WO2023225988A1 - 一种维持纳米孔测序速度的方法 - Google Patents

一种维持纳米孔测序速度的方法 Download PDF

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WO2023225988A1
WO2023225988A1 PCT/CN2022/095500 CN2022095500W WO2023225988A1 WO 2023225988 A1 WO2023225988 A1 WO 2023225988A1 CN 2022095500 W CN2022095500 W CN 2022095500W WO 2023225988 A1 WO2023225988 A1 WO 2023225988A1
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atp
double
helicase
pore
sequencing
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PCT/CN2022/095500
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French (fr)
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季州翔
鲁敬雄
郑荣荣
章文蔚
董宇亮
黎宇翔
曾涛
徐讯
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深圳华大生命科学研究院
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    • 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

Definitions

  • the present invention relates to a method for maintaining nanopore sequencing speed. Further, a method for sequencing a double-stranded target polynucleotide and a kit for sequencing a double-stranded target polynucleotide are provided.
  • nanopore sequencing technology which can realize nucleic acid sequence reading at the single molecule level, has developed rapidly. This technology is superior to next generation sequencing technology in terms of portability, sequencing read length, and sequencing speed. Has obvious advantages.
  • scientists used alpha-hemolysin protein to realize the recognition of different bases for the first time Kernowicz, John J., et al. Proceedings of the National Academy of Sciences 93.24 (1996): 13770-13773.), creating a nanopore-based The first detection principle for DNA sequencing.
  • helicase uses the energy generated by the hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to move along the nucleic acid backbone in a specific direction (5'-3' or 3'-5') to remove hydrogen-bonded nucleic acids. Double-stranded DNA unwinds into single-stranded DNA.
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • the inventor of this application discovered that there is a problem in the above sequencing process. That is, the nanopore sequencing speed decreased as sequencing progressed. The reduction in sequencing speed will affect sequencing throughput and sequencing accuracy. Therefore, if you want to maintain high throughput and high accuracy during the sequencing process, you need to provide a method that can maintain the speed of nanopore sequencing.
  • the inventor of the present application found through a large number of experiments that the nanopore sequencing speed is related to the composition of the conductivity buffer on one or both sides of the membrane. Furthermore, the inventor of the present application realized for the first time through extensive experiments and repeated exploration that during the above-mentioned sequencing process, controlling the amounts of ADP and ATP at the same time is the key to affecting the sequencing rate, and thus completed the application of the present invention.
  • the present application provides a method for sequencing a double-stranded target polynucleotide, including:
  • nucleotides e.g., A, T, C, G
  • the transmembrane pore is able to distinguish nucleotides with similar structures .
  • individual nucleotides can be identified at the single molecule level based on their current amplitude or the duration of the interaction when they interact with a transmembrane pore. If a characteristic current associated with the nucleotide is detected flowing through the pore, the type of nucleotide passing through the pore (e.g., A, T, C, G) can be determined, that is, sequencing can be achieved. By sequentially identifying nucleotides in a target polynucleotide, the sequence of the target polynucleotide can be estimated or determined.
  • the method further includes measuring the current through the pore during interaction with the nucleotide.
  • the methods also provide circuitry capable of applying an electrical potential and measuring electrical signals across the membrane and pores.
  • the methods also provide patch clamping or voltage clamping.
  • the helicase separates the double strands of the double-stranded target polynucleotide to form a single-stranded target polynucleotide.
  • the helicase uses energy generated from the hydrolysis of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to move in a specific direction (5' to 3' or 3' to 5') along the nucleic acid backbone, Unwinds hydrogen-bonded double-stranded DNA into single-stranded DNA.
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • the helicase controls movement of the single-stranded polynucleotide through the pore along a field generated by an applied voltage. In certain embodiments, the helicase acts as a brake, preventing the single-stranded polynucleotide from moving too quickly through the pore under the influence of an applied voltage.
  • the method further includes: (d) reducing the voltage applied across the pore such that the single-stranded polynucleotide moves through the pore in the opposite direction as in step (b), and A portion of the nucleotides in the polynucleotide interacts with the pore, and the current passing through the pore during each interaction is measured to perform a corrected reading of the sequence of the target polynucleotide obtained in step (c). .
  • the single-stranded polynucleotide can interact with the pore on either side of the membrane.
  • the single-stranded polynucleotide can interact with the pore in any way and at any site.
  • the ATP-generating enzyme is selected from: pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nuclear Glycoside diphosphate kinase, or any combination thereof.
  • the ATP-generating enzyme is pyruvate kinase and the ATP-generating substrate is phosphoenolpyruvate.
  • the phosphoenolpyruvate is 5 mM and the pyruvate kinase is 0.02 U/mL.
  • phosphoenolpyruvate is 5 mM and pyruvate kinase is 0.2 U/mL.
  • the phosphoenolpyruvate is 5 mM and the pyruvate kinase is 2 U/mL.
  • the ATP-generating enzyme is acetate kinase and the ATP-generating substrate is lithium potassium acetylphosphate.
  • the lithium potassium acetyl phosphate is 5 mM and the acetate kinase is 0.02 U/mL.
  • the ATP-generating enzyme is creatine phosphokinase and the ATP-generating substrate is creatine phosphate disodium.
  • the ATP-generating enzyme is FoF1-ATPase and the ATP-generating substrate is inorganic phosphate.
  • the ATP-generating enzyme is creatine phosphokinase and the ATP-generating substrate is creatine phosphate.
  • the ATP-generating enzyme is polyphosphate kinase and the ATP-generating substrate is polyphosphate.
  • the ATP-generating enzyme is a nucleoside diphosphate kinase and the ATP-generating substrate is a nucleoside triphosphate.
  • the amounts of ATP-generating enzyme and ATP-generating substrate used are not limited to the specific concentrations and amounts used in the examples. As long as the concentration of ATP can be kept relatively constant during the sequencing process, the sequencing rate can be better kept stable. In fact, during the sequencing process, under the action of ATP-generating enzymes, the substrate will react and the amount will gradually decrease. Those skilled in the art have the ability to appropriately adjust the concentrations and amounts of ATP-generating enzymes and ATP-generating substrates in the sequencing system according to experimental purposes to obtain appropriate concentrations and amounts of ATP-generating enzymes and ATP-generating substrates.
  • the helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof.
  • the helicase is further linked to an additional polypeptide selected from a tag, a cleavage site, a signal or guide peptide, a detectable label, or any combination thereof.
  • the helicase is wild-type Dda or a mutant thereof.
  • the helicase has the amino acid sequence set forth in SEQ ID NO:2.
  • the transmembrane pore is a transmembrane protein pore or a solid state pore.
