WO2023060420A1 - Mutant de monomère de porine, pore protéique et leur utilisation - Google Patents

Mutant de monomère de porine, pore protéique et leur utilisation Download PDF

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WO2023060420A1
WO2023060420A1 PCT/CN2021/123210 CN2021123210W WO2023060420A1 WO 2023060420 A1 WO2023060420 A1 WO 2023060420A1 CN 2021123210 W CN2021123210 W CN 2021123210W WO 2023060420 A1 WO2023060420 A1 WO 2023060420A1
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seq
mutant
protein
porin
mutated
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PCT/CN2021/123210
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Chinese (zh)
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刘少伟
岳飞飞
谢馥励
赵帅
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成都齐碳科技有限公司
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Priority to PCT/CN2021/123210 priority Critical patent/WO2023060420A1/fr
Publication of WO2023060420A1 publication Critical patent/WO2023060420A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/28—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Vibrionaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material

Definitions

  • the invention belongs to the technical field of characterization of target analyte characteristics, and particularly relates to a mutant of a porin monomer, a protein pore containing the same and its application for detecting target analytes.
  • nucleic acid sequencing technology continues to develop, becoming the core field of life science research, and playing a huge role in promoting the development of technology in the fields of biology, chemistry, electricity, life science, and medicine.
  • Using nanopores to develop a new type of rapid, accurate, low-cost, high-precision and high-throughput nucleic acid sequencing technology is one of the hot spots of the post-human genome project.
  • Nanopore sequencing technology also known as the fourth-generation sequencing technology, is a single-stranded nucleic acid molecule as a sequencing unit, using a nanopore that can provide an ion current channel, so that the single-stranded nucleic acid molecule is driven by electrophoresis. Through this nanopore, when the nucleic acid passes through the nanopore, the current of the nanopore is reduced, and the gene sequencing technology reads the sequence information in real time for the different signals generated.
  • Nanopore sequencing can not only realize natural DNA and RNA sequencing, but also directly obtain the base modification information of DNA and RNA.
  • the use of bisulfite treatment provides a great impetus for the direct study of epigenetic-related phenomena at the genomic level.
  • nanopore detection technology has the advantages of low cost, high throughput, and label-free.
  • Nanopore analysis technology originated from the invention of the Coulter counter and the recording technology of single-channel current.
  • Physiology and Medicine Nobel Prize winners Neher and Sakamann used patch clamp technology to measure membrane potential in 1976 to study membrane proteins and ion channels, which promoted the practical application of nanopore sequencing technology.
  • Kasianowicz et al. proposed a new idea of using ⁇ -hemolysin to sequence DNA, which was a milestone in the sequencing of biological nanopore single molecules.
  • biological nanopores such as MspA porin and phage Phi29 connector enriched the research on nanopore analysis technology.
  • Li et al. opened a new era of solid-state nanopore research. Limited by the development of the semiconductor and material industries, solid-state nanopore sequencing progresses slowly.
  • nanopore sequencing technology lies in the design of a special biological nanopore.
  • the read head structure formed by the constriction area in the hole can cause the channel current when single-stranded nucleic acid (such as ssDNA) molecules pass through the nanopore.
  • the blockage of the nanopore briefly affects the current intensity flowing through the nanopore (the magnitude of the current change affected by each base is different), and finally the highly sensitive electronic device detects these changes to identify the base passing through.
  • protein pores are used as nanopores for sequencing, and porins are mainly derived from Escherichia coli.
  • nanopore sequencing technology there is a single nanopore protein, and it is necessary to develop alternative nanopore proteins to realize nanopore sequencing technology. Porins are also closely related to sequencing accuracy, and porins are also involved in changes in the interaction mode with rate-controlling proteins. Further optimizing the stability of the interaction interface between porins and rate-controlling proteins is crucial to improving the consistency and stability of sequencing data. have a positive impact. The accuracy of nanopore sequencing technology also needs to be improved. Therefore, it is necessary to develop improved nanopore proteins to further improve the resolution of nanopore sequencing.
  • the purpose of the embodiments of the present invention is to provide a mutant of an alternative porin monomer, a protein pore comprising the same, and an application thereof.
  • an embodiment of the present invention provides a mutant of a porin monomer, wherein the amino acid of the mutant of the porin monomer includes the sequence shown in SEQ ID NO: 1 or has at least 99%, 98 %, 97%, 96%, 95%, 90%, 80%, 70%, 60% or 50% identity sequence, or consists of it, and the amino acid of the mutant of the porin monomer is included in the corresponding A mutation at one or more of positions Q68, N72, and F73 of SEQ ID NO:1;
  • Q68, N72, and F73 are specifically: (1) Q68; (2) N72; (3) F73; (4) Q68 and N72; (5) Q68 and F73; (6) N72 and F73 ; (7) Q68, N72 and F73.
  • the amino acid of the mutant of the porin monomer is included in 68-73, 68-218, 61-73, 61-77, 61-218, 66-73, or 66- Mutations at one or more positions of 218.
  • amino acids of the mutant of the porin monomer include:
  • one or more positions corresponding to Q68, N72, and F73 of SEQ ID NO:1 have amino acid insertions, deletions, and/or substitutions; (2) Q61, K66, Q68, Q68, and F73 corresponding to SEQ ID NO:1 There are insertions, deletions and/or substitutions of amino acids at one or more positions of N72 and F73N; or (3) one or more of K66, Q68, N72, F73, D120, and E218 corresponding to SEQ ID NO:1 Positions have insertions, deletions and/or substitutions of amino acids.
  • amino acid mutation of the mutant of the porin monomer is selected from the following:
  • Q68 corresponding to SEQ ID NO: 1 is mutated to 0 to 5 of G, A, V, L, and I; N72 is mutated to 0 to 5 of G, A, V, L, and I; F73 is mutated 0 to 4 of N, E, D, Q;
  • Q61 corresponding to SEQ ID NO: 1 is mutated to 0 to 5 of G, A, V, L, and I; K66 is mutated to 0 to 3 of R, H, and K; Q68 is mutated to G, A 0 to 5 of , V, L, I; N72 is mutated to 0 to 5 of G, A, V, L, I; F73 is mutated to 0 to 4 of N, E, D, Q; and
  • K66 corresponding to SEQ ID NO: 1 is mutated to 0 to 3 of R, H, and K; Q68 is mutated to 0 to 5 of G, A, V, L, and I; N72 is mutated to G, A 0 to 5 of , V, L, and I; F73 is mutated to 0 to 4 of N, E, D, and Q; D120 is mutated to 0 to 4 of N, E, D, and Q; E218 is mutated to 0 to 5 of G, A, V, L, I.
  • amino acid mutation of the mutant of the porin monomer is selected from the following:
  • Q68 corresponding to SEQ ID NO:1 is mutated to G, A, V, L, or I; N72 is mutated to G, A, V, L, or I; F73 is mutated to N, E, D, or Q;
  • Q61 corresponding to SEQ ID NO:1 is mutated to G, A, V, L, or I; K66 is mutated to R, or H; Q68 is mutated to G, A, V, L, or I; N72 is mutated to G , A, V, L, or I; F73 is mutated to N, E, D, or Q; and
  • K66 corresponding to SEQ ID NO: 1 is mutated to R, or H; Q68 is mutated to G, A, V, L, or I; N72 is mutated to G, A, V, L, or I; F73 is mutated to N , E, D, or Q; D120 is mutated to N, E, or Q; E218 is mutated to G, A, V, L, or I.
