CN113651876A

CN113651876A - Mutant of porin monomer, protein pore and application thereof

Info

Publication number: CN113651876A
Application number: CN202110949862.9A
Authority: CN
Inventors: 刘少伟; 何京雄; 李倩雯; 岳飞飞
Original assignee: Chengdu Qitan Technology Ltd
Current assignee: Chengdu Qitan Technology Ltd
Priority date: 2021-08-18
Filing date: 2021-08-18
Publication date: 2021-11-16
Anticipated expiration: 2041-08-18
Also published as: CN113651876B

Abstract

The invention belongs to the technical field of characterization of target analyte characteristics, and particularly provides a mutant of a porin monomer, a protein pore comprising the mutant, and application of the mutant to detection of a target analyte, wherein the amino acid of the mutant of the porin monomer comprises mutations at one or more positions corresponding to A74, P75, G76, N77, A78, T79, N80 and F81 of SEQ ID NO. 1.

Description

Mutant of porin monomer, protein pore and application thereof

Technical Field

The invention belongs to the technical field of characterization of target analyte characteristics, and particularly relates to a mutant of a pore protein monomer, a protein pore containing the mutant and application of the mutant in detection of a target analyte.

Background

With the research on the structure and sequence of nucleic acid, the nucleic acid sequencing technology is continuously developed, becomes the core field of life science research, and plays a great role in promoting the technical development in the fields of biology, chemistry, electricity, life science, medicine and the like. The research of a novel rapid, accurate, low-cost, high-precision and high-throughput nucleic acid sequencing technology by using the nanopore is one of the hot spots of the subsequent human genome project.

Nanopore (Nanopore) sequencing technology, also known as fourth generation sequencing technology, is a gene sequencing technology that takes a single-stranded nucleic acid molecule as a sequencing unit, utilizes a Nanopore capable of providing an ion current channel, allows the single-stranded nucleic acid molecule to pass through the Nanopore under electrophoretic drive, reduces the current of the Nanopore when nucleic acid passes through the Nanopore, and reads sequence information in real time for different generated signals.

The nanopore sequencing is mainly characterized in that: the reading length is long, the accuracy rate is high, and most error regions occur in the homopolymeric oligonucleotide region. Nanopore sequencing can not only realize natural DNA and RNA sequencing, but also directly acquire base modification information of DNA and RNA, for example, methylated cytosine can be directly read without need of bisulfite (bisufite) treatment on genome in advance like a second generation sequencing method, which greatly promotes the direct study of epigenetic correlation phenomenon at genome level. As a novel platform, the nanopore detection technology has the advantages of low cost, high flux, no mark and the like.

Nanopore analysis techniques originated from the invention of the Coulter counter and the recording technique of single-channel currents. In 1976, Neher and Sakamann who obtained Nobel prize in physiology and medicine use the patch clamp technology to measure membrane potential and study membrane protein and ion channels, thus promoting the practical application process of nanopore sequencing technology. In 1996, Kasiaanowicz et al proposed a new idea of DNA sequencing using α -hemolysin, which is a landmark marker for single molecule sequencing of biological nanopores. Subsequently, the research on biological nanopores such as MspA porins and phage Phi29 connectors is reported, and the research on nanopore analysis technology is enriched. Li et al in 2001 opened a new era of solid-state nanopore research. Solid state nanopore sequencing has been slow progressing, limited by advances in the semiconductor and materials industries.

One of the key points of nanopore sequencing technology lies in the design of a special biological nanopore, a reading head structure formed in a constriction zone in the nanopore can cause the blockage of channel current when a single-stranded nucleic acid (such as ssDNA) molecule passes through the nanopore, so that the current intensity flowing through the nanopore is influenced transiently (the amplitude of current change influenced by each base is different), and finally, high-sensitivity electronic equipment detects the changes so as to identify the passed base. Currently, protein pores are used as nanopores for sequencing, and the pore proteins mainly use escherichia coli as a source.

At present, the nanopore protein is single, and a substitute nanopore protein needs to be developed to realize a nanopore sequencing technology. The porin is also closely related to sequencing precision, and the porin also relates to mode change of interaction with the rate control protein, so that the stability of an interaction interface of the porin and the rate control protein is further optimized, and the consistency and the stability of sequencing data are positively influenced. The accuracy of nanopore sequencing technology is also in need of improvement, and therefore, there is a need to develop improved nanopore proteins to further improve the resolution of nanopore sequencing.

Disclosure of Invention

To solve the above problems, it is an object of embodiments of the present invention to provide an alternative mutant of a porin monomer, a protein pore comprising the same, and uses thereof.

In a first aspect, embodiments of the invention provide a mutant of a porin monomer, wherein the amino acids of the mutant of a porin monomer comprise or consist of the sequence set forth in SEQ ID No. 1 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60% or 50% identity thereto, and the amino acids of the mutant of a porin monomer comprise a mutation at one or more positions corresponding to a74, P75, G76, N77, a78, T79, N80, and F81 of SEQ ID No. 1.

Preferably, the amino acids of the mutant of porin monomers comprise:

(1) (ii) having insertions, deletions and/or substitutions of amino acids at one or more positions corresponding to A74, P75, G76, N77, A78, T79, N80 and F81 of SEQ ID NO 1; or (2) having an insertion, deletion and/or substitution of an amino acid at one or more positions corresponding to A74, P75, G76, N77, A78, T79, N80, F81, S82 and T83 of SEQ ID NO. 1.

In one embodiment, the amino acid mutation of the mutant of porin monomers is selected from the group consisting of:

(a) the amino acids APGNATNF corresponding to positions 74-81 of SEQ ID NO. 1 are mutated to M₁M₂M₃M₄M₅M₆Wherein M is₁0 to 1 selected from P; m ₂0 to 3 selected from Y, F, W; m ₃0 to 1 selected from P; m ₄0 to 5 selected from A, G, V, L, I; m ₅0 to 4 selected from N, D, E, Q; m ₆0 to 5 selected from A, G, V, L, I;

(b) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₇M₈M₉M₁₀M₁₁M₁₂M₁₃M₁₄Wherein M is₇0 to 5 selected from A, G, V, L, I; m ₈0 to 3 selected from H, K, R; m ₉0 to 1 selected from P; m ₁₀0 to 5 selected from A, G, V, L, I; m ₁₁0 to 5 selected from A, G, V, L, I; m ₁₂0 to 5 selected from T, S, C, U, M; m ₁₃0 to 5 selected from S, C, U, T, M; m ₁₄0 to 1 selected from P;

(c) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₁₅M₁₆M₁₇M₁₈M₁₉M₂₀M₂₁M₂₂Wherein M is₁₅0 to 1 selected from P; m ₁₆0 to 5 selected from A, G, V, L, I; m ₁₇0 to 5 selected from A, G, V, L, I; m ₁₈0 to 5 selected from S, C, U, T, M; m ₁₉0 to 5 selected from S, C, U, T, M; m ₂₀0 to 5 selected from L, G, A, V, I; m ₂₁0 to 5 selected from S, C, U, T, M; m ₂₂0 to 1 selected from P; and

(d) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₂₃M₂₄M₂₅M₂₆M₂₇M₂₈M₂₉M₃₀Wherein M is₂₃0 to 1 selected from P; m ₂₄0 to 3 selected from Y, F, W; m ₂₅0 to 1 selected from P; m ₂₆0 to 5 selected from A, G, V, L, I; m ₂₇0 to 4 selected from N, D, E, Q; m ₂₈0 to 5 selected from A, G, V, L, I; m ₂₉0 to 5 selected from S, C, U, T, M; m ₃₀0 to 1 selected from P.

(a) the amino acids APGNATNF corresponding to positions 74-81 of SEQ ID NO. 1 are mutated to M₁M₂M₃M₄M₅M₆Wherein M is₁Is selected from P; m₂Selected from Y, F or W; m₃Is selected from P; m₄Selected from A, G, V, L or I; m₅Selected from N, D, E or Q; m₆Selected from A, G, V, L or I;

(b) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₇M₈M₉M₁₀M₁₁M₁₂M₁₃M₁₄Wherein M is₇Selected from A, G, V, L or I; m₈Selected from H, K or R; m₉Is selected from P; m₁₀Selected from A, G, V, L or I; m₁₁Selected from A, G, V, L or I; m₁₂Selected from T, S, C, U or M; m₁₃Selected from S, C, U, T or M; m₁₄Is selected from P;

(c) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₁₅M₁₆M₁₇M₁₈M₁₉M₂₀M₂₁M₂₂Wherein M is₁₅Is selected from P; m₁₆Selected from A, G, V, L or I; m₁₇Selected from A, G, V, L or I; m₁₈Selected from S, C, U, T or M; m₁₉Is selected from SC, U, T or M; m₂₀Selected from L, G, A, V or I; m₂₁Selected from S, C, U, T or M; m₂₂Is selected from P; and

(d) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₂₃M₂₄M₂₅M₂₆M₂₇M₂₈M₂₉M₃₀Wherein M is₂₃Is selected from P; m₂₄Selected from Y, F or W; m₂₅Is selected from P; m₂₆Selected from A, G, V, L or I; m₂₇Selected from N, D, E or Q; m₂₈Selected from A, G, V, L or I; m₂₉Selected from S, C, U, T or M; m₃₀Is selected from P.

(a) the amino acid APGNATNF corresponding to the 74-81 position of SEQ ID NO. 1 is mutated into PYPANA;

(b) amino acid APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 is mutated to AHPAATSP;

(c) amino acid APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 is mutated to PASSSLSP; and

(d) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to PYPANASP.

In a second aspect, embodiments of the invention provide a protein pore comprising at least one mutant of a porin monomer.

In a third aspect, embodiments of the present invention provide a complex for characterising a target analyte, characterised in that: the protein pore and the rate-controlling protein combined with the protein pore.

In a fourth aspect, embodiments of the invention provide nucleic acids encoding a mutant, protein pore, or complex of a porin monomer.

In a fifth aspect, embodiments of the invention provide vectors or genetically engineered host cells comprising the nucleic acids.

In a sixth aspect, embodiments of the present invention provide the use of a mutant of a porin monomer, a protein well, complex, nucleic acid, vector or host cell thereof, in detecting the presence, absence or one or more characteristics of a target analyte, or in the manufacture of a product for detecting the presence, absence or one or more characteristics of a target analyte.

In a seventh aspect, the embodiments of the present invention provide a method for producing a protein pore or a polypeptide thereof, comprising transforming the host cell with the vector, and inducing the host cell to express the protein pore or the polypeptide thereof.

In an eighth aspect, embodiments of the present invention provide a method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

a. contacting a target analyte with a protein pore, complex, or protein pore in a complex such that the target analyte moves relative to the protein pore; and

b. obtaining one or more measurements while the target analyte is moving relative to the protein pore, thereby determining the presence, absence or one or more characteristics of the target analyte.

In one embodiment, the method comprises: the target analyte interacts with the protein pores present in the membrane such that the target analyte moves relative to the protein pores.

In one embodiment, the target analyte is a nucleic acid molecule.

In one embodiment, a method for determining the presence, absence or one or more characteristics of a target analyte comprises coupling the target analyte to a membrane; and the target analyte interacts with the protein pores present in the membrane such that the target analyte moves relative to the protein pores.

In a ninth aspect, embodiments of the present invention provide a kit for determining the presence, absence or one or more characteristics of a target analyte, comprising a mutant of said porin monomer, said protein pore, said complex, said nucleic acid, or said vector or host, and components of said membrane. .

In a tenth aspect, embodiments of the present invention provide a device for determining the presence, absence or one or more characteristics of a target analyte, comprising said protein pore or said complex, and said membrane.

In one embodiment, the target analyte comprises a polysaccharide, a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a drug, a diagnostic agent, an explosive, or an environmental contaminant;

preferably, the target analyte comprises a polynucleotide,

more preferably, the polynucleotide comprises DNA or RNA; and/or, the one or more characteristics are selected from (i) the length of the polynucleotide; (ii) identity of the polynucleotides; (iii) the sequence of the polynucleotide; (iv) (iv) the secondary structure of the polynucleotide and (v) whether the polynucleotide is modified; and/or, the rate controlling protein in the complex comprises a polynucleotide binding protein.

Drawings

The drawings described are only schematic and are non-limiting.

Fig. 1 illustrates the basic working principle of a nanopore according to one embodiment.

Figure 2 shows a schematic diagram of DNA sequencing according to one embodiment.

FIG. 3 shows the corresponding pore blocking signal when a nucleotide passes through a protein pore according to one embodiment.

Fig. 4A and 4B illustrate a wild-type protein pore channel surface structure and a bandmap model according to one embodiment. Fig. 4A is a surface structure model, and fig. 4B is a streamer structure model.

