A nanopore sequencing method mediated by liposome vesicles

Abstract

The present invention discloses a nanopore sequencing method mediated by liposome vesicles, which relates to the field of genomic sequencing. First, a closed circuit is formed in two half-pools within a flow cell. Then, multiple vesicles formed by liposomes are slowly pipetted into the organic phase using a syringe. The inner pore protein of the syringe and the sequencing sample are encapsulated in each vesicle. After the vesicles are driven by an electric field to float near the channel pore, they are punctured, and the single lipid molecular layer around the channel becomes a bilayer lipid membrane. The translocation voltage is controlled to insert the pore protein into the bilayer lipid membrane to form a nanopore in the middle of the two half-pools. A speed control protein is added near the nanopore, thereby driving the nucleic acid of the sequencing sample near the nanopore to continuously pass through the pore for sequencing at a set translocation speed. The method of the present invention can achieve a relatively high sequencing speed by regulating the speed of the protein, and the nanopore sequencing process is stable, with simple operation, high efficiency, and can achieve a relatively high capture rate.

Description

Technical Field

[0001] The present invention belongs to the field of genome sequencing, and particularly relates to a nanopore sequencing method mediated by liposome vesicles. Background Art

[0002] Nanopore sequencing technology is a new technology for rapidly determining an individual's genome and has received wide attention in the medical field. Compared with traditional gene technologies, it greatly reduces the current cost of DNA sequence analysis and can determine genomic sequences more rapidly, sensitively, and accurately. As an emerging fourth-generation DNA sequencing technology, solid-state nanopore sequencing technology has characteristics such as low cost, long read length, and easy integration, and has great advantages in terms of cost, speed, etc.

[0003] The measurement of nanopore analysis technology usually uses the method of the patch clamp direct current constant potential mode to record the ionic conductance current. When the analyte passes through the nanopore, it will cause a change in the internal electrolyte environment of the pore and generate a change in the pulsed ionic conductance current. The magnitude and duration of this change in conductance current reflect the information of the analyte passing through the nanopore, and the detection of the analyte can be achieved through the statistical analysis of a large number of pulsed currents.

[0004] However, currently, nanopore sequencing technology faces the problem of low capture rate when polynucleotides in the form of DNA or RNA pass through the nanopore, which results in low sequence coverage of the read, affects the accuracy and integrity of the sequencing result, and thus it is difficult to obtain an accurate electrical signal reflecting the sequence information.

[0005] In order to improve the capture rate of sequencing, the most commonly used existing method is to change the bias voltage that drives the DNA molecule to pass through the nanopore during the nanopore sequencing process. However, by this method, while changing the driving voltage, it will also change other sequencing factors such as the sequencing current value, which will further affect the sequencing result, and it is also very likely to affect the stability and reliability of nanopore sequencing, resulting in deviation or uncertainty of the sequencing result.

[0006] Therefore, there is an urgent need for a method that can further improve the nucleic acid capture rate on the premise of ensuring the stability and accuracy of nanopore sequencing. Summary of the Invention

[0007] In view of the low capture rate faced by current nanopore sequencing technology and the defect and problem that the existing methods for improving the capture rate are prone to affecting the stability and reliability of nanopore sequencing, thereby affecting the accuracy of the sequencing result, the present invention provides a nanopore sequencing method mediated by liposome vesicles.

[0008] The solution adopted by the present invention to solve its technical problems is: A nanopore sequencing method mediated by liposome vesicles, comprising the following steps:

[0009] S1. Separate two half-pools with the same volume in a fluid cell, and there is a 100-μm channel hole between them. Add an organic phase solution to one half-pool and an aqueous phase to the other half-pool. The organic phase is mixed with an amphiphilic lipid solution, so that a single lipid molecular layer is formed near the channel hole. The aqueous phase is a conductive buffer solution. The two half-pools are respectively connected to separate Ag / AgCl electrodes and electrically connected to an amplifier to form a closed loop.

[0010] S2. Add the sequencing sample and the pore-forming protein into a syringe, and slowly pipette multiple vesicles formed by liposomes into the organic phase with the syringe. The pore protein and the sequencing sample are encapsulated in each vesicle.

[0011] S3. After the vesicles are free near the channel hole under the drive of an electric field, pierce them. Then the single lipid molecular layer around the channel becomes a double lipid molecular layer. Control the translocation voltage to insert the pore protein into the double lipid molecular layer to form a nanopore in the middle of the two half-pools.

[0012] S4. Add a speed control protein near the nanopore, so as to drive the nucleic acid of the sequencing sample near the nanopore to continuously pass through the pore from the organic phase half-pool to the aqueous phase half-pool at a set translocation speed to achieve sequencing.