  • the transmembrane protein pore is selected from the group consisting of hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29connector, T7connector, GspD, InvG, or thereof random combination.
  • the transmembrane pore is also connected to an additional polypeptide selected from a tag, an enzyme cleavage site, a signal or guide peptide, a detectable label, or any combination thereof.
  • the transmembrane pore is a wild-type CsgG protein or a mutant thereof. In certain embodiments, the transmembrane pore has the amino acid sequence set forth in SEQ ID NO: 1.
  • the membrane is an amphiphilic layer (eg, phospholipid bilayer) or a high molecular polymer membrane (eg, di-block, tri-block).
  • the membrane is a phospholipid bilayer, wherein the transmembrane pore is inserted into the phospholipid bilayer.
  • the methods are generally performed using a membrane: (i) an artificial bilayer containing pores, (ii) an isolated naturally occurring pore-containing lipid bilayer, or (iii) cells with pores inserted therein .
  • the method is preferably performed using an artificial bilayer (eg, an artificial phospholipid bilayer).
  • the double-stranded target polynucleotide is a DNA duplex and/or a DNA-RNA hybrid duplex.
  • the double-stranded target polynucleotide is naturally occurring and/or synthetic.
  • the double-stranded target polynucleotide is obtained from a biological sample extracted from a virus, a prokaryote (e.g., a bacterium), a eukaryote (e.g., a plant (e.g., a cereal, a legume) , fruit or vegetable), mammal (e.g., horse, cow, rat or human), or any combination thereof.
  • a prokaryote e.g., a bacterium
  • a eukaryote e.g., a plant (e.g., a cereal, a legume) , fruit or vegetable
  • mammal e.g., horse, cow, rat or human
  • the two strands of the double-stranded target polynucleotide are connected at or near one end of the target polynucleotide by a bridging moiety; the bridging moiety is selected from a polymer linker, Chemical linker, polynucleotide or polypeptide.
  • the double-stranded target polynucleotide is circular or linear.
  • the double-stranded target polynucleotide can be sequenced using methods as described above.
  • the double-stranded target polynucleotide can be of any length.
  • the double-stranded target 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 nucleotide pairs in length.
  • the double-stranded target polynucleotide can be 1,000 or more nucleotide pairs, 5,000 or more nucleotide pairs, or 100,000 or more nucleotide pairs in length.
  • the double-stranded target polynucleotide may be naturally occurring or synthetic.
  • the method can be used to verify the sequence of artificially synthesized oligonucleotides, and the method is usually performed in vitro.
  • the double-stranded target polynucleotide comprises at least one single-stranded overhang (e.g., a 5' overhang and/or a 3' overhang) that contains a leader sequence,
  • the leader sequence leads the nucleic acid strand to which it is linked into the pore.
  • step (b) the double-stranded target polynucleotide is contacted with a helicase to form a complex, and then the complex is contacted with a transmembrane pore.
  • methods as described above are generally performed in the presence of nanopore sequencing reagents.
  • step (b) a reagent for nanopore sequencing is contacted with the helicase.
  • the reagents for nanopore sequencing are selected from the group consisting of ATP, inorganic salts (e.g., chloride salts, e.g., sodium chloride, potassium chloride, lithium chloride), buffers (HEPES and/or or Tris-HCl), EDTA, metal ions (e.g., Mn 2+ , Mg 2+ , Co + , Zn 2+ , Cu 2+ , Cu + , Ni + , Fe 2+ , Fe 3+ ), or any of them combination.
  • inorganic salts e.g., chloride salts, e.g., sodium chloride, potassium chloride, lithium chloride
  • buffers HEPES and/or or Tris-HCl
  • EDTA e.g., Mn 2+ , Mg 2+ , Co + , Zn 2+ , Cu 2+ , Cu + , Ni + , Fe 2+ , Fe 3+
  • metal ions e.g., Mn 2+
  • the chloride salt concentration is saturated.
  • the chloride salt concentration is 0.1-2.5M, 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, or 1M-1.4M.
  • the methods described above are performed at a pH of 4.0-12.0, 4.5-10.0, 5.0-9.0, 5.5-8.8, 6.0-8.7, or 7.0-8.8 or 7.5-8.5. In certain embodiments, the methods described above are performed at a pH of 7.5.
  • the present application provides a kit, which includes: a membrane containing a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; optionally, the kit also includes a nanoparticle for nanoparticles.
  • a kit which includes: a membrane containing a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; optionally, the kit also includes a nanoparticle for nanoparticles.
  • Well sequencing reagents are well sequencing reagents.
  • the reagents for nanopore sequencing are selected from the group consisting of ATP, inorganic salts (e.g., chloride salts, e.g., sodium chloride, potassium chloride, lithium chloride), buffers (HEPES and/or or Tris-HCl), EDTA, metal ions (e.g., Mn 2+ , Mg 2+ , Co + , Zn 2+ , Cu 2+ , Cu + , Ni + , Fe 2+ , Fe 3+ ), or any of them combination.
  • inorganic salts e.g., chloride salts, e.g., sodium chloride, potassium chloride, lithium chloride
  • buffers HEPES and/or or Tris-HCl
  • EDTA e.g., Mn 2+ , Mg 2+ , Co + , Zn 2+ , Cu 2+ , Cu + , Ni + , Fe 2+ , Fe 3+
  • metal ions e.g., Mn 2+
  • the ATP-generating enzyme is selected from: pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nuclear Glycoside diphosphate kinase, or any combination thereof.
  • the kit comprises: a membrane comprising a transmembrane pore, a helicase, ATP, (I) pyruvate kinase and phosphoenolpyruvate, (II) acetate kinase and lithium potassium acetylphosphate, (III) Creatine phosphokinase and creatine phosphate, (IV) FoF1-ATPase and inorganic phosphate; (V) Creatine phosphokinase and creatine phosphate; (VI) Polyphosphokinase and polyphosphate; (VII) Nuclear Glycoside diphosphate kinase and nucleoside triphosphate, or any one or more combinations of (I) to (VII).
  • the kit is used to sequence a double-stranded target polynucleotide.
  • the helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof.
  • the helicase is further linked to an additional polypeptide selected from a tag, a cleavage site, a signal or guide peptide, a detectable label, or any combination thereof.
  • the helicase is wild-type Dda or a mutant thereof.
  • the helicase has the amino acid sequence set forth in SEQ ID NO:2.
  • the transmembrane pore is a transmembrane protein pore or a solid state pore.
  • the transmembrane protein pore is selected from the group consisting of hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29connector, T7connector, GspD, InvG, or any thereof combination.
  • the transmembrane pore is also connected to an additional polypeptide selected from a tag, an enzyme cleavage site, a signal or guide peptide, a detectable label, or any combination thereof.