Fig. 5 shows a wild-type protein pore constriction zone amino acid residue distribution and constriction zone diameter, according to one embodiment.

Figure 6A shows a surface potential map of a wild-type protein monomer according to one embodiment.

Fig. 6B shows a stick model of a monomer streamer model and its constriction zone amino acid residue distribution, according to one embodiment.

Fig. 7A shows the constriction zone amino acid residue distribution characteristics and constriction zone diameters of mutant well 1, according to one embodiment.

Fig. 7B shows the banderol graph of fig. 7A.

FIG. 8 shows a cartoon representation of a mutant hole 1 based on homology modeling according to one embodiment.

FIG. 9 shows the structure of the DNA construct BS7-4C3-SE1 according to one embodiment.

FIG. 10 shows the structure of the DNA construct BS7-4C3-PLT according to one embodiment.

FIG. 11A shows the opening current and its gating characteristics at a voltage of 180mV for a mutant opening 1 according to one embodiment.

FIG. 11B shows a nucleic acid via scenario with a mutant well 1 at +180mV voltage, according to one embodiment.

12A, 12B, 12C, and 12D show example current trajectories when helicase Mph-MP1-E105C/A362C controls the translocation of DNA construct BS7-4C3-SE1 through mutation well 1, according to one embodiment.

FIG. 13 is an enlarged view of a single signal region of the embodiment of FIG. 12B.

Figure 14A shows the opening current and its gating characteristics at ± 180mV voltage for protein pore mutant 2 according to one embodiment.

Figure 14B shows a nucleic acid via at +180mV voltage for protein pore mutant 2, according to one embodiment.

FIGS. 15A and 15B show example current trajectories when helicase Mph-MP1-E105C/A362C controls translocation of DNA construct BS7-4C3-PLT through mutant pore 2, according to one embodiment.

FIG. 16 is an enlarged view of a single signal in the embodiment of FIG. 15A.

Figure 17A shows the opening current and its gating characteristics at ± 180mV voltage for protein pore mutant 3 according to one embodiment.

Figure 17B shows a nucleic acid via scenario for protein pore mutant 3 at +180mV voltage, according to one embodiment.

18A, 18B, 18C, 18D, 18E, and 18F show example current trajectories when helicase Mph-MP1-E105C/A362C controls the translocation of DNA construct BS7-4C3-PLT through mutation pore 3, according to one embodiment.

FIG. 19 is an enlarged view of a single signal region of the embodiment of FIG. 18B.

Figure 20A shows the opening current and its gating characteristics at ± 180mV voltage for protein pore mutant 4 according to one embodiment.

Figure 20B shows a nucleic acid via situation for protein pore mutant 4 at a voltage of +180mV, according to one embodiment.

21A, 21B, 21C, 21D, 21E, and 21F show example current trajectories when helicase Mph-MP1-E105C/A362C controls the translocation of DNA construct BS7-4C3-PLT through mutation pore 4, according to one embodiment.

FIG. 22 is an enlarged area display of a single signal of the embodiment of FIG. 21.

FIG. 23 shows the result of SDS-PAGE electrophoretic detection of mutant 1 according to one embodiment.

Detailed Description

It is understood that the unused applications of the disclosed products and methods may be adapted according to the specific needs of the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

Also, as used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a nucleotide" includes two or more nucleotides and reference to "a helicase" includes two or more helicases.

As used herein, the term "comprising" means that any of the listed elements must be included, and that other elements may also optionally be included. "consisting of" means not including all the elements not listed. Embodiments defined by each of these terms are within the scope of the invention.

As used herein, a "nucleotide sequence", "DNA sequence" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length (ribonucleotides or deoxyribonucleotides). The term refers only to the primary structure of the molecule. Thus, the term includes double-and single-stranded DNA and RNA.

The term "nucleic acid" as used herein refers to a single-or double-stranded covalently linked sequence of nucleotides in which the 3 'and 5' ends of each nucleotide are linked by a phosphodiester linkage. A nucleotide may consist of a deoxyribonucleotide base or a ribonucleotide base. Nucleic acids may include DNA and RNA, and may be synthetically prepared in vitro or isolated from natural sources. Nucleic acids may further include modified DNA or RNA, e.g., methylated DNA or RNA, or RNA that has been post-translationally modified, e.g., 5 '-capping with 7-methylguanosine, 3' -end processing, e.g., cleavage and polyadenylation, and splicing. Nucleic acids may also include synthetic nucleic acids (XNA), such as Hexitol Nucleic Acids (HNA), cyclohexene nucleic acids (CeNA), Threose Nucleic Acids (TNA), Glycerol Nucleic Acids (GNA), Locked Nucleic Acids (LNA) and Peptide Nucleic Acids (PNA). The size of a nucleic acid (or polynucleotide) is typically expressed in terms of the number of base pairs (bp) of a double-stranded polynucleotide, or in the case of a single-stranded polynucleotide, the number of nucleotides (nt). 1 kilobase or nt equals one kilobase pair (kb). Polynucleotides less than about 40 nucleotides in length are commonly referred to as "oligonucleotides" and may comprise primers for use in DNA manipulation, e.g., by Polymerase Chain Reaction (PCR).

Polynucleotides, such as nucleic acids, are macromolecules comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides may be naturally occurring or synthetic. One or more nucleotides in the polynucleotide may be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For example, the polynucleotide may comprise a pyrimidine dimer. This dimer is often associated with damage caused by ultraviolet light and is the major cause of cutaneous melanoma. One or more nucleotides in the polynucleotide may be modified, for example with a conventional label or tag. The polynucleotide may comprise one or more nucleotides that are abasic (i.e., lack nucleobases), or lack nucleobases and sugars (i.e., are C3).

The nucleotides in the polynucleotide may be linked to each other in any manner. The nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides may be linked by their nucleobases, as in the guanine dimers.

The polynucleotide may be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double stranded. The polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide may comprise an RNA strand that is hybridized to a DNA strand. The polynucleotide may be any synthetic nucleic acid known in the art, such as Peptide Nucleic Acid (PNA), Glycerol Nucleic Acid (GNA), Threose Nucleic Acid (TNA), Locked Nucleic Acid (LNA) or other synthetic polymers having nucleotide side chains. The PNA backbone is composed of repeating N- (2-aminoethyl) -glycine units linked by peptide bonds. The GNA backbone is composed of repeating ethylene glycol units linked by phosphodiester bonds. The TNA skeleton is composed of resuscitated glycosyl connected together through phosphodiester bonds. LNAs are formed from the ribonucleic acids described above, with an additional bridging structure connecting the 2 'oxygen and the 4' carbon in the ribose moiety. The Bridged Nucleic Acid (BNA) is a modified RNA nucleotide. They may also be referred to as restricted or inaccessible RNA13BNA monomers that may contain a 5-, 6-or even 7-membered bridging structure and have an "immobilized" C3 '-internal sugar folding structure (C3' -endo sugar tucking). The bridging structure is synthetically introduced into the 2 ', 4' -position of the ribose to produce the 2 ', 4' -BNA monomer.

The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The polynucleotide may be of any length. For example, the polynucleotide may be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide may be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs or 100000 or more nucleotides or nucleotide pairs in length.

Any number of polynucleotides may be studied. For example, the methods of the embodiments can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polynucleotides. If two or more polynucleotides are characterized, they may be the case for different polynucleotides or the same polynucleotide.

Polynucleotides may be naturally occurring or synthetic. For example, the method can be used to verify the sequence of the prepared oligonucleotides. The method is typically performed in vitro.

In the context of the present disclosure, the term "amino acid" is used in its broadest sense and is meant to include amine (NH) -containing compounds₂) And a Carboxyl (COOH) functional group and a side chain (e.g., R group) specific to each amino acid. In some embodiments, an amino acid refers to a naturally occurring L α -amino acid or residue. The commonly used single and three letter abbreviations for naturally occurring amino acids are used herein: a ═ Ala; c ═ Cys; d ═ Asp; e is Glu; f ═ Phe; g ═ Gly; h ═ His; i ═ Ile; k ═ Lys; l ═ Leu; m is Met; n ═ Asn; p ═ Pro; q ═ Gln; r ═ Arg; s is Ser; t ═ Thr; v is Val; w ═ Trp; and Y-Tyr (leining e r, a.l. (1975) BioChemis try, 2 nd edition, pages 71-92, Worth Publishers, New York). The generic term "amino acid" also includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids that are not normally incorporated into proteins (such as norleucine), and chemically synthesized compounds (such as β -amino acids) that have properties known in the art to be characteristic of amino acids. For example, included within the definition of amino acid are analogs or mimetics of phenylalanine or proline that allow the same conformational restriction of a peptide compound as a native Phe or Pro. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids. Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer editions, Vol.5, page 341, Academic Press, Inc., N.Y.1983, which are incorporated herein by reference, list additional examples of amino acids.

The terms "protein," "polypeptide," and "peptide" are further used interchangeably herein to refer to polymers of amino acid residues as well as variants and synthetic analogs of amino acid residues. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The polypeptide may also undergo maturation or post-translational modification processes, which may include, but are not limited to: glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc.

"homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified or wild-type protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which the sequences are identical on an amino acid-amino acid basis over a comparison window. Thus, the "percent sequence identity" is calculated by: the two optimally aligned sequences are compared over a comparison window, the number of positions in the two sequences at which the same amino acid residue occurs (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, gin, Cys, and Met) is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to yield the percentage of sequence identity.

Sequence identity may also be a fragment or portion of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity to a full-length reference sequence, but the sequence of a particular region, domain or subunit may have 80%, 90% or up to 99% sequence identity to a reference sequence.

The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. The wild-type gene is the gene most commonly observed in a population and is therefore arbitrarily designed as the "normal" or "wild-type" form of the gene. Conversely, the terms "modified," "mutation," or "variant" refer to a gene or gene product that exhibits a sequence modification (e.g., substitution, truncation, or insertion), post-translational modification, and/or functional property (e.g., altered characteristics) as compared to the wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered characteristics compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutated monomer. Methods of introducing or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-trnas in the IVTT system for expressing mutant monomers. Alternatively, the non-naturally occurring amino acids may be introduced by expressing a mutant monomer in vibrio thermophilus (thermolesulfovibrio sp.n1), which is auxotrophic for particular amino acids in the presence of synthetic (i.e., non-naturally occurring) analogues of those particular amino acids. If the mutated monomers are generated using partial peptide synthesis, they may also be generated by naked ligation. Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties, or similar side chain volumes. The amino acids introduced may have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge similar to the amino acids they replace. Alternatively, a conservative substitution may introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected based on the properties of the 20 major amino acids defined in table 1 below. In the case of amino acids with similar polarity, this can also be determined with reference to the hydrophilicity scale for the amino acid side chains in table 2.

TABLE 1 chemical Properties of amino acids

TABLE 2 hydrophilicity Scale

Side chains	Hydrophilicity
		Ile,I	4.5
Val,V	4.2
		Leu,L	3.8
Phe,F	2.8
		Cys,C	2.5
Met,M	1.9
		Ala,A	1.8
Gly,G	-0.4
		Thr,T	-0.7
Ser,S	-0.8
		Trp,W	-0.9
Tyr,Y	-1.3
		Pro,P	-1.6
His,H	-3.2
		Glu,E	-3.5
Gln,Q	-3.5
		Asp,D	-3.5
Asn,N	-3.5
		Lys,K	-3.9
Arg,R	-4.5

It is well known that conservative substitutions of amino acids with similar properties to each other, as shown in Table 3, do not generally affect the activity of the peptide sequence.

TABLE 3 conservative amino acid substitutions

The mutated or modified protein, monomer or peptide may also be chemically modified at any site in any manner. The mutated or modified monomer or peptide is preferably chemically modified by attachment of the molecule to one or more cysteines (cysteine linkage), attachment of the molecule to one or more lysines, attachment of the molecule to one or more unnatural amino acids, enzymatic modification of an epitope or modification of the terminus. Suitable methods for making such modifications are well known in the art. Mutants of modified proteins, monomers or peptides may be chemically modified by attachment of any molecule. For example, mutants of modified proteins, monomers or peptides may be chemically modified by attachment of dyes or fluorophores. In some embodiments, the mutant or modified monomer or peptide is chemically modified with a molecular adaptor that facilitates interaction between the pore comprising the monomer or peptide and the target nucleotide or target polynucleotide sequence. The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, a substance capable of hybridizing, a DNA binding agent or intercalator, a peptide or peptide analogue, a synthetic polymer, an aromatic planar molecule, a positively charged small molecule or a small molecule capable of hydrogen bonding.