[0013] Furthermore, the channel hole is made of any one of silicon-based or Teflon materials. The shift and docking signals adopt a 5-kHz fourth-order Bessel low-pass filter, and the sampling frequency is 100 kHz. The amplifier is a patch clamp amplifier Axopatch200B, which is used to measure the change of ionic current in the loop.

[0014] Furthermore, the organic phase solution is prepared by dissolving ionized cations and / or permanent cationic lipids, amphiphilic phospholipids, steroids, and surfactants in an organic solvent.

[0015] Furthermore, the ionizable cationic lipid and / or permanent cationic lipid is one or more of 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl ester, 4-(hydroxybutyl)aza-dialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 4-(N,N-dimethylamino)butyric acid (dilinoleoyl)methyl ester, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-(2,2-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethylamine, heptadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate), and methoxypolyethylene glycol ditetradecylacetamide.

[0016] Furthermore, the amphiphilic phospholipid is one or more of dierucoyl phosphatidylcholine, diphytanoyl phosphatidylcholine (1,2-diphytanoyl-sn-glycero-3-phosphocholine, DPhPC), 1,2-distearoyl-sn-glycerophosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, or (2,3-dioleoyl-propyl)-trimethylammonium sulfate; the solvent for dissolving the amphiphilic phospholipid molecules is a hydrocarbon-containing substance, an oily substance, or a mixture of the two, including one or several of silicone oil, AR20, hexadecane, or pentane.

[0017] Furthermore, the steroid compound is one or more of cholesterol, lanosterol, sitosterol, stigmasterol, and ergosterol.

[0018] Furthermore, the aqueous phase is a phosphate buffer solution, a HEPES buffer solution containing KCl or NaCl, or a CAPS buffer solution containing KCl or NaCl.

[0019] Furthermore, the sequencing sample is a DNA, RNA, or protein biomolecule; the porin is an αHL, MspA, or OmpG pore-forming membrane protein.

[0020] Furthermore, the rate-limiting protein in S4 is phi29 DNAP, the translocation rate is controlled by the rate-limiting protein phi29 DNAP, and the translocation voltage is +180 mV.

[0021] Furthermore, for the vesicle liposome, the amphiphilic phospholipid accounts for 10% - 25% of the vesicle lipid phase.

[0022] Furthermore, the amphiphilic phospholipid molecules are first dissolved in an organic solvent, and the solvent is a hydrocarbon-containing substance, an oily substance, or a mixture of the two, specifically, it can be one or more of silicone oil, AR20, hexadecane, or pentane.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The present invention provides a nanopore sequencing method mediated by liposome vesicles. The nanopore sequencing technology of the present invention uses an electrochemical method to capture and detect proteins and DNA molecules in single cells, so it can achieve efficient single-cell protein and DNA sequencing without relying on traditional fluorescence labeling detection.

[0025] The pore protein used in the nanopore sequencing of the present invention has a novel structure and can be orderly replaced, which enables flexible adjustment and optimization of the performance of the pore protein during the sequencing process to meet the sequencing requirements of different types of single-cell samples.

[0026] The nanopore sequencing method of the present invention adopts the method of rupturing vesicles, which can achieve high-throughput DNA sequencing, and can quickly replace new vesicles after the sequencing is completed to continue the subsequent sequencing experiment. This high-efficiency sequencing method helps to improve the data output and experimental efficiency of single-cell sequencing.

[0027] The nanopore sequencing method provided by the present invention can achieve single-molecule level detection, with high sensitivity; and by regulating the speed of the protein, a high sequencing speed can be achieved, and the process of nucleic acid can also be monitored in real time to obtain the sequencing results in a timely manner. Moreover, the nanopore sequencing process is stable. Compared with traditional sequencing methods, it is simple to operate, has high efficiency, and can achieve a high capture rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the connection structure of the sequencing device of the present invention.

[0029] Figure 2 It is a schematic diagram of the nucleic acid sequencing process of the present invention.

[0030] Figure 3 It is a schematic diagram of the original sequence signal of the sequencing sample of the present invention.

[0031] Figure 4 It is a sequence list of the primers used in the present invention. EMBODIMENTS

[0032] The present invention will be further described below with reference to the drawings and embodiments.

[0033] Please refer to Figures 1-4, the present invention provides a technical solution for a nanopore sequencing method mediated by liposome vesicles: Example

[0034] This example provides a nanopore sequencing method mediated by liposome vesicles, which specifically includes the following steps:

[0035] S1. Connect the sequencing device. As Figure 1 shown, first divide the fluid cell into two half-cells with the same volume. There is a 100-μm channel pore between the two half-cells, and the channel pore is made of either silicon-based or Teflon material. An organic phase is added to one of the half-cells, which is the Cis end, and an aqueous phase is added to the other half-cell, which is the Trans end.

