Method for maintaining nanopore sequencing speed

Abstract

According to the method for sequencing the double-stranded target polynucleotide, disclosed by the invention, ATP (adenosine triphosphate) can be kept at a relatively constant concentration in a sequencing process, so that the sequencing rate can be better kept stable and unchanged. The present invention also provides a kit for sequencing a double-stranded target polynucleotide, comprising a transmembrane pore membrane, a helicase, an ATP-generating enzyme, and an ATP-generating substrate.

Description

A method to maintain nanopore sequencing speed Field of the Invention

[0001] The present invention relates to a method for maintaining nanopore sequencing speed and further provides a method for sequencing a double-stranded target polynucleotide and a kit for sequencing a double-stranded target polynucleotide.

[0002] Background of the Invention

[0003] With the continuous advancement of nanotechnology, nanopore sequencing technology, which can read nucleic acid sequences at the single-molecule level, has rapidly developed. This technology offers significant advantages over next-generation sequencing in terms of portability, read length, and speed. In 1996, scientists first used the alpha-hemolysin protein to identify different bases (Kasianowicz, John J., et al. Proceedings of the National Academy of Sciences 93.24(1996):13770-13773.), pioneering DNA sequencing based on the nanopore detection principle.

[0004] Oxford Nanopore Technologies, a UK company, has launched a number of sequencers with different throughputs. The principle behind this technology is that a transmembrane protein with a nanometer diameter is inserted into a polymer membrane, forming a nanopore channel on the membrane. Two electrodes are placed on both sides of the membrane, and a conductivity buffer is placed on both sides of the membrane. When electricity is applied, ions pass through the transmembrane protein to generate an electric current. The double-stranded DNA in the sequencing library is unwound into single-stranded DNA by the action of the helicase. The single-stranded DNA passes through the transmembrane protein under the action of the electric field, generating a characteristic current signal. The current characteristic signal is analyzed by an algorithm to achieve sequence reading. The 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'), unwinding the hydrogen-bonded double-stranded DNA into single-stranded DNA.

[0005] Summary of the Invention

[0006] The inventors of this application discovered a problem with the aforementioned sequencing process. Specifically, as sequencing progresses, the nanopore sequencing speed decreases. This reduction in sequencing speed affects both sequencing throughput and accuracy. Therefore, if high throughput and accuracy are to be maintained throughout the sequencing process, a method for maintaining the nanopore sequencing speed is required.

[0007] To address the above issues, the inventors of this application discovered through extensive experimentation that the nanopore sequencing speed is related to the composition of the conductivity buffer on one or both sides of the membrane. Furthermore, through extensive experimentation and repeated exploration, the inventors of this application first realized that simultaneously controlling the amounts of ADP and ATP during the sequencing process is key to influencing the sequencing rate, leading to the completion of this application.

[0008] Therefore, the present application provides a method for sequencing a double-stranded target polynucleotide, comprising:

[0009] (a) providing a double-stranded target polynucleotide, a membrane comprising a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; wherein the ATP-generating substrate can react with ADP to generate ATP under the catalysis of the ATP-generating enzyme;

[0010] (b) contacting the double-stranded target polynucleotide with a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate, wherein the helicase separates the double strands of the double-stranded target polynucleotide to form a single-stranded target polynucleotide; and the helicase moves the single-stranded polynucleotide in the transmembrane pore, so that a portion of the nucleotides in the single-stranded polynucleotide interacts with the transmembrane pore;

[0011] (c) measuring the current passing through the transmembrane pore during each interaction to thereby determine the sequence of the double-stranded target polynucleotide.

[0012] In the method herein, because different nucleotides (e.g., A, T, C, G) have different effects on the current passing through the transmembrane pore, and the transmembrane pore can distinguish nucleotides with similar structures. Therefore, in certain embodiments, each nucleotide can be identified at the single molecule level based on their current amplitude or the duration of interaction when the nucleotide interacts with the transmembrane pore. If a characteristic current associated with the nucleotide is detected flowing through the pore, the type of nucleotide (e.g., A, T, C, G) passing through the pore can be determined, i.e., sequencing is achieved. By continuously identifying the nucleotides in the target polynucleotide, it is possible to estimate or determine the sequence of the target polynucleotide.

[0013] In certain embodiments, the method further comprises measuring the current through the pore during interaction with the nucleotide. Thus, in certain embodiments, the method further comprises providing a circuit capable of applying an electric potential and measuring the electrical signal across the membrane and pore. In certain embodiments, the method further comprises patch clamping or voltage clamping.

