WO2023123370A1 - Protéine à nanopore et son utilisation associée en séquençage - Google Patents

Protéine à nanopore et son utilisation associée en séquençage Download PDF

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WO2023123370A1
WO2023123370A1 PCT/CN2021/143722 CN2021143722W WO2023123370A1 WO 2023123370 A1 WO2023123370 A1 WO 2023123370A1 CN 2021143722 W CN2021143722 W CN 2021143722W WO 2023123370 A1 WO2023123370 A1 WO 2023123370A1
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nanoporin
nanopore
protein
sequencing
electrode
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PCT/CN2021/143722
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Chinese (zh)
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董宇亮
胡兆龙
王子
郭斐
刘欢欢
吴蔚
季州翔
曾涛
章文蔚
徐讯
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深圳华大生命科学研究院
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Priority to CN202180104098.3A priority Critical patent/CN118234741A/zh
Priority to PCT/CN2021/143722 priority patent/WO2023123370A1/fr
Publication of WO2023123370A1 publication Critical patent/WO2023123370A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of single-molecule sequencing, in particular to a nanopore protein and related applications in sequencing.
  • Biomacromolecules such as DNA, RNA, and protein are the basic substances that constitute life, and their primary sequence structure and group post-modification determine their biological functions.
  • the abundance of biological macromolecules in organisms is also closely related to biological phenotype.
  • the technology of sequencing and quantifying biological macromolecules is a core tool for understanding the laws of life.
  • the existing technologies cannot completely restore the sequence, modification, abundance and other information of these biomacromolecules.
  • nucleic acids neither the early Sanger sequencing method nor the current mainstream sequencing-by-synthesis sequencing method can achieve continuous and accurate detection of long-segment DNA sequences and direct measurement of original base modifications and RNA sequences.
  • the necessary PCR Amplification will introduce errors and biases; for proteins, the detection of their primary amino acid sequence and modification can only be satisfied in an indirect way, and no matter the Edman (Edman) degradation sequencing method or mass spectrometry sequencing method, the additional processing steps are not enough. Avoid reducing the resolution and introducing errors.
  • the traditional biomacromolecular sequencing measurement technology is ensemble measurement, which obtains averaged information and cannot reflect the individual state differences of individual molecules, which often determine the fate of cells. The direct and complete determination of the sequence, modification and abundance of a single biological macromolecule can truly restore the details of the state of the biological system, which is an important challenge for future biotechnology. On the basis of this demand, single-molecule sequencing technology emerged as the times require.
  • Single-molecule sequencing technologies are mainly divided into two categories: one is optical-based zero-mode waveguide sequencing, represented by Pacific Biosciences (Pacbio) in the United States; the other is electrical-based nanopore sequencing, represented by Oxford Nanotechnology Co. Represented by Oxford Nanopore Technologies (ONT).
  • the former is applied to DNA sequencing, and the sequence and modification information of the template strand can be inferred based on the pulsed fluorescent signal generated when the polymerase extends the DNA primer chain to synthesize new bases; the latter can be applied to both DNA and RNA sequencing.
  • the base unit on the RNA molecule is analyzed and sequenced by analyzing the continuous current signal generated when the base unit passes through the nanoporin one by one.
  • Nanopore sequencing has more advantages in sequencing speed, throughput, portability, and direct RNA sequencing, and has gained widespread attention in recent years.
  • Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products.
  • natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection.
  • the properties of the amino acid residues in the pore walls of natural nanoporins, especially the charge properties do not necessarily meet the properties of a specific analyte.
  • the main purpose of the present invention is to provide a nanoporin and its related application in sequencing, so as to solve the problem of poor channel stability of the nanoporin in the prior art.
  • a kind of nanopore protein comprising (a) MP964, MP964 is the protein with the amino acid sequence shown in SEQ ID NO: 1; Or (b) MP964Mut, MP964Mut is a protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, and 127th , No. 143, No.
  • 148 a protein with a pore structure after substitution and/or deletion and/or addition of one or several amino acids; or (d) and any of (a), (b) and (c)
  • the defined amino acid sequence has more than 80% homology and has the same function protein.
  • the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/S/H; No. 127 Bit: K/H/Y; No. 143: K/H/Y; No. 148: R/H/Y; Among them, "/" represents "or”;
  • any defined amino acid sequence has more than 85%, preferably more than 90%, more preferably more than 95%, more preferably more than 99% homology and have the same function protein; preferably, Nanoporins are derived from mycobacteria.