The presence of the adapter improves the host-guest chemistry of the pore and nucleotide or polynucleotide sequence, thereby improving the sequencing capability of the pore formed by the mutated monomer. The principles of host-guest chemistry are well known in the art. The adaptors have an effect on the physical or chemical properties of the pore, which improves the interaction of the pore with the nucleotide or polynucleotide sequence. The adapter may alter the charge of the barrel or channel of the pore, or specifically interact or bind with a nucleotide or polynucleotide sequence, thereby facilitating its interaction with the pore.

A "protein pore" is a transmembrane protein structure that defines a channel or pore that allows molecules and ions to translocate from one side of the membrane to the other. The translocation of ionic species through the pore may be driven by a potential difference applied to either side of the pore. A "nanopore" is a protein pore in which the smallest diameter of a channel through which a molecule or ion passes is on the order of nanometers (10)^-9Rice). In some embodiments, the protein pore may be a transmembrane protein pore. The transmembrane protein structure of a protein pore may be monomeric or oligomeric in nature. Typically, the well comprisesA plurality of polypeptide subunits arranged around a central axis, thereby forming a protein-lined channel extending substantially perpendicular to a membrane in which the nanopore resides. The number of polypeptide subunits is not limited. Typically, the number of subunits is from 5 to 30, suitably from 6 to 10. Alternatively, the number of subunits is not defined as in the case of perfringolysin (perfringolysin) or related large membrane pores. The portion of the protein subunit within the nanopore that forms the protein lining channel typically comprises a secondary structural motif that may include one or more transmembrane β -barrel and/or α -helical portions.

In one embodiment, the protein pore comprises one or more pore protein monomers. Each porin monomer may be from vibrio thermophilus. In one embodiment, the protein pore comprises a mutant of one or more pore protein monomers (i.e., a monomer mutated in one or more pore proteins).

In one embodiment, the porin is from a wild-type protein, wild-type homolog, or mutant thereof of kingdom biologies. The mutant may be a modified porin or a porin mutant. Modifications in the mutants include, but are not limited to, any one or more of the modifications disclosed herein or combinations of such modifications. In one embodiment, the wild-type protein of kingdom biologics is a protein from vibrio thermophilus.

In one embodiment, porin homologue refers to a polypeptide having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% complete sequence identity to the protein set forth in SEQ ID NO. 1.

In one embodiment, a porin homologue refers to a polynucleotide having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% complete sequence identity to the polynucleotide encoding the protein set forth in SEQ ID NO. 2. The polynucleotide sequence may comprise a sequence that differs from SEQ ID NO. 2 based on the degeneracy of the genetic code.

Polynucleotide sequences can be derived and replicated using methods standard in the art. Chromosomal DNA encoding the wild-type porin can be extracted from a pore-producing organism, such as Vibrio thermophilus. The gene encoding the pore subunit can be amplified using PCR including specific primers. The amplified sequence may then be subjected to site-directed mutagenesis. Suitable methods for site-directed mutagenesis are known in the art and include, for example, combinatorial chain reactions. The constructed polynucleotides encoding the embodiments can be prepared using techniques well known in the art, such as those described in Sambrook, J.and Russell, D. (2001). Molecular Cloning A Laboratory Manual,3rd edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y..

The resulting polynucleotide sequence may then be integrated into a recombinant replicable vector, such as a cloning vector. The vectors can be used to replicate the polynucleotides in compatible host cells. Thus a polynucleotide sequence may be prepared by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which cause the vector to replicate. The vector may be recovered from the host cell.

Fundamental working principle of nanopore or protein pore

In one embodiment, within the electrolyte filled chamber 100, an insulating film 102 with nanoscale pores divides the chamber into 2 cells, as shown in fig. 1, and when a voltage is applied to the electrolyte chamber, ions or other small molecule species pass through the pores under the force of an electric field, creating a stable detectable ionic current. Different types of biomolecules can be detected by grasping the size and surface characteristics of the nanopore, the applied voltage and the solution conditions.

Because the four bases of adenine (A), guanine (G), cytosine (C) and thymine (T) which form DNA have different molecular structures and sizes, when single-stranded DNA (ssDNA) passes through a nano-scale pore under the drive of a speed-control enzyme and an electric field, the change amplitude of current caused by the difference of chemical properties of different bases when the single-stranded DNA passes through a nano-scale pore or a protein pore is different, and thus the sequence information of the detected nucleic acid such as DNA is obtained.

FIG. 2 shows a schematic 200 of DNA sequencing. As shown in fig. 2, in a typical nanopore/protepore sequencing experiment, the nanopore is the only channel for ions to pass through on both sides of the phospholipid membrane. Rate controlling proteins, such as polynucleotide binding proteins, act as motor proteins for nucleic acid molecules, such as DNA, pulling DNA strands to pass through the nanopore/protein pore in single nucleotide steps. Whenever one nucleotide passes through the nanopore/protein pore, the corresponding pore blocking signal is recorded (fig. 3). By analyzing the current signals associated with these sequences by a corresponding algorithm, sequence information of nucleic acid molecules such as DNA can be deduced back.

In the examples, porins are screened from different species in nature (mainly bacteria and archaea) by bioinformatics means and evolutionary points of view. In one embodiment, the porin is from any organism, preferably from vibrio thermophilus, and the proteins homologous to the e.coli amyloid secretion channel (e.coli CsgG) have less than 40% sequence identity, preferably 20% -40% sequence identity. By sequence analysis, porins have a complete functional domain. And (3) utilizing a structural biology means to predict and analyze a porin 3D structural model, and selecting a channel protein with a proper reading head architecture form. And then, modifying, testing and optimizing the candidate channel protein (or porin) by means of genetic engineering, protein directed evolution, computer-aided protein design and the like, and obtaining a plurality of homologous protein mutants, preferably four homologous protein mutants (different homologous protein frameworks) with different signal characteristics and signal distribution modes through several iterations.

The porins of the examples are applicable to fourth generation sequencing technologies. In one embodiment, the porin is a nanopore. In one embodiment, the porin may be applied to solid-state wells for sequencing.

In one embodiment, a new protein backbone is employed, forming a new constriction zone (read head zone) structure, thereby providing a completely new mode of action during sequencing. The porins of the examples have good skip-edge distribution and efficiency of recombination with phospholipid membranes.

In one embodiment, genetic mutation of a wild-type porin monomer modifies a mutant that forms a porin monomer. In one embodiment, the amino acids of the mutant of the porin monomer comprise the sequence set forth in SEQ ID No. 1 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identity thereto, and the amino acids of the mutant of the porin monomer have a mutation corresponding to position 74-81 or position 74-83 of SEQ ID No. 1.

In one embodiment, the mutation comprises an insertion, deletion and/or substitution of an amino acid. In one embodiment, the mutations at positions 74-81 or 74-83 of SEQ ID NO. 1 are (1) amino acid insertions, deletions and/or substitutions at one or more of positions 74-81 of SEQ ID NO. 1; or (2) SEQ ID NO 1 with insertions, deletions and/or substitutions of amino acids at one or more of positions 74-83.

In one embodiment, the mutant of the porin monomer has amino acid (1) with an insertion, deletion and/or substitution of an amino acid at one or more positions corresponding to a74, P75, G76, N77, a78, T79, N80 and F81 of SEQ ID NO: 1; or (2) having an insertion, deletion and/or substitution of an amino acid at one or more positions corresponding to A74, P75, G76, N77, A78, T79, N80, F81, S82 and T83 of SEQ ID NO. 1.

In one embodiment, the mutant of the porin monomer has only mutations at amino acids corresponding to positions 74-81 of SEQ ID NO:1 (i.e. a74, P75, G76, N77, a78, T79, N80, and F81).

In one embodiment, the amino acids of the mutant of porin monomer have insertions, deletions and/or substitutions of amino acids only at one or more positions corresponding to positions 74-81 of SEQ ID NO:1 (i.e. a74, P75, G76, N77, a78, T79, N80 and F81).

In one embodiment, the position corresponding to SEQ ID NO. 1 is such that the numbering of the sequence of SEQ ID NO. 1 is used regardless of whether the relative position is unchanged by amino acid insertion or deletion or by using an identity sequence such that the numbering of the sequence is changed. For example, it is within the scope of the present invention that A74 corresponding to SEQ ID NO. 1 may be mutated to A74P and that even if the SEQ ID NO. 1 sequence number is changed or a sequence having the identity as defined herein with SEQ ID NO. 1 is used, the amino acid A corresponding to position 74 of SEQ ID NO. 1 (even if position 74 is not in the other sequence) may be mutated to P.

In one embodiment, the amino acids of the mutant of the porin monomer consist of the sequence set forth in SEQ ID No. 1, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% or 70%, 65%, 60%, 55%, or 50% identity thereto, and the porin has a mutation corresponding to position 74-81 or 74-83 of SEQ ID No. 1.

In one embodiment, the SEQ ID NO 1 sequence of the porin monomer is from Vibrio thermophilus. The nucleotide sequence of the amino acid of the SEQ ID NO. 1 is SEQ ID NO. 2.

In one embodiment, the mutant of the protein monomer has a mutation at positions 74-81, or 74-83 of the sequence of SEQ ID NO. 1.

In one embodiment, the amino acids corresponding to positions 74-81 of SEQ ID NO 1 are mutated or substituted with M₁M₂M₃M₄M₅M₆Wherein M is₁Is selected from P; m₂Selected from Y, F or W; m₃Is selected from P; m₄Selected from A, G, V, L or I; m₅Selected from N, D, E or Q; m₆Selected from A, G, V, L or I.

In one embodiment, the amino acids corresponding to positions 74-83 of SEQ ID NO. 1 are mutated or substituted with M₇M₈M₉M₁₀M₁₁M₁₂M₁₃M₁₄Wherein M is₇Selected from A, G, V, L or I; m₈Selected from H, K or R; m₉Is selected from P; m₁₀Selected from A, G, V, L or I; m₁₁Selected from A, G, V, L or I; m₁₂Selected from T, S, C, U or M; m₁₃Selected from S, C, U, T or M; m₁₄Is selected from P.

In one embodiment, the amino acids corresponding to positions 74-83 of SEQ ID NO. 1 are mutated or substituted with M₁₅M₁₆M₁₇M₁₈M₁₉M₂₀M₂₁M₂₂Wherein M is₁₅Is selected from P; m₁₆Selected from A, G, V, L or I; m₁₇Selected from A, G, V, L or I; m₁₈Selected from S, C, U, T or M; m₁₉Selected from S, C, U, T or M; m₂₀Selected from L, G, A, V or I; m₂₁Selected from S, C, U, T or M; m₂₂Is selected from P.

In one embodiment, the amino acids corresponding to positions 74-83 of SEQ ID NO. 1 are mutated or substituted with M₂₃M₂₄M₂₅M₂₆M₂₇M₂₈M₂₉M₃₀Wherein M is₂₃Is selected from P; m₂₄Selected from Y, F or W; m₂₅Is selected from P; m₂₆Selected from A, G, V, L or I; m₂₇Selected from N, D, E or Q; m₂₈Selected from A, G, V, L or I; m₂₉Selected from S, C, U, T or M; m₃₀Is selected from P.

In one embodiment, the mutant of a porin monomer, wherein the amino acid mutation is selected from the group consisting of:

(a) the amino acid APGNATNF corresponding to the positions 74-81 of SEQ ID NO. 1 is mutated or replaced by PYPANA;

(b) (ii) amino acid APGNATNFST mutation or substitution to AHPAATSP corresponding to positions 74-83 of SEQ ID No. 1;

(c) (ii) a mutation or substitution of amino acid APGNATNFST corresponding to positions 74-83 of SEQ ID No. 1 to PAASSLSP; and

(d) the amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated or replaced with PYPANASP.

In one embodiment, the amino acid sequence of the mutant of the porin monomer comprises or consists of SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 27, or SEQ ID No. 28.

In one embodiment, the protein pore comprises a mutant of at least one porin monomer (or porin mutated monomer). In one embodiment, the protein pore comprises mutants of at least two, three, four, five, six, seven, eight, nine or ten or more pore protein monomers. In one embodiment, the protein pore comprises a mutant of at least two porin monomers, which may beAre the same or different. In one embodiment, the protein pore comprises a mutant of two or more pore protein monomers, preferably the mutants of two or more monomers are identical. In one embodiment, the constriction zone pore diameter of the protein pore is 0.7nm to 2.2nm, 0.9nm to 1.6nm, 1.4 to 1.6nm, or

Use of a mutant of a porin monomer or a protein well comprising the same for detecting the presence, absence or one or more characteristics of a target analyte. In one embodiment, mutants of porin monomers or protein pores are used to detect the sequence of a nucleic acid molecule, or to characterize a polynucleotide sequence, e.g., sequence a polynucleotide sequence, as they can distinguish different nucleotides with high sensitivity. Mutants of porin monomers or protein wells comprising them can distinguish between four nucleotides in DNA and RNA, even methylated and unmethylated nucleotides, with unexpectedly high resolution. Mutant of porin monomers or protein wells showed almost complete separation of all four DNA/RNA nucleotides. Deoxycytidine monophosphate (dCMP) and methyl-dCMP are further distinguished based on the residence time in the protein pore and the current flowing through the protein pore.