[0014] In the methods herein, the helicase separates the double strands of the double-stranded target polynucleotide to form a single-stranded target polynucleotide. In certain embodiments, the helicase utilizes 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' to 3' or 3' to 5'), thereby unwinding the hydrogen-bonded double-stranded DNA into single-stranded DNA.

[0015] In certain embodiments, the helicase controls the movement of the single-stranded polynucleotide along the field generated by the applied voltage to pass through the hole. In certain embodiments, the helicase acts as a brake to prevent the single-stranded polynucleotide from moving through the hole too quickly under the influence of the applied voltage. In certain embodiments, the method further comprises: (d) reducing the voltage applied across the hole so that the single-stranded polynucleotide moves through the hole in the opposite direction to that in step (b), and a portion of the nucleotides in the polynucleotide interacts with the hole, measuring the current passing through the hole during each interaction, thereby correcting the sequence of the target polynucleotide obtained in step (c).

[0016] In certain embodiments, the single stranded polynucleotide may interact with the pore on either side of the membrane.The single stranded polynucleotide may interact with the pore at any site in any manner.

[0017] In certain embodiments, the ATP generating enzyme is selected from the group consisting of: pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nucleoside diphosphate kinase, or any combination thereof.

[0018] In certain embodiments, the ATP generating enzyme is pyruvate kinase, and the ATP generating substrate is phosphoenolpyruvate. In certain embodiments, phosphoenolpyruvate is 5mM and pyruvate kinase is 0.02U / mL. In certain embodiments, phosphoenolpyruvate is 5mM and pyruvate kinase is 0.2U / mL. In certain embodiments, phosphoenolpyruvate is 5mM and pyruvate kinase is 2U / mL.

[0019] In certain embodiments, the ATP generating enzyme is acetate kinase, and the ATP generating substrate is lithium potassium acetyl phosphate. In certain embodiments, the lithium potassium acetyl phosphate is 5 mM and the acetate kinase is 0.02 U / mL.

[0020] In certain embodiments, the ATP-generating enzyme is creatine phosphokinase, and the ATP-generating substrate is creatine phosphate disodium.

[0021] In certain embodiments, the ATP generating enzyme is FoF1-ATPase and the ATP generating substrate is inorganic phosphate.

[0022] In certain embodiments, the ATP-generating enzyme is creatine phosphokinase, and the ATP-generating substrate is creatine phosphate.

[0023] In certain embodiments, the ATP-generating enzyme is a polyphosphate kinase and the ATP-generating substrate is polyphosphate.

[0024] In certain embodiments, the ATP-generating enzyme is a nucleoside diphosphate kinase and the ATP-generating substrate is a nucleoside triphosphate.

[0025] Herein, the amounts of the 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 ATP concentration can be maintained at a relatively constant level during the sequencing process, thereby better maintaining a stable sequencing rate, it will suffice. In fact, during the sequencing process, under the action of the ATP generating enzyme, the substrate reacts and the amount gradually decreases. Those skilled in the art are capable of appropriately adjusting the concentrations and amounts of the ATP generating enzyme and ATP generating substrate in the sequencing system according to the experimental objectives to obtain appropriate concentrations and amounts of the ATP generating enzyme and ATP generating substrate.

[0026] In certain embodiments, the helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof.

[0027] In certain embodiments, the helicase is further linked to an additional polypeptide selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0028] In certain embodiments, the helicase is wild-type Dda or a mutant thereof.

[0029] In certain embodiments, the helicase has the amino acid sequence shown in SEQ ID NO:2.

[0030] In certain embodiments, the transmembrane pore is a transmembrane protein pore or a solid-state pore.

[0031] In certain embodiments, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector, T7 connector, GspD, InvG, or any combination thereof.

[0032] In certain embodiments, the transmembrane pore is further linked to an additional polypeptide, wherein the additional polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0033] In certain embodiments, the transmembrane pore is a wild-type CsgG protein or a mutant thereof. In certain embodiments, the transmembrane pore has the amino acid sequence shown in SEQ ID NO: 1.

[0034] In certain embodiments, the membrane is an amphiphilic layer (eg, a phospholipid bilayer) or a high molecular weight polymer membrane (eg, di-block, tri-block).