  • the pore diameter of the nanoporin is 1.2-1.6nm; preferably, the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170pA ⁇ 190pA; Preferably, the conductance of the nanoporin is 0.8 ⁇ 1.2nS.
  • a kit which includes the above-mentioned nanoporin.
  • the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; preferably, the nanoporin
  • the assay buffer is 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • an isolated DNA molecule has (a) a nucleotide sequence encoding the above-mentioned nanoporin; or (b) under stringent conditions with (a) the nucleotide sequence of hybridization of the defined DNA molecule; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) with (a) to (c) Any one of the nucleotide sequences defined in has more than 70% homology and encodes a DNA molecule with the same functional protein.
  • it has more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% homology with any of the nucleotide sequences defined in (a) to (c), and the encoding has the same function Protein DNA molecule.
  • a recombinant vector which comprises the above DNA molecule.
  • a host cell is provided, the host cell is transformed with the above recombinant vector.
  • a nanoporous biofilm which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when crossing the membrane When an electric field force is applied, conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin.
  • the membrane layer includes a lipid layer; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include a phospholipid bilayer; preferably, the lipid layer includes a planar membrane layer or a liposome; Preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • the nanoporin is mobile in the membrane layer; preferably, when an electric field force is applied across the membrane layer, the nanopore biomembrane can displace the biomolecules to be tested through the pores; preferably, the biomolecules to be tested include DNA, RNA, polypeptide or protein; Preferably, the biomolecule to be tested has a modified group molecule, more preferably the group molecule is selected from cholesterol, polyethylene glycol, biotin or fluorescent group molecule; Preferably, DNA and /or RNA includes any one or more of the following modified bases: 5-methylcytosine, 6-methyladenine, 7-methylguanine or pseudouracil.
  • a nanopore sequencing device includes the above-mentioned nanopore biofilm.
  • the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell, and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and The second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two receiving electrodes respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the receiving electrode and signal processing
  • the chip is connected; preferably, the electrolyte is a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; more preferably,
  • the nanoporin assay buffer includes 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; preferably, the first electrode and the second electrode include metal or composite
  • a sequencing method utilizes the above-mentioned nanopore protein, or the above-mentioned nanopore biofilm, or the above-mentioned nanopore sequencing device by analyzing the biomolecules to be tested through The electrical signal generated by the pores of nanoporins is used to sequence the biomolecules to be tested.
  • the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin to generate electrical signals; preferably ground, the electrical signal includes a blocking current amplitude.
  • a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biofilm, and the nanoporin is sequenced using the structure of the nanoporous biofilm.
  • the pore stability of the mutated nanoporin is improved, which facilitates the improvement of sequencing accuracy and data throughput in subsequent sequencing.
  • Figure 1 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964 according to Example 5 of the present invention
  • Figure 2 shows a schematic diagram of the SDS-PAGE purification results of nanoporin MP964Mut according to Example 6 of the present invention
  • Fig. 3 shows a schematic diagram of the homology modeling three-dimensional structure of nanoporin MP964 according to Example 7 of the present invention
  • Fig. 4 shows a schematic diagram of the homology modeling three-dimensional structure of the nanoporin MP964Mut according to Example 7 of the present invention
  • Fig. 5 shows the current retardation percentage distribution graph of the oligonucleotide sample of nanoporin MP964 according to Example 8 of the present invention
  • Fig. 6 shows the current retardation percentage distribution diagram of the oligonucleotide sample of nanoporin MP964Mut according to Example 8 of the present invention
  • Figure 7 shows a statistical schematic diagram of the DNA capture rate of nanoporins MP964 and MP964Mut according to Example 8 of the present invention
  • Figure 8 shows a schematic diagram of the conductance distribution of the nanoporin MP964Mut in different experiments according to Example 9 of the present invention
  • Figure 9 shows a schematic diagram of the current characteristics of the oligonucleotide sample A according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
  • Fig. 10 shows a schematic diagram of the percentage distribution diagram of current blockage of oligonucleotide sample A according to Example 9 of the present invention
  • Figure 11 shows a schematic diagram of the current characteristics of the oligonucleotide sample B according to Example 9 of the present invention passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV;
  • Figure 12 shows a schematic diagram of the current block percentage distribution diagram of oligonucleotide sample B according to Example 9 of the present invention.
