H183F, or H183W; K185 mutation is K185A, K185G, K185V, K185L, K185I, K185Y, K185F, or K185W; R188 mutation to R188A, R188G, R188S, R188T, R188N, or R188Q; K219 mutation is K219A, K219G, K219V, K219L, K219I, K219Y, K219F, or K219W; R222 mutated to R222A, R222G, R222S, R222T, R222N or R222Q; S223 mutation to S223A, S223G, S223V, S223L, S223I, S223Y, S223F, or S223W; R230 mutated to R230A, R230G, R230S, R230T, R230N, or R230Q; K232 mutation is K232A, K232G, K232S, K232T, K232N, or K232Q; E238 mutation is E238A, E238G, E238S, E238T, E238N, or E238Q; R239 is mutated to R239A, R239G, R239V, R239L, R239I, R239Y, R239F or R239W. The porin monomer according to claim 1 or 2 is characterized in that the porin monomer comprises a protein having any amino acid sequence of SEQ ID NO: 2-SEQ ID NO: 4. A protein construct, characterized in that the protein construct is formed by covalently or non-covalently linking two or more porin monomers according to any one of claims 1 to 3. A porin, characterized in that the porin is composed of 7 to 11 porin monomers according to any one of claims 1 to 3, connected covalently or non-covalently, preferably 9 porin monomers. The porin according to claim 5 is characterized in that the porin is composed of 9 porin monomers connected non-covalently, and the porin monomers include proteins having any amino acid sequence in SEQ ID NO: 1-SEQ ID NO: 4. The porin according to claim 5 or 6, characterized in that the pore diameter of the porin is 0.5 to 3 nm. A kit, characterized in that the kit comprises the porin monomer according to any one of claims 1 to 3, or the protein construct according to claim 4, or the porin according to any one of claims 5 to 7. The kit according to claim 8 is characterized in that the kit further comprises a membrane layer, and the membrane layer comprises a lipid layer or an artificial polymer membrane. The kit according to claim 9, characterized in that the kit further comprises at least one of the following: a sequencing buffer, a nuclease, a polymerase, a topoisomerase, a ligase, a helicase, and a single-stranded DNA linked to cholesterol. An isolated DNA molecule, characterized in that the DNA molecule has: A nucleotide sequence encoding a porin monomer according to any one of claims 1 to 3; or a nucleotide sequence encoding a protein construct according to claim 4, or a nucleotide sequence encoding a porin according to any one of claims 5 to 7. The DNA molecule according to claim 11 is characterized in that it has more than 70%, preferably more than 80%, more preferably more than 90%, further preferably more than 99%, and most preferably more than 99% identity with the nucleotide sequence shown in SEQ ID NO: 5 and encodes a DNA molecule with the same functional protein. A recombinant vector, characterized in that the recombinant vector comprises the DNA molecule according to claim 11 or 12. A host cell, characterized in that the host cell is transformed with the recombinant vector according to claim 13. A nanopore sensor, characterized in that the nanopore sensor comprises: film layer; and a porin protein inserted into the membrane layer and forming a pore that generates an electric current when a voltage is applied across the membrane layer; Wherein, the porin comprises the porin according to any one of claims 5 to 7. The nanopore sensor according to claim 15, characterized in that the membrane layer comprises a lipid layer or an artificial polymer membrane; Preferably, the lipid layer comprises amphiphilic lipids; Preferably, the amphiphilic lipid comprises a phospholipid bilayer; Preferably, the lipid layer comprises a planar membrane layer or a liposome; Preferably, the liposomes comprise multilamellar liposomes or unilamellar liposomes; Preferably, the lipid layer comprises a phospholipid bilayer composed of diphytylphosphatidylcholine. The nanopore sensor according to claim 15, characterized in that When a voltage is applied across the membrane layer, the biomolecule to be detected passes through the pore in the nanopore sensor and shifts, and the pore generates a changing current; Preferably, the biomolecule to be detected includes DNA, RNA or polypeptide; Preferably, the DNA and/or RNA comprises any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine, pseudouracil. A nanopore sequencing device, characterized in that the nanopore sequencing device comprises the nanopore sensor according to any one of claims 15 to 17. The nanopore sequencing device according to claim 18, characterized in that the nanopore sequencing device comprises: an electrolytic cell containing a sequencing buffer; A nanopore sensor, wherein the nanopore sensor is located in the center of the electrolytic cell and divides the electrolytic cell and the sequencing buffer into a positive electrode electrolyte region and a negative electrode electrolyte region; A first electrode and a second electrode, wherein the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte region and the negative electrode electrolyte region, and the first electrode and the second electrode are connected to a signal processing chip; Preferably, the first electrode and the second electrode comprise metal or composite electrode materials; Preferably, the first electrode and the second electrode are different, namely silver and silver chloride, respectively; or the first electrode and the second electrode are the same, including gold, platinum, graphene or titanium nitride. A sequencing method, characterized in that the sequencing method uses the porin described in any one of 5 to 7, or the nanopore sensor described in any one of claims 15 to 17, or the nanopore sequencing device described in claim 18 or 19 to determine the sequence of the biological molecule to be tested by detecting and analyzing the electrical signal generated when the biological molecule to be tested passes through the pore of the porin. The sequencing method according to claim 20, characterized in that the biomolecule to be detected includes modified or unmodified DNA, RNA or polypeptide; Preferably, the electrical signal comprises an electric current. The sequencing method according to claim 20, characterized in that the biological molecule to be detected is a target nucleic acid sequence, and the sequencing method comprises: (a) contacting the nucleic acid sequence with a porin and a nucleic acid binding protein according to any one of claims 5 to 7, so that the nucleic acid binding protein controls the speed at which the target nucleic acid sequence moves through the pore of the porin, wherein the nucleic acid binding protein is selected from any one or more of a nuclease, a polymerase, a topoisomerase, a ligase, a helicase or a single-stranded binding protein; (b) When a voltage is applied across the pore, when the nucleic acid sequence moves through the pore, an electrical signal passing through the pore is measured, wherein different types of nucleotides generate different electrical signals when passing through the pore, thereby determining the sequence information of the nucleic acid based on the electrical signal. Use of the porin monomer according to any one of claims 1 to 3, or the porin according to any one of claims 5 to 7, or the kit according to any one of claims 8 to 10, or the DNA molecule according to claim 11 or 12, or the recombinant vector according to claim 13, or the host cell according to claim 14, or the nanopore sensor according to any one of claims 15 to 17, or the nanopore sequencing device according to claim 18 or 19, or the sequencing method according to any one of claims 20 to 22 in biological small molecule detection, nucleic acid sequencing or polypeptide sequencing.

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