Mutants of porin monomers or protein pores can also distinguish between different nucleotides under a range of conditions. In particular, mutants of the porin monomers or protein pores distinguish nucleotides under conditions that facilitate nucleic acid characterization, such as sequencing. By varying the applied potential, salt concentration, buffer, temperature and the presence of additives such as urea, betaine and DTT, the extent to which mutants of porin monomers or protein wells distinguish between different nucleotides can be controlled. This allows mutants of porin monomers or protein pore functions to be finely controlled, especially in sequencing. Mutants of porin monomers or protein pores may also be used to identify polynucleotide polymers by interaction with one or more monomers rather than on nucleotide-based nucleotides.

A mutant of a porin monomer or protein pore may be isolated, substantially isolated, purified, or substantially purified. The mutant of the porin monomer or protein pore of the examples is isolated or purified if it is completely free of any other components, such as liposomes or other protein pores/porins. A mutant of a porin monomer or protein pore is substantially isolated if it is mixed with a carrier or diluent that does not interfere with its intended use. For example, a mutant of a porin monomer or a protein pore is substantially isolated or substantially purified if it is present in a form comprising less than 10%, less than 5%, less than 2%, or less than 1% of other components such as triblock copolymers, liposomes, or other protein pores/porins. Alternatively, a mutant of a porin monomer or protein pore may be present in the membrane.

For example, the membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed of amphiphilic molecules, for example, phospholipids, which have hydrophilicity and lipophilicity. The amphiphilic molecules may be synthetic or naturally occurring. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is generally planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported. The membrane may be a lipid bilayer. The lipid bilayer is formed by two opposing layers of lipid. The two layers of lipids are arranged such that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of the lipid face outward toward the aqueous environment on each side of the bilayer. The membrane includes a solid layer. The solid-state layer may be formed of organic and inorganic materials. If the membrane comprises a solid layer, the pores are typically present in the amphiphilic membrane or in a layer comprised within the solid layer, e.g. in holes, wells, gaps, channels, trenches or slits within the solid layer.

Characterization of analytes

Embodiments provide a method of determining the presence, absence or one or more characteristics of a target analyte. The method involves contacting the target analyte with a mutant or protein well of a pore protein monomer such that the target analyte moves relative to, e.g., through, the mutant or protein well of the pore protein monomer and taking one or more measurements as the target analyte moves relative to the mutant or protein well of the pore protein monomer, thereby determining the presence, absence or one or more characteristics of the target analyte. The target analyte may also be referred to as a template analyte or analyte of interest.

The target analyte is preferably a polysaccharide, metal ion, inorganic salt, polymer, amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide, polynucleotide, dye, drug, diagnostic agent, explosive or environmental contaminant. The methods can involve determining the presence, absence, or one or more characteristics of two or more target analytes of the same class, e.g., two or more proteins, two or more nucleotides, or two or more drugs. Alternatively, the method can involve determining the presence, absence, or one or more characteristics of two or more different classes of target analytes, e.g., one or more proteins, one or more nucleotides, and one or more drugs.

The method comprises contacting the target analyte with a mutant of a porin monomer or a protein well such that the target analyte moves through the mutant of the porin monomer or the protein well. The protein pore typically comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 monomers from which the porin is mutated, e.g., 7, 8, 9, or 10 monomers. The protein pores comprise the same monomer or different porin monomers, preferably comprising 8 or 9 of the same monomer. One or more of the monomers, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10, are preferably chemically modified as discussed above. In one embodiment, the amino acids of each monomer include SEQ ID NO 1 and the above-described mutants thereof. In one embodiment, the amino acid of each monomer consists of SEQ ID NO 1 and its above mutants.

The methods of the embodiments can measure two, three, four, or five or more characteristics of the polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether or not the polynucleotide is modified. In one embodiment, any combination of (i) to (v) may be measured.

For (i), the length of the polynucleotide can be measured, for example, by determining the number of interactions or the duration of interactions between the polynucleotide and the mutant/protein pore of the protein monomer.

For (ii), the identity of the polynucleotide may be measured in a number of ways, which may be measured in conjunction with or without measurement of the polynucleotide sequence. The former is simpler; the polynucleotide is sequenced and then identified. The latter can be done in several different ways. For example, the presence of a particular motif in a polynucleotide can be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, measurement of a particular electrical and/or optical signal in the method may identify the polynucleotide as being from a particular source.

For (iii), the sequence of the polynucleotide may be determined as previously described. Suitable sequencing methods, particularly those using electrical measurement methods, are described in Stoddart D et al, ProC Natl Acad Sci, 12; 106(19)7702-7, Lieberman KR et al, J Am Chem SoC.2010; 132(50)17961-72, and in International application W02000/28312.

For (iv), secondary structure can be measured using a variety of methods. For example, if the method involves an electrical measurement method, the secondary structure may be measured using changes in dwell time or changes in current flowing through the hole. This allows for distinguishing regions of single-stranded and double-stranded polynucleotides.

For (v), the presence or absence of any modification can be measured. The method preferably comprises determining whether the polynucleotide has been modified by methylation, oxidation, damage, by one or more proteins or by one or more labels, tags or by the absence or absence of nucleobases and sugars. Specific modifications will result in specific interactions with the pore, which can be measured using the methods described below. For example, methylcytosine can be distinguished from cytosine based on the current flowing through the pore during its interaction with each nucleotide.

The target polynucleotide is contacted with a mutant/protein pore of a protein monomer, for example a protein monomer as in the examples. Mutants/protein pores of the protein monomer are usually present in the membrane. Suitable membranes are as described hereinbefore. The method may be carried out using any device suitable for studying membrane/protein pores or mutant systems of porin monomers, in which a mutant of a protein monomer/protein pore is present in a membrane. The method may be performed using any device suitable for trans-membranous pore sensing. For example, the device comprises a chamber containing an aqueous solution and a barrier dividing the chamber into two parts. The barrier typically has a hole in which a membrane containing a pore is formed. Or the barrier forms a membrane in which mutants/protein pores of the protein monomer are present. The process may be carried out using the apparatus described in International application No. PCT/GB08/000562(WO 2008/102120).

Various different types of measurements may be made. This includes, but is not limited to, electrical and optical measurements. Electrical measurements include voltage measurements, capacitance measurements, current measurements, impedance measurements, tunneling measurements (Ivanov AP et al, Nano lett.2011jan 12; 11(I):279-85) and FET measurements (international application TO 2005/124888). Optical measurements can be combined with electrical measurements (Soni GV et al, Rev Sci Instrum.2010Jan; 81(1) 014301). The measurement may be a transmembrane current measurement, for example a measurement of the ionic current flowing through the pore. In one embodiment, the electrical or optical measurements may employ conventional electrical or optical measurements.

Electrical measurements can be made using the techniques described in Stoddart D et al, ProC Natl Acad Sci, 12; 106(19)7702-7, Lieberman KR et al, J Am Chem SoC.2010; 132(50)17961-72 and the standard single-channel recording device of international application WO 2000/28312. Alternatively, the electrical measurements may be made using a multichannel system, for example as described in international application W02009/077734 and international application WO 2011/067559.

The method is preferably carried out using an electrical potential applied across the membrane. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is the use of a salt gradient across the membrane, for example an amphiphilic molecule layer. Salt gradients are disclosed in Holden et al, J Am Chem soc.2007jul 11; 129(27) 8650-5. In some cases, the current flowing through the mutant/protein pore of the protein monomer as the polynucleotide moves relative to the mutant/protein pore of the protein monomer is used to estimate or determine the sequence of the polynucleotide. This is strand sequencing.

The method may comprise measuring the current flowing through the pore as the polynucleotide moves relative to the pore. The apparatus used in the method may therefore also comprise a circuit capable of applying an electrical potential and measuring an electrical signal across the membrane and the pores. The method may be performed using patch-clamp or voltage clamp.

May comprise measuring the current flowing through the pore as the polynucleotide moves relative to the pore. Suitable conditions for measuring ion flow through a transmembrane protein pore are known in the art and are disclosed in the examples. The method is typically performed by applying a voltage across the membrane and the pores. The voltage used is typically from +5V to-5V, for example from +4V to-4V, from +3V to-3V or from +2V to-2V. The voltages used are generally from-600 mV to +600V or-400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from-400 mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300nA P +400 mV. The voltage used is more preferably in the range of 100mV to 240mV and most preferably in the range of 120mV to 220 mV. By using an increased applied potential, the recognition of different nucleotides by the wells can be increased.

The process is typically carried out in the presence of any charge carrier, for example a metal salt such as an alkali metal salt, a halide salt such as a chloride salt, for example an alkali metal chloride salt. The charge carrier may comprise an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazole chloride. In the above exemplary apparatus, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is generally used. KCl, NaCl and mixtures of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric on the membrane. For example, the type and/or concentration of charge carriers may be different on each side of the membrane.

The concentration of the salt may be saturated. The concentration of the salt may be 3M or less, and is usually 0.1 to 2.5M, 0.3 to 1.9M, 0.5 to 1.8M, 0.7 to 1.7M, 0.9 to 1.6M or 1M to 1.4M. The concentration of the salt is preferably 150mM to 1M. The process is preferably carried out using a salt concentration of at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least 2.0M, at least 2.5M or at least 3.0M. High salt concentrations provide a high signal-to-noise ratio and allow the presence of nucleotides to be identified in the context of normal current fluctuations to be indicated by the current.

The method is typically carried out in the presence of a buffer. In the above exemplary device, the buffer is present in an aqueous solution in the chamber. Any buffer may be used in the methods of the invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES or Tris-HCl buffers. The process is typically carried out at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

The process may be carried out at a temperature of from 0 ℃ to 100 ℃, from 15 ℃ to 95 ℃, from 16 ℃ to 90 ℃, from 17 ℃ to 85 ℃, from 18 ℃ to 80 ℃, from 19 ℃ to 70 ℃ or from 20 ℃ to 60 ℃. The process is typically carried out at room temperature. The process is optionally carried out at a temperature that supports enzyme function, for example about 37 ℃.

In one embodiment, a method for determining the presence, absence or one or more characteristics of a target analyte (e.g., a polynucleotide) comprises coupling the target analyte to a membrane; and the target analyte interacts with (e.g., contacts) the protein pore present in the membrane such that the target analyte moves relative to (e.g., through) the protein pore. In one embodiment, the current through the protein pore is measured as the target analyte moves relative to the protein pore, thereby determining the presence, absence or one or more characteristics (e.g., sequence of polynucleotides) of the target analyte.

Polynucleotide binding proteins

The characterization methods of the embodiments preferably comprise contacting the polynucleotide with a polynucleotide binding protein such that the protein controls movement of the polynucleotide relative to, e.g., through, a mutant/protein pore of a protein monomer.

More preferably, the method comprises (a) contacting the polynucleotide with the mutant/protein pore of the protein monomer and the polynucleotide binding protein such that the protein controls movement of the polynucleotide relative to the mutant/protein pore of the protein monomer, e.g., through the mutant/protein pore of the protein monomer, and (b) taking one or more measurements as the polynucleotide moves relative to the mutant/protein pore of the protein monomer, wherein the measurements are indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.

More preferably, the method comprises (a) contacting the polynucleotide with the mutant/protein pore of the protein monomer and the polynucleotide binding protein such that the protein controls movement of the polynucleotide relative to the mutant/protein pore of the protein monomer, e.g., through the mutant/protein pore of the protein monomer, and (b) measuring a current through the mutant/protein pore of the protein monomer as the polynucleotide moves relative to the mutant/protein pore of the protein monomer, wherein the current is indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.

The polynucleotide binding protein may be any protein that is capable of binding to a polynucleotide and controlling its movement through a pore. Polynucleotide binding proteins typically interact with and modify at least one property of a polynucleotide. Proteins may be modified by cleaving polynucleotides to form individual nucleotides or short strands of nucleotides (e.g., dinucleotides or trinucleotides). A protein may modify a polynucleotide by orienting it or moving it to a specific location, i.e., controlling its movement.