  • Fig. 13 shows a schematic diagram of controlling DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and the current amplitudes of different amplitudes are generated as the DNA moves, wherein, graph (A) is a current characteristic graph. Figure (B) is an enlarged current diagram;
  • Fig. 14 shows that when the DNA is controlled to pass through the nanoporin mutant MP964Mut under an applied voltage of 150mV, current amplitude changes of different magnitudes are produced as the DNA moves, wherein, graph (A) is a current characteristic graph.
  • Figure (B) is an enlarged current diagram.
  • Natural nanoporins generally have the ability to form pores, but in the in vitro expression and purification system of recombinant proteins, the stability of the pores of recombinant nanoporins may not meet the needs of single-molecule detector-related instrument products. At the same time, natural nanoporins have a wide range of pore size distributions, which may not necessarily meet the needs of single-molecule detection.
  • a nanopore protein including (a) MP964, MP964 is a protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) MP964Mut, MP964Mut is A protein having the amino acid sequence shown in SEQ ID NO: 2; or (c) at least one of the following positions of the amino acid sequence in (a) or (b): 97th, 98th, 127th, 143rd, 148th, a protein having a pore structure through substitution and/or deletion and/or addition of one or several amino acids; or any one of (d) and (a), (b) and (c) defined
  • the amino acid sequence has more than 80% homology and has the same function protein.
  • the nanoporin defined in (a) or (b) above has a pore structure, and when applied to nanopore sequencing, it can allow the biomolecules to be tested to pass through the pore one by one to generate a current signal.
  • MP964Mut is a nanopore protein obtained by mutation based on MP964. On the basis of (a) or (b) sequence, the protein is mutated, for example, on the basis of retaining the mutation site of MP964Mut, after substitution and/or deletion and/or addition of one or several amino acids at other positions , can still obtain and maintain the pore structure and function of the above-mentioned nanoporin.
  • Nanopore proteins may affect the stability of proteins and aggregates, the inner diameter of pores, and the amino acid residues on the inner wall of pores, thereby affecting their physical and chemical properties and the passage performance of biomolecules to be tested.
  • the methods for proteins with nanopore structure and functional activity are known to those skilled in the art, or can be realized by further combining with the similar screening steps of MP964Mut in this application.
  • amino acid sequence of MP964 is as follows: SEQ ID NO: 1:
  • the amino acid sequence of MP964Mut is as follows: SEQ ID NO: 2:
  • the types of amino acids substituted at each site are independently selected from the following: No. 97: Q/Y/A/S/H; No. 98: Q/Y/ S/H; No. 127: /K/H/Y; No. 143: K/H/Y; No.
  • homologous proteins with the same function, their properties such as protein and aggregate stability, pore inner diameter, pore inner wall amino acid residues, and the passing performance of biomolecules to be tested are all It has the same probability as the protein provided by the sequence (a) or (b), and is a homologous protein obtained by amino acid mutation.
  • the nanoporin is derived from mycobacteria.
  • the pore diameter of the nanoporin is 1.2-1.6 nm.
  • the pore diameter range is smaller than that of existing nanoporins, so the pore stability is better and the sequencing accuracy is also higher.
  • the current amplitude value of the nanoporin at a voltage of 150mV is 145-155pA; preferably, the current amplitude value of the nanoporin at a voltage of 180mV is 170-190pA; preferably, the conductance of the nanoporin is 0.8-1.2 nS.
  • the smaller the pore diameter of the nanoporin the higher the accuracy when it is used for sequencing. If the pore diameter of the nanoporin is too large (more than one molecule may pass through the pore at a time), it is difficult to meet the needs of single-molecule sequencing. When the biomolecule to be tested passes through the too large pore, the current signal generated may be missed or generated Errors lead to low sequencing accuracy. In single-molecule sequencing, the same molecule is sequenced multiple times to obtain accurate sequencing results. Therefore, the higher the accuracy of sequencing, the shorter the number and time required for sequencing. Using nanopore proteins with high sequencing accuracy for sequencing can greatly reduce the time and cost of sequencing, and this advantage is especially obvious in high-throughput sequencing.
  • a kit which includes the above-mentioned nanoporin.
  • the kit containing the above-mentioned nanoporin for sequencing, because the pore diameter of the above-mentioned nanoporin is relatively smaller, the pore stability is better, and the sequencing accuracy is relatively higher, so the sequencing time and cost can be reduced.