[0035] The method described above can be performed using any suitable membrane. In certain embodiments, the membrane is a phospholipid bilayer, wherein the transmembrane pore is inserted into the phospholipid bilayer.

[0036] In certain embodiments, the method is typically performed using a membrane that is: (i) an artificial bilayer comprising a pore, (ii) an isolated naturally occurring pore-containing lipid bilayer, or (iii) a cell having a pore inserted therein. The method is preferably performed using an artificial bilayer (e.g., an artificial phospholipid bilayer).

[0037] In certain embodiments, the double-stranded target polynucleotide is a double-stranded DNA and / or a double-stranded DNA-RNA hybrid.

[0038] In certain embodiments, the double-stranded target polynucleotide is naturally occurring and / or artificially synthesized.

[0039] In certain embodiments, the double-stranded target polynucleotide is obtained from a biological sample extracted from a virus, a prokaryotic organism (e.g., a bacterium), a eukaryotic organism (e.g., a plant (e.g., a cereal, a legume, a fruit, or a vegetable), a mammal (e.g., a horse, a cow, a mouse, or a human), or any combination thereof.

[0040] In certain embodiments, 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, a chemical linker, a polynucleotide or a polypeptide.

[0041] In certain embodiments, the double-stranded target polynucleotide is circular or linear.

[0042] In certain embodiments, all or only a portion of the double-stranded target polynucleotide can be sequenced using the methods described above. The double-stranded target polynucleotide can be of any length. For example, 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 can be naturally occurring or artificially synthesized. For example, the method can be used to verify the sequence of an artificially synthesized oligonucleotide, and the method is typically performed in vitro.

[0043] In certain embodiments, the double-stranded target polynucleotide comprises at least one single-stranded overhang (eg, a 5' overhang and / or a 3' overhang), the single-stranded overhang comprising a leader sequence that guides the attached nucleic acid strand into the pore.

[0044] In certain embodiments, in 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.

[0045] In certain embodiments, the methods described above are typically performed in the presence of nanopore sequencing reagents.

[0046] In certain embodiments, in step (b), reagents for nanopore sequencing are contacted with the helicase.

[0047] In certain embodiments, the reagents for nanopore sequencing are selected from ATP, inorganic salts (e.g., chloride salts, such as sodium chloride, potassium chloride, lithium chloride), buffers (HEPES and / 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 combination thereof.

[0048] In certain embodiments, the chloride salt concentration is saturating.

[0049] In certain embodiments, 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.

[0050] In certain embodiments, the method described above is 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 method described above is performed at a pH of 7.5.

[0051] In another aspect, the present application provides a kit comprising: a membrane comprising a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; optionally, the kit further comprises reagents for nanopore sequencing.

[0052] In certain embodiments, the reagents for nanopore sequencing are selected from ATP, inorganic salts (e.g., chloride salts, such as sodium chloride, potassium chloride, lithium chloride), buffers (HEPES and / 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 combination thereof.

[0053] In certain embodiments, the ATP generating enzyme is selected from the group consisting of: pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nucleoside diphosphate kinase, or any combination thereof.

[0054] In certain embodiments, the kit comprises: a membrane comprising a transmembrane pore, a helicase, ATP, (I) pyruvate kinase and phosphoenolpyruvate, (II) acetate kinase and potassium lithium acetyl phosphate, (III) creatine phosphokinase and creatine phosphate, (IV) FoF1-ATPase and inorganic phosphate; (V) creatine phosphokinase and creatine phosphate; (VI) polyphosphate kinase and polyphosphate; (VII) nucleoside diphosphate kinase and nucleoside triphosphate, or any one or more combinations of (I) to (VII).

[0055] In certain embodiments, the kit is used to sequence a double-stranded target polynucleotide.

[0056] In certain embodiments, the helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof.

[0057] In certain embodiments, the helicase is further linked to an additional polypeptide selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0058] In certain embodiments, the helicase is wild-type Dda or a mutant thereof.

[0059] In certain embodiments, the helicase has the amino acid sequence shown in SEQ ID NO:2.

[0060] In certain embodiments, the transmembrane pore is a transmembrane protein pore or a solid-state pore. In certain embodiments, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector, T7 connector, GspD, InvG, or any combination thereof.

[0061] In certain embodiments, the transmembrane pore is further linked to an additional polypeptide, wherein the additional polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof.

[0062] In certain embodiments, the transmembrane pore is a wild-type CsgG protein or a mutant thereof. In certain embodiments, the transmembrane pore has the amino acid sequence shown in SEQ ID NO: 1.