  • the transmembrane pore is a wild-type CsgG protein or a mutant thereof. In certain embodiments, the transmembrane pore has the amino acid sequence set forth in SEQ ID NO: 1.
  • the membrane is an amphiphilic layer (eg, phospholipid bilayer) or a high molecular polymer membrane (eg, di-block, tri-block).
  • the kit as described above is used for preparing a sequencing device for sequencing double-stranded target polynucleotides.
  • polynucleotide is a large molecule containing one, two, or more than two nucleotides.
  • a polynucleotide may comprise any combination of any nucleotides, which may be naturally occurring or artificial. Nucleotides usually contain nucleobases, sugars, and at least one phosphate group.
  • transmembrane pore is a structure that allows the flow of hydrated ions driven by an applied electric potential from one side of the membrane to the other side of the membrane, which transmembrane pores are typically inserted into the membrane (e.g., lipid bis layer).
  • the transmembrane pore is preferably a transmembrane protein pore, which is a polypeptide or collection of polypeptides that allows the flow of hydrated ions from one side of the membrane to the other side of the membrane, said transmembrane protein pore allowing multiple nuclei The nucleotide moves through the pore.
  • Transmembrane protein pores typically contain barrels or channels through which ions can flow.
  • Transmembrane protein pores typically contain amino acids that facilitate interaction with target nucleotides, and these amino acids are preferably located near the constriction of the barrel or channel.
  • the term "moving" is the movement of a single-stranded polynucleotide from one side of the pore to the other. Movement of the single-stranded polynucleotide through the pore can be subject to electrical potential, enzymatic action, as well as electrical potential and enzymatic action. The movement may be one-way or two-way.
  • ATP-generating enzyme refers to an enzyme that can catalyze a substrate in a system to generate ATP.
  • ATP-generating substrate refers to a substance that can be converted or generated into ATP under the catalysis of an ATP-generating enzyme.
  • the ATP-generating substrate is capable of reacting with ADP to generate ATP catalyzed by the ATP-generating enzyme.
  • This application provides a method that can always maintain a stable sequencing speed during the sequencing process, further maintaining high throughput and high accuracy of sequencing.
  • the method and kit of the present invention can maintain a relatively constant concentration of ATP during the sequencing process, thereby better maintaining the stability of the sequencing rate. .
  • Figure 1 shows a schematic diagram of the principle of the method of the present application.
  • Figure 2 shows the effect of adding ADP on sequencing rate.
  • Figure 2A shows the results of the control group (no ADP added).
  • Figure 2B shows the results of experimental group 1 (added 15mM ADP).
  • Figure 2C shows the results of experimental group 2 (added 15mM ADP). 30mM ADP).
  • Figure 3 shows the impact of the control group and experimental group 1 (containing PEP and PK) on the sequencing rate in Example 2.
  • Figure 3A is the attenuation result of the sequencing rate
  • Figure 3B is the change of the sequencing rate percentage over time (initial rate is 100%).
  • Figure 4 shows the impact of Experimental Group 2 (containing PEP and PK) on the sequencing rate in Example 2.
  • Figure 4A is the attenuation result of the sequencing rate
  • Figure 4B is the change of the sequencing rate percentage over time (the initial rate is 100% ).
  • Figure 5 shows the impact of experimental group 3 (containing PEP and PK) on the sequencing rate in Example 2.
  • Figure 5A is the attenuation result of the sequencing rate
  • Figure 5B is the change of the sequencing rate percentage over time (the initial rate is 100% ).
  • Figure 6 shows the impact of the control group and the experimental group (containing Ac and AcK) on the sequencing rate in Example 3.
  • Figure 6A is the attenuation result of the sequencing rate
  • Figure 6B is the change of the sequencing rate percentage over time (the initial rate is 100 %).
  • Figure 7 shows the impact of the control group and the experimental group (containing Cre and CK) on the sequencing rate in Example 4.
  • Figure 7A is the attenuation result of the sequencing rate
  • Figure 7B is the change in the sequencing rate percentage over time (the initial rate is 100 %).
  • the molecular biology experimental methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al. , Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995.
  • Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.
  • the inventor of the present application conducted various explorations into the sequencing system, and finally unexpectedly discovered that adding ADP to the sequencing buffer of the system would reduce the sequencing rate.
  • the CsgG transmembrane protein mutant (the wild-type amino acid sequence is shown in SEQ ID NO.1, the mutation site is: Y51A/F56Q/R97W/R192D) was used as a nanopore, and the nanopore detection method was used for detection.
  • fuel (ATP)-free buffer (470mM KCl, 25mM HEPES, 1mM EDTA, pH 8.0) is used to flow through the system to remove excess transmembrane protein.
  • the constructed pUC57 sequencing library (SEQ ID NO.3) containing T4Dda helicase (the wild-type encoding amino acid sequence is shown in SEQ ID NO.2, the mutation site is E94C/C109A/C136A/A360C) and the sequencing buffer
  • the liquid was premixed and added to the nanopore experimental system, and the CsgG transmembrane protein sequencing was detected at a voltage of 0.18V. in,
  • the sequencing buffer in the control group ( Figure 2A) is 470mM KCl, 25mM HEPES, 30mM ATP, 25mM MgCl 2 , 1mM EDTA, pH 8.0;
  • the sequencing buffer of experimental group 1 ( Figure 2B) is 470mM KCl, 25mM HEPES, 30mM ATP, 15mM ADP, 25mM MgCl 2 , 1mM EDTA, pH 8.0;
  • the sequencing buffer of experimental group 2 was 470mM KCl, 25mM HEPES, 30mM ATP, 30mM ADP, 25mM MgCl 2 , 1mM EDTA, pH 8.0;
  • the applicant further explored the sequencing system, that is, adding components to the sequencing buffer of the system to reduce the amount of ADP generated by the system during the sequencing process, and at the same time increase the ATP in the system during the sequencing process. amount.
  • the CsgG transmembrane protein mutant (the wild-type amino acid sequence is shown in SEQ ID NO.1, the mutation site is: Y51A/F56Q/R97W/R192D) was used as a nanopore, and the nanopore detection method was used for detection.
  • fuel (ATP)-free buffer (470mM KCl, 25mM HEPES, 1mM EDTA, pH 8.0) is used to flow through the system to remove excess transmembrane protein.
  • the constructed pUC57 sequencing library (SEQ ID NO.3) containing T4Dda helicase (the wild-type encoding amino acid sequence is shown in SEQ ID NO.2, the mutation site is E94C/C109A/C136A/A360C) and the sequencing buffer
  • the liquid was premixed and added to the nanopore experimental system, and the CsgG transmembrane protein sequencing was detected at a voltage of 0.18V. in,
  • the sequencing buffer in the control group is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0;
  • the experimental group sets 3 concentrations:
  • the sequencing buffer of experimental group 1 is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0, 5mM PEP (phosphoenolpyruvate, Roche, product number: 10108294001), 0.02U/mL PK (pyruvate kinase) , Roche, item number: 10109045001).