  • the kit also includes a lipid layer or an artificial polymer membrane; preferably, the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include phospholipids bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilamellar liposome or a unilamellar liposome; preferably, the lipid layer comprises a diphytylphosphatidyl Phospholipid bilayers composed of choline.
  • the lipid layer includes amphiphilic lipids; preferably, the amphiphilic lipids include phospholipids bilayer; preferably, the lipid layer comprises a planar membrane layer or a liposome; preferably, the liposome comprises a multilamellar liposome or a unilamellar liposome; preferably, the lipid layer comprises a diphytylphosphatidyl Phospholipid bilayers composed of choline.
  • Artificial polymer films include but are not limited to polysiloxane, polyolefin, perfluoropolyether, perfluoroalkyl polyether, polystyrene, polyoxypropylene, polyvinyl acetate, polyoxybutylene, polyisoprene , polybutadiene, polyvinyl chloride, polyalkylacrylate, polyalkylmethacrylate, polyacrylonitrile, polypropylene, PTHF, polymethacrylate, polyacrylate, polysulfone, polyvinyl ether, Poly(propylene oxide) and its copolymers, radical-substituted C1-C6 alkyl acrylates and methacrylates, acrylamides, methacrylamides, (C1-C6 alkyl)acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethylene glycol monomethacrylate and polyethylene glycol mono
  • the kit also includes a nanoporin experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer
  • the solution contains 0.1-1.0M KCl; preferably, the nanopore assay buffer includes 0.5M KCl, 10mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 1mM ethylenediaminetetraacetic acid (EDTA), pH 7.8.
  • nanoporin lipid layer and nanoporin experimental buffer in the above kit
  • one or more nanoporins can be inserted into the lipid layer in the nanoporin experimental buffer to form a nanoporous organism membrane.
  • the nanopore experimental buffer can provide a neutral environment to maintain the stability of the nanopore protein and lipid layer, and the metal ions contained in it make the nanopore experimental buffer have good conductivity.
  • lipid layer There are many options for the choice of lipid layer.
  • nanoporin can be inserted to form a nanopore biomembrane.
  • an isolated DNA molecule has (a) a nucleotide sequence encoding the aforementioned nanoporin; or (b) under stringent conditions with (a ) the nucleotide sequence of the hybridized DNA molecule as defined; or (c) has the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; or (d) is defined in (a) to (c) Any nucleotide sequence has more than 70% (preferably more than 80%, more preferably more than 85%, more preferably more than 90%, most preferably more than 95%, such as 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, Even more than 99.9%) homologous DNA molecules that encode proteins with the same function.
  • the above-mentioned DNA molecule can encode the nanopore protein having the above-mentioned structure and function of the present application.
  • the gene shares more than 75%, preferably more than 85%, more preferably more than 95%, and more preferably more than 99% of any nucleotide sequence defined in (a) to (c). DNA molecules that are homologous and encode proteins with the same function.
  • DNA molecules with 75%, 85%, 90%, 95%, 99% or more homology and encoding nanopore proteins with the same function, the active sites, active pockets, and active mechanisms of the encoded proteins are equal to (a)
  • the genes provided by the sequences have the same high probability and are homologous genes obtained through nucleotide mutation.
  • isolated in this application means altered “by the hand of man” from its natural state, ie, if it occurs in nature, it is altered and/or separated from its original environment.
  • a polynucleotide or polypeptide naturally occurring in a living organism is not “isolated”, whereas the same polynucleotide or polypeptide separated from its natural state coexistence is “isolated” (as used herein the term).
  • the DNA molecule in the present invention hybridizes with the gene encoding the nanoporin of the present invention under "stringent conditions", which refers to the conditions under which the presence of the gene encoding the nanopore protein of the present invention can be identified by means of nucleic acid hybridization.
  • stringent conditions refers to the conditions under which the presence of the gene encoding the nanopore protein of the present invention can be identified by means of nucleic acid hybridization.
  • two DNA molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two DNA molecules can specifically hybridize to each other.
  • One DNA molecule is said to be the "complement” of the other if two DNA molecules exhibit perfect complementarity.
  • two DNA molecules are said to be "complementary” if they are capable of hybridizing to each other with sufficient stability so that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure.
  • a DNA molecule In order for a DNA molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
  • a substantially homologous sequence is a DNA molecule that can specifically hybridize with a matching complementary strand of another DNA molecule under highly stringent conditions.
  • Suitable stringent conditions to promote DNA hybridization for example, treatment with 6.0 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0 ⁇ SSC at 50° C., are known to those skilled in the art. is well known.