  • amino acid mutation of the mutant of the porin monomer is selected from the following:
  • an embodiment of the present invention provides a mutant of a porin monomer, wherein the amino acid of the mutant of the porin monomer includes the sequence shown in SEQ ID NO: 1 or has at least 99%, 98 %, 97%, 96%, 95%, 90%, 80%, 70%, 60% or 50% identical sequences, and mutants of said porin monomers include:
  • (5) have a mutation at Q68I/Q68L/Q68V, N72A/N72G, and/or F73Q/F73N corresponding to SEQ ID NO:1, and additionally have a mutation at at least one position of Q61L, K66R, D120N, and E218I .
  • Q61 is mutated to 0 to 5 of G, A, V, L, I; K66 is mutated to R 0 to 3 of , H, and K; Q68 is mutated to 0 to 5 of G, A, V, L, and I; N72 is mutated to 0 to 5 of G, A, V, L, and I; F73 Mutated to 0 to 4 of N, E, D, Q; D120 mutated to 0 to 4 of N, E, D, Q; E218 mutated to 0 to 5 of G, A, V, L, I kind.
  • 0 to N types include 0, 1, 2, 3, 4...N types.
  • Q61 is mutated to 0 to 5 of G, A, V, L, and I, it means that Q61 is mutated to 0, 1, 2, 3, or 4 of G, A, V, L, and I. or 5 amino acids.
  • the amino acids before and after the mutation are different.
  • D120 to be mutated to 0 to 4 of N, E, D, Q
  • this is 1 D120 is not mutated to D, but can only be mutated to any of N, E, Q species; when there are two species, D120 is mutated into any two of N, E, D, Q, and so on.
  • the mutation when the mutation is 0 amino acid, it refers to the deletion of this amino acid.
  • Q61 when Q61 is mutated to 0 of G, A, V, L, I, it means Q61 deletion.
  • an embodiment of the present invention provides a protein pore, including at least one mutant of a porin monomer.
  • the embodiment of the present invention provides a complex for characterizing a target analyte, characterized in that: the protein pore and the rate-controlling protein used in conjunction with it.
  • embodiments of the present invention provide nucleic acids encoding mutants of porin monomers, protein pores, or complexes.
  • the embodiments of the present invention provide a vector or a genetically engineered host cell comprising the nucleic acid.
  • the embodiments of the present invention provide mutants of porin monomers, their protein pores, complexes, nucleic acids, vectors or host cells in detecting the presence, absence or one or more characteristics of target analytes or preparing detection targets. Application in a product of the presence, absence, or one or more characteristics of an analyte.
  • 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 a polypeptide thereof.
  • embodiments of the present invention provide a method for determining the presence, absence or one or more characteristics of a target analyte, comprising:
  • the method comprises: said target analyte interacting with said protein pore present in a membrane such that said target analyte moves relative to said protein pore.
  • the target analyte is a nucleic acid molecule.
  • a method for determining the presence, absence, or one or more characteristics of a target analyte comprises coupling said target analyte to a membrane; The protein pore interacts so that the target analyte moves relative to the protein pore.
  • an embodiment of the present invention provides a kit for determining the presence, absence or one or more characteristics of a target analyte, including the mutant of the porin monomer, the protein pore, The complex, the nucleic acid, or the vector or host, and the membrane components.
  • the embodiments of the present invention provide a device for determining the presence, absence or one or more characteristics of a target analyte, including the protein pore or the complex, and the membrane.
  • the target analytes include polysaccharides, metal ions, inorganic salts, polymers, amino acids, peptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, drugs, diagnostic agents, explosives substances or environmental pollutants;
  • said target analyte comprises a polynucleotide
  • said polynucleotide comprises DNA or RNA; and/or, said one or more characteristics are selected from (i) the length of said polynucleotide; (ii) the identity of said polynucleotide; (iii) the sequence of the polynucleotide; (iv) the secondary structure of the polynucleotide and (v) whether the polynucleotide is modified; and/or, the rate-controlling protein in the complex Including polynucleotide binding proteins.
  • Figure 1 illustrates the basic working principle of a nanopore according to one embodiment.
  • Fig. 2 shows a schematic diagram of DNA sequencing according to one embodiment.
  • Figure 3 shows the corresponding pore blocking signal when nucleotides pass through the protein pore according to one embodiment.
  • FIGS. 4A, 4B and 4C show a wild-type protein pore channel surface structure and ribbon diagram model according to one embodiment.
  • 4A is a side view of the surface structure model
  • FIG. 4B is a top view of the surface structure model
  • FIG. 4C is a streamer structure model.
  • Figure 5 shows the constriction region amino acid residue distribution and constriction region diameter of wild-type channels according to one embodiment.
  • Figure 6A shows a surface potential map of a wild-type channel monomer according to one embodiment
  • Figure 6B shows a monomer streamer model and a stick model of the amino acid residue distribution in its constriction region.
  • Fig. 7 shows the distribution characteristics of the amino acid residues in the constriction zone of mutation hole 1 and the diameter of the constriction zone according to one embodiment.
  • Fig. 8 shows a cartoon schematic diagram of homology-based modeling of mutant pore 1 according to an embodiment.
  • Figure 9 shows the structure of the DNA construct BS7-4C3-PLT according to one embodiment.
  • FIG. 10A shows the opening current and gating characteristics of the mutation hole 1 at a voltage of ⁇ 180 mV according to one embodiment.
  • FIG. 10B shows the situation of nucleic acid passing through the mutation hole 1 under the voltage of +180mV according to one embodiment.
  • 11A and 11B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 1, according to one embodiment.
  • FIG. 12 is an enlarged display diagram of a single signal area in the embodiment of FIG. 11A .
  • 14A and 14B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 2, according to one embodiment.
  • FIG. 15A shows the opening current and gating characteristics of the mutation hole 3 at a voltage of ⁇ 180 mV according to one embodiment.
  • FIG. 15B shows the condition of nucleic acid passing through the mutation hole 3 under the voltage of +180mV according to one embodiment.
  • Figures 16A and 16B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 3, according to one embodiment.
  • Fig. 17 is an enlarged display diagram of a single signal area in the embodiment of Fig. 16A.
  • FIG. 19A shows the opening current and gating characteristics of the mutation hole 4 at a voltage of ⁇ 180 mV according to one embodiment.
  • FIG. 19B shows the situation of nucleic acid passing through the mutation hole 4 under the voltage of +180mV according to one embodiment.
  • Figure 21 shows the protein purification results of Mutant 1 according to an embodiment
  • lanes 1-5 show the SDS-PAGE electrophoresis detection results of different fractions separated.
  • Fig. 22 shows the result of molecular sieve purification of the protein of mutant 1 according to an embodiment, and the position indicated by the arrow is the oligomeric state distribution of the peak of the target protein.