  • the sequencing buffer of experimental group 2 is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0, 5mM PEP (phosphoenolpyruvate), 0.2U/mL PK (pyruvate kinase).
  • the sequencing buffer of experimental group 3 is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0, 5mM PEP (phosphoenolpyruvate), 2U/mL PK (pyruvate kinase).
  • the sequencing buffer formula of the control group is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0;
  • the sequencing buffer formula of the experimental group is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0, 5mM Ac (lithium acetyl potassium phosphate, sigma, 01409-500MG), 0.02U/mL AcK (acetate kinase, sigma) , A7437-250UN).
  • the sequencing buffer formula of the control group is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0;
  • the experimental group sequencing buffer formula is 470mM KCl, 25mM HEPES, 5mM ATP, 25mM MgCl2, 1mM EDTA, pH 8.0, 5mM Cre (creatine phosphate disodium, sigma, P7936-1G)/0.02U/mL CK (creatine phosphate Kinase, sigma, C3755-3.5KU).

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Abstract

本发明提供一种对双链靶多核苷酸进行测序的方法，所述方法可以使测序过程中ATP保持相对恒定的浓度，从而能够更好的保持测序速率稳定不变。本发明还提供一种用于对双链靶多核苷酸进行测序的试剂盒，包含跨膜孔的膜、解旋酶、ATP生成酶以及ATP生成底物。

Description

一种维持纳米孔测序速度的方法 发明领域
本发明涉及一种维持纳米孔测序速度的方法。进一步的，提供一种对双链靶多核苷酸进行测序的方法，以及一种用于对双链靶多核苷酸进行测序的试剂盒。
发明背景
随着纳米技术的不断发展，可以在单分子水平实现核酸序列读取的纳米孔测序技术迅猛发展，该技术较下一代测序技术(Next generation sequencing)在便携性、测序读长、测序速度等方面具有明显优势。1996年，科学家首次利用alpha-hemolysin蛋白实现了不同碱基的识别(Kasianowicz,John J.,et al.Proceedings of the National Academy of Sciences 93.24(1996):13770-13773.)，开创了基于纳米孔检测原理进行DNA测序的先河。
英国牛津纳米孔公司(Oxford Nanopore Technologies)推出了多款不同通量的测序仪；该原理为：直径为纳米尺度的跨膜蛋白插入到高分子聚合物膜中，在膜上形成纳米孔通道。在膜两侧放置两个电极，膜的两侧为电导缓冲液。通电后，离子通过跨膜蛋白产生电流。测序文库双链DNA在解旋酶作用下解旋为单链DNA，单链DNA在电场力作用下穿过跨膜蛋白，产生特征电流信号；通过算法对电流特征信号进行分析，实现序列读取。其中，解旋酶利用三磷酸腺苷(ATP)或三磷酸鸟苷(GTP)水解产生的能量，沿着核酸骨架往特定方向(5‘-3’或3‘-5’)移动，将氢键结合的双链DNA解旋为单链DNA。
发明内容
本申请的发明人发现：在上述测序过程中存在着一个问题。即，随着测序的进行，纳米孔的测序速度有所下降。而测序速度的降低，则会影响测序通量和测序的准确率。因此，如果想要在测序过程中始终保持高通量以及高准确率，就需要提供一种能够维持纳米孔测序速度的方法。
为了解决上述问题，本申请的发明人通过大量实验发现了纳米孔测序速度与膜一侧或两侧的电导缓冲液组成成分有关。进一步的，本申请的发明人通过大量实验以及反复摸索首次意识到，在上述测序过程中，同时控制ADP和ATP的量是影响测序速率的关键，并由此完成了本发明申请。
因此，本申请提供了一种对双链靶多核苷酸进行测序的方法，包括：
(a)提供双链靶多核苷酸、包含跨膜孔的膜、解旋酶、ATP生成酶以及ATP生成底物；其中，所述ATP生成底物能够在所述ATP生成酶的催化下与ADP反应以生成ATP；
(b)使所述双链靶多核苷酸与跨膜孔、解旋酶、ATP生成酶和ATP生成底物接触，所述解旋酶使所述双链靶多核苷酸的双链分开，形成单链靶多核苷酸；并且，所述解旋酶使所述单链多核苷酸在所述跨膜孔中移动，使得所述单链多核苷酸中的一部分核苷酸与所述跨膜孔相互作用；
(c)测量每次相互作用期间通过所述跨膜孔的电流，从而确定所述双链靶多核苷酸的序列。
在本文的方法中，由于不同的核苷酸(例如，A，T，C，G)对通过跨膜孔的电流具有不同的效应，且所述跨膜孔能够区分具有相似结构的核苷酸。因此，在某些实施方案中，根据各个核苷酸与跨膜孔相互作用时它们的电流振幅或相互作用的持续时间，可以在单分子水平上鉴定各个核苷酸。如果检测到与该核苷酸相关的特征性电流 流过所述孔，那么可以判断通过所述孔的核苷酸的类型(例如，A，T，C，G)，即实现测序。通过连续鉴定靶多核苷酸中的核苷酸，使得能够估计或确定所述靶多核苷酸的序列。
在某些实施方案中，所述方法还包括测量与核苷酸相互作用期间通过所述孔的电流。因此，在某些实施方案中，所述方法还提供能够施加电势和测量跨越所述膜和孔的电信号的电路。在某些实施方案中，所述方法还提供膜片钳或电压钳。
