Protein sequencing method based on nano-motion platform and nano-pore

Abstract

The invention relates to the technical field of protein detection, and discloses a protein sequencing method based on a nano motion platform and a nanopore, which comprises the following steps: step 1, processing a nano through hole, step 2, constructing a detection platform, step 3, electrically driving and embedding a functional protein into a solid nanopore, and detecting the protein by adopting a protein sequencing system. According to the protein sequencing method based on the nano-motion platform and the nano-pore, the distance between the driving protein in the maintaining device and the reading protein in the chip film is adjusted by using the sub-nano single-molecule control system, and the problem that the length reading is difficult due to the limitation of a nano-space during protein molecule detection at present is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein detection, and in particular to a protein sequencing method based on a nano motion platform and a nanopore. Background Art

[0002] Modern proteomics relies heavily on tandem mass spectrometry (MS), which is valued for its high precision and ability to identify and quantify proteins in complex mixtures. However, most mass spectrometers are bulky, have high investment costs, are expensive to maintain, and require professional personnel to operate. With the growing demand for high-throughput proteomics research and personalized medicine, there is an urgent need to develop scalable and low-cost protein analysis technologies.

[0003] Compared with mass spectrometry equipment, nanopore-based detection technology provides a low-cost and high-throughput platform that can adapt to the native environment. In nanopore analysis, when the analyte passes through a single nanopore, the ionic current passing through the nanopore is blocked under the action of an applied potential. Importantly, the magnitude of the current blockage is mainly proportional to the volume excluded by the analyte, which allows size differentiation of chemically similar (bio)polymers such as PEG chains, DNA, proteins, and peptides. In addition, nanopores can accurately detect a variety of molecules, including proteins and DNA, and have the advantages of being label-free, rapid, and highly accurate at the single-molecule level.

[0004] At present, biological nanopore detection technology has been proven to achieve highly sensitive detection and resolution of 20 amino acids. Recently, Martin-Baniandres et al. used an engineered charged selective nanopore to use electroosmosis to achieve non-enzymatic capture, unfolding and transport of single peptide chains, and to detect post-translational modifications within peptide chains. The length of these peptide chains can exceed 1200 amino acid residues, providing a possibility for nanopores to achieve long-read protein sequencing. Nova et al. used Hel308 helicase to control the peptide chain through the sensing area of ​​the nanopore, realizing the detection of phosphorylation modifications at the single molecule level, and can distinguish peptide sequences with one or two closely phosphorylation sites with an accuracy of 95%. However, due to the spatial limitation of the nanopore, the detection length of this technology can only be 15-20 amino acids. Motone et al. used ClpX helicase nanopores to pull proteins through CsgG, which can perform long-distance, single-molecule reading of single protein molecules and achieve sequencing of complete protein chains. However, this method involves the pre-treatment of the target peptide chain and the binding efficiency of ClpX helicase, which limits the application of this technology to a certain extent. Therefore, protein (peptide) sequencing still faces challenges such as short read length, poor stability, and low accuracy. Therefore, achieving stable, long read length, and high-accuracy protein sequencing is of great significance to life science research.

[0005] Therefore, a protein sequencing method based on a nano-motion platform and nanopores is proposed. A solid-state nanopore channel is processed on a chip membrane, and a protein with high-resolution reading capabilities for various molecules including proteins and DNA is embedded in the solid-state nanopore to form a reading part for peptide chain sequence information; a holding device is fixed on the upper end of the chip film through a sub-nanometer single-molecule manipulation system, and the holding device can be connected or combined with a protein with a biomolecular motor function. The sub-nanometer single-molecule manipulation system is used to control the holding device to move up and down with nanometer-level precision, so that the distance between the functional protein fixed on the holding device and the functional protein embedded in the nanopore of the chip film can be adjusted at the nanometer level, which can effectively solve the problem of short read length caused by space limitations in protein sequencing.

[0006] [1]Martin-Baniandres, P., Lan, WH., Board, S. et al. Enzyme-lessnanopore detection of post-translational modifications within longpolypeptides. Nat. Nanotechnol. 18, 1335–1340 (2023). [2]Nova, IC, Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol 42, 710–714 (2024). [3]Motone, K., Kontogiorgos-Heintz, D., Wee, J. et al. Multi-pass, single-molecule nanopore reading of long protein strands. Nature 633, 662–669(2024). Summary of the invention

[0007] The purpose of the present invention is to provide a protein sequencing method based on a nano-motion platform and a nanopore, which utilizes a sub-nanometer single-molecule manipulation system to adjust the distance between the driving protein in the holding device and the reading protein in the chip film, thereby overcoming the problem of difficulty in reading length due to the limitation of nano-space during current protein molecule detection.

[0008] To achieve the above object, the present invention provides a protein sequencing method based on a nanomotion platform and a nanopore, comprising the following steps: 1) Construction of protein sequencing system: Step 1, processing nano-through holes: manufacturing solid-state nano-holes on the chip film and the holding device respectively; Step 2, detection platform construction: combining the holding device connected to the functional protein with the sub-nanometer single-molecule manipulation system, and assembling it with the chip film with nanopores processed; Step 3, electrically driven embedding of functional proteins into solid-state nanopores: two functional proteins with the ability to read proteins and DNA and control translocation speed are respectively embedded into solid-state nanopores to obtain a protein sequencing system based on a nanomotion platform and nanopores; 2) Perform protein sequencing: Step 4: Under the action of the functional protein with the ability to control the translocation speed, the peptide chain-modified DNA molecule can achieve complete reading of the peptide chain information through the functional protein with the reading ability, and the distance between the chip film and the holding device is adjusted by the sub-nanometer single-molecule manipulation system.

[0009] Furthermore, the method for preparing nanopores on the chip film in step 1 includes electrical breakdown, focused ion beam, focused electron beam; the holding device is connected to the functional protein, and the holding device includes carbon nanotubes, glass nanotubes, functionalized materials with a tip of 1-100nm, etc.

[0010] Furthermore, in step 2, the sub-nanometer single-molecule manipulation system is a nano-displacement motion platform, and the distance between the holding device and the nanopores of the chip film is adjusted by the sub-nanometer single-molecule manipulation system.

[0011] Furthermore, in step 3, the functional protein with reading capability is embedded in the solid nanopore of the chip film, and the functional protein with translocation speed control capability is embedded in the solid nanopore of the holding device.

[0012] The detection principle of the present invention is: The peptide-modified DNA molecules, under the action of functional proteins with the ability to control the translocation speed, pass through functional proteins with reading capabilities, and at the same time, use a sub-nanometer single-molecule manipulation system to control the up and down movement of the holding device with nanometer-level precision, so that the distance between the functional protein fixed on the holding device and the functional protein embedded in the nanopores of the chip film can be adjusted at the nanometer level, thereby achieving complete reading of the peptide chain information.

[0013] The advantages and positive effects of the protein sequencing method based on the nanomotion platform and nanopore described in the present invention are: The present invention processes a solid-state nanopore channel on a chip film, embeds a protein with high-resolution reading capability for various molecules including protein and DNA into the solid-state nanopore, and forms a reading portion for peptide chain sequence information; a holding device is fixed at the upper end of the chip film by a sub-nanometer single-molecule manipulation system, and the holding device can be connected or combined with a protein having a biomolecular motor function, and the holding device is controlled to move up and down with nanometer-level precision by the sub-nanometer single-molecule manipulation system, so that the distance between the functional protein fixed on the holding device and the functional protein embedded in the nanopore of the chip film can be adjusted at the nanometer level, effectively solving the problem of short read length due to space limitations in protein sequencing.

[0014] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic diagram of a protein sequencing system according to an embodiment of the present invention; Figure 2 Schematic diagram of protein molecule sequencing according to an embodiment of the present invention.

[0016] Reference numerals 1 and 2 are chip films; 3 is a protein with reading capability; 4 is a protein with translocation speed control capability; 5 is a glass nanotube. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0018] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0019] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0020] A protein sequencing method based on a nanomotion platform and a nanopore comprises the following steps: 1. Construction of protein sequencing system: Step 1, processing nano-through holes: using the focused helium ion beam method to prepare solid-state nano-holes in the chip film, using the laser drawing method to prepare glass nano-holes and fix them on the holding device, and the functional protein is fixed or connected to the tip of the glass nano-hole.

[0021] Step 2, detection platform construction: combine the holding device connected to the functional protein with the sub-nano single-molecule manipulation system, and assemble them with the chip film with nanopores processed; the sub-nano single-molecule manipulation system is a nano-displacement motion platform, and the distance between the holding device and the nanopores of the chip film is adjusted by the sub-nano single-molecule manipulation system.

[0022] Step 3, electrically driven embedding of functional proteins into solid-state nanopores: two functional proteins with the ability to read proteins and DNA and the ability to control translocation speed are respectively embedded into solid-state nanopores to obtain a protein sequencing system based on a nanomotion platform and nanopores. Among them, the functional protein with the ability to read (such as MspA) is embedded in the solid-state nanopore of the chip film, and the functional protein with the ability to control translocation speed (such as phi29-DNAP) is embedded in the solid-state nanopore of the holding device.

[0023] 2. Peptide chain read length detection: Step 4: Under the action of the functional protein with the ability to control the translocation rate, the peptide chain-modified DNA molecule can read the peptide chain information completely through the functional protein with reading ability embedded in the solid nanopore of the chip film.

[0024] The following is a detailed description using MspA with reading ability, phi29-DNAP with translocation speed control ability, and glass nanotubes as examples.

[0025] Example A protein sequencing method based on a nanomotion platform and a nanopore comprises the following steps: 1. Construction of protein sequencing system (such as Figure 1 shown): (1) Nanopore processing on chip films and glass nanotubes: Use the focused helium ion beam method to prepare solid-state nanopores on chip films, and use the laser drawing method to prepare glass nanopores. One solid-state nanopore is made on the chip film and one on the glass nanotube respectively. (2) Construction of the detection platform: The glass nanotube with nanopores is combined with the sub-nanometer single-molecule manipulation system, and placed in a KCl solution together with the chip film with nanopores, and electrodes are applied at both ends of the solution. The distance between the glass nanopore and the chip film nanopore is adjusted by the sub-nanometer single-molecule manipulation system; (3) Electrically driven embedding of functional proteins into solid-state nanopores: A solution containing MspA protein is added to the end of the chip film, and a certain bias voltage is applied to allow MspA to embed into the solid-state nanopores of the chip film; a solution containing phi29-DNAP is injected into the glass nanotube. Under the action of the electric field force of the bias voltage, the protein moves to the tip of the glass nanotube and embeds into the solid-state nanopore at that position.

[0026] 2. Peptide chain read length detection (detection process as follows Figure 2 shown): (4) Read length detection of the peptide-modified portion of the DNA molecule: When phi29-DNAP uses a single-stranded DNA molecule as a template to synthesize a double-stranded DNA molecule, it drives the peptide-modified portion of the DNA through the MspA embedded in the solid-state nanopore at the end of the chip film to start reading the peptide sequence. The distance between the chip film position and the glass nanotube is adjusted by a sub-nanometer single-molecule manipulation system to avoid the phenomenon of termination of reading due to the movement of the peptide-modified end of the DNA molecule to the phi29-DNAP position.

[0027] The present invention processes a solid-state nanopore channel on a chip film, embeds a protein with high-resolution reading capability for various molecules including protein and DNA into the solid-state nanopore, and forms a reading portion for peptide chain sequence information; a holding device is fixed at the upper end of the chip film by a sub-nanometer single-molecule manipulation system, and the holding device can be connected or combined with a protein having a biomolecular motor function, and the holding device is controlled to move up and down with nanometer-level precision by the sub-nanometer single-molecule manipulation system, so that the distance between the functional protein fixed on the holding device and the functional protein embedded in the nanopore of the chip film can be adjusted at the nanometer level, effectively solving the problem of short read length due to space limitations in protein sequencing.

[0028] Therefore, the present invention adopts the above-mentioned protein sequencing method based on a nano-motion platform and a nanopore, and utilizes a sub-nanometer single-molecule manipulation system to adjust the distance between the driving protein in the holding device and the reading protein in the chip film, thereby overcoming the problem of difficulty in reading length due to the limitation of nano-space during current protein molecule detection.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A protein sequencing method based on a nanomotion platform and a nanopore, characterized in that: The following steps are involved: 1) Construction of protein sequencing system: Step 1, processing nano-through holes: manufacturing solid-state nano-holes on the chip film and the holding device respectively; Step 2, detection platform construction: combining the holding device connected to the functional protein with the sub-nanometer single-molecule manipulation system, and assembling it with the chip film with nanopores processed; Step 3, electrically driven embedding of functional proteins into solid-state nanopores: two functional proteins with the ability to read proteins and DNA and control translocation speed are respectively embedded into solid-state nanopores to obtain a protein sequencing system based on a nanomotion platform and nanopores; 2) Perform protein sequencing: Step 4: Under the action of the functional protein with the ability to control the translocation speed, the peptide chain-modified DNA molecule can achieve complete reading of the peptide chain information through the functional protein with the reading ability, and the distance between the chip film and the holding device is adjusted by the sub-nanometer single-molecule manipulation system.

2. A protein sequencing method based on a nanomotion platform and a nanopore according to claim 1, characterized in that: The method for preparing nanopores on the chip film in step 1 includes electrical breakdown, focused ion beam, and focused electron beam; the holding device is connected to the functional protein, and the holding device includes carbon nanotubes, glass nanotubes, and functionalized materials with a tip of 1-100nm.

3. A protein sequencing method based on a nanomotion platform and a nanopore according to claim 1, characterized in that: In step 2, the sub-nanometer single-molecule manipulation system is a nano-displacement motion platform, and the distance between the holding device and the nanopores of the chip film is adjusted by the sub-nanometer single-molecule manipulation system.

4. The protein sequencing method based on a nanomotion platform and a nanopore according to claim 1, characterized in that: In step 3, the functional protein with reading capability is embedded in the solid nanopore of the chip film, and the functional protein with translocation speed control capability is embedded in the solid nanopore of the holding device.

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