  • the salt concentration in the washing step can be selected from about 2.0 ⁇ SSC, 50°C for low stringency conditions to about 0.2 ⁇ SSC, 50°C for high stringency conditions.
  • the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions.
  • Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed.
  • the stringent conditions in the present invention can specifically hybridize with the nucleotide sequence encoding the nanoporin of the present application in 6 ⁇ SSC, 0.5% SDS solution at 65° C., and then use 2 ⁇ SSC, Wash the membrane once with 0.1% SDS, 1 ⁇ SSC, and 0.1% SDS.
  • a recombinant vector comprises the above-mentioned DNA molecule, that is, a nanoporin expression gene.
  • the nanoporin expression gene is inserted into the recombinant vector, and the nanoporin expression gene is copied in large quantities by utilizing the self-replicating function of the recombinant vector.
  • "Recombinant” herein refers to genetically engineered DNA prepared by transplanting or splicing genes from one species into cells of a host organism of a different species. This DNA becomes part of the host's genetic makeup and is replicated.
  • a host cell transformed with the above-mentioned recombinant vector is provided.
  • the above recombinant vector is transformed into a host cell, and the host cell is used to replicate, transcribe, and translate the nanoporin expression gene on the recombinant vector, so that a large amount of nanoporin can be produced.
  • Host cells include common host cells such as Escherichia coli, yeast, mammalian cells, and insect cells. The host cells are used to fold the nanoporin to form a correct three-dimensional structure, and obtain a nanoporin with normal structure and function.
  • a nanoporous biofilm which includes: a membrane layer; and a nanoporin inserted into the middle of the membrane layer to form a channel, when an electric field is applied across the membrane layer When the force is applied, the conduction occurs in the channel; wherein, the nanoporin includes the above-mentioned nanoporin.
  • the nanoporous biomembrane in this application specifically refers to the membrane layer with nanoporin inserted.
  • This kind of nanoporous biomembrane inserts nanoporin with pores in the membrane layer, which can fix the direction of the pores of the nanoporin.
  • the diameter of the pores is perpendicular to the direction of the electric field, and the pores Conductance occurs to generate an electrical signal.
  • the membrane layer comprises a lipid layer; preferably, the lipid layer comprises amphiphilic lipids; preferably, the amphiphilic lipid comprises a phospholipid bilayer; preferably, the lipid layer comprises a planar membrane layer or liposome; preferably, the liposome includes multilamellar liposomes or unilamellar liposomes; preferably, the lipid layer includes a phospholipid bilayer composed of diphytylphosphatidylcholine.
  • lipid layer There are many options for the choice of lipid layer.
  • nanoporin can be inserted to form a nanopore biomembrane.
  • the nanoporin is mobile within the membrane layer; preferably, the nanoporous biomembrane is capable of translocating the biomolecules to be tested through the pores when an electric field force is applied across the membrane layer; preferably , the biomolecules to be tested include DNA, RNA, polypeptide or protein; preferably, the biomolecules to be tested have modified group molecules, more preferably the group molecules are selected from cholesterol, polyethylene glycol with different degrees of polymerization, biotin or Fluorophore molecules; preferably, DNA and/or RNA include any one or more of the following modified bases: 5-methylcytosine (5mC), 6-methyladenine (m6A), 7-methylguanine Purine (m7G), pseudouracil (pseudouridine, ⁇ ).
  • 5mC 5-methylcytosine
  • m6A 6-methyladenine
  • m7G 7-methylguanine Purine
  • pseudouracil pseudouracil
  • the nanoporin is movable in the membrane layer, and can automatically adjust its position and orientation under the action of an electric field.
  • the biomolecules to be tested pass through the nanoporin through the pores under the action of the electric field force.
  • the biomolecules to be tested include biomacromolecules such as DNA, RNA, polypeptide or protein that carry biological genetic information.
  • the biomolecules to be tested can have moieties for modification. Molecules include, but are not limited to, cholesterol, polyethylene glycols of different degrees of polymerization, biotin, or fluorophore molecules.
  • a nanopore sequencing device is provided, and the nanopore sequencing device includes the above-mentioned nanopore biofilm.
  • the nanopore sequencing device can be used to perform single-molecule sequencing of the biomolecules to be tested.