在本文的方法中，所述解旋酶使所述双链靶多核苷酸的双链分开，形成单链靶多核苷酸。在某些实施方案中，解旋酶利用三磷酸腺苷(ATP)或三磷酸鸟苷(GTP)水解产生的能量，沿着核酸骨架往特定方向(5’至3’或3’至5’)移动，将氢键结合的双链DNA解旋为单链DNA。
在某些实施方案中，所述解旋酶控制所述单链多核苷酸顺着施加电压产生的场移动，以通过所述孔。在某些实施方案中，所述解旋酶充当制动器，防止所述单链多核苷酸在施加电压的影响下过快的移动通过所述孔。在某些实施方案中，所述方法还包括：(d)降低跨越所述孔施加的电压，使得所述单链多核苷酸以与步骤(b)中相反的方向移动通过所述孔，并且所述多核苷酸中的一部分核苷酸与所述孔相互作用，测量每次相互作用期间通过所述孔的电流，从而对步骤(c)中获得的靶多核苷酸的序列进行校正读取。
在某些实施方案中，所述单链多核苷酸可在所述膜的任一侧与所述孔相互作用。所述单链多核苷酸可以以任何方式在任何位点与所述孔相互作用。
在某些实施方案中，所述ATP生成酶选自：丙酮酸激酶、乙酸激酶、肌酸磷酸激酶、丝氨酸激酶、苏氨酸激酶、酪氨酸激酶，FoF1-ATP酶，聚磷酸激酶，核苷二磷酸激酶，或其任意组合。
在某些实施方案中，所述ATP生成酶为丙酮酸激酶，且所述ATP生成底物为磷酸烯醇丙酮酸。在某些实施方案中，磷酸烯醇丙酮酸为5mM，丙酮酸激酶为0.02U/mL。在某些实施方案中，磷酸烯醇丙酮酸为5mM，丙酮酸激酶为0.2U/mL。在某些实施方案中，磷酸烯醇丙酮酸为5mM，丙酮酸激酶为2U/mL。
在某些实施方案中，所述ATP生成酶为乙酸激酶，且所述ATP生成底物为乙酰磷酸钾锂。在某些实施方案中，乙酰磷酸钾锂为5mM，乙酸激酶为0.02U/mL。
在某些实施方案中，所述ATP生成酶为肌酸磷酸激酶，且所述ATP生成底物为磷酸肌酸二钠。
在某些实施方案中，所述ATP生成酶为FoF1-ATP酶，且所述ATP生成底物为无机磷酸盐。
在某些实施方案中，所述ATP生成酶为肌酸磷酸激酶，且所述ATP生成底物为磷酸肌酸。
在某些实施方案中，所述ATP生成酶为聚磷酸激酶，且所述ATP生成底物为聚磷酸。
在某些实施方案中，所述ATP生成酶为核苷二磷酸激酶，且所述ATP生成底物为核苷三磷酸。
在本文中，所使用的ATP生成酶与ATP生成底物的量并不局限于实施例所使用的具体浓度与量。只要能够使测序过程中ATP保持相对恒定的浓度，从而能够更好的保持测序速率稳定不变即可。事实上，在测序过程中，在ATP生成酶的作用下，底物会发生反应而量逐渐减少。本领域技术人员有能力根据实验目的，对测序体系中ATP生成酶与ATP生成底物的浓度和量进行适当的调整，以获得合适的ATP生成酶与ATP生成底物的浓度和量。
在某些实施方案中，所述解旋酶选自Dda、UvrD、Rep、RecQ、PcrA、eIF4A、 NS3、Rep、gp41、T7gp4，或其任意组合。
在某些实施方案中，所述解旋酶还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合。
在某些实施方案中，所述解旋酶是野生型Dda或其突变体。
在某些实施方案中，所述解旋酶具有如SEQ ID NO:2所示的氨基酸序列。
在某些实施方案中，所述跨膜孔是跨膜蛋白孔或固态孔。
在某些实施方案中，，所述跨膜蛋白孔选自hemolysin、MspA、MspB、MspC、MspD、Frac、ClyA、PA63、CsgG、CsgD、XcpQ、SP1、Phi29connector、T7connector、GspD、InvG，或其任意组合。
在某些实施方案中，所述跨膜孔还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合。
在某些实施方案中，所述跨膜孔是野生型CsgG蛋白或其突变体。在某些实施方案中，所述跨膜孔具有如SEQ ID NO:1所示的氨基酸序列。
在某些实施方案中，所述膜是两亲性层(例如，磷脂双分子层)或高分子聚合物膜(例如，di-block，tri-block)。
如上所述方法可使用任何合适的膜进行。在某些实施方案中，所述膜为磷脂双分子层，其中，所述跨膜孔被插入所述磷脂双分子层中。
在某些实施方案中，所述方法通常使用如下膜进行：(i)包含孔的人造双层，(ii)分离的天然存在的含孔脂双层，或(iii)有孔插入其中的细胞。所述方法优选地使用人造双层(例如，人造磷脂双分子层)进行。
在某些实施方案中，所述双链靶多核苷酸为DNA双链和/或DNA-RNA杂交的双链。
在某些实施方案中，所述双链靶多核苷酸是天然存在的和/或人工合成的。
在某些实施方案中，所述双链靶多核苷酸获自生物样品，所述生物样品提取自病毒，原核生物(例如，细菌)，真核生物(例如，植物(例如，谷类，豆类，水果或蔬菜)，哺乳动物(例如，马，牛，鼠或人)，或其任意组合。
在某些实施方案中，所述双链靶多核苷酸的两条链在所述靶多核苷酸的一个末端处或其附近通过桥连部分连接；所述桥连部分选自聚合物接头，化学接头，多核苷酸或多肽。
在某些实施方案中，所述双链靶多核苷酸是环状的或线性的。
在某些实施方案中，可使用如上所述的方法对所述双链靶多核苷酸的全部或仅一部分进行测序。所述双链靶多核苷酸可为任意长度。例如，所述双链靶多核苷酸可为至少10、至少50、至少100、至少150、至少200、至少250、至少300、至少400或至少500个核苷酸对的长度。所述双链靶多核苷酸可为1000或更多个核苷酸对，5000或更多个核苷酸对或者100000或更多个核苷酸对的长度。所述双链靶多核苷酸可为天然存在的或人工合成的。例如，所述方法可用于验证人工合成的寡核苷酸的序列，所述方法通常在体外进行。
在某些实施方案中，所述双链靶多核苷酸包含至少一个单链悬突(例如，5’端悬突和/或3’端悬突)，所述单链悬突含有前导序列，所述前导序列引领与其衔接的核酸链进入所述孔。
在某些实施方案中，在步骤(b)中，使所述双链靶多核苷酸与解旋酶接触，以形成复合物，然后，使所述复合物与跨膜孔接触。
在某些实施方案中，如上所述的方法通常是在纳米孔测序的试剂的存在下进行。
在某些实施方案中，在步骤(b)中，使用于纳米孔测序的试剂与所述解旋酶接触。
在某些实施方案中，所述用于纳米孔测序的试剂选自ATP、无机盐(例如，氯化物盐，例如，氯化钠、氯化钾、氯化锂)、缓冲液(HEPES和/或Tris-HCl)、EDTA、金属离子(例如，Mn 2+、Mg 2+、Co +、Zn 2+、Cu 2+、Cu +、Ni +、Fe 2+、Fe 3+)，或其任意组合。
在某些实施方案中，所述氯化物盐浓度为饱和的。
在某些实施方案中，所述氯化物盐浓度为0.1-2.5M、0.3-1.9M、0.5-1.8M、0.7-1.7M、0.9-1.6M或1M-1.4M。