[0063] In certain embodiments, the membrane is an amphiphilic layer (eg, a phospholipid bilayer) or a high molecular weight polymer membrane (eg, di-block, tri-block).

[0064] The kit as described above is used for preparing a sequencing device for sequencing a double-stranded target polynucleotide.

[0065] Definition of terms

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents, applications, and other publications mentioned herein are incorporated herein by reference in their entirety. If a definition set forth herein conflicts or is inconsistent with a definition set forth in the patents, applications, and other publications incorporated herein by reference, the definition set forth herein shall prevail.

[0067] As used herein, the term "polynucleotide" is a macromolecule comprising one, two, or more nucleotides. A polynucleotide may comprise any combination of nucleotides, which may be naturally occurring or artificial. Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group.

[0068] As used herein, the term "transmembrane pore" is a structure that allows the hydrated ions driven by an applied potential to flow from one side of the film to the other side of the film, and the transmembrane pore is typically inserted into a membrane (e.g., lipid bilayer). Transmembrane pore is preferably a transmembrane protein pore, and the " transmembrane protein pore" is a polypeptide or polypeptide set that allows hydrated ions to flow from one side of the film to the other side of the film, and the transmembrane protein pore allows polynucleotides to move and pass through the hole. Transmembrane protein pore typically includes a barrel or channel, and ions can flow through the barrel or channel. Transmembrane protein pore typically includes an amino acid that promotes interaction with the target nucleotide, and these amino acids are preferably located near the constriction of the barrel or channel.

[0069] As used herein, the term "mobilize" refers to the movement of a single-stranded polynucleotide from one side of the pore to the other. The movement of the single-stranded polynucleotide through the pore can be induced by an electric potential, an enzymatic effect, or both. The movement can be unidirectional or bidirectional.

[0070] As used herein, the term "ATP-generating enzyme" refers to an enzyme that can catalyze a substrate in a system to generate ATP.

[0071] As used herein, the term "ATP generating substrate" refers to a substance that can be converted or generated into ATP under the catalysis of an ATP generating enzyme. In certain embodiments, the ATP generating substrate can react with ADP to generate ATP under the catalysis of the ATP generating enzyme.

[0072] Beneficial technical effects of the present invention

[0073] This application provides a method for maintaining a stable sequencing speed throughout the sequencing process, further maintaining high sequencing throughput and accuracy. Compared to other methods (e.g., replacing the entire sequencing buffer or adding additional ATP), the method and kit of the present invention can maintain a relatively constant ATP concentration during the sequencing process, thereby better maintaining a stable sequencing rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG1 shows a schematic diagram of the principle of the method of the present application.

[0075] Figure 2 shows the effect of adding ADP on the sequencing rate, where Figure 2A is the result of the control group (no ADP added), Figure 2B is the result of experimental group 1 (15mM ADP added), and Figure 2C is the result of experimental group 2 (30mM ADP added).

[0076] Figure 3 shows the effects of the control group and experimental group 1 (containing PEP and PK) on the sequencing rate in Example 2, wherein Figure 3A shows the attenuation results of the sequencing rate, and Figure 3B shows the change in the sequencing rate percentage over time (the initial rate is 100%).

[0077] Figure 4 shows the effect of experimental group 2 (containing PEP and PK) in Example 2 on the sequencing rate, wherein Figure 4A shows the attenuation result of the sequencing rate, and Figure 4B shows the change in the sequencing rate percentage over time (the initial rate is 100%).

[0078] Figure 5 shows the effect of experimental group 3 (containing PEP and PK) in Example 2 on the sequencing rate, wherein Figure 5A is the attenuation result of the sequencing rate, and Figure 5B is the change in the sequencing rate percentage over time (the initial rate is 100%).

[0079] FIG6 shows the effects of the control group and experimental group (containing Ac and AcK) of Example 3 on the sequencing rate, wherein FIG6A shows the attenuation results of the sequencing rate, and FIG6B shows the change in the sequencing rate percentage over time (the initial rate is 100%).

[0080] FIG7 shows the effects of the control group and experimental group (containing Cre and CK) of Example 4 on sequencing rate, wherein FIG7A shows the attenuation results of sequencing rate, and FIG7B shows the change of sequencing rate percentage over time (initial rate is 100%).

[0081] Sequence information

[0082] The information of some sequences involved in this application is shown in Table 1 below.