  • a nucleotide includes two or more nucleotides
  • a helicase includes two or more helicases.
  • nucleotide sequence refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA and RNA.
  • nucleic acid refers to a single- or double-stranded covalently linked sequence of nucleotides wherein the 3' and 5' ends on each nucleotide are linked by a phosphodiester bond.
  • Nucleotides can consist of deoxyribonucleotide bases or ribonucleotide bases.
  • Nucleic acids can include DNA and RNA, and can be prepared synthetically in vitro or isolated from natural sources.
  • the nucleic acid may further comprise modified DNA or RNA, such as methylated DNA or RNA, or RNA that has been post-translationally modified, such as 5'-capping with 7-methylguanosine, 3'-end processing, such as cleavage and polyadenylation, and splicing.
  • Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA).
  • HNA hexitol nucleic acid
  • CeNA cyclohexene nucleic acid
  • TAA threose nucleic acid
  • GNA glycerol nucleic acid
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • nucleic acid or polynucleotide
  • bp base pairs
  • nt nucleotides
  • kb kilobase pair
  • Polynucleotides of less than about 40 nucleotides in length are commonly referred to as “oligonucleotides” and may contain primers used in DNA manipulations, such as 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 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.
  • the polynucleotide may comprise a pyrimidine dimer. This dimer is often associated with damage caused by UV light and is a major cause of skin melanoma.
  • One or more nucleotides in the polynucleotide may be modified, for example with conventional labels or tags.
  • the polynucleotide may comprise one or more abasic (ie, lacking a nucleobase), or lacking a nucleobase and sugar (ie, being C3) nucleotides.
  • the nucleotides in the polynucleotide may be linked to each other in any manner.
  • the nucleotides are usually linked by their sugar and phosphate groups, as in nucleic acids.
  • the nucleotides may be linked via their nucleobases, as in pyridine dimers.
  • a polynucleotide can be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded.
  • a polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • a polynucleotide may comprise an RNA strand 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 nucleic acid having nucleotide side chains.
  • PNA peptide nucleic acid
  • GNA glycerol nucleic acid
  • TNA locked nucleic acid
  • LNA locked nucleic acid 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 backbone is composed of repeating threosyl groups linked together by phosphodiester bonds.
  • LNA is formed from ribonucleic acid as described above, with an additional bridge connecting the 2' oxygen and 4' carbon in the ribose moiety.
  • Bridged nucleic acids are modified RNA nucleotides. They can also be called restricted or inaccessible RNA13BNA monomers can contain 5-membered, 6-membered or even 7-membered bridges with "fixed" C3'-endo sugar puckering .
  • the bridging structure is synthetically introduced into the 2',4'-position of ribose to generate a 2',4'-BNA monomer.
  • the polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • a polynucleotide can be of any length.
  • a polynucleotide can 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 cores in length nucleotide pair.
  • any number of polynucleotides can be studied.
  • the methods of the embodiments may 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 different polynucleotides or in the case of the same polynucleotide.
  • Polynucleotides can be naturally occurring or synthetic.
  • the methods can be used to verify the sequence of prepared oligonucleotides.
  • the methods are typically performed in vitro.
  • amino acid is used in its broadest sense and is meant to include amino acids containing amine ( NH2 ) and carboxyl (COOH) functional groups as well as side chains (such as R groups) that are unique to each amino acid. of organic compounds.
  • amino acid refers to a naturally occurring L ⁇ -amino acid or residue.
  • amino acid also includes D-amino acids, retro-inverse amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids (such as norleucine) that are not usually incorporated into proteins, and amino acids that are known in the art.
  • Chemically synthesized compounds such as ⁇ -amino acids with properties known to be characteristic of amino acids.
  • analogs or mimetics of phenylalanine or proline are included in the definition of amino acid which allow the same conformational constraints on the peptide compound as native Phe or Pro. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids.
  • protein 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. Accordingly, these terms apply to amino acid polymers in which one or more amino acid residues are a synthetic non-naturally occurring amino acid, such as a chemical analog of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Polypeptides 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, and the like.
  • “Homologues” of a protein encompass peptides, oligonucleotides, oligonucleotides 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 to the unmodified protein from which they were derived. Peptides, polypeptides, proteins and enzymes.
  • amino acid identity refers to the degree to which sequences are identical on an amino acid-by-amino acid basis over a comparison window.
  • percent sequence identity is calculated by comparing two optimally aligned sequences over a comparison window and determining the occurrence of identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to get the number of matching positions, divide the number of matching positions by the positions in the comparison window total (i.e., window size), and multiply the result by 100 to obtain percent sequence identity.
  • amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met
  • Sequence identity may also be to fragments or portions of full-length polynucleotides or polypeptides. Thus, a sequence may have only 50% overall sequence identity to the full-length reference sequence, but the sequence of a particular region, domain or subunit may have 80%, 90%, or as much as 99% sequence identity to the reference sequence.
  • wild-type refers to a gene or gene product isolated from a naturally occurring source.
  • a wild-type gene is the most frequently observed gene in a population, and thus is arbitrarily designed to be the "normal” or "wild-type” form of that gene.
  • modified refers to a gene or gene product that exhibits sequence modifications (for example, substitutions, truncations or insertions), post-translational modifications and/or A gene or gene product that has a functional property (eg, altered property). 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.
  • arginine (R) can be substituted for methionine (M) by substituting a codon for arginine (CGT) for methionine (ATG) at the relevant position in the polynucleotide encoding the mutated monomer.
  • CCT codon for arginine
  • ATG methionine
  • non-naturally occurring amino acids can be introduced by including a synthetic aminoacyl-tRNA in the IVTT system used to express the mutated monomer.
  • non-naturally occurring amino acids can be introduced by expressing mutated monomers in marine Vibrio that are sensitive to specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. It is nutritionally deficient.
  • Mutant monomers can also be produced by naked ligation if they are produced using partial peptide synthesis. Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume.
  • the introduced amino acids may have a similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.
  • a conservative substitution can 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 can 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 by reference to the hydrophilicity scale for amino acid side chains in Table 2.
  • Mutated or modified proteins, monomers or peptides may also be chemically modified at any point in any manner.
  • the mutated or modified monomer or peptide is preferably 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 chemical modification by attachment of an unnatural amino acid, enzymatic modification of the epitope, or modification of the terminus. Suitable methods for making such modifications are well known in the art.
  • Modified mutants of proteins, monomers or peptides can be chemically modified by attachment of any molecule.
  • mutants of modified proteins, monomers, or peptides can be chemically modified by the attachment of dyes or fluorophores.
  • a mutated or modified monomer or peptide is chemically modified with a molecular adapter that facilitates the interaction between a pore comprising the monomer or peptide and a target nucleotide or target polynucleotide sequence.
  • Molecular adapters are preferably cyclic molecules, cyclodextrins, substances capable of hybridization, DNA binding or intercalating agents, peptides or peptide analogs, synthetic polymers, aromatic planar molecules, positively charged small molecules or capable of hydrogen bonding bonded small molecules.