在某些实施方案中，如上所述方法在4.0-12.0、4.5-10.0、5.0-9.0、5.5-8.8、6.0-8.7或者7.0-8.8或7.5-8.5的pH下进行。在某些实施方案中，如上所述方法在7.5的pH下进行。
在另一方面，本申请提供了一种试剂盒，其包含：包含跨膜孔的膜、解旋酶、ATP生成酶以及ATP生成底物；任选地，所述试剂盒还包含用于纳米孔测序的试剂。
在某些实施方案中，所述用于纳米孔测序的试剂选自ATP、无机盐(例如，氯化物盐，例如，氯化钠、氯化钾、氯化锂)、缓冲液(HEPES和/或Tris-HCl)、EDTA、金属离子(例如，Mn 2+、Mg 2+、Co +、Zn 2+、Cu 2+、Cu +、Ni +、Fe 2+、Fe 3+)，或其任意组合。
在某些实施方案中，所述ATP生成酶选自：丙酮酸激酶、乙酸激酶、肌酸磷酸激酶、丝氨酸激酶、苏氨酸激酶、酪氨酸激酶，FoF1-ATP酶，聚磷酸激酶，核苷二磷酸激酶，或其任意组合。
在某些实施方案中，所述试剂盒包含：包含跨膜孔的膜、解旋酶、ATP，(I)丙酮酸激酶和磷酸烯醇丙酮酸，(II)乙酸激酶和乙酰磷酸钾锂，(III)肌酸磷酸激酶和磷酸肌酸，(Ⅳ)FoF1-ATP酶和无机磷酸盐；(Ⅴ)肌酸磷酸激酶和磷酸肌酸；(Ⅵ)聚磷酸激酶和聚磷酸；(Ⅶ)核苷二磷酸激酶和核苷三磷酸，或(I)至(Ⅶ)中任意一组或几组的组合。
在某些实施方案中，所述试剂盒用于对双链靶多核苷酸进行测序。
在某些实施方案中，所述解旋酶选自Dda、UvrD、Rep、RecQ、PcrA、eIF4A、NS3、Rep、gp41、T7gp4，或其任意组合。
在某些实施方案中，所述解旋酶还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合。
在某些实施方案中，所述解旋酶是野生型Dda或其突变体。
在某些实施方案中，所述解旋酶具有如SEQ ID NO:2所示的氨基酸序列。
在某些实施方案中，所述跨膜孔是跨膜蛋白孔或固态孔。在某些实施方案中，所述跨膜蛋白孔选自hemolysin、MspA、MspB、MspC、MspD、Frac、ClyA、PA63、CsgG、CsgD、XcpQ、SP1、Phi29connector、T7connector、GspD、InvG，或其任意组合。
在某些实施方案中，所述跨膜孔还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合。
在某些实施方案中，所述跨膜孔是野生型CsgG蛋白或其突变体。在某些实施方案中，所述跨膜孔具有如SEQ ID NO:1所示的氨基酸序列。
在某些实施方案中，所述膜是两亲性层(例如，磷脂双分子层)或高分子聚合物膜(例如，di-block，tri-block)。
如前所述的试剂盒用于制备测序装置中的用途，所述测序装置用于对双链靶多核苷酸进行测序。
术语定义
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。本文提及的所有专利、申请和其他出版物均通过引用整体并入本文。如果本文中提出的定义与通过引用并入本文的专利、申请和其他出版物中所述的定义相抵触或不一致，则以本文所述的定义为准。
如本文中所使用的，术语“多核苷酸”是包含一个、两个或两个以上核苷酸的大分子。多核苷酸可包含任何核苷酸的任意组合，其可以是天然存在的或人工的。核苷酸通常含有核碱基、糖和至少一个磷酸基团。
如本文中所使用的，术语“跨膜孔”是允许外加电势驱动的水合离子从膜的一侧流动到膜的另一侧的结构，所述跨膜孔通常被插入膜(例如，脂双层)中。跨膜孔优选地为跨膜蛋白孔，所述“跨膜蛋白孔”为允许水合离子从膜的一侧流动到膜的另一侧的多肽或多肽的集合，所述跨膜蛋白孔允许多核苷酸移动并通过所述孔。跨膜蛋白孔通常包含桶状体或通道，离子可通过所述桶状体或通道流动。跨膜蛋白孔通常包含促进与靶核苷酸相互作用的氨基酸，这些氨基酸优选的位于所述桶状体或通道的缩窄处附近。
如本文中所使用的，术语“移动”是使单链多核苷酸从所述孔的一侧移动到另一侧。所述单链多核苷酸通过所述孔的移动可受电势作用、酶促作用，以及电势和酶促作用。所述移动可以是单向的，或双向的。
如本文中所使用的，术语“ATP生成酶”是指可以催化体系中的底物以生成ATP的酶。
如本文中所使用的，术语“ATP生成底物”是指在ATP生成酶的催化下，可以转化或生成ATP的物质。在某些实施方案中，所述ATP生成底物能够在所述ATP生成酶的催化下与ADP反应以生成ATP。
本发明的有益技术效果
本申请提供了一种能够在测序过程中始终保持测序速度稳定的方法，进一步保持测序的高通量以及高精确度。与其它方法相比(例如，整体更换测序缓冲液，或额外添加ATP)，本发明的方法以及试剂盒可以使测序过程中ATP保持相对恒定的浓度，从而能够更好的保持测序速率稳定不变。
附图说明
图1显示了本申请方法的原理示意图。
图2显示了添加ADP对测序速率的影响结果，其中，图2A为对照组(未添加ADP)的结果，图2B为实验组1(添加15mM ADP)的结果，图2C为实验组2(添加30mM ADP)的结果。
图3显示了实施例2中对照组和实验组1(含有PEP和PK)对测序速率的影响，其中，图3A为测序速率的衰减结果，图3B为测序速率百分数随时间的变化(初始速率为100％)。
图4显示了实施例2中实验组2(含有PEP和PK)对测序速率的影响，其中，图4A为测序速率的衰减结果，图4B为测序速率百分数随时间的变化(初始速率为100％)。
图5显示了实施例2中实验组3(含有PEP和PK)对测序速率的影响，其中，图5A为测序速率的衰减结果，图5B为测序速率百分数随时间的变化(初始速率为100％)。
图6显示了实施例3对照组和实验组(含有Ac和AcK)对测序速率的影响，其中，图6A为测序速率的衰减结果，图6B为测序速率百分数随时间的变化(初始速率 为100％)。
图7显示了实施例4对照组和实验组(含有Cre和CK)对测序速率的影响，其中，图7A为测序速率的衰减结果，图7B为测序速率百分数随时间的变化(初始速率为100％)。
序列信息
本申请所涉及的部分序列的信息如下面的表1所示。
表1.部分序列的信息
Figure PCTCN2022095500-appb-000001
Figure PCTCN2022095500-appb-000002
实施例
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1