The polynucleotide binding protein is preferably derived from a polynucleotide processing enzyme. A polynucleotide processive enzyme is a polypeptide that is capable of interacting with a polynucleotide and modifying at least one property of the polynucleotide. The enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or short strands of nucleotides (e.g., dinucleotides or trinucleotides). The enzyme may modify the polynucleotide by orienting it or moving it to a specific location. The polynucleotide-processing enzyme need not exhibit enzymatic activity as long as it is capable of binding a polynucleotide and controlling its movement through the pore. For example, the enzyme may be modified to remove its enzymatic activity, or may be used under conditions that prevent its use as an enzyme.

The polynucleotide processing enzyme is preferably a polymerase, exonuclease, helicase and topoisomerase, e.g., gyrase. In one embodiment, the enzyme is preferably a helicase, such as Hel308Mbu, Hel308Csy, Hel308Tga, Hel308Mhu, Tral Eco, XPD Mbu, Dda, or variants thereof. Any helicase may be used in the examples.

In one embodiment, any number of helicases may be used. For example, I, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used. In some embodiments, a different number of helicases may be used.

The methods of the embodiments preferably comprise contacting the polynucleotide with two or more helicases. The two or more helicases are typically the same helicase. The two or more helicases may be different helicases.

The two or more helicases may be any combination of the helicases described above. The two or more helicases may be two or more Dda helicases. The two or more helicases may be one or more Dda helicases and one or more TrwC helicases. The two or more helicases may be different variants of the same helicase.

The two or more helicases are preferably linked to each other. The two or more helicases are more preferably covalently linked to each other. Helicases may be ligated in any order and using any method.

Reagent kit

The invention also provides a kit for characterizing a target analyte (e.g., a target polynucleotide). The kit contains the components of the wells and membranes of the examples. The film is preferably formed from the components. Pores are preferably present in the membrane. The kit may comprise the components of any of the membranes disclosed above (e.g., an amphiphilic layer or a triblock copolymer membrane). The kit may further comprise a polynucleotide binding protein. Any of the polynucleotide binding proteins discussed above may be used.

In one embodiment, the membrane is an amphiphilic layer, a solid state layer, or a lipid bilayer.

The kit may further comprise one or more anchors for coupling the polynucleotide to the membrane.

The kit is preferably for characterizing double-stranded polynucleotides and preferably comprises a Y adaptor and a hairpin loop adaptor.

The Y adaptor preferably has one or more helicases ligated, and the hairpin loop adaptor preferably has one or more molecular brakes ligated. The Y adaptor preferably comprises one or more first anchors for coupling the polynucleotide to the membrane, the hairpin loop adaptor preferably comprises one or more second anchors for coupling the polynucleotide to the membrane, and the strength of coupling of the hairpin loop adaptor to the membrane is preferably greater than the strength of coupling of the Y adaptor to the membrane.

The kit may additionally comprise one or more other reagents or instruments that enable performance of any of the embodiments mentioned above. Such reagents or instruments include one or more of the following: suitable buffers (aqueous solutions), means for obtaining a sample from an individual (e.g., a container or instrument containing a needle), means for amplifying and/or expressing a polynucleotide, or a voltage or patch clamp device. The reagents may be present in the kit in a dry form such that the fluid sample re-suspends the reagents. The kit may also optionally contain instructions to enable use of the kit with the methods of the invention or details as to which organism may use the method.

Equipment (or device)

The invention also provides a device for characterizing a target analyte (e.g., a target polynucleotide). The device comprises a mutant/protein pore of single or multiple protein monomers, and single or multiple membranes. Mutants/pores of the protein monomer are preferably present in the membrane. The number of pores and membranes is preferably equal. Preferably, there is a single hole in each membrane.

The apparatus preferably further comprises instructions for implementing the method of the embodiments. The device may be any conventional device for analyte analysis, for example an array or chip. Any of the embodiments discussed in connection with the method of the embodiment are equally applicable to the device. The device may also include any of the features present in the kits described herein. The apparatus used in the examples may be embodied as a QNome-9604.

The above mentioned prior art is incorporated herein by reference in its entirety.

The following examples are given to illustrate the invention without limiting it.

Example 1

In the examples, the wild-type porin is from Vibrio thermophilus and the amino acid sequence of the wild-type porin is SEQ ID NO 1 and the nucleotide sequence encoding this amino acid sequence is represented by SEQ ID NO 2. Mutant 1 of porin monomer is wild-type porin with mutation at position 74-81 corresponding to SEQ ID NO. 1, specifically APGNATNF at position 74-81 is replaced by PYPANA. The protein pore of mutant 1 comprising a porin monomer is mutant pore 1. The amino acid sequence of the mutant 1 of the protein monomer is shown as SEQ ID NO. 24, and the nucleic acid sequence is shown as SEQ ID NO. 25.

Example 2

In the examples, the wild-type porin is from Vibrio thermophilus and the amino acid sequence of the wild-type porin is SEQ ID NO. 1 and the nucleotide sequence of the sequence encoding this amino acid is represented by SEQ ID NO. 2. Mutant 2 of porin monomer wild-type porin has mutations in positions 74-83 corresponding to SEQ ID NO:1, in particular APGNATNFST at positions 74-83 is replaced with AHPAATSP. The protein pore of mutant 2, which includes a porin monomer, is mutant pore 2. The amino acid sequence of the mutant 2 of the protein monomer is shown as SEQ ID NO. 26.

Example 3

In the examples, the wild-type porin is from Vibrio thermophilus and the amino acid sequence of the wild-type porin is SEQ ID NO. 1 and the nucleotide sequence of the sequence encoding this amino acid is represented by SEQ ID NO. 2. Mutant 3 of the porin monomer is wild-type porin with mutations at positions 74-83 corresponding to SEQ ID NO. 1, in particular APGNATNFST at positions 74-83 being replaced by PASSLPs. The protein pore of mutant 3, which includes a porin monomer, is mutant pore 3. The amino acid sequence of mutant 3 of the protein monomer is shown as SEQ ID NO. 27.

Example 4

In the examples, the wild-type porin is from Vibrio thermophilus and the amino acid sequence of the wild-type porin is SEQ ID NO. 1 and the nucleotide sequence of the sequence encoding this amino acid is represented by SEQ ID NO. 2. Mutant 4 of the porin monomer is wild-type porin with a mutation at position 74-83 corresponding to SEQ ID NO:1, in particular APGNATNFST at position 74-83 being replaced by PYPANASP. The protein pore of mutant 4 comprising a porin monomer is mutant pore 4. The amino acid sequence of the mutant 4 of the protein monomer is shown as SEQ ID NO. 28.

Example 5

The wild-type porin is subjected to homologous modeling by SWISS MODEL, and the amino acid of a wild-type porin monomer is shown in SEQ ID NO. 1. FIG. 4A is a surface map 400 of a predicted protein structure, wherein the darker portions are shown as a single protein monomer 402. Fig. 4B is a representation 404 of a ribbon model with a darker portion of protein monomers 406.

FIG. 5 shows a graph of surface potentials of two

protein monomers

502 and 504, wherein the intensity of the color indicates the intensity of the electric property. The middle part shows the amino acid composition of the structure of the constriction zone and the diameter of the pore channel of the constriction zone. Maximum diameter of

Secondly, it is

Minimum diameter of

Fig. 6A shows a plot of the surface potential of wild-type protein monomers, and fig. 6B shows the monomer secondary structure constituent elements and their constriction zone stick models, with the constriction zone loop amino acid composition and numbering magnified, where portion 602 is the amino acid residue directed to the central region of the protein channel.

Mutant wells 1 were homologously modeled using SWISS MODEL. FIG. 7A shows the constriction zone amino acid residue distribution characteristics and constriction zone diameters of mutant well 1 (wild-type amino acid residues APGNATNF from positions 74-81 were replaced with PYPANA). Fig. 7A is a surface potential diagram. The electrical characteristics of the porin are shown primarily relative to the monomer surface and the read head region. The surface charge characteristic of the porin is closely related to the efficiency of nucleic acid recruitment and via hole increase, and the electric property near the reading head is closely related to whether the nucleic acid can effectively pass through the hole constriction zone so as to realize sequencing. FIG. 7B is a ribbon diagram showing the amino acid composition and constriction zone diameter relative to the vicinity of the monomer read head. The key amino acid residue distribution of the narrow region of the protein channel of the mutant 1 is shown, the mutant structure reduces the thickness of a constriction zone, and the amino acid residue pointing to the center of the protein channel is asparagine at position 78. The hydrogen bonding interactions formed by the amino acid residues at positions 72-85 are closely related to the proper assembly of the protein pore channel complex. The predicted diameter of the narrowest region is about

Fig. 8 shows the overall structural features of mutant pore 1 based on homologous modeling, with region 1 corresponding to the coronal-forming region, region 2 corresponding to the constriction and annuli (constrictions and loops) region or the channel neck region, and region 3 corresponding to the transmembrane β -barrel region.

Example 6 preparation of DNA constructs

Two DNA constructs, BS7-4C3-SE1 and BS7-4C3-PLT, respectively, were made. The structure of BS7-4C3-SE1 is shown in FIG. 9, and the sequence information is shown below:

a:30*C3

5'-TTTTT TTTTT-3' (i.e. SEQ ID NO:3)

c, rate controlling protein

d:4*C18

e:5’-AATGT ACTTC GTTCA GTTAC GTATTGCT-3' (i.e. SEQ ID NO:4)

f:

(i.e., SEQ ID NO:5)

g cholesterol label

h:

(i.e., SEQ ID NO:6)

i: 5'-AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAAAA AAGCA ATACG TAACT GAACG AAGTA CATTA AAAAA AAAAA AAAAA AAAA-3' (i.e., SEQ ID NO:7)

5'-ATCCT TTTTT TTTTT TTTTT TTTT-3' (i.e., SEQ ID NO:8)

k: 5'-AATGT ACTTC GTTCA GTTAC GTATT GCTTT TTTTT TTTTT TTTTT TTT-3' (i.e., SEQ ID NO:9)

l:dSpacer

m: 5'-TTTTT TTTTT TTTTT TTTTT-3' (i.e., SEQ ID NO:10)

The structure of BS7-4C3-PLT is shown in FIG. 10, and the sequence information is as follows:

a:30*C3

5'-TTTTT TTTTT-3' (i.e. SEQ ID NO:11)

c, rate controlling protein

d:4*C18

e: 5'-AATGT ACTTC GTTCA GTTAC GTATT GCT-3' (i.e., SEQ ID NO:12)

f 5 'P-GC AATAC GTAAC TGAAC GAAGT TCACTATCGCATTCTCATGA-3' (i.e. SEQ ID NO:13)

g cholesterol label

h: 5'-TCATG AGAAT GCGAT AGTGA-3' (i.e., SEQ ID NO:14)

i 5 '-AAAAAAAAAAAAAAAAAAAAAAAAAAAA (i.e., SEQ ID NO:15)/dSpacer/AAAAAAAAAA (i.e., SEQ ID NO:16)/dSpacer/AAAAAAAAAAAAAATCTCTGAATCTCTGAATCTCTGAATCTCTAAAAAAAAAAAAGAAAAAAAAAAAACAAAAAAAAAAAATAAAAAAAAAAAAAGCAATACGTAACTGAACGAAGTACATTAAAAAAAAAA (i.e., SEQ ID NO:17) -3'

j：5’

-ATCCTTTTTTTTTTAATGTACTTCGTTCAGTTACGTATTGCT-3’

(i.e., SEQ ID NO:18)

k 5 'P-TTTTTTTTTTTTATTTTTTTTTTTTGTTTTTTTTTTTTCTTTTTTTTTTTTAGAGATTCAGAGATTCAGAGATTCAGAGATTTTTTTTTTTTTT (i.e., SEQ ID NO:19)/dSpacer/TTTTTTTTTTTT (i.e., SEQ ID NO:20)/iSPC3/TTTTTTTTTTTTTTTTTTTTTTTTTTTT (i.e., SEQ ID NO:21) -3'

C3, C18, dSpacer and iSpC3 are marker (marker) sequences introduced indicating the resolution characteristics of pore sequencing.

In this example, the c-tachykinin protein in FIGS. 9 and 10 is helicase Mph-MP1-E105C/A362C (with mutation E105C/A362C), the amino acid sequence is SEQ ID NO:22, and the nucleic acid sequence is SEQ ID NO: 23.

Example 7

The mutant hole 1 is used as a protein hole and is detected by adopting a technical method of single-hole sequencing. After insertion of a single porin with the amino acid sequence mutant 1 into the phospholipid bilayer, buffer (625mM KCl, 10mM HEPES pH 8.0, 50mM MgCl)₂) Flow through the system to remove any excess mutant 1 nanopores. Adding DNA construct BS7-4C3-SE1 or BS7-4C3-PLT (1-2 nM final concentration) into the mutant 1 nanopore experimental system, mixing uniformly, and adding buffer (625mM KCl, 10mM HEPES pH 8.0, 50mM MgCl)₂) Flow through the system to remove any excess of the DNA construct BS7-4C3-SE1 or BS7-4C 3-PLT. A premix of helicase (Mph-MP1-E105C/A362C, 15nM final concentration), fuel (ATP 3mM final concentration) was then added to the single mutant 1 nanopore assay system and the sequencing of mutant 1 pore proteins was monitored at +180mV voltage.