  • adapters improves the host-guest chemistry of the pore and the nucleotide or polynucleotide sequence, thereby improving the sequencing capability of pores formed from mutated monomers.
  • the principles of host-guest chemistry are well known in the art.
  • Adapters have an effect on the physical or chemical properties of the pore that improves the interaction of the pore with the nucleotide or polynucleotide sequence.
  • Adapters can alter the charge of the barrel or channel of the pore, or specifically interact or bind to 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 the translocation of molecules and ions from one side of the membrane to the other. The translocation of ionic species through the pore can 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 the pathway through which molecules or ions pass is on the nanometer scale (10 -9 meters).
  • the protein pore may be a transmembrane protein pore.
  • the transmembrane protein structure of the protein pore can be monomeric or oligomeric in nature.
  • a pore comprises multiple polypeptide subunits arranged around a central axis, forming a protein-lined channel extending substantially perpendicular to the 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 as defined as in the case of perfringolysin or related large membrane pores.
  • the portion of the protein subunit that forms the protein-lined channel within the nanopore typically contains secondary structural motifs that may include one or more transmembrane ⁇ -barrel and/or ⁇ -helical portions.
  • the protein pore comprises one or more porin monomers.
  • Each porin monomer may be from a marine Vibrio.
  • the protein pore comprises a mutant of one or more porin monomers (ie, one or more porin mutated monomers).
  • the porin is from a wild-type protein from the kingdom of Bacteria, a wild-type homologue, or a mutant thereof. Mutants can be modified porins or porin mutants. Modifications in a mutant include, but are not limited to, any one or more modifications or combinations of modifications disclosed herein.
  • the Bacteria wild-type protein is a protein from Vibrio maritimus.
  • the wild-type protein of the Kingdom of Biology is a protein from Vibrio maritima (Gene: JCM19240_1936).
  • a porin homologue refers to a protein having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% complete sequence identity of the polypeptides.
  • a porin homologue refers to a polynucleotide having at least 99%, 98%, 97%, 96%, 95%, 94%, 93% of the polynucleotide encoding the protein shown in SEQ ID NO:2 , 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% complete sequence identity.
  • 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 standard methods in the art. Chromosomal DNA encoding wild-type porins can be extracted from pore-producing organisms such as Vibrio maritima. The gene encoding the pore subunit can be amplified using PCR including specific primers. The amplified sequence can then be subjected to site-directed mutagenesis. Suitable methods of site-directed mutagenesis are known in the art and include, for example, combinatorial chain reactions. The constructed polynucleotides encoding the embodiments can be prepared by techniques known in the art, for example, in Sambrook, J. and Russell, D. (2001). Molecular Cloning A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Those described in Harbor, NY.
  • polynucleotide sequences can then be incorporated into recombinant replicable vectors, such as cloning vectors.
  • the vector can be used to replicate the polynucleotide in a compatible host cell.
  • polynucleotide sequences can 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 that cause replication of the vector. The vector can be recovered from the host cell.
  • an insulating film 102 with nanoscale pores divides the cavity into two small chambers, as shown in FIG. 1 , when a voltage acts on the electrolyte chamber, ions or other Small molecules pass through the pores under the action of an electric field, forming a stable and detectable ionic current.
  • ions or other Small molecules pass through the pores under the action of an electric field, forming a stable and detectable ionic current.
  • Different types of biomolecules can be detected by knowing the size and surface properties of the nanopores, the applied voltage, and the solution conditions.
  • ssDNA single-stranded DNA
  • FIG. 2 shows a schematic diagram 200 of DNA sequencing.
  • the nanopore is the only channel through which ions on both sides of the phospholipid membrane pass.
  • Rate-controlling proteins such as polynucleotide binding proteins act as motor proteins for nucleic acid molecules such as DNA, pulling DNA strands sequentially through the nanopore/protein pore in steps of single nucleotides.
  • the corresponding pore blocking signal is recorded ( Figure 3).
  • porins are screened from different species (mainly bacteria and archaea) in nature by means of bioinformatics and evolution.
  • the porin is from any organism, preferably from Vibrio maritima.
  • sequence analysis the porin has a complete functional domain.
  • structural biology methods to predict and analyze the 3D structure model of the porin, select the channel protein with a suitable reading head architecture.
  • genetic engineering, protein engineering, protein directed evolution, and computer-aided protein design are used to transform, test, and optimize candidate channel proteins (or porins).
  • multiple homologous protein mutants are obtained, preferably Two (different homologous protein backbones), with different signal characteristics and signal distribution patterns.
  • porins in the examples can be applied to the fourth generation sequencing technology.
  • the porin is a nanoporin.
  • porins can be applied to solid state pores for sequencing.
  • a new protein backbone is used to form a new constriction region (reading head region) structure, thereby providing a new mode of action during the sequencing process.
  • the porins of the examples have good edge-hopping distribution and recombination efficiency with phospholipid membranes.
  • the wild-type porin monomer is genetically mutated to form a mutant of the porin monomer.
  • the amino acid of the mutant of the porin monomer comprises the sequence shown in SEQ ID NO: 1 or comprises at least 99%, 98%, 97%, 96%, 95%, 94%, 93% , 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identical sequence, and the mutant of the porin monomer Amino acids have mutations at one or more positions corresponding to Q68, N72, and F73 of SEQ ID NO:1.
  • mutations include insertions, deletions and/or substitutions of amino acids.
  • having a mutation at one or more positions of Q68, N72, and F73 of SEQ ID NO:1 is having a mutation at one or more positions of Q68, N72, and F73 of SEQ ID NO:1 Amino acid insertions, deletions and/or substitutions.
  • the amino acids of the mutant of the porin monomer correspond to (1) 68-73, (2) 68-218, (3) 61-73, (4) 61- Mutations at one or more of positions 218, (5)66-73, or (6)66-218.
  • the amino acids of the mutant of the porin monomer correspond to (1) 68-73, (2) 68-218, (3) 61-73, (4) 61- One or more of positions 218, (5)66-73, or (6)66-218 have amino acid insertions, deletions and/or substitutions.
  • the amino acid of the mutant of the porin monomer has mutations only at positions Q68, N72, and F73 corresponding to SEQ ID NO: 1, or has amino acid insertions or deletions at one or more positions and/or replace.
  • the amino acid of the mutant of the porin monomer has mutations only at positions Q61, K66, Q68, N72, and F73N corresponding to SEQ ID NO: 1, or has amino acids at one or more positions insertions, deletions and/or substitutions.
  • the amino acid of the mutant of the porin monomer has mutations only at positions K66, Q68, N72, F73, D120, and E218 corresponding to SEQ ID NO: 1, or at one or more positions With amino acid insertions, deletions and/or substitutions.
  • At one or more positions means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10... or up to all positions. For example, at one or more positions of 5 amino acids is at 1, 2, 3, 4 or 5 positions.
  • the position corresponding to SEQ ID NO: 1 means that no matter whether the sequence number is changed by amino acid insertion or deletion or the use of an identity sequence, the relative position remains unchanged, and the sequence of SEQ ID NO: 1 can still be used number.