本申请的发明人对测序体系进行了多方面的探索，并最终出人意料的发现，向体系测序缓冲液中添加ADP，会降低测序速率。
具体来说，使用CsgG跨膜蛋白突变体(野生型氨基酸序列由SEQ ID NO.1所示，突变位点为：Y51A/F56Q/R97W/R192D)作为纳米孔，采用纳米孔检测方法进行检测。单个CsgG跨膜蛋白插入磷脂双分子层后，使用不含燃料(ATP)的缓冲液(470mM KCl，25mM HEPES，1mM EDTA，PH 8.0)流经该系统以除去多余的跨膜蛋白。将构建好的含有T4Dda解旋酶(野生型编码氨基酸序列由SEQ ID NO.2所示，突变位点为E94C/C109A/C136A/A360C)的pUC57测序文库(SEQ ID NO.3)和测序缓冲液预混后加入到纳米孔实验系统中，在0.18V电压下检测CsgG跨膜蛋白测序情况。其中，
对照组(图2A)测序缓冲液为470mM KCl，25mM HEPES，30mM ATP，25mM MgCl 2，1mM EDTA，PH 8.0；
实验组1(图2B)测序缓冲液为470mM KCl，25mM HEPES，30mM ATP，15mM ADP，25mM MgCl 2，1mM EDTA，PH 8.0；
实验组2(图2C)测序缓冲液为470mM KCl，25mM HEPES，30mM ATP，30mM ADP，25mM MgCl 2，1mM EDTA，PH 8.0；
实验结果如图2所示，图2的横坐标为测序速度，纵坐标为测序速度对应的频数，由图2可以看出，与对照组相比，向测序体系缓冲液中添加ADP，可降低测序速度。并且，ADP添加量更大的实验组2在实验组1的基础上测序速率进一步下降。
实施例2
在上述实施例的基础上，申请人进一步对测序体系进行探索，即在体系测序缓冲液中添加组分，以降低测序过程中的体系产生的ADP的量，同时增加测序过程中的体系中ATP的量。
具体来说，使用CsgG跨膜蛋白突变体(野生型氨基酸序列由SEQ ID NO.1所示，突变位点为：Y51A/F56Q/R97W/R192D)作为纳米孔，采用纳米孔检测方法进行检测。单个CsgG跨膜蛋白插入磷脂双分子层后，使用不含燃料(ATP)的缓冲液(470mM KCl，25mM HEPES，1mM EDTA，PH 8.0)流经该系统以除去多余的跨膜蛋白。将构建好的含有T4Dda解旋酶(野生型编码氨基酸序列由SEQ ID NO.2所示，突变位点为E94C/C109A/C136A/A360C)的pUC57测序文库(SEQ ID NO.3)和测序缓冲液预混后加入到纳米孔实验系统中，在0.18V电压下检测CsgG跨膜蛋白测序情况。其中，
对照组测序缓冲液为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0；
实验组设置3种浓度：
实验组1测序缓冲液为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0，5mM PEP(磷酸烯醇丙酮酸，Roche，货号：10108294001)，0.02U/mL PK(丙酮酸激酶，Roche，货号：10109045001)。
实验组2测序缓冲液为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0，5mM PEP(磷酸烯醇丙酮酸)，0.2U/mL PK(丙酮酸激酶)。
实验组3测序缓冲液为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0，5mM PEP(磷酸烯醇丙酮酸)，2U/mL PK(丙酮酸激酶)。
实验组1、2、3的实验结果分别如图3、4、5所示，与对照组相比，本申请的测序方法以及测序缓冲液在测序过程中明显表现的更加稳定，在测序后期，几乎比对照高出20％的测序速率。而在测序过程中，本申请的测序速度几乎无降低。
并且，图4和图5的实验结果显示，体系中PEP或PK(丙酮酸激酶)的浓度改变并不影响测序的速率，可达到测序速度无明显衰减。
实施例3
本申请的实验条件与步骤和实施例2相同，不同之处在于，
对照组测序缓冲液配方为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0；
实验组测序缓冲液配方为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0，5mM Ac(乙酰磷酸钾锂，sigma，01409-500MG)，0.02U/mL AcK(乙酸激酶，sigma，A7437-250UN)。
实验结果显示(图6)，与对照组相比，本申请的测序方法以及测序缓冲液在测序过程中明显表现的更加稳定，在测序后期，几乎比对照高出10％的测序速率。
实施例4
本申请的实验条件与步骤和实施例2相同，不同之处在于，
对照组测序缓冲液配方为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0；
实验组测序缓冲液配方为470mM KCl，25mM HEPES，5mM ATP，25mM MgCl2，1mM EDTA，PH 8.0，5mM Cre(磷酸肌酸二钠，sigma，P7936-1G)/0.02U/mL CK(肌酸磷酸激酶，sigma，C3755-3.5KU)。
实验结果显示(图7)，与对照组相比，本申请的测序方法以及测序缓冲液在测序过程中明显表现的更加稳定，在测序后期，几乎比对照高出20％的测序速率。而在测序过程中，本申请的测序速度几乎无降低。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (10)

  1. 一种对双链靶多核苷酸进行测序的方法，包括：
    (a)提供双链靶多核苷酸、包含跨膜孔的膜、解旋酶、ATP生成酶以及ATP生成底物；其中，所述ATP生成底物能够在所述ATP生成酶的催化下与ADP反应以生成ATP；
    (b)使所述双链靶多核苷酸与跨膜孔、解旋酶、ATP生成酶和ATP生成底物接触，所述解旋酶使所述双链靶多核苷酸的双链分开，形成单链靶多核苷酸；并且，所述解旋酶使所述单链多核苷酸在所述跨膜孔中移动，使得所述单链多核苷酸中的一部分核苷酸与所述跨膜孔相互作用；
    (c)测量每次相互作用期间通过所述跨膜孔的电流，从而确定所述双链靶多核苷酸的序列。
  2. 权利要求1的方法，其中，权利要求1的方法具有选自下列的一项或多项特征：
    (1)所述ATP生成酶选自：丙酮酸激酶、乙酸激酶、肌酸磷酸激酶、丝氨酸激酶、苏氨酸激酶、酪氨酸激酶，FoF1-ATP酶，聚磷酸激酶，核苷二磷酸激酶，或其任意组合；