The mutant wells 1 were opened at a voltage of + -180 mV. FIG. 11A shows that a bias voltage is applied to complete the pore embedding in the system, the upper and lower base lines show the current levels (about 270pA and-350 pA, respectively) of the positive and negative openings, and the pore channel is found to have no gate control. FIG. 11B is a nucleic acid via signal shown by the downward line in the case of a nucleic acid via in mutant well 1 at +180mV voltage after addition of single stranded nucleic acid. This demonstrates that mutant pore 1 can stably open pores and can pass through this nucleic acid.

And (3) sequencing the DNA construct BS7-4C3-SE1 through the mutant hole 1 by adopting a single-hole sequencing technical method, and adding a nucleic acid sequencing signal appearing in a sequencing system after hole embedding. FIGS. 12A-D show exemplary current trajectories when helicase Mph-MP1-E105C/A362C controls translocation of DNA construct BS7-4C3-SE1 through mutation well 1. According to the signal characteristics, the related characteristics of the mutant hole 1 such as sequencing resolution, stability, signal consistency and the like can be obtained. The hole has clear steps, obvious jump distribution and high-precision sequencing capability. From the four signal characteristics, the sequencing signals are highly consistent.

Fig. 13 is an enlarged view of the portion of fig. 12B showing the current trajectory. The graph with the dotted line frame and the arrow (middle graph) is the result of the filtering process of the original signal (y-axis coordinate of the two traces is current (pA) and x-axis coordinate is time (s)). The dotted arrow indicates a portion showing the result of enlargement of the current trace. The area of this single signal is shown in an enlarged scale, further demonstrating that the mutant well has high resolution for nucleic acid sequencing.

Example 8

Similar to example 7, example 8 uses the bump hole 2 for the empty test and the via hole test.

FIG. 14A shows the opening current at 180mV for mutant hole 2 and its gating characteristics. FIG. 14B shows a single stranded nucleic acid via at +180mV voltage for mutant well 2. The open pore has certain instability and is gated in the reverse direction, and nucleic acid can pass through the pore. Comparison with mutant 1 demonstrates that mutation of the core amino acid in this region can have a significant impact on signal characteristics and sequencing.

And (3) sequencing the DNA construct BS7-4C3-PLT by using a single-hole sequencing technology through the mutant hole 2, and adding a nucleic acid sequencing signal appearing in a sequencing system after hole embedding. FIGS. 15A and 15B show example current trajectories when helicase Mph-MP1-E105C/A362C controls translocation of the DNA construct BS7-4C3-PLT through the mutation pore 2. According to the signal characteristics, the mutant wells 1 have higher resolution and lower noise than the mutant wells 2.

Fig. 16 is an enlarged view of the portion of fig. 15A showing the current trajectory. The graph with the dotted line frame and the arrow shows the result of the filtering process of the original signal (the y-axis coordinate of the two traces is current (pA) and the x-axis coordinate is time (s)). The dotted arrow indicates a portion showing the result of enlargement of the current trace. The area of this single signal is shown in an enlarged scale, indicating that mutant well 1 has a higher resolution than mutant well 2.

Mutant well 2 can also achieve sequencing, but the sequencing accuracy is worse than mutant well 1.

Example 9

Similar to example 7, example 9 uses the bump hole 3 for the empty test and the via hole test.

FIG. 17A shows the opening current and its gating characteristics for mutant hole 3 at 180 mV. FIG. 17B shows single stranded nucleic acid via scenario for mutant well 3 at +180mV voltage. The nucleic acid may be passed through a pore. Comparison with mutant 1 demonstrates that mutation of the core amino acid in this region can have a significant impact on signal characteristics and sequencing.

And sequencing the DNA construct BS7-4C3-PLT through the mutant hole 3 by adopting a single-hole sequencing technical method, and adding a nucleic acid sequencing signal appearing in a sequencing system after hole embedding. FIGS. 18A-18F show example current trajectories when helicase Mph-MP1-E105C/A362C controls translocation of the DNA construct BS7-4C3-PLT through the mutation pore 3. According to the signal characteristics, the mutant hole 1 has higher resolution and less noise than the mutant hole 3.

Fig. 19 is an enlarged view of the current trajectory shown in fig. 18B. The graph with the dotted line frame and the arrow shows the result of the filtering process of the original signal (the y-axis coordinate of the two traces is current (pA) and the x-axis coordinate is time (s)). The dotted arrow indicates a portion showing the result of enlargement of the current trace. The area of this single signal is shown in an enlarged scale, further demonstrating that mutant well 1 is more resolved than mutant well 3.

Mutant well 3 can also achieve sequencing, but the sequencing accuracy is worse than mutant well 1.

Example 10

Similar to example 7, example 10 uses the bump hole 4 for the empty test and the via hole test.

FIG. 20A shows the opening current at a voltage of + -180 mV for mutant hole 4 and its gating characteristics. FIG. 20A shows a single stranded nucleic acid via with a mutant pore 4 at +180mV voltage. The nucleic acid may be passed through a pore. Comparison with mutant 1 demonstrates that mutation of the core amino acid in this region can have a significant impact on signal characteristics and sequencing.

And (3) sequencing the DNA construct BS7-4C3-PLT through the mutant pore 4 by adopting a single-pore sequencing technical method, and adding a nucleic acid sequencing signal appearing in a sequencing system after pore embedding. FIGS. 21A-21F show example current trajectories when helicase Mph-MP1-E105C/A362C controls translocation of the DNA construct BS7-4C3-PLT through the mutation pore 4. According to the signal characteristics, the mutant wells 1 have higher resolution and lower noise than the mutant wells 4.

Fig. 22 is an enlarged view of the embodiment of fig. 21 partially showing the current trace. The graph with the dotted line frame and the arrow shows the result of the filtering process of the original signal (the y-axis coordinate of the two traces is current (pA) and the x-axis coordinate is time (s)). The dotted arrow indicates a portion showing the result of enlargement of the current trace. The area of this single signal is shown magnified and indicates that mutant well 1 is more resolved than mutant well 4.

Mutant wells 4 can also be sequenced, but the sequencing accuracy is worse than mutant wells 1.

Example 11

The recombinant plasmid containing mutant 1 nucleic acid sequence (SEQ ID NO:25) of porin monomer is transformed into BL21(DE3) competent cells by a heat shock method, 0.5ml of LB culture medium is added to be cultured for 1h at 30 ℃, then a proper amount of bacterial liquid is taken and coated on an ampicillin resistant solid LB plate, overnight culture is carried out at 37 ℃, a single colony is picked the next day and inoculated into 50ml of liquid LB culture medium containing ampicillin resistance to be cultured overnight at 37 ℃. Transferred to ampicillin-resistant TB liquid medium at an inoculum size of 1% for scale-up culture, cultured at 37 ℃ and 220rpm, and continuously measured for OD 600. When OD600 ═ 2.0-2.2, the culture broth in TB medium was cooled to 16 ℃ and expression was induced by addition of Isopropylthiogalactoside (IPTG) to reach a final concentration of 0.015 mM. After the induction expression is carried out for 20-24h, the thalli are collected by centrifugation. The thalli is crushed under high pressure after being resuspended by crushing buffer solution, purified by a Ni-NTA affinity chromatography method, and a target elution sample is collected. Mutant 2-4 of porin monomers was purified as above.

Illustratively, FIG. 23 shows the protein purification results of mutant 1, and lanes 1-6 show SDS-PAGE electrophoretic detection of the different fractions separated.

SEQUENCE LISTING

<110> Chengdu carbon technology Co., Ltd

Mutant of <120> porin monomer, protein pore and application thereof

<130> SPI213792-53

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 312

<212> PRT

<213> Thermodesulfovibrio

<400> 1

Met Phe Asn Phe Ile Lys Phe Lys Phe Ile Leu Ile Leu Phe Leu Pro

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Pro Ile Leu Phe Gly Cys Met Ala Met Thr Pro Asn Ile Ala Ser Ile

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Arg Ala Thr Ser Gly Pro Glu Tyr Ile Thr Ser Ile His Arg Asp Leu

35 40 45

Val Ser Leu Pro Lys Pro Glu Asn Pro Ile Pro Val Ala Val Tyr Lys

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Phe Arg Asp Gln Thr Gly Gln Tyr Lys Ala Pro Gly Asn Ala Thr Asn

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Phe Ser Thr Ala Val Thr Gln Gly Ala Thr Ser Ile Leu Ile Lys Ala

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Leu Glu Asp Ser Gly Trp Phe Leu Pro Val Glu Arg Glu Gly Leu Ala

100 105 110

Asn Leu Leu Gln Glu Arg Lys Ile Val Leu Gln Met Arg Glu Leu Tyr

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Leu Thr Glu Glu Gln Lys Lys Gln Phe Glu Pro Leu Pro Pro Leu Leu

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Tyr Ala Gly Ile Ile Phe Glu Gly Gly Ile Ile Gly Tyr Asp Ser Asn

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Val Thr Thr Gly Gly Ile Gly Ala Lys Tyr Phe Gly Ala Gly Gly Ser

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Ala Glu Tyr Arg Val Asp Lys Val Ser Ile Tyr Leu Arg Ala Val Ser

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Val Lys Asn Gly Ala Val Leu Lys Thr Val Gln Thr Ser Lys Thr Val

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Leu Ser Gln Met Leu Ser Leu Gly Ile Phe Arg Phe Val Arg Leu Asn

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Arg Leu Leu Glu Ala Glu Ala Gly Ile Ala Ala Asn Glu Pro Val Glu

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Met Ala Val Gln Glu Ala Ile Glu Lys Ala Val Tyr Asp Ile Ile Ile

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Glu Gly Val Lys Thr Gly Ile Trp Lys Pro Lys Asp Thr Lys Glu Phe

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Glu Lys Trp Leu Ser Lys Tyr Glu Asn Glu Leu Lys Glu Lys Thr Phe

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Ala Ser Glu Ser Ile Ser Lys Leu Ser Lys Gln Lys Glu Ser Lys Asp

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Ile Val Lys Glu Leu Phe Lys Tyr

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<213> Thermodesulfovibrio

<400> 2

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ggttgcatgg cgatgacccc gaacattgcg agcatccgtg cgaccagcgg tccggagtac 120

attaccagca tccaccgtga cctggtgagc ctgccgaaac cggaaaatcc gatcccggtg 180

gcggtttaca aattccgtga tcagaccggt caatataagg cgccgggcaa cgcgaccaat 240

tttagcaccg cggttaccca gggtgcgacc agcattctga tcaaagcgct ggaggacagc 300

ggttggttcc tgccggtgga gcgtgaaggc ctggcgaacc tgctgcagga gcgcaagatc 360

gttctgcaaa tgcgtgaact gtacctgacc gaggaacaga agaaacaatt tgagccgctg 420

ccgccgctgc tgtatgcggg tatcattttt gaaggtggca tcattggcta tgacagcaac 480

gtgaccaccg gtggcattgg tgcgaaatat tttggtgcgg gtggcagcgc ggagtaccgt 540

gtggataaag ttagcatcta tctgcgtgct gtgagcgtta agaatggtgc ggtgctgaaa 600

accgttcaga ccagcaagac cgtgctgagc caaatgctga gcctgggtat tttccgtttt 660

gttcgcctga accgtctgct ggaggcggaa gcgggtattg cggcgaatga gccggtggaa 720

atggcggttc aggaagcgat cgaaaaagcg gtgtacgaca tcattatcga aggtgttaag 780

accggcatct ggaagccgaa agataccaag gagttcgaaa aatggctgag caagtatgag 840

aacgaactga aggagaaaac ctttgcgagc gaaagcatta gcaagctgag caagcaaaaa 900

gagagcaaag atatcgttaa ggaactgttc aaatattaa 939

<210> 3

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<220>

<223> part of BS7-4C3-SE1

<400> 3

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<213> Artificial Sequence

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<400> 4

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<213> Artificial Sequence

<220>

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<400> 5

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<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-SE1

<400> 6

tcatgagaat gcgatagtga 20

<210> 7

<211> 89

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-SE1

<400> 7

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagcaatacg taactgaacg 60

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<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-SE1

<400> 8

atcctttttt tttttttttt tttt 24

<210> 9

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-SE1

<400> 9

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<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-SE1