  • the nanopore sequencing device includes: an electrolytic cell containing an electrolyte; a nanopore biofilm located in the center of the electrolytic cell and dividing the electrolytic cell and the electrolyte into a positive electrolyte area and a negative electrolyte area; the first electrode and the second electrode, the first electrode and the second electrode are respectively arranged in the positive electrode electrolyte area and the negative electrode electrolyte area; the receiving electrode includes two and are respectively located in the positive electrode liquid area and the negative electrode electrolyte area, the The receiving electrode is connected to the signal processing chip; preferably, the electrolyte is a nanopore experimental buffer; preferably, the nanoporin experimental buffer is a HEPES buffer; preferably, the nanoporin experimental buffer contains 0.1-1.0M KCl; More preferably, nanopore experiment buffer comprises 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8; Preferably, first electrode and second electrode comprise metal or composite electrode material; Preferably,
  • the nanopore sequencing device includes an electrolytic cell containing electrolyte, a nanopore biomembrane, a first electrode and a second electrode. Put the nanoporous biofilm into the center of the electrolytic cell containing the electrolyte, and decompose the electrolytic cell to form the positive electrolyte area and the negative electrolyte area. The two areas are respectively equipped with two electrodes, and the two electrodes are used to form and apply to the nanoporous biofilm. The electric field on the membrane.
  • the biomolecules to be tested pass through the nanopore proteins on the membrane, generating a current amplitude. The current amplitude can be received by the receiving electrode, and the current amplitude is transmitted to a signal processing chip connected to the receiving electrode. According to the difference in current amplitude, the signal processing chip, that is, the nanopore sequencing device including the signal processing chip, can perform data analysis and determination on the sequence of the biomolecules to be tested.
  • a sequencing method uses the above-mentioned nanopore protein, or nanopore biomembrane, or nanopore sequencing device to analyze the biomolecules passing through the nanopore protein.
  • the electrical signal generated during the pore channel is used to sequence the biomolecules to be tested.
  • the biomolecules to be tested include modified or unmodified DNA, RNA, polypeptide or protein; preferably, under the action of an electric field force, a single molecule of the biomolecules to be tested passes through the pores of the nanoporin , generating an electrical signal; preferably, the electrical signal includes a blocking current amplitude.
  • a single nanoporin is inserted into a lipid layer, preferably a phospholipid bilayer, to form a nanoporous biomembrane, and the nanoporin is sequenced using the structure of the nanoporous biomembrane.
  • the accuracy will not be affected by the accumulation of errors, so extremely long read lengths can be achieved.
  • it can make up for the unavoidable gap (Gap) problem in the assembly of short sequencing fragments in traditional sequencing, determine whether there are deletions, duplications, inversions, and translocations of long fragments in the chromosome, and cover the entire length of the transcriptome with a typical length of several kb, so as to provide Scientific research such as genome assembly, structural variation, and alternative splicing provides new solutions.
  • Gap unavoidable gap
  • nanopore sequencing does not require PCR amplification, the original base modification information on the nucleic acid molecule to be tested can be retained, and then the type, site and abundance of the modified base can be directly obtained through one-time sequencing. Therefore, the nanoporin of the present application can also detect several nucleic acid molecules with DNA/RNA modified bases: including 5-methylcytosine (5mC), 6-methyladenine (m6A), 7- Methylguanine (m7G), pseudouracil (pseudouridine, ⁇ ), etc.
  • 5mC 5-methylcytosine
  • m6A 6-methyladenine
  • m7G 7- Methylguanine
  • pseudouracil pseudouracil
  • nanopore sequencing has the characteristics of long read length, high portability, fast sequencing speed and real-time readout, so it is suitable for major epidemic monitoring and rapid detection of pathogens (for example, Zika virus ) virus, Ebola virus (Ebola virus), Dengue virus (Dengue virus) and novel coronavirus (Coronavirus) and other large-scale epidemic operations), very time-sensitive.
  • pathogens for example, Zika virus
  • Ebola virus Ebola virus
  • Dengue virus Dengue virus
  • coronavirus novel coronavirus
  • nanopore sequencing can also be used for the rapid detection of other pathogens such as bacteria and fungi.
  • the nanopore sequencing platform Based on the common composition of proteins and nucleic acid molecules, the nanopore sequencing platform also has great application potential in the field of protein sequencing. For example, according to the explorations that have been carried out so far, it can be known that by using protein unfolding enzyme as a rate-controlling tool, the characteristic signal of protein was successfully observed, and the preliminary identification of protein type and modification state was realized, which verified the possibility of nanopore protein sequencing. In future development, by further optimizing the rate control system and developing adapted nanopore proteins and signal analysis algorithms, the fingerprinting and even sequence identification of proteins at the single-molecule level can be finally realized.