[0083] Table 1. Information of partial sequences

[0084]

[0085] Example

[0086] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0087] Unless otherwise specified, the molecular biology experimental methods used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0088] Example 1

[0089] The inventors of the present application have conducted multi-faceted explorations of the sequencing system and ultimately unexpectedly discovered that adding ADP to the sequencing buffer of the system would reduce the sequencing rate.

[0090] Specifically, a CsgG transmembrane protein mutant (wild-type amino acid sequence shown in SEQ ID NO. 1, mutations: Y51A / F56Q / R97W / R192D) was used as a nanopore for detection. After a single CsgG transmembrane protein was inserted into the phospholipid bilayer, a fuel (ATP)-free buffer (470 mM KCl, 25 mM HEPES, 1 mM EDTA, pH 8.0) was passed through the system to remove excess transmembrane protein. A pre-mixed pUC57 sequencing library (SEQ ID NO. 3) containing the T4Dda helicase (wild-type encoding amino acid sequence shown in SEQ ID NO. 2, mutations: E94C / C109A / C136A / A360C) and sequencing buffer were added to the nanopore experimental system, and the CsgG transmembrane protein sequencing was detected at 0.18 V. Specifically,

[0091] The sequencing buffer for the control group ( Figure 2A ) was 470 mM KCl, 25 mM HEPES, 30 mM ATP, 25 mM MgCl , 1 mM EDTA, pH 8.0;

[0092] For experimental group 1 ( Fig. 2B ), the sequencing buffer consisted of 470 mM KCl, 25 mM HEPES, 30 mM ATP, 15 mM ADP, 25 mM MgCl , 1 mM EDTA, pH 8.0;

[0093] For experimental group 2 ( Fig. 2C ), the sequencing buffer was 470 mM KCl, 25 mM HEPES, 30 mM ATP, 30 mM ADP, 25 mM MgCl , 1 mM EDTA, pH 8.0;

[0094] The experimental results are shown in Figure 2, where the horizontal axis represents sequencing speed and the vertical axis represents the frequency corresponding to the sequencing speed. As shown in Figure 2, adding ADP to the sequencing buffer reduces sequencing speed compared to the control group. Furthermore, the sequencing speed of Experimental Group 2, which added a larger amount of ADP, further decreased compared to Experimental Group 1.

[0095] Example 2

[0096] Based on the above examples, the applicant further explored the sequencing system, namely, adding components to the system sequencing buffer to reduce the amount of ADP generated by the system during the sequencing process, while increasing the amount of ATP in the system during the sequencing process.

[0097] Specifically, a CsgG transmembrane protein mutant (wild-type amino acid sequence shown in SEQ ID NO. 1, mutations: Y51A / F56Q / R97W / R192D) was used as a nanopore for detection. After a single CsgG transmembrane protein was inserted into the phospholipid bilayer, a fuel (ATP)-free buffer (470 mM KCl, 25 mM HEPES, 1 mM EDTA, pH 8.0) was passed through the system to remove excess transmembrane protein. A pre-mixed pUC57 sequencing library (SEQ ID NO. 3) containing the T4Dda helicase (wild-type encoding amino acid sequence shown in SEQ ID NO. 2, mutations: E94C / C109A / C136A / A360C) and sequencing buffer were added to the nanopore experimental system, and the CsgG transmembrane protein sequencing was detected at 0.18 V. Specifically,

[0098] The sequencing buffer for the control group was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0;

[0099] The experimental group was set up with 3 concentrations:

[0100] The sequencing buffer of experimental group 1 was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0, 5 mM PEP (phosphoenolpyruvate, Roche, catalog number: 10108294001), and 0.02 U / mL PK (pyruvate kinase, Roche, catalog number: 10109045001).

[0101] The sequencing buffer of experimental group 2 was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0, 5 mM PEP (phosphoenolpyruvate), and 0.2 U / mL PK (pyruvate kinase).

[0102] The sequencing buffer of experimental group 3 was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0, 5 mM PEP (phosphoenolpyruvate), and 2 U / mL PK (pyruvate kinase).

[0103] The experimental results for Experimental Groups 1, 2, and 3 are shown in Figures 3, 4, and 5, respectively. Compared to the control group, the sequencing method and sequencing buffer of the present application were significantly more stable during the sequencing process. In the late stage of sequencing, the sequencing rate was almost 20% higher than that of the control. During the entire sequencing process, the sequencing speed of the present application was almost unchanged.