<400> 10

tttttttttt tttttttttt 20

<210> 11

<211> 10

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 11

tttttttttt 10

<210> 12

<211> 28

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<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 12

aatgtacttc gttcagttac gtattgct 28

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

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<400> 13

gcaatacgta actgaacgaa gttcactatc gcattctcat ga 42

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

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<400> 14

tcatgagaat gcgatagtga 20

<210> 15

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 15

aaaaaaaaaa aaaaaaaaaa aaaaaaaa 28

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<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 16

aaaaaaaaaa aa 12

<210> 17

<211> 132

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 17

aaaaaaaaaa aaaatctctg aatctctgaa tctctgaatc tctaaaaaaa aaaaagaaaa 60

aaaaaaaaca aaaaaaaaaa ataaaaaaaa aaaaagcaat acgtaactga acgaagtaca 120

ttaaaaaaaa aa 132

<210> 18

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> part of BS7-4C3-PLT

<400> 18

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<210> 19

<211> 94

<212> DNA

<213> Artificial Sequence

<220>

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Met Ile Thr Ile Asp Gln Leu Thr Glu Gly Gln Phe Asp Ser Leu Gln

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Arg Ala Lys Val Leu Ile Gln Glu Ala Thr Lys Asn Asp Gly Asn Trp

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Asn His Arg Thr Lys His Leu Thr Ile Asn Gly Pro Ala Gly Thr Gly

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Lys Thr Thr Met Met Lys Phe Leu Val Ser Trp Leu Arg Asp Glu Gly

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Ile Thr Gly Val Ala Leu Ala Ala Pro Thr His Ala Ala Lys Lys Val

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Leu Ala Asn Ala Val Gly Glu Glu Val Ser Thr Ile His Ser Ile Leu

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Lys Ile Asn Pro Thr Thr Tyr Glu Cys Lys Gln Phe Phe Glu Gln Ser

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Ala Pro Pro Asp Leu Ser Lys Ile Arg Ile Leu Ile Cys Glu Glu Cys

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Ala Asp Asp Lys Gly Ile Ser Arg Phe Phe Thr Asp Gln Arg Phe Glu