[0036] The organic phase and the aqueous phase respectively provide the environments at the Cis end and the Trans end. The two half-cells are respectively connected to separate Ag / AgCl electrodes and electrically connected to an Axopatch 200B patch clamp amplifier to form a closed loop. The Axopatch 200B patch clamp can record and amplify the ionic current signal generated when a single DNA molecule passes through the nanopore. The translocation and docking signals are filtered by a 5-kHz fourth-order Bessel low-pass filter, and the sampling frequency is 100 kHz. And the whole device is placed in a Faraday cage containing a copper box to minimize the coupling of external electromagnetic noise.

[0037] S2. Prepare the organic phase solution and the aqueous phase solution, including:

[0038] (1) Organic phase solution

[0039] A certain amount of amphiphilic lipid solution is mixed in the organic phase. In this example, 50% of 1,2-dioleoyl-3-dimethylammonium-propane (DODAP) is selected as the ionizable cationic lipid, 10% of 1,2-distearoyl-sn-glycero-phosphatidylcholine (DSPC) is selected as the amphiphilic phospholipid, 40% of cholesterol is selected as the steroid compound and Tween 80 are simultaneously dissolved in absolute ethanol as the organic phase solution. The total concentration of lipid substances is 20 mg / mL, and the mass fraction of Tween 80 is 0.1%.

[0040] (2) Aqueous phase solution

[0041] The aqueous phase solution is a conductive buffer solution, including phosphate buffer solution, HEPES buffer solution containing KCl or NaCl, or CAPS buffer solution containing KCl or NaCl, etc. The conductive buffer solution in this example is composed of 1 M KCl, 25 mM HEPES, and 500 mM MgCl2·6H2O, and 50 mL is prepared for use.

[0042] Mix the above organic phase solution into the Cis-end half cell, and mix the aqueous phase solution into the Trans-end half cell. The liquid levels of the solutions in the two half cells are exactly close to the vicinity of the center of the channel pore. And because the organic phase is mixed with a lipid membrane, a single lipid molecular layer will be formed by the lipid membrane near the pore.

[0043] S3. Perform a sequencing experiment using the above sequencing device. The specific process is as follows:

[0044] 1) Prepare the sequencing sample:

[0045] Prepare the synthesized map C Primer, C-Blocker, and deBruijin Primer into a 100 μM stock solution according to the instructions using TE buffer. The corresponding sequences of the above primers are as Figure 4 shown; respectively take 1 μL of the above stock solution, 4 μL of dNTP, and 13 μL of annealing buffer, and anneal on a PCR thermal cycler. Heat at 95 °C for 3 minutes, and then program the temperature to drop to 25 °C within 14 minutes; take 2 μL of the annealed sample, 10 μL of sequencing buffer, and 8 μL of nuclease-free water to prepare a 20 μL sequencing sample. The sequencing data signal of this sample is as Figure 3 shown.

[0046] Among them, the annealing buffer is prepared by mixing 1 M KCl and 10 mM HEPES, and prepare 50 mL for later use; the sequencing buffer is prepared by mixing 300 mM KCl, 10 mM HEPES, 10 mM MgCl₂·6H₂O, 10 mM (NH₄)₂SO₄, 4 mM DTT, and 5 mM ATP, and prepare 50 mL for later use.

[0047] 2) Add the above-prepared 10 μL sequencing sample and the pore-forming protein with a final concentration of 0.25 ng / mL to a syringe, and slowly blow multiple vesicles into the organic phase with the syringe. In this way, the pore protein and the detection sample in the syringe are wrapped in individual vesicles. The vesicles are formed by liposomes, and the amphiphilic phospholipids account for 10%-20% of the lipid phase of the vesicles.

[0048] 3) After the vesicles are free near the channel pore under the drive of an electric field, use a pointed tool to pierce the vesicles. Then the single lipid molecular layer near the pore becomes a double lipid molecular layer. Continuously control the translocation voltage at +180 mV to insert the pore protein into the lipid bilayer to form a nanopore in the middle of the two half cells. At this time, the detected nucleic acid also aggregates near the pore. Then add a certain amount of speed control protein phi29 DNAP near the pore to control the translocation speed. At this time, the nucleic acid continuously passes through the pore for sequencing at a certain translocation speed to obtain a sequence signal. Later, the base signal can be distinguished through data conversion, effectively improving the detection capture rate.

[0049] Further, the above-mentioned detection sample can be a biomolecule such as DNA, RNA or protein; the above-mentioned porin can be a pore-forming membrane protein such as αHL, MspA or OmpG.