  • Q68 corresponding to SEQ ID NO: 1 can be mutated to Q68I, even if the sequence number of SEQ ID NO: 1 changes or adopts a sequence having identity defined herein with SEQ ID NO: 1, corresponding to SEQ ID NO: 1 Amino acid Q at position 68 (even if it is not position 68 in another sequence) can be mutated into I, which is still within the protection scope of the present invention.
  • the amino acids of the mutant of the porin monomer consist of the sequence shown in SEQ ID NO: 1, or have at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, or 70%, 65%, 60%, 55%, or 50% identity sequence composition, and the porin monomer
  • the amino acid of the mutant corresponds to one or more positions of Q68, N72, and F73 of SEQ ID NO:1.
  • the sequence of SEQ ID NO: 1 of the porin monomer is from Vibrio maritima.
  • the nucleotide sequence encoding the amino acid of SEQ ID NO:1 is SEQ ID NO:2.
  • Q68 corresponding to SEQ ID NO: 1 is 0 to 5 in G, A, V, L, I; N72 is 0 to 5 in G, A, V, L, I ; F73 is mutated into 0 to 4 of N, E, D, Q.
  • Q61 corresponding to SEQ ID NO:1 is mutated into 0 to 5 of G, A, V, L, and I; K66 is mutated into 0 to 3 of R, H, and K; Q68 is mutated into 0 to 5 of G, A, V, L, I; N72 is mutated to 0 to 5 of G, A, V, L, I; F73 is mutated to 0 to 4 of N, E, D, Q kind.
  • the K66 corresponding to SEQ ID NO: 1 is mutated into 0 to 3 of R, H, and K; the Q68 is mutated into 0 to 5 of G, A, V, L, and I; the N72 is mutated into 0 to 5 of G, A, V, L, and I; F73 is mutated to 0 to 4 of N, E, D, and Q; D120 is mutated to 0 to 4 of N, E, D, and Q; E218 is mutated into 0 to 5 of G, A, V, L, and I.
  • a mutant of a porin monomer wherein the amino acid mutation is selected from the following:
  • the amino acid sequence of the mutant of the porin monomer comprises, or consists of, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
  • the protein pore comprises at least one mutant of a porin monomer (or a mutated monomer of a porin). In one embodiment, the protein pore comprises at least two, three, four, five, six, seven, eight, nine, or ten or more mutants of the porin monomer. In one embodiment, the protein pore comprises mutants of at least two porin monomers, which may be the same or different. In one embodiment, the protein pore comprises mutants of two or more monomers of the porin, preferably the mutants of the two or more monomers are the same. In one embodiment, the protein pore comprises mutants of nine porin monomers. In one embodiment, the diameter of the constricted region of the protein pore is 0.7nm-2.2nm, 0.9nm-1.6nm, 1.4-1.6nm or
  • mutants of porin monomers or protein pores comprising the same for detecting the presence, absence or one or more characteristics of a target analyte.
  • mutants of porin monomers or protein pores are used to detect the sequence of a nucleic acid molecule, or characterize a polynucleotide sequence, eg, sequence a polynucleotide sequence, because they can distinguish between different nucleotides with high sensitivity.
  • Mutants of porin monomers or protein pores that include them can discriminate between four nucleotides in DNA and RNA, and even between methylated and unmethylated nucleotides, with unexpectedly high resolution .
  • dCMP deoxycytosine monophosphate
  • Mutants of porin monomers or protein pores can also discriminate between different nucleotides under a range of conditions.
  • mutants of said porin monomers or protein pores discriminate between nucleotides under conditions favorable for nucleic acid characterization, such as sequencing.
  • the degree to which mutants of porin monomers or protein pores discriminate between different nucleotides can be controlled by varying the applied potential, salt concentration, buffer, temperature and the presence of additives such as urea, betaine and DTT. This allows mutants of porin monomers or the function of the protein pore to be finely tuned, especially when sequenced.
  • Mutants of porin monomers or protein pores can also be used to identify polynucleotide polymers by interacting with one or more monomers rather than at nucleotide-based nucleotides.
  • a mutant of a porin monomer or a protein pore may be isolated, substantially isolated, purified or substantially purified. Mutants of the porin monomers or protein pores of the examples are isolated or purified if they are completely free of any other components, such as liposomes or other protein pores/porins. A mutant porin monomer or protein pore is substantially isolated if the mutant porin monomer or protein pore is mixed with a carrier or diluent that does not interfere with its intended use.
  • the mutant of the porin monomer or the protein pore can contain 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/pores If the mutant porin monomer or protein pore exists in the form of a protein, the porin monomer is substantially isolated or substantially purified. Alternatively, mutants of porin monomers or protein pores can be present in the membrane.
  • the membrane is preferably an amphiphilic layer.
  • the amphiphilic layer is a layer formed of amphiphilic molecules, for example, phospholipids, which have both hydrophilic and lipophilic properties.
  • Amphiphiles can be synthetic or naturally occurring.
  • the amphiphilic layer can be monolayer or bilayer.
  • the amphiphilic layer is usually planar.
  • the amphiphile may be curved.
  • the amphiphilic layer may be supported.
  • the membrane can be a lipid bilayer.
  • a lipid bilayer is formed by two opposing layers of lipids. The two layers of lipids are aligned such that their hydrophobic tail groups face each other to form a hydrophobic interior.
  • the hydrophilic headgroups of the lipids face outward towards the aqueous environment on each side of the bilayer.
  • the membrane includes a solid state layer. Solid layers can be formed from organic and inorganic materials. If the membrane includes a solid state layer, the pores are typically present in the amphiphilic membrane or in a layer comprised within the solid state layer, eg, in holes, wells, gaps, channels, trenches or slits within the solid state layer.
  • Embodiments provide a method of determining the presence, absence, or one or more properties of a target analyte.
  • the method involves contacting the target analyte with a mutant porin monomer or protein pore such that the target analyte moves relative to, e.g., through, the mutant porin monomer or protein pore, and One or more measurements are taken as the target analyte moves relative to the mutant porin monomer or protein pore, thereby determining the presence, absence, or one or more properties of the target analyte.
  • the target analyte may also be referred to as a template analyte or an analyte of interest.
  • Target analytes are preferably polysaccharides, metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, dyes, drugs, diagnostic agents, explosives or environmental pollutants .
  • the method may involve determining the presence, absence or one or more properties of two or more target analytes of the same class, eg, two or more proteins, two or more nucleotides, or two or more drugs.
  • the method may involve determining the presence, absence or one or more properties of two or more different classes of target analytes, e.g., one or more proteins, one or more nucleotides and one or more medications.
  • the method comprises contacting the target analyte with a mutant porin monomer or protein pore such that the target analyte moves through the mutant porin monomer or protein pore.
  • 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 porin mutant monomers, e.g. , 7, 8, 9 or 10 monomers.
  • the protein pores comprise identical monomers or different porin monomers, preferably 8 or 9 identical monomers. One or more of said monomers, eg 2, 3, 4, 5, 6, 7, 8, 9 or 10, are preferably chemically modified as discussed above.
  • the amino acid of each monomer comprises SEQ ID NO: 1 and the above-mentioned mutants thereof.