165 170 175

Gln Thr Tyr Leu Thr Glu Ile Lys Arg Ser Asn Met Pro Ile Ile Glu

180 185 190

Val Ala Thr Glu Ile Arg Asn Gly Gly Trp Ile Arg Glu Asn Ile Ile

195 200 205

Asp Asp Leu Gly Val Lys Gln Asp Lys Ser Val Ser Glu Phe Met Thr

210 215 220

Asn Tyr Phe Lys Val Val Lys Ser Ile Asp Asp Leu Tyr Glu Thr Arg

225 230 235 240

Met Tyr Ala Tyr Thr Asn Asn Ser Val Asp Thr Leu Asn Lys Ile Ile

245 250 255

Arg Lys Lys Leu Tyr Glu Thr Glu Gln Asp Phe Ile Val Gly Glu Pro

260 265 270

Ile Val Met Gln Glu Pro Leu Ile Arg Asp Ile Asn Tyr Glu Gly Lys

275 280 285

Arg Phe Gln Glu Ile Val Phe Asn Asn Gly Glu Tyr Leu Glu Val Ser

290 295 300

Glu Ile Lys Pro Met Glu Ser Val Leu Lys Cys Arg Asn Ile Asp Tyr

305 310 315 320

Gln Leu Val Leu His Tyr Tyr Gln Leu Lys Val Lys Ser Ile Asp Thr

325 330 335

Gly Glu Ser Gly Leu Ile Asn Thr Ile Ser Asp Lys Asn Glu Leu Asn

340 345 350

Lys Phe Tyr Met Phe Leu Gly Lys Val Cys Gln Asp Tyr Lys Ser Gly

355 360 365

Thr Ile Lys Ala Phe Trp Asp Asp Phe Trp Lys Ile Lys Asn Asn Tyr

370 375 380

His Arg Val Lys Pro Leu Pro Val Ser Thr Ile His Lys Gly Gln Gly

385 390 395 400

Ser Thr Val Asp Asn Ser Phe Leu Tyr Thr Pro Cys Ile Thr Lys Tyr

405 410 415

Ala Glu Pro Asp Leu Ala Ser Gln Leu Leu Tyr Val Gly Val Thr Arg

420 425 430

Ala Arg His Asn Val Asn Phe Val Gly

435 440

<210> 23

<211> 1326

<212> DNA

<213> Artificial Sequence

<220>

<223> helicase

<400> 23

atgatcacca tcgaccagct gaccgaaggt cagttcgact ctctgcagcg tgctaaagtt 60

ctgatccagg aagctaccaa aaacgacggt aactggaacc accgtaccaa acacctgacc 120

atcaacggtc cggctggtac cggtaaaacc accatgatga aattcctggt ttcttggctg 180

cgtgacgaag gtatcaccgg tgttgctctg gctgctccga cccacgctgc taaaaaagtt 240

ctggctaacg ctgttggtga agaagtttct accatccact ctatcctgaa aatcaacccg 300

accacctacg aatgcaaaca gttcttcgaa cagtctgctc cgccggacct gtctaaaatc 360

cgtatcctga tctgcgaaga atgctctttc tacgacatca aactgttcga aatcctgatg 420

aactctatcc agccgtggac catcatcatc ggtatcggtg accgtgctca gctgcgtccg 480

gctgacgaca aaggtatctc tcgtttcttc accgaccagc gtttcgaaca gacctacctg 540

accgaaatca aacgttctaa catgccgatc atcgaagttg ctaccgaaat ccgtaacggt 600

ggttggattc gtgaaaacat catcgacgac ctgggtgtta aacaggacaa atctgtttct 660

gaatttatga ccaactactt caaagttgtt aaatctatcg acgacctgta cgaaacccgt 720

atgtacgctt acaccaacaa ctctgttgac accctgaaca aaatcatccg taaaaaactg 780

tacgaaaccg aacaggactt catcgttggt gaaccgatcg ttatgcagga accgctgatc 840

cgtgacatca actacgaagg taaacgtttc caggaaatcg ttttcaacaa cggtgaatac 900

ctggaagttt ctgaaatcaa accgatggaa tctgttctga aatgccgtaa catcgactac 960

cagctggttc tgcactacta ccagctgaaa gttaaatcta tcgacaccgg tgaatctggt 1020

ctgatcaaca ccatctctga caaaaacgaa ctgaacaaat tctacatgtt cctgggtaaa 1080

gtttgccagg actacaaatc tggtaccatc aaagcgttct gggacgactt ctggaaaatc 1140

aaaaacaact accaccgtgt taaaccgctg ccggtttcta ccatccacaa aggtcagggt 1200

tctaccgttg acaactcttt cctgtacacc ccgtgcatca ccaaatacgc tgaaccggac 1260

ctggcttctc agctgctgta cgttggtgtt acccgtgctc gtcacaacgt taacttcgtt 1320

ggttaa 1326

<210> 24

<211> 310

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 1

<400> 24

Met Phe Asn Phe Ile Lys Phe Lys Phe Ile Leu Ile Leu Phe Leu Pro

1 5 10 15

Pro Ile Leu Phe Gly Cys Met Ala Met Thr Pro Asn Ile Ala Ser Ile

20 25 30

Arg Ala Thr Ser Gly Pro Glu Tyr Ile Thr Ser Ile His Arg Asp Leu

35 40 45

Val Ser Leu Pro Lys Pro Glu Asn Pro Ile Pro Val Ala Val Tyr Lys

50 55 60

Phe Arg Asp Gln Thr Gly Gln Tyr Lys Pro Tyr Pro Ala Asn Ala Ser

65 70 75 80

Thr Ala Val Thr Gln Gly Ala Thr Ser Ile Leu Ile Lys Ala Leu Glu

85 90 95

Asp Ser Gly Trp Phe Leu Pro Val Glu Arg Glu Gly Leu Ala Asn Leu

100 105 110

Leu Gln Glu Arg Lys Ile Val Leu Gln Met Arg Glu Leu Tyr Leu Thr

115 120 125

Glu Glu Gln Lys Lys Gln Phe Glu Pro Leu Pro Pro Leu Leu Tyr Ala

130 135 140

Gly Ile Ile Phe Glu Gly Gly Ile Ile Gly Tyr Asp Ser Asn Val Thr

145 150 155 160

Thr Gly Gly Ile Gly Ala Lys Tyr Phe Gly Ala Gly Gly Ser Ala Glu

165 170 175

Tyr Arg Val Asp Lys Val Ser Ile Tyr Leu Arg Ala Val Ser Val Lys

180 185 190

Asn Gly Ala Val Leu Lys Thr Val Gln Thr Ser Lys Thr Val Leu Ser

195 200 205

Gln Met Leu Ser Leu Gly Ile Phe Arg Phe Val Arg Leu Asn Arg Leu

210 215 220

Leu Glu Ala Glu Ala Gly Ile Ala Ala Asn Glu Pro Val Glu Met Ala

225 230 235 240

Val Gln Glu Ala Ile Glu Lys Ala Val Tyr Asp Ile Ile Ile Glu Gly

245 250 255

Val Lys Thr Gly Ile Trp Lys Pro Lys Asp Thr Lys Glu Phe Glu Lys

260 265 270

Trp Leu Ser Lys Tyr Glu Asn Glu Leu Lys Glu Lys Thr Phe Ala Ser

275 280 285

Glu Ser Ile Ser Lys Leu Ser Lys Gln Lys Glu Ser Lys Asp Ile Val

290 295 300

Lys Glu Leu Phe Lys Tyr

305 310

<210> 25

<211> 933

<212> DNA

<213> Artificial Sequence

<220>

<223> mutant 1

<400> 25

atgtttaatt ttattaagtt taaattcata cttatcttgt tcctgccgcc tattctcttt 60

ggctgcatgg caatgacccc gaatattgcg tccattcgtg ctactagtgg tccggagtac 120

attacctcga ttcatcgtga cctggttagc ctgccgaaac cggaaaaccc gattccggta 180

gccgtttata aattccgcga ccagaccggt cagtataaac cgtacccggc taatgcttcg 240

accgcagtga cccagggtgc gacctctatc ctgatcaaag cactagaaga tagcggttgg 300

tttctcccag tggaacgcga gggcctggcg aatctgttgc aagagcgcaa aatcgtgctg 360

cagatgcgtg aactgtactt gaccgaagag cagaagaaac aatttgagcc gctcccaccg 420

ttgttgtacg caggcatcat cttcgaaggt ggcattatcg gctatgatag caacgtgacc 480

actggtggta tcggtgctaa gtattttggc gcgggtggta gcgcggaata ccgtgttgat 540

aaagtttcca tatatctgag agcggtgagc gttaagaacg gtgcggttct taagaccgtt 600

caaacctcga agacggtcct gtcccagatg ctgagcctgg gcatcttccg ctttgttcgt 660

ctgaaccgtc tgttagaggc cgaagctggc atcgctgcca atgaaccggt cgaaatggcg 720

gtgcaagagg cgattgaaaa agcggtctac gacattatta tcgagggcgt taagacgggt 780

atttggaaac cgaaagatac caaagagttc gaaaaatggc tgagcaagta cgagaacgag 840

ctgaaagaga agacgtttgc gtccgaatct atcagcaagc tgtcaaagca gaaggagagc 900

aaggacatcg tgaaggagct gttcaaatac tga 933

<210> 26

<211> 310

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 2

<400> 26

Met Phe Asn Phe Ile Lys Phe Lys Phe Ile Leu Ile Leu Phe Leu Pro

1 5 10 15

Pro Ile Leu Phe Gly Cys Met Ala Met Thr Pro Asn Ile Ala Ser Ile

20 25 30

Arg Ala Thr Ser Gly Pro Glu Tyr Ile Thr Ser Ile His Arg Asp Leu

35 40 45

Val Ser Leu Pro Lys Pro Glu Asn Pro Ile Pro Val Ala Val Tyr Lys

50 55 60

Phe Arg Asp Gln Thr Gly Gln Tyr Lys Ala His Pro Ala Ala Thr Ser

65 70 75 80

Pro Ala Val Thr Gln Gly Ala Thr Ser Ile Leu Ile Lys Ala Leu Glu

85 90 95

Asp Ser Gly Trp Phe Leu Pro Val Glu Arg Glu Gly Leu Ala Asn Leu

100 105 110

Leu Gln Glu Arg Lys Ile Val Leu Gln Met Arg Glu Leu Tyr Leu Thr

115 120 125

Glu Glu Gln Lys Lys Gln Phe Glu Pro Leu Pro Pro Leu Leu Tyr Ala

130 135 140

Gly Ile Ile Phe Glu Gly Gly Ile Ile Gly Tyr Asp Ser Asn Val Thr

145 150 155 160

Thr Gly Gly Ile Gly Ala Lys Tyr Phe Gly Ala Gly Gly Ser Ala Glu

165 170 175

Tyr Arg Val Asp Lys Val Ser Ile Tyr Leu Arg Ala Val Ser Val Lys

180 185 190

Asn Gly Ala Val Leu Lys Thr Val Gln Thr Ser Lys Thr Val Leu Ser

195 200 205

Gln Met Leu Ser Leu Gly Ile Phe Arg Phe Val Arg Leu Asn Arg Leu

210 215 220

Leu Glu Ala Glu Ala Gly Ile Ala Ala Asn Glu Pro Val Glu Met Ala

225 230 235 240

Val Gln Glu Ala Ile Glu Lys Ala Val Tyr Asp Ile Ile Ile Glu Gly

245 250 255

Val Lys Thr Gly Ile Trp Lys Pro Lys Asp Thr Lys Glu Phe Glu Lys

260 265 270

Trp Leu Ser Lys Tyr Glu Asn Glu Leu Lys Glu Lys Thr Phe Ala Ser

275 280 285

Glu Ser Ile Ser Lys Leu Ser Lys Gln Lys Glu Ser Lys Asp Ile Val

290 295 300

Lys Glu Leu Phe Lys Tyr

305 310

<210> 27

<211> 310

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 3

<400> 27

Met Phe Asn Phe Ile Lys Phe Lys Phe Ile Leu Ile Leu Phe Leu Pro

1 5 10 15

Pro Ile Leu Phe Gly Cys Met Ala Met Thr Pro Asn Ile Ala Ser Ile

20 25 30

Arg Ala Thr Ser Gly Pro Glu Tyr Ile Thr Ser Ile His Arg Asp Leu

35 40 45

Val Ser Leu Pro Lys Pro Glu Asn Pro Ile Pro Val Ala Val Tyr Lys

50 55 60

Phe Arg Asp Gln Thr Gly Gln Tyr Lys Pro Ala Ala Ser Ser Leu Ser

65 70 75 80

Pro Ala Val Thr Gln Gly Ala Thr Ser Ile Leu Ile Lys Ala Leu Glu

85 90 95

Asp Ser Gly Trp Phe Leu Pro Val Glu Arg Glu Gly Leu Ala Asn Leu

100 105 110

Leu Gln Glu Arg Lys Ile Val Leu Gln Met Arg Glu Leu Tyr Leu Thr

115 120 125

Glu Glu Gln Lys Lys Gln Phe Glu Pro Leu Pro Pro Leu Leu Tyr Ala

130 135 140

Gly Ile Ile Phe Glu Gly Gly Ile Ile Gly Tyr Asp Ser Asn Val Thr

145 150 155 160

Thr Gly Gly Ile Gly Ala Lys Tyr Phe Gly Ala Gly Gly Ser Ala Glu

165 170 175

Tyr Arg Val Asp Lys Val Ser Ile Tyr Leu Arg Ala Val Ser Val Lys

180 185 190

Asn Gly Ala Val Leu Lys Thr Val Gln Thr Ser Lys Thr Val Leu Ser

195 200 205

Gln Met Leu Ser Leu Gly Ile Phe Arg Phe Val Arg Leu Asn Arg Leu

210 215 220

Leu Glu Ala Glu Ala Gly Ile Ala Ala Asn Glu Pro Val Glu Met Ala

225 230 235 240

Val Gln Glu Ala Ile Glu Lys Ala Val Tyr Asp Ile Ile Ile Glu Gly

245 250 255

Val Lys Thr Gly Ile Trp Lys Pro Lys Asp Thr Lys Glu Phe Glu Lys

260 265 270

Trp Leu Ser Lys Tyr Glu Asn Glu Leu Lys Glu Lys Thr Phe Ala Ser

275 280 285

Glu Ser Ile Ser Lys Leu Ser Lys Gln Lys Glu Ser Lys Asp Ile Val

290 295 300

Lys Glu Leu Phe Lys Tyr

305 310

<210> 28

<211> 310

<212> PRT

<213> Artificial Sequence

<220>

<223> mutant 4

<400> 28

Met Phe Asn Phe Ile Lys Phe Lys Phe Ile Leu Ile Leu Phe Leu Pro

1 5 10 15

Pro Ile Leu Phe Gly Cys Met Ala Met Thr Pro Asn Ile Ala Ser Ile

20 25 30

Arg Ala Thr Ser Gly Pro Glu Tyr Ile Thr Ser Ile His Arg Asp Leu

35 40 45

Val Ser Leu Pro Lys Pro Glu Asn Pro Ile Pro Val Ala Val Tyr Lys

50 55 60

Phe Arg Asp Gln Thr Gly Gln Tyr Lys Pro Tyr Pro Ala Asn Ala Ser

65 70 75 80

Pro Ala Val Thr Gln Gly Ala Thr Ser Ile Leu Ile Lys Ala Leu Glu

85 90 95

Asp Ser Gly Trp Phe Leu Pro Val Glu Arg Glu Gly Leu Ala Asn Leu

100 105 110

Leu Gln Glu Arg Lys Ile Val Leu Gln Met Arg Glu Leu Tyr Leu Thr

115 120 125

Glu Glu Gln Lys Lys Gln Phe Glu Pro Leu Pro Pro Leu Leu Tyr Ala

130 135 140

Gly Ile Ile Phe Glu Gly Gly Ile Ile Gly Tyr Asp Ser Asn Val Thr

145 150 155 160

Thr Gly Gly Ile Gly Ala Lys Tyr Phe Gly Ala Gly Gly Ser Ala Glu

165 170 175

Tyr Arg Val Asp Lys Val Ser Ile Tyr Leu Arg Ala Val Ser Val Lys

180 185 190

Asn Gly Ala Val Leu Lys Thr Val Gln Thr Ser Lys Thr Val Leu Ser

195 200 205

Gln Met Leu Ser Leu Gly Ile Phe Arg Phe Val Arg Leu Asn Arg Leu

210 215 220

Leu Glu Ala Glu Ala Gly Ile Ala Ala Asn Glu Pro Val Glu Met Ala

225 230 235 240

Val Gln Glu Ala Ile Glu Lys Ala Val Tyr Asp Ile Ile Ile Glu Gly

245 250 255

Val Lys Thr Gly Ile Trp Lys Pro Lys Asp Thr Lys Glu Phe Glu Lys

260 265 270

Trp Leu Ser Lys Tyr Glu Asn Glu Leu Lys Glu Lys Thr Phe Ala Ser

275 280 285

Glu Ser Ile Ser Lys Leu Ser Lys Gln Lys Glu Ser Lys Asp Ile Val

290 295 300

Lys Glu Leu Phe Lys Tyr

305 310

Claims

1. A mutant of a porin monomer, wherein the amino acids of the mutant of a porin monomer comprise the sequence set forth in SEQ ID No. 1 or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60% or 50% identity thereto, and the amino acids of the mutant of a porin monomer comprise mutations at one or more positions corresponding to a74, P75, G76, N77, a78, T79, N80 and F81 of SEQ ID No. 1.

2. A mutant of a porin monomer of claim 1, wherein the amino acids of said mutant of a porin monomer comprise:

3. A mutant of a porin monomer as claimed in any one of the preceding claims, wherein said sequence set forth in SEQ ID NO 1 is derived from Vibrio thermophilus.

4. A mutant of a porin monomer as claimed in any one of the preceding claims, wherein said mutant of a porin monomer has an amino acid mutation selected from the group consisting of:

(a) the amino acids APGNATNF corresponding to positions 74-81 of SEQ ID NO. 1 are mutated to M₁M₂M₃M₄M₅M₆Wherein M is₁0 to 1 selected from P; m₂0 to 3 selected from Y, F, W; m₃0 to 1 selected from P; m₄0 to 5 selected from A, G, V, L, I; m₅0 to 4 selected from N, D, E, Q; m₆0 to 5 selected from A, G, V, L, I;

(b) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₇M₈M₉M₁₀M₁₁M₁₂ M₁₃M₁₄Wherein M is₇0 to 5 selected from A, G, V, L, I; m₈0 to 3 selected from H, K, R; m₉0 to 1 selected from P; m₁₀0 to 5 selected from A, G, V, L, I; m₁₁0 to 5 selected from A, G, V, L, I; m₁₂0 to 5 selected from T, S, C, U, M; m₁₃0 to 5 selected from S, C, U, T, M; m₁₄0 to 1 selected from P;

(c) 74-corresponding to SEQ ID NO:1Amino acid APGNATNFST at position 83 was mutated to M₁₅M₁₆M₁₇M₁₈M₁₉M₂₀ M₂₁M₂₂Wherein M is₁₅0 to 1 selected from P; m₁₆0 to 5 selected from A, G, V, L, I; m₁₇0 to 5 selected from A, G, V, L, I; m₁₈0 to 5 selected from S, C, U, T, M; m₁₉0 to 5 selected from S, C, U, T, M; m₂₀0 to 5 selected from L, G, A, V, I; m₂₁0 to 5 selected from S, C, U, T, M; m₂₂0 to 1 selected from P; and

(d) amino acids APGNATNFST corresponding to positions 74-83 of SEQ ID NO. 1 were mutated to M₂₃M₂₄M₂₅M₂₆M₂₇M₂₈ M₂₉M₃₀Wherein M is₂₃0 to 1 selected from P; m₂₄0 to 3 selected from Y, F, W; m₂₅0 to 1 selected from P; m₂₆0 to 5 selected from A, G, V, L, I; m₂₇0 to 4 selected from N, D, E, Q; m₂₈0 to 5 selected from A, G, V, L, I; m₂₉0 to 5 selected from S, C, U, T, M; m₃₀0 to 1 selected from P.

5. A mutant of a porin monomer as claimed in any one of the preceding claims, wherein said mutant of a porin monomer has an amino acid mutation selected from the group consisting of:

6. A mutant of a porin monomer as claimed in any one of the preceding claims, wherein the amino acid sequence of said mutant of a porin monomer comprises or consists of SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 27 or SEQ ID NO 28.

7. A protein pore comprising at least one mutant of a porin monomer of any one of the preceding claims.

8. A protein pore according to claim 7, wherein the protein pore comprises at least two mutants of the porin monomer.

9. The protein pore of any one of claims 7-8, wherein the constriction zone pore diameter of the protein pore is from 0.7nm to 2.2nm, from 0.9nm to 1.6nm, from 1.4nm to 1.6nm, or

10. A complex for characterizing a target analyte, characterized by: comprising a protein pore according to any one of claims 7 to 9 and rate-controlling protein bound thereto.

11. A nucleic acid encoding a mutant of a porin monomer of any one of claims 1-6, a protein pore of any one of claims 7-9, or a complex of claim 10.

12. The nucleic acid of claim 11, wherein the nucleotide sequence of the porin monomer is the sequence set forth in SEQ ID NO 2.

13. A vector or a genetically engineered host cell comprising the nucleic acid of any one of claims 11-12.

14. Use of a mutant of a porin monomer of any one of claims 1 to 6, a protein pore of any one of claims 7 to 9, a complex of claim 10, a nucleic acid of any one of claims 11 to 12, or a vector or host cell of claim 13, for detecting the presence, absence or one or more characteristics of a target analyte or for the manufacture of a product for detecting the presence, absence or one or more characteristics of a target analyte.

15. A method of producing a protein pore or polypeptide thereof, comprising transforming a host cell according to claim 13 with a vector comprising claim 13, and inducing the host cell to express a protein pore or polypeptide thereof according to any one of claims 7 to 9.

16. A method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

a. contacting a target analyte with a protein pore according to any one of claims 7-9, a complex according to claim 10, or the protein pore in a complex according to claim 10, such that the target analyte moves relative to the protein pore; and

17. The method of claim 16, wherein the method comprises:

the target analyte interacts with the protein pores present in the membrane such that the target analyte moves relative to the protein pores.

18. A kit for determining the presence, absence or one or more characteristics of a target analyte comprising a mutant of a porin monomer of any one of claims 1 to 6, a protein well of any one of claims 7 to 9, a complex of claim 10, a nucleic acid of any one of claims 11 to 12, or a vector or host of claim 13, and a component of a membrane of claim 17.

19. A device for determining the presence, absence or one or more characteristics of a target analyte comprising a protein pore according to any one of claims 7 to 9 or a complex according to claim 10, and a membrane according to claim 17.

20. The use, method, kit or device of any one of claims 14-19, wherein the target analyte comprises a polysaccharide, a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a drug, a diagnostic agent, an explosive, or an environmental contaminant;

preferably, the target analyte comprises a polynucleotide,