[0104] Furthermore, the experimental results in FIG4 and FIG5 show that changes in the concentration of PEP or PK (pyruvate kinase) in the system do not affect the sequencing rate, and the sequencing speed can be achieved without significant attenuation.

[0105] Example 3

[0106] The experimental conditions and steps of this application are the same as those of Example 2, except that:

[0107] The sequencing buffer formula for the control group was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0;

[0108] The sequencing buffer formula of the experimental group was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0, 5 mM Ac (lithium potassium acetyl phosphate, Sigma, 01409-500MG), and 0.02 U / mL AcK (acetate kinase, Sigma, A7437-250UN).

[0109] The experimental results show ( FIG. 6 ) that, compared with the control group, the sequencing method and sequencing buffer of the present application are significantly more stable during the sequencing process. In the later stage of sequencing, the sequencing rate is almost 10% higher than that of the control.

[0110] Example 4

[0111] The experimental conditions and steps of this application are the same as those of Example 2, except that:

[0112] The sequencing buffer formula for the control group was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0;

[0113] The sequencing buffer formula of the experimental group was 470 mM KCl, 25 mM HEPES, 5 mM ATP, 25 mM MgCl2, 1 mM EDTA, pH 8.0, 5 mM Cre (creatine phosphate disodium, Sigma, P7936-1G) / 0.02 U / mL CK (creatine phosphokinase, Sigma, C3755-3.5 KU).

[0114] The experimental results (Figure 7) show that compared with the control group, the sequencing method and sequencing buffer of the present application were significantly more stable during the sequencing process. In the late stage of sequencing, the sequencing rate was almost 20% higher than that of the control group. During the entire sequencing process, the sequencing speed of the present application was almost unchanged.

[0115] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for sequencing a double-stranded target polynucleotide, comprising: (a) providing a double-stranded target polynucleotide, a membrane comprising a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; wherein the ATP-generating substrate can react with ADP to generate ATP under the catalysis of the ATP-generating enzyme; (b) contacting the double-stranded target polynucleotide with a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate, wherein the helicase separates the double strands of the double-stranded target polynucleotide to form a single-stranded target polynucleotide; and the helicase moves the single-stranded polynucleotide in the transmembrane pore, so that a portion of the nucleotides in the single-stranded polynucleotide interacts with the transmembrane pore; (c) measuring the current passing through the transmembrane pore during each interaction to thereby determine the sequence of the double-stranded target polynucleotide.

2. The method of claim 1, wherein The method of claim 1 has one or more features selected from the group consisting of: (1) The ATP generating enzyme is selected from the group consisting of pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nucleoside diphosphate kinase, or any combination thereof; (2) the ATP generating enzyme is pyruvate kinase, and the ATP generating substrate is phosphoenolpyruvate; (3) The ATP generating enzyme is acetate kinase, and the ATP generating substrate is lithium potassium acetyl phosphate; (4) The ATP generating enzyme is creatine phosphokinase, and the ATP generating substrate is creatine disodium phosphate; (5) The ATP generating enzyme is FoF1-ATPase, and the ATP generating substrate is inorganic phosphate; (6) The ATP generating enzyme is creatine phosphokinase, and the ATP generating substrate is creatine phosphate; (7) The ATP generating enzyme is polyphosphate kinase, and the ATP generating substrate is polyphosphate; (8) The ATP generating enzyme is a nucleoside diphosphate kinase, and the ATP generating substrate is a nucleoside triphosphate.

3. The method of claim 1 or 2, wherein The helicase has one or more characteristics selected from the group consisting of: (1) The helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof; (2) The helicase is further linked to another polypeptide, wherein the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof; (3) The helicase is wild-type Dda or a mutant thereof; preferably, the helicase has an amino acid sequence as shown in SEQ ID NO:

2.

4. The method of any one of claims 1 to 3, wherein The transmembrane pore has one or more characteristics selected from the following: (1) The transmembrane pore is a transmembrane protein pore or a solid-state pore; preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector, T7 connector, GspD, InvG, or any combination thereof; (2) the transmembrane pore is further connected to another polypeptide, wherein the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof; (3) The transmembrane pore is a wild-type CsgG protein or a mutant thereof; preferably, the transmembrane pore has the amino acid sequence shown in SEQ ID NO:

1.