  • the amino acid of each monomer consists of SEQ ID NO: 1 and its above-mentioned mutants.
  • the methods of the embodiments may measure two, three, four or five or more characteristics of a 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 the polynucleotide has been modified. In one embodiment, any combination of (i) to (v) may be measured.
  • (i) for example, by determining the number of interactions between the polynucleotide and the mutant/protein pore of the protein monomer or the duration of the interaction between the polynucleotide and the mutant/protein pore of the protein monomer vs. The length of the polynucleotide is measured.
  • the identity of the polynucleotide can be measured in a variety of ways, either in conjunction with or without measurement of the polynucleotide sequence.
  • the former is simpler; the polynucleotides are 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 rest of the sequence of the polynucleotide).
  • measurement of specific electrical and/or optical signals in the method can identify the polynucleotide as coming from a specific source.
  • the sequence of the polynucleotide can be determined as previously described. Suitable sequencing methods, in particular those using electrical measurements, 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.
  • secondary structure can be measured in a number of ways. For example, if the method involves an electrical measurement method, the secondary structure can be measured using a change in residence time or a change in the current flowing through the pore. This allows distinguishing between regions of single- and double-stranded polynucleotides.
  • the presence or absence of any modification can be measured.
  • the method comprises determining whether the polynucleotide is abasic or lacks nucleobases and sugars by methylation, oxidation, damage, with one or more proteins or with one or more labels, tagging or grooming. Certain 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 monomer/protein pore, such as a mutant protein monomer/protein pore as in the examples.
  • Mutants/protein pores of said protein monomers are usually present in membranes. Suitable membranes are described above.
  • the method can be performed using any device suitable for studying membranes/protein pores or mutant systems of porin monomers - where mutants of protein monomers/protein pores are present in membranes.
  • the method can be performed using any device suitable for use with the transmembrane porosity side.
  • the device comprises a chamber containing an aqueous solution and a barrier dividing the chamber into two parts.
  • the barrier typically has pores in which a membrane comprising pores is formed.
  • the barrier forms a membrane in which mutants/protein pores of protein monomers are present.
  • the method can be performed using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120).
  • Electrical measurements include voltage measurement, capacitance measurement, current measurement, impedance measurement, tunneling measurement (Ivanov AP et al., Nano Lett.2011Jan 12; 11(I):279-85) and FET measurement (International Application WO 2005/124888).
  • Optical measurements can be combined with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1) 014301).
  • the measurement may be a transmembrane current measurement, eg measurement of ionic current flowing through the pore.
  • the electrical or optical measurements may employ conventional electrical or optical measurements.
  • Electrical measurements can be used as 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 international application WO Standard single channel recording equipment in 2000/28312.
  • electrical measurements can be performed using a multi-channel system, for example as described in International Application WO 2009/077734 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.
  • the applied potential may be a chemical potential.
  • An example of this is using a salt gradient across a membrane, such as an amphiphilic layer. Salt gradients are disclosed in Holden et al., J Am Chem SoC. 2007 Jul 11;129(27):8650-5.
  • 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 chain 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 thus also include circuitry capable of applying an electrical potential and measuring electrical signals across the membrane and pores.
  • the method can be performed using patch clamp or voltage clamp.
  • the method may include measuring the current flowing through the pore as the polynucleotide moves relative to the pore. Suitable conditions for measuring ion flux through transmembrane protein pores are known in the art and disclosed in the Examples.
  • the method is generally carried out by applying a voltage across the membrane and the pore.
  • the voltage used is typically from +5V to -5V, eg from +4V to -4V, from +3V to -3V or from +2V to -2V.
  • the voltage used is typically from -600mV to +600V or -400mV to +400mV.
  • the voltage used preferably has a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300nA ⁇ P+400mV upper limit range.
  • the voltage used is more preferably in the range of 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV.
  • the process is generally carried out in the presence of any charge carrier, such as a metal salt such as an alkali metal salt, a halide salt such as a chloride salt, such as an alkali metal chloride salt.
  • Charge carriers may include ionic liquids or organic salts such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride.
  • the salt is present in the aqueous solution in the chamber.
  • potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide is used.
  • KCl, NaCl and mixtures of potassium ferrocyanide and potassium ferricyanide are preferred.
  • 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 typically 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 from 150 mM to 1M.
  • the method is preferably performed using a salt concentration of at least 0.3M, eg 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.
  • a high salt concentration provides a high signal-to-noise ratio and allows the passing of the current to indicate the presence of the nucleotide to be identified against the background of normal current fluctuations.
  • the methods are generally performed in the presence of a buffer.
  • the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the methods of the invention.
  • the buffer is a phosphate buffer.
  • Other suitable buffers are HEPES or Tris-HCl buffer.
  • the process is typically performed 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 method can be carried out at a temperature of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C to 80°C, 19°C to 70°C or 20°C to 60°C.
  • the method is generally carried out at room temperature.
  • the method is optionally performed at a temperature that supports enzyme function, eg, about 37°C.
  • 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 combining the target analyte with The protein pores present in the membrane interact (eg, contact) such that the target analyte moves relative to (eg, passes through) the protein pore.
  • the current flow through the protein pore as the target analyte moves relative to the protein pore is measured to determine the presence, absence or one or more characteristics of the target analyte (e.g., multinucleated). nucleotide sequence).
  • a rate-controlling protein is one that can control the speed at which a target analyte (e.g., a polynucleotide) moves relative to a protein pore (e.g., slow it down) so that this speed is able to respond to the presence, absence, or one or more characteristics of the target analyte.
  • a target analyte e.g., a polynucleotide
  • Proteins for detection eg, sequencing of polynucleotides.
  • Protein pores are used in conjunction with rate-controlling proteins to characterize target analytes.
  • the rate-controlling protein slows down the passage of polynucleotides through the protein pore to enable sequencing.
  • Rate-controlling proteins include the polynucleotide binding proteins described below.
  • 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 the mutant/protein pore of the protein monomer, e.g. Mutant/protein pores.
  • the method comprises (a) contacting the polynucleotide with a mutant/protein pore of a protein monomer and a polynucleotide binding protein such that the protein controls the relative relation of the polynucleotide to the mutant/pore of the protein monomer. Movement of the protein pore, e.g., through the mutant/protein pore of the protein monomer, and (b) obtaining one or more measurements as the polynucleotide moves relative to the mutant/protein pore of the protein monomer, wherein, Such measurements are indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.
  • the method comprises (a) contacting the polynucleotide with a mutant/protein pore of a protein monomer and a polynucleotide binding protein such that the protein controls the relative relation of the polynucleotide to the mutant/pore of the protein monomer. Movement of the protein pore, e.g., through the mutant/protein pore of the protein monomer, and (b) measuring 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.
  • a polynucleotide binding protein can be any protein capable of binding a polynucleotide and controlling its movement through a pore.
  • a polynucleotide binding protein generally interacts with and modifies at least one property of a polynucleotide. Proteins can modify polynucleotides by cleaving them to form individual nucleotides or short chains of nucleotides (eg, dinucleotides or trinucleotides).
  • a protein can modify a polynucleotide by orienting it or moving it to a specific position, ie, controlling its movement.