[0050] As Figure 2 shown, where phi29 DNAP is the speed-controlling protein, and the porin MspA has a conical structure. As a nano-trap, it analyzes the structure of the protein to be detected in a state of partially accommodating it. It has the advantages of being easy to modify, having strong stability, and being able to withstand acidic and alkaline environments, high temperatures and high voltages. In the figure, the test sample moves from the Cis side (the side where DNA is loaded) to the Trans side (the side where DNA exits). The translocation speed is controlled by phi29 DNAP, so that the nucleic acid of the sample to be detected continuously passes through the pore for sequencing at a set translocation speed, thus effectively improving the detection capture rate.

[0051] The nanopore sequencing method provided by the present invention can achieve single-molecule level detection, with high sensitivity; and by regulating the speed of the protein, a high sequencing speed can be achieved, and the process of nucleic acid can also be monitored in real time to obtain the sequencing results in a timely manner. Moreover, the nanopore sequencing process is stable. Compared with traditional sequencing methods, it is simple to operate, has high efficiency, and can achieve a high capture rate.

[0052] The above are only the preferred embodiments of the present invention and do not limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nanopore sequencing method mediated by liposome vesicles, characterized in that: The following steps are involved: S1. Separate two half-pools of the same volume in the fluid pool, and set a channel hole of 100 μm between the two. Add an organic phase solution into one half-pool and add an aqueous phase into the other half-pool. The organic phase is prepared by dissolving ionizable cations and / or permanent cationic lipids, amphiphilic phospholipids, steroid compounds, and surfactants in an organic solvent, so as to form a single lipid molecule layer near the channel hole. The aqueous phase is a conductive buffer solution. The two half cells were connected to separate Ag / AgCl electrodes and electrically connected to the amplifier to form a closed loop; S2, adding the sequencing sample and the pore-forming protein into a syringe, and slowly blowing a plurality of vesicles formed by liposomes into the organic phase with the syringe, wherein the pore protein and the sequencing sample are encapsulated in the vesicles; S3, after the vesicles are freed near the channel pore under the driving of the electric field, they are punctured one by one, and the single molecular layer around the channel is transformed into a double lipid molecular layer. The translocation voltage is controlled to make the porin insert into the double lipid molecular layer to form a nanopore in the middle of the two half pools; S4. Add a rate-controlling protein near the nanopore to drive the sequencing sample nucleic acid near the nanopore to continuously pass from the organic phase half-cell to the aqueous phase half-cell at a set translocation speed to achieve sequencing.

2. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The channel hole is made of either silicon or Teflon material; the displacement and docking signals use a 5kHz quadrupole Bessel low-pass filter with a sampling frequency of 100kHz; the amplifier is a patch clamp amplifier Axopatch 200B, which is used to measure the change of ion current in the loop.

3. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The ionizable cationic lipid and / or permanent cationic lipid are 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleyl N, N-dimethyl-3-aminopropane (DODMA), 4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl lipid, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dimyristoyl One or more of coryl-rac-glycerol-3-methoxypolyethylene glycol, 2-(2,2-bis((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethylamine, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((decyloxy)hexyl)amino)octanoate), and methoxypolyethylene glycol ditetradecyl acetamide.

4. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The amphiphilic phospholipid is one or more of dierucylphosphatidylcholine, diphytanoylphosphatidylcholine (1,2-diphytanoyl-sn-glycero-3-phosphocholine, DPhPC), 1,2-distearoyl-sn-glycerophosphatidylcholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine or (2,3-dioleoyl-propyl)-trimethylamine sulfate; the solvent for dissolving the amphiphilic phospholipid molecules is a hydrocarbon-containing substance, an oily substance or a mixture of the two; the hydrocarbon-containing substance is one or both of hexadecane and pentane; and the oily substance is silicone oil.

5. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The steroidal compound is one or more of cholesterol, lanosterol, sitosterol, stigmasterol and ergosterol.

6. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The aqueous phase is a phosphate buffer solution, a HEPES buffer solution containing KCl or NaCl, or a CAPS buffer solution containing KCl or NaCl.

7. The method of nanopore sequencing mediated by liposome vesicles according to claim 1, characterized in that: The sequencing sample is a DNA, RNA or protein biological molecule; the porin is an αHL, MspA or OmpG pore-forming membrane protein.

8. The liposome vesicle-mediated nanopore sequencing method according to claim 1, characterized in that: The rate-controlling protein in the S4 is phi29 DNAP, the translocation speed is controlled by the rate-controlling protein phi29 DNAP, and the translocation voltage is +180 mV.

Images