    (2)所述ATP生成酶为丙酮酸激酶，且所述ATP生成底物为磷酸烯醇丙酮酸；
    (3)所述ATP生成酶为乙酸激酶，且所述ATP生成底物为乙酰磷酸钾锂；
    (4)所述ATP生成酶为肌酸磷酸激酶，且所述ATP生成底物为磷酸肌酸二钠；
    (5)所述ATP生成酶为FoF1-ATP酶，且所述ATP生成底物为无机磷酸盐；
    (6)所述ATP生成酶为肌酸磷酸激酶，且所述ATP生成底物为磷酸肌酸；
    (7)所述ATP生成酶为聚磷酸激酶，且所述ATP生成底物为聚磷酸；
    (8)所述ATP生成酶为核苷二磷酸激酶，且所述ATP生成底物为核苷三磷酸。
  3. 权利要求1或2的方法，其中，所述解旋酶具有选自下列的一项或多项特征：
    (1)所述解旋酶选自Dda、UvrD、Rep、RecQ、PcrA、eIF4A、NS3、Rep、gp41、T7gp4，或其任意组合；
    (2)所述解旋酶还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号 肽或导肽、可检测的标记，或其任意组合；
    (3)所述解旋酶是野生型Dda或其突变体；优选地，所述解旋酶具有如SEQ ID NO:2所示的氨基酸序列。
  4. 权利要求1-3任一项的方法，其中，所述跨膜孔具有选自下列的一项或多项特征：
    (1)所述跨膜孔是跨膜蛋白孔或固态孔；优选地，所述跨膜蛋白孔选自hemolysin、MspA、MspB、MspC、MspD、Frac、ClyA、PA63、CsgG、CsgD、XcpQ、SP1、Phi29 connector、T7 connector、GspD、InvG，或其任意组合；
    (2)所述跨膜孔还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合；
    (3)所述跨膜孔是野生型CsgG蛋白或其突变体；优选地，所述跨膜孔具有如SEQ ID NO:1所示的氨基酸序列。
  5. 权利要求1-4任一项的方法，其中，所述膜是两亲性层(例如，磷脂双分子层)或高分子聚合物膜(例如，di-block，tri-block)。
  6. 权利要求1-5任一项的方法，其中，所述双链靶多核苷酸具有选自下列的一项或多项特征：
    (1)所述双链靶多核苷酸为DNA双链和/或DNA-RNA杂交的双链；
    (2)所述双链靶多核苷酸是天然存在的和/或人工合成的；
    (3)所述双链靶多核苷酸获自生物样品，所述生物样品提取自病毒，原核生物(例如，细菌)，真核生物(例如，植物(例如，谷类，豆类，水果或蔬菜)，哺乳动物(例如，马，牛，鼠或人)，或其任意组合；
    (4)所述双链靶多核苷酸的两条链在所述靶多核苷酸的一个末端处或其附近通过桥连部分连接；所述桥连部分选自聚合物接头，化学接头，多核苷酸或多肽；
    (5)所述双链靶多核苷酸是环状的或线性的。
  7. 权利要求1-6任一项的方法，其中，所述方法具有选自下列的一项或多项特征：
    (1)所述双链靶多核苷酸包含至少一个单链悬突(例如，5’端悬突和/或3’端悬突)，所述单链悬突含有前导序列，所述前导序列引领与其衔接的核酸链进入所述孔；
    (2)在步骤(b)中，使所述双链靶多核苷酸与解旋酶接触，以形成复合物，然后，使所述复合物与跨膜孔接触；
    (3)在步骤(b)中，使用于纳米孔测序的试剂与所述解旋酶接触；优选地，所述用于纳米孔测序的试剂选自ATP、无机盐(例如，氯化物盐，例如，氯化钠、氯化钾、氯化锂)、缓冲液(HEPES和/或Tris-HCl)、EDTA、金属离子(例如，Mn 2+、Mg 2+、Co +、Zn 2+、Cu 2+、Cu +、Ni +、Fe 2+、Fe 3+)，或其任意组合。
  8. 一种试剂盒，其包含：包含跨膜孔的膜、解旋酶、ATP生成酶以及ATP生成底物；任选地，所述试剂盒还包含用于纳米孔测序的试剂；
    优选地，所述用于纳米孔测序的试剂选自ATP、无机盐(例如，氯化物盐，例如，氯化钠、氯化钾、氯化锂)、缓冲液(HEPES和/或Tris-HCl)、EDTA、金属离子(例如，Mn 2+、Mg 2+、Co +、Zn 2+、Cu 2+、Cu +、Ni +、Fe 2+、Fe 3+)，或其任意组合；
    优选地，所述ATP生成酶选自：丙酮酸激酶、乙酸激酶、肌酸磷酸激酶、丝氨酸激酶、苏氨酸激酶、酪氨酸激酶，FoF1-ATP酶，聚磷酸激酶，核苷二磷酸激酶，或其任意组合；
    优选地，所述试剂盒包含：包含跨膜孔的膜、解旋酶、ATP，以及(I)丙酮酸激酶和磷酸烯醇丙酮酸，(II)乙酸激酶和乙酰磷酸钾锂，(III)肌酸磷酸激酶和磷酸肌酸，(Ⅳ)FoF1-ATP酶和无机磷酸盐；(Ⅴ)肌酸磷酸激酶和磷酸肌酸；(Ⅵ)聚磷酸激酶和聚磷酸；(Ⅶ)核苷二磷酸激酶和核苷三磷酸，或(I)至(Ⅶ)中任意一组或几组的组合；
    优选地，所述试剂盒用于对双链靶多核苷酸进行测序。
  9. 权利要求的8的试剂盒，其中，所述试剂盒具有选自下列的一项或多项特征：
    (1)所述解旋酶选自Dda、UvrD、Rep、RecQ、PcrA、eIF4A、NS3、Rep、gp41、T7gp4，或其任意组合；
    (2)所述解旋酶还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合；
    (3)所述解旋酶是野生型Dda或其突变体；优选地，所述解旋酶具有如SEQ ID NO: 2所示的氨基酸序列；
    (4)所述跨膜孔是跨膜蛋白孔或固态孔；优选地，所述跨膜蛋白孔选自hemolysin、MspA、MspB、MspC、MspD、Frac、ClyA、PA63、CsgG、CsgD、XcpQ、SP1、Phi29 connector、T7 connector、GspD、InvG，或其任意组合；
    (5)所述跨膜孔还连接有另外的多肽，所述另外的多肽选自标签、酶切位点、信号肽或导肽、可检测的标记，或其任意组合；
    (6)所述跨膜孔是野生型CsgG蛋白或其突变体；优选地，所述跨膜孔具有如SEQ ID NO:1所示的氨基酸序列；
    (7)所述膜是两亲性层(例如，磷脂双分子层)或高分子聚合物膜(例如，di-block，tri-block)。
  10. 权利要求的8或9的试剂盒用于制备测序装置中的用途，所述测序装置用于对双链靶多核苷酸进行测序。
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