  • the polynucleotide binding protein is preferably derived from a polynucleotide handling enzyme.
  • a polynucleotide-handling enzyme is a polypeptide capable of interacting with and modifying at least one property of a polynucleotide.
  • the enzymes can modify polynucleotides by cleaving them to form individual nucleotides or short chains of nucleotides (eg, dinucleotides or trinucleotides).
  • the enzyme can modify the polynucleotide by orienting it or moving it to a specific position.
  • a polynucleotide-handling enzyme need not exhibit enzymatic activity as long as it is capable of binding polynucleotides and controlling their movement through the pore.
  • the enzyme may be modified to remove its enzymatic activity, or may be used under conditions that prevent its use as an enzyme.
  • Polynucleotide-handling enzymes are preferably polymerases, exonucleases, helicases and topoisomerases, e.g., gyrase.
  • the enzyme is preferably a helicase, such as Hel308Mbu, Hel308Csy, Hel308Tga, Hel308Mhu, Tral Eco, XPD Mbu, Dda or variants thereof. Any helicase can be used in the embodiments.
  • any number of helicases can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used. In some embodiments, different numbers of helicases can 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 aforementioned helicases.
  • 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.
  • the helicases can be linked in any order and using any method.
  • the invention also provides a kit for characterizing a target analyte (eg, a target polynucleotide).
  • a target analyte eg, a target polynucleotide
  • the kit contains the well and membrane components of the examples.
  • the film is preferably formed from components. Pores are preferably present in the membrane.
  • the kit may comprise the components of any of the membranes disclosed above, such as amphiphilic or triblock copolymer membranes.
  • the kit may further comprise a polynucleotide binding protein. Any of the polynucleotide binding proteins discussed above can be used.
  • 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 Y adapters and hairpin loop adapters.
  • Y adapters preferably have one or more helicases attached, and hairpin loop adapters preferably have one or more molecular brakes attached.
  • Y adapters preferably comprise one or more first anchors for coupling the polynucleotide to the membrane
  • hairpin loop adapters preferably comprise one or more second anchors for coupling the polynucleotide to the membrane Anchor, and the strength of coupling of the hairpin loop adapter to the membrane is preferably greater than that of the Y adapter to the membrane.
  • the kit may additionally comprise one or more other reagents or instruments that enable performance of any of the above-mentioned embodiments.
  • reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), a device for obtaining a sample from an individual (such as a container or instrument containing a needle), a device for amplifying and/or expressing a polynucleotide device, or voltage or patch clamp equipment.
  • the reagents may be present in the kit in a dry form such that the fluid sample resuspends the reagents.
  • the kit may also optionally contain instructions enabling use of the kit with the methods of the invention or details as to which organisms may use the methods.
  • the invention also provides an apparatus for characterizing a target analyte (eg, a target polynucleotide).
  • the device includes single or multiple protein monomer mutants/protein pores, and single or multiple membranes.
  • a mutant/protein pore of said protein monomer is preferably present in said membrane.
  • the number of pores and membranes is preferably equal. Preferably there is a single pore in each membrane.
  • the apparatus preferably also includes instructions for implementing the methods of the embodiments.
  • the device can be any conventional device for analyte analysis, eg, an array or a chip. Any of the embodiments discussed in connection with the method of an embodiment applies equally to the device.
  • the device may also include any of the features present in the kits described herein.
  • the equipment used in the embodiment can specifically be the gene sequencer QNome-9604 of Qitan Technology.
  • the wild-type porin is from Vibrio maritima
  • the amino acid sequence of the wild-type porin is SEQ ID NO: 1
  • the nucleotide sequence encoding the amino acid sequence is shown in SEQ ID NO: 2.
  • Mutant 1 of the porin monomer is that the wild-type porin has mutations at multiple positions corresponding to SEQ ID NO: 1, specifically Q68I, N72A, and F73Q.
  • the protein pore of mutant 1 that includes a porin monomer is mutant pore 1.
  • the amino acid sequence of mutant 1 of the protein monomer is shown in SEQ ID NO:16.
  • the wild-type porin is from Vibrio maritima
  • the amino acid sequence of the wild-type porin is SEQ ID NO: 1
  • the nucleotide sequence of the sequence encoding the amino acid is shown in SEQ ID NO: 2.
  • Mutant 2 of the porin monomer is that the wild-type porin has multiple mutations corresponding to SEQ ID NO: 1, specifically Q61L, K66R, Q68L, N72G and F73N.
  • the protein pore of mutant 2 that includes a porin monomer is mutant pore 2.
  • the amino acid sequence of mutant 2 of the protein monomer is shown in SEQ ID NO:17.
  • the wild-type porin is from Vibrio maritima
  • the amino acid sequence of the wild-type porin is SEQ ID NO: 1
  • the nucleotide sequence of the sequence encoding the amino acid is shown in SEQ ID NO: 2.
  • Mutant 3 of the porin monomer is that the wild-type porin has mutations at multiple positions corresponding to SEQ ID NO: 1, specifically Q68V, N72A, and F73Q.
  • the protein pore of mutant 3 that includes a porin monomer is mutant pore 3.
  • the amino acid sequence of mutant 3 of the protein monomer is shown in SEQ ID NO:18.
  • the wild-type porin is from Vibrio maritima
  • the amino acid sequence of the wild-type porin is SEQ ID NO: 1
  • the nucleotide sequence of the sequence encoding the amino acid is shown in SEQ ID NO: 2.
  • Mutant 4 of the porin monomer is that the wild-type porin has mutations at multiple positions corresponding to SEQ ID NO: 1, specifically K66R, Q68V, N72A, F73N, D120N, and E218I.
  • the protein pore of mutant 4 that includes a porin monomer is mutant pore 4.
  • the amino acid sequence of mutant 4 of the protein monomer is shown in SEQ ID NO:19.
  • FIG. 4A is a side view 400 of a predicted protein structure model, where a protein monomer 402 is shown in a darker color.
  • FIG. 4B is a top view 404 of the surface structure model, where a darker part shows a protein monomer 406 .
  • FIG. 4C is a streamer structure model diagram 408 , and the darker part is the protein monomer 410 .
  • Figure 5 shows the distribution of amino acid residues and the diameter of the constriction zone of the wild-type channel.
  • the diameter of the channel in the constriction zone between the two porin monomers 502 and 504 is at most about followed by approximately Minimum diameter of approx. Shown in the middle is the amino acid composition of the constriction structure, Q68, N72 and F73.
  • Figure 6A shows the surface potential map of the wild-type channel monomer, where the color depth represents the electrical strength.
  • Figure 6B shows the stick model of the monomer streamer model and the distribution of amino acid residues in the constriction region. The amino acid composition and numbering of the loop in the constriction region are enlarged, and part 602 is the amino acid residue pointing to the central region of the protein pore.
  • Fig. 7 shows the distribution characteristics of amino acid residues in the constriction zone of mutation hole 1 and the diameter of the constriction zone.
  • the stick model shows the distribution of key amino acid residues in the narrow region of the mutant pore, and the amino acid residues pointing to the center of the pore are isoleucine I at position 68, alanine A at position 72, and glutamine Q at position 73.