  • the nanopore platform can also be used as a basic detection platform, combined with sensing means, to complete the metabolomic detection of various small molecules and macromolecules. Combining genomics, proteomics, and metabolomics, the nanopore platform can eventually develop into a general-purpose measurement platform that meets the needs of full-omics analysis, providing a powerful research tool for a deeper understanding of the law of life and the mechanism of disease occurrence .
  • the gene sequence (SEQ ID NO: 3) encoded by nanoporin MP964 was inserted into the cloning region of the vector pET24a after digestion with NdeI and XhoI.
  • the six His at the N-terminal of the amino acid sequence of MP964 were used as purification tags, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964.
  • the Agilent site-directed mutagenesis kit was used, and the expression vector of nanoporin MP964 was used as a template to construct the expression gene of the corresponding mutant protein MP964Mut.
  • the N-terminal 6 His of the amino acid sequence of the nanoporin MP964 mutant was used as a purification tag, the screening tag in the expression vector was kanamycin, and the constructed vector was named pET24a-MP964Mut.
  • Embodiment 3 expresses the cultivation and induction of MP964 bacterial strain
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET24a-MP964 was transformed into Escherichia coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 ⁇ g/mL kanamycin, and cultured overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterial solution obtained above into 50 mL LB (containing 50 ⁇ g/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours.
  • OD600 value reaches about 0.6-0.8
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • Embodiment 4 expresses the cultivation and induction of MP964Mut bacterial strain
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L.
  • the recombinant expression vector pET24a-MP964Mut was transformed into E. coli expression strain E.coli BL21(DE3), and the bacterial solution was evenly spread on a plate with 50 ⁇ g/mL kanamycin, and cultivated overnight at 37°C. Pick a single colony and culture it in 5 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at 37°C, 200 rpm, and culture overnight. Inoculate the bacterium solution obtained above into 50 mL LB liquid medium (containing 50 ⁇ g/mL kanamycin) at a ratio of 1:100 and culture at 37°C, 200 rpm, for 4 hours.
  • Buffer A equilibration buffer 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol, pH 7.9.
  • Buffer B elution buffer 20mM Tris-HCl+250mM NaCl+0.5% Tween-20+5% glycerol+500mM imidazole, pH 7.9.
  • Buffer C equilibration buffer 20mM Tris-HCl+50mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer D elution buffer 20mM Tris-HCl+1000mM NaCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • Buffer E dilution 20mM Tris-HCl+0.5% Tween-20+5% glycerol, pH 6.5.
  • the single-channel current detection in this experiment is based on Axon Digidata 1550B low-noise data acquisition system and Axopatch 200B patch clamp amplifier.
  • a phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) was formed in the center of the electrolytic cell containing 150 ⁇ m small pores of Teflon material; the Ag and AgCl electrodes were respectively Placed in the positive electrolyte area and the negative electrolyte area of the electrolytic cell separated by the phospholipid bilayer membrane, and the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8).
  • nanopore experimental buffer 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • DNA passes through the nanoporin under the action of an electric field force, resulting in a blocking current amplitude.
  • FIG. 5 for the percentage distribution of the current blockade of the nanoporin MP964 to the oligonucleotide sample
  • FIG. 6 for the percentage distribution of the current blockade of the nanopore protein MP964Mut to the oligonucleotide sample.
  • Figure 7 for the statistical comparison of the DNA capture rates of nanoporins MP964 and MP964Mut. It shows that compared with MP964, the mutated nanopore protein MP964Mut has better DNA capture ability.
  • G-quadruplex is a high-level structure folded by DNA containing tandem repeated guanine. It exists widely in prokaryotic and eukaryotic cells and participates in multiple functions such as gene replication, recombination, and regulation. Plays an important role in the life activities of cells. Therefore, the basic research on the structure and biological function of G-quadruplex is of great significance.
  • the single channel current detection of this embodiment is based on Axon Digidata 1550B low noise data acquisition system and Axopatch200B patch clamp amplifier.
  • a phospholipid bilayer composed of diphytylphosphatidylcholine (DPhPC, 1,2-diphytanoyl-sn-glycero-3-phosphocholine) is formed in the center of the electrolytic cell containing 150 ⁇ m small pores of Teflon material; Ag/AgCl electrodes are placed In the positive and negative electrolyte areas of the electrolyzer separated by a phospholipid bilayer membrane, the two areas are filled with nanopore experimental buffer (0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8).