5. The method of any one of claims 1 to 4, wherein The membrane is an amphiphilic layer (eg, phospholipid bilayer) or a high molecular polymer membrane (eg, di-block, tri-block).

6. The method of any one of claims 1 to 5, wherein The double-stranded target polynucleotide has one or more characteristics selected from the following: (1) The double-stranded target polynucleotide is a double-stranded DNA and / or a double-stranded DNA-RNA hybrid; (2) the double-stranded target polynucleotide is naturally occurring and / or artificially synthesized; (3) the double-stranded target polynucleotide is obtained from a biological sample, wherein the biological sample is extracted from a virus, a prokaryotic organism (e.g., a bacterium), a eukaryotic organism (e.g., a plant (e.g., a cereal, a legume, a fruit or a vegetable), a mammal (e.g., a horse, a cow, a mouse or a human), or any combination thereof; (4) the two chains 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, a chemical linker, a polynucleotide or a polypeptide; (5) The double-stranded target polynucleotide is circular or linear.

7. The method of any one of claims 1 to 6, wherein The method has one or more characteristics selected from the following: (1) the double-stranded target polynucleotide comprises at least one single-stranded overhang (e.g., a 5'-end overhang and / or a 3'-end overhang), wherein the single-stranded overhang comprises a leader sequence, and the leader sequence guides the nucleic acid strand connected thereto into the pore; (2) in step (b), contacting the double-stranded target polynucleotide with a helicase to form a complex, and then contacting the complex with a transmembrane pore; (3) In step (b), a reagent for nanopore sequencing is contacted with the helicase; preferably, the reagent for nanopore sequencing is selected from ATP, an inorganic salt (e.g., a chloride salt, such as sodium chloride, potassium chloride, lithium chloride), a buffer (HEPES and / or Tris-HCl), EDTA, a metal ion (e.g., Mn 2+ Mg 2+ 、Co + 、Zn 2+ 、Cu 2+ 、Cu + 、Ni + 、Fe 2+ 、Fe 3+ ), or any combination thereof.

8. A kit comprising: a membrane comprising a transmembrane pore, a helicase, an ATP-generating enzyme, and an ATP-generating substrate; optionally, the kit further comprises reagents for nanopore sequencing; Preferably, the reagents for nanopore sequencing are selected from ATP, inorganic salts (e.g., chloride salts, such as sodium chloride, potassium chloride, lithium chloride), buffers (HEPES and / 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 combination thereof; Preferably, the ATP generating enzyme is selected from the group consisting of: pyruvate kinase, acetate kinase, creatine phosphokinase, serine kinase, threonine kinase, tyrosine kinase, FoF1-ATPase, polyphosphate kinase, nucleoside diphosphate kinase, or any combination thereof; Preferably, the kit comprises: a membrane comprising a transmembrane pore, a helicase, ATP, and (I) pyruvate kinase and phosphoenolpyruvate, (II) acetate kinase and potassium lithium acetyl phosphate, (III) creatine phosphokinase and creatine phosphate, (IV) FoF1-ATPase and inorganic phosphate; (V) creatine phosphokinase and creatine phosphate; (VI) polyphosphate kinase and polyphosphate; (VII) nucleoside diphosphate kinase and nucleoside triphosphate, or any one or more combinations of (I) to (VII); Preferably, the kit is used for sequencing a double-stranded target polynucleotide.

9. The kit of claim 8, wherein The kit has one or more features selected from the following: (1) The helicase is selected from Dda, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, Rep, gp41, T7gp4, or any combination thereof; (2) The helicase is further linked to another polypeptide, wherein the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof; (3) The helicase is wild-type Dda or a mutant thereof; preferably, the helicase has the amino acid sequence shown in SEQ ID NO: 2; (4) The transmembrane pore is a transmembrane protein pore or a solid-state pore; preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, Frac, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector, T7 connector, GspD, InvG, or any combination thereof; (5) The transmembrane pore is further connected to another polypeptide, wherein the other polypeptide is selected from a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable label, or any combination thereof; (6) The transmembrane pore is a wild-type CsgG protein or a mutant thereof; preferably, the transmembrane pore has the amino acid sequence shown in SEQ ID NO: 1; (7) The membrane is an amphiphilic layer (e.g., phospholipid bilayer) or a high molecular polymer membrane (e.g., di-block, tri-block).

10. Use of the kit according to claim 8 or 9 for preparing a sequencing device for sequencing a double-stranded target polynucleotide.

Images