  • the hydrogen bond interaction formed by amino acid residues 61-77 is closely related to the correct assembly of the channel complex.
  • the diameter of the narrowest region of the constriction zone between the two porin monomers 702 and 704 is about The widest area diameter is approx. Median diameter approx.
  • Figure 8 shows a cartoon schematic diagram of mutation hole 1 based on homology modeling.
  • Region 1 corresponds to the crown formation region
  • region 2 corresponds to the constriction and loops region
  • region 3 corresponds to the transmembrane ⁇ barrel region .
  • the DNA construct BS7-4C3-PLT was prepared.
  • the structure of BS7-4C3-PLT is shown in Figure 9, and the sequence information is as follows:
  • C3, C18, dSpacer and iSpC3 are marker sequences introduced to indicate the resolution characteristics of well sequencing.
  • the c rate-controlling protein in Fig. 9 is the helicase Mph-MP1-E105C/A362C (with mutation E105C/A362C), the amino acid sequence is SEQ ID NO: 14, and the nucleic acid sequence is SEQ ID NO: 15 .
  • Mutation hole 1 was used as a protein hole, and was detected by single-hole sequencing technology. After insertion of a single porin with amino acid sequence Mutant 1 into the phospholipid bilayer, buffer (625 mM KCl, 10 mM HEPES pH 8.0, 50 mM MgCl 2 ) was passed through the system to remove any excess Mutant 1 nanopores . The DNA construct BS7-4C3-PLT (1-2nM final concentration was added to the mutant 1 nanopore experimental system, after mixing, the buffer solution (625mM KCl, 10mM HEPES pH 8.0, 50mM MgCl 2 ) was flowed through the system to remove any excess DNA construct BS7-4C3-PLT.
  • buffer solution (625mM KCl, 10mM HEPES pH 8.0, 50mM MgCl 2 ) was flowed through the system to remove any excess DNA construct BS7-4C3-PLT.
  • helicase Mph-MP1-E105C/A362C, 15nM final concentration
  • fuel ATP 3mM final concentration
  • Mutation hole 1 opens at a voltage of ⁇ 180mV.
  • Fig. 10A shows the opening current and gating characteristics of mutant pore 1 at a voltage of ⁇ 180 mV.
  • FIG. 10B shows the situation of the single-stranded nucleic acid passing through the mutation hole 1 under the voltage of +180mV. Nucleic acids can pass through the pores. After adding ssNucleic acid, the downward line shows the signal of the nucleic acid through the hole.
  • the DNA construct BS7-4C3-PLT was sequenced through the mutant hole 1 by single-hole sequencing technology, and the nucleic acid sequencing signal generated by the sequencing system was added after the hole insertion was completed.
  • 11A and 11B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 1 . According to the signal characteristics, mutation hole 1 can be used for nucleic acid sequencing.
  • FIG. 12 is an enlarged result showing the current trace in the part of FIG. 11A .
  • the portion indicated by the dotted arrow shows the enlarged result of the current trace.
  • Figure 13 shows the chip test current trace when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant hole 1. These further indicate that mutant pore 1 can be used for nucleic acid sequencing.
  • the embodiment 8 uses the mutation hole 2 to perform the empty detection and via detection.
  • FIGS. 14A and 14B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 2. According to the signal characteristics, mutation hole 2 can be used for nucleic acid sequencing.
  • the embodiment 9 uses the mutation hole 3 to perform the empty detection and via detection.
  • FIG. 15A shows the opening current and gating characteristics of mutant pore 3 at a voltage of ⁇ 180 mV.
  • FIG. 15B shows the condition of single-stranded nucleic acid passing through mutation hole 3 at +180mV voltage. Nucleic acids can pass through the pores. After adding ssnucleic acid, the downward line shows the signal of the nucleic acid passing through the hole.
  • FIGS. 16A and 16B show exemplary current traces when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct BS7-4C3-PLT through mutant pore 3. According to the signal characteristics, mutation hole 3 can be used for nucleic acid sequencing.
  • Figure 17 shows a zoomed-in result of part of the current trace.
  • the portion indicated by the dotted arrow shows the enlarged result of the current trace.
  • Figure 18 shows the ChIP test current trace when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct phage10k through mutant pore 3. These indicated that mutant pore 3 could be used for nucleic acid sequencing.
  • Example 10 Similar to Example 7, in Example 10, the mutation hole 4 is used for empty testing and via testing.
  • FIG. 19A shows the opening current and gating characteristics of mutant pore 4 at a voltage of ⁇ 180 mV.
  • FIG. 19B shows the condition of single-stranded nucleic acid passing through mutant hole 4 at +180mV voltage. Nucleic acids can pass through the pores. After adding ssnucleic acid, the downward line shows the signal of the nucleic acid passing through the hole.
  • Figure 20 shows the ChIP test current trace when the helicase Mph-MP1-E105C/A362C controls the translocation of the DNA construct phage10k through mutant pore 4. These indicated that mutation hole 4 could be used for nucleic acid sequencing.
  • 1% inoculum was transferred to ampicillin-resistant TB liquid medium for expanded culture, cultured at 37°C and 220 rpm, and its OD600 value was continuously measured.
  • IPTG Isopropyl ⁇ -D-Thiogalactoside
  • the cells were collected by centrifugation. The cells were resuspended in the disruption buffer and then crushed under high pressure, purified by Ni-NTA affinity chromatography, and the target eluted samples were collected. Mutants 2-4 of the porin monomer were purified as above.
  • FIG. 21 shows the protein purification results of mutant 1, and lanes 1-5 show the SDS-PAGE electrophoresis detection results of different fractions separated.
  • Figure 22 shows the results of molecular sieve purification of the protein of mutant 1, and the position indicated by the arrow is the oligomeric state distribution of the target protein peak.

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Abstract

La présente invention concerne un mutant d'un monomère de porine, un pore protéique le contenant et leur utilisation pour détecter un analyte cible. L'acide aminé du mutant du monomère de porine comprend une séquence représentée dans SEQ ID NO : 1, et l'acide aminé du mutant du monomère de porine comprend une mutation à une ou plusieurs positions correspondant à Q68, N72 et F73 de SEQ ID NO : 1.
PCT/CN2021/123210 2021-10-12 2021-10-12 Mutant de monomère de porine, pore protéique et leur utilisation WO2023060420A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110914290A (zh) * 2017-06-30 2020-03-24 弗拉芒区生物技术研究所 新颖蛋白孔
CN113195736A (zh) * 2018-11-08 2021-07-30 牛津纳米孔科技公司 孔
CN113480620A (zh) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113651876A (zh) * 2021-08-18 2021-11-16 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113754743A (zh) * 2021-10-12 2021-12-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110914290A (zh) * 2017-06-30 2020-03-24 弗拉芒区生物技术研究所 新颖蛋白孔
CN113195736A (zh) * 2018-11-08 2021-07-30 牛津纳米孔科技公司 孔
CN113480620A (zh) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113651876A (zh) * 2021-08-18 2021-11-16 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113754743A (zh) * 2021-10-12 2021-12-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

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