  • nanopore experimental buffer 0.5M KCl, 10mM HEPES, 1mM EDTA, pH 7.8.
  • Figure 8 shows the conductance distribution of the nanopore protein MP964Mut in different batches of experiments, which is used to investigate the uniformity of the nanopore.
  • the distribution of conductivity in Figure 8 has an obvious main peak, which reflects the uniformity of the opening current in the single-molecule experiment. The more concentrated the distribution of the opening current, the more uniform the formed pores, and the more stable the pores. good.
  • Oligonucleotide sample A (SEQ ID NO: 5: ggttggtgtggttgg) or sample B (SEQ ID NO: 6: ggttggtgtggttggttttttttttttt) was dissolved in nanopore buffer and annealed, cooled to 4°C before use. After a single nanoporin is inserted into the phospholipid bilayer, add an appropriate amount of oligonucleotide sample A or oligonucleotide sample B to the positive electrolyte area of the electrolytic cell. DNA passes through the nanopore under the action of an electric field force, generating a blocking current amplitude.
  • Quadruplex DNA polymers with different structures produce different amplitudes of blocking currents, so the current amplitudes can be used to distinguish G-quadruplexes composed of different sequences.
  • the percent current block is the ratio of the blocked current (Ib) to the open pore current (Io) associated with the porosity event. The results are shown in Figure 9, Figure 10, Figure 11 and Figure 12.
  • Example 10 The nanopore biofilm was constructed using nanopore protein MP964Mut and used for DNA sequencing.
  • Plasmid pUC57 was digested with EcoRI and HindIII restriction enzymes at 37°C for 2 hours; then purified using 0.4-0.6X AMPure XP magnetic beads (Beckman) to obtain double-stranded DNA fragments with high purity.
  • a sequencing library was constructed for the target DNA fragments using the ligation sequencing 109 kit (SQK-LSK109, Oxford Nanopore Technologies Ltd). Sequencing buffer: 0.5M KCl, 10 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 2 mM ATP, pH 7.8.
  • the sequencing library containing the pUC57 sequence and the single-stranded DNA with cholesterol (FLT reagent) were mixed with the sequencing buffer and added to the nanopore sequencing device; after applying an applied voltage of 150mV or 180mV, it was observed that the DNA was captured by the nanopore, resulting in characteristic The amplitude value of the blocking current. And as the helicase moves the DNA through the nanopore, the amplitude of the current changes stepwise. Different DNA sequences produce different blocking current amplitude values.
  • the single-stranded DNA with cholesterol can be combined with the phospholipid bilayer, which helps the nanopore capture the sequencing library and reduces the loading amount of the sequencing library.
  • FIG. 13 shows that the helicase controls DNA passing through the nanoporin mutant MP964Mut under an applied voltage of 180mV, and current amplitudes of different magnitudes are generated as the DNA moves.
  • FIG. 13(A) is a current characteristic diagram
  • FIG. 13(B) is an enlarged current diagram.
  • FIG. 14 shows that the helicase controls DNA to pass through the nanoporin mutant MP964Mut under an applied voltage of 150 mV, and the current amplitude changes with different amplitudes are produced as the DNA moves.
  • FIG. 14(A) is a current characteristic diagram
  • FIG. 14(B) is an enlarged current diagram.
  • nanopore protein MP964Mut can construct a successful nanopore biofilm, and can realize the sequencing of DNA fragments.

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Abstract

La présente invention concerne une protéine à nanopore et son utilisation associée en séquençage. La protéine à nanopore comprend : (a) une protéine MP964 ; ou (b) une protéine MP964 MUT ; ou (c) une protéine soumise à la substitution et/ou à la délétion et/ou à l'addition d'un ou de plusieurs acides aminés à au moins l'une des positions suivantes de la séquence d'acides aminés de la protéine dans (a) ou (b) et ayant une structure de canal poreux : position 97, position 98, position 127, position 143 et position 148 ; ou (d) une protéine ayant 80 % ou plus d'homologie avec la séquence d'acides aminés de la protéine définie dans l'un quelconque de (a), (b) et (c) et ayant la même fonction que la protéine.
PCT/CN2021/143722 2021-12-31 2021-12-31 Protéine à nanopore et son utilisation associée en séquençage WO2023123370A1 (fr)

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