A protein sequencing method based on a nano-motion platform and a nanopore

Abstract

The present invention relates to the technical field of protein detection, and discloses a protein sequencing method based on a nano-motion platform and a nanopore, comprising the following steps: Step 1, fabricating a nano-through hole; Step 2, building a detection platform; Step 3, electrically driving a functional protein to be embedded into a solid-state nanopore, and performing protein detection by using a protein sequencing system. By adopting the above-mentioned protein sequencing method based on a nano-motion platform and a nanopore, the present invention utilizes a sub-nanometer single-molecule manipulation system to adjust and maintain the distance between the driving protein in the holding device and the reading protein in the chip film, overcoming the problem of difficult read length in current protein molecule detection due to the limitation of the nano space.

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 largely relies on tandem mass spectrometry (MS) technology, which is valued for its high precision and the ability to identify and quantify proteins in complex mixtures. However, most mass spectrometers are bulky, have high investment costs, expensive maintenance fees, and require professional operation. 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 and can adapt to the native environment. In nanopore analysis, when an analyte passes through a single nanopore, an applied potential will block the ionic current passing through the nanopore. Importantly, the magnitude of the current blockage is mainly proportional to the volume excluded by the analyte, which allows size discrimination of chemically similar (bio)polymers such as PEG chains, DNA, proteins, and peptides. In addition, nanopores can accurately detect various molecules including proteins and DNA and have the advantages of label-free, fast, and high precision at the single-molecule level.

[0004] Currently, it has been proven that biological nanopore detection technology can achieve highly sensitive detection and resolution of 20 amino acids. Recently, Martin-Baniandres et al. used an engineered charge-selective nanopore to utilize the electroosmotic phenomenon to achieve non-enzymatic capture, unfolding, and translocation of a single peptide chain, and realized the detection of post-translational modifications within the peptide chain. 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 the Hel308 helicase to control the passage of peptide chains through the sensing region of the nanopore, achieving the detection of phosphorylation modifications at the single-molecule level and being able to distinguish peptide sequences with one or two closely spaced phosphorylation sites with 95% accuracy. 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 the ClpX helicase nanopore to pull proteins through CsgG, enabling long-distance, single-molecule reading of single protein molecules and realizing the sequencing of complete protein chains. However, this method involves the pretreatment of the target peptide chain and the problem of the binding efficiency of the ClpX helicase, which somewhat limits the application of this technology. Therefore, in protein (peptide segment) sequencing, there are still challenges such as short read lengths, poor stability, and low accuracy. Therefore, achieving stable, long-read, and highly accurate protein sequencing is of great significance for 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 fabricated on a chip membrane, and a protein with high-resolution reading ability for various molecules including proteins and DNA is embedded into the solid-state nanopore to form a reading part for peptide chain sequence information. At the upper end of the chip thin film, a holding device is fixed by a sub-nanometer single-molecule manipulation system. The holding device can be connected or combined with a protein having the function of a biomolecular motor. The sub-nanometer single-molecule manipulation system is used to control the up and down movement of the holding device with nanometer-level precision, so as to realize nanometer-level adjustment of the distance between the functional protein fixed on the holding device and the functional protein embedded in the nanopore of the chip thin film, effectively solving the problem of short read lengths caused by space limitations in protein sequencing.

[0006] [1] Martin-Baniandres, P., Lan, WH., Board, S. et al. Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nat. Nanotechnol. 18, 1335–1340 (2023).

[0007] [2] Nova, I.C., Ritmejeris, J., Brinkerhoff, H. et al. Detection of phosphorylation post-translational modifications along single peptides with nanopores. Nat Biotechnol 42, 710–714 (2024).

[0008] [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

[0009] The object of the present invention is to provide a protein sequencing method based on a nano motion platform and a nanopore, which uses a sub-nano single molecule manipulation system to adjust the distance between the kinesin in the holding device and the reading protein in the chip film, overcoming the problem of difficult read length due to the limitation of the nano space during the current protein molecule detection.

[0010] To achieve the above object, the present invention provides a protein sequencing method based on a nano motion platform and a nanopore, comprising the following steps:

[0011] 1) Construct a protein sequencing system:

[0012] Step 1, fabricate a nano through-hole: respectively fabricate solid-state nanopores on the chip film and the holding device;

[0013] Step 2, build a detection platform: combine the holding device connected with the functional protein and the sub-nano single molecule manipulation system, and assemble them with the chip film processed with nanopores;

[0014] Step 3, electrically drive and embed the functional proteins into the solid-state nanopores: respectively embed two functional proteins with the ability to read proteins and DNA and the ability to control the translocation speed into the solid-state nanopores to obtain a protein sequencing system based on a nano motion platform and a nanopore;

[0015] 2) Perform protein sequencing:

[0016] Step 4, under the action of the functional protein with the ability to control the translocation speed, the DNA molecule modified with a peptide chain passes through the functional protein with the ability to read, realizing the complete reading of the peptide chain information. The distance between the chip film and the holding device is adjusted by the sub-nano single molecule manipulation system.

[0017] Further, the method for preparing the nanopore on the chip film in Step 1 includes electrical breakdown, focused ion beam, and focused electron beam; the holding device is connected with the functional protein, and the holding device includes carbon nanotubes, glass nanotubes, functionalized materials with a tip of 1 - 100 nm, etc.

[0018] Further, in Step 2, the sub-nano single molecule manipulation system is a nano displacement motion platform, and the distance between the holding device and the nanopore of the chip film is adjusted by the sub-nano single molecule manipulation system.

[0019] Further, in Step 3, the functional protein with the ability to read is embedded into the solid-state nanopore of the chip film, and the functional protein with the ability to control the translocation speed is embedded into the solid-state nanopore of the holding device.

[0020] The detection principle of the present invention is:

[0021] The DNA molecule modified by peptide chain, under the action of a functional protein with translocation speed control ability, passes through a functional protein with reading ability, and at the same time, the up and down movement of the holding device is controlled with nanoscale precision through a 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 thin film is adjusted at the nanoscale, realizing the complete reading of peptide chain information.

[0022] The advantages and positive effects of a protein sequencing method based on a nanomotion platform and nanopore according to the present invention are as follows:

[0023] In the present invention, a solid-state nanopore channel is processed on a chip thin film, and a protein with high-resolution reading ability 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 at the upper end of the chip thin film through a sub-nanometer single-molecule manipulation system. The holding device can be connected or combined with a protein with the function of a biomolecular motor. The up and down movement of the holding device is controlled with nanoscale precision through 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 thin film is adjusted at the nanoscale, effectively solving the problem of short read length caused by space limitation in protein sequencing.

[0024] The technical solution of the present invention will be further described in detail below with reference to the drawings and embodiments. Description of the Drawings

[0025] Figure 1 Schematic diagram of the protein sequencing system according to the embodiment of the present invention;

[0026] Figure 2 Schematic diagram of protein molecule sequencing according to the embodiment of the present invention.

[0027] Reference Signs

[0028] 1, 2 are chip thin films; 3 is a protein with reading ability; 4 is a protein with translocation speed control ability; 5 is a glass nanotube. Detailed Embodiments

[0029] The technical solution of the present invention will be further described below with reference to the drawings and embodiments.

[0030] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0031] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are all commercially available as conventional products.

[0032] A protein sequencing method based on a nanomotion platform and nanopore includes the following steps:

[0033] 1. Construct a protein sequencing system:

[0034] Step 1, fabricate nano-through holes: Use the focused helium ion beam method to fabricate solid-state nanopores in the chip thin film, use the laser pulling method to fabricate glass nanopores and fix them on the holding device, and fix or connect functional proteins to the tips of the glass nanopores.

[0035] Step 2, build a detection platform: Combine the holding device connected to the functional protein with the sub-nanometer single-molecule manipulation system, and assemble it with the chip thin film with fabricated nanopores; the sub-nanometer single-molecule manipulation system is a nano-displacement motion platform, and the distance between the holding device and the nanopores in the chip thin film is adjusted through the sub-nanometer single-molecule manipulation system.

[0036] Step 3, electro-drive the embedding of functional proteins into solid-state nanopores: Embed two functional proteins with the ability to read proteins and DNA and control the translocation speed into the solid-state nanopores respectively to obtain a protein sequencing system based on the nano-motion platform and nanopores. Among them, the functional protein with the ability to read (such as MspA) is embedded into the solid-state nanopores of the chip thin film, and the functional protein with the ability to control the translocation speed (such as phi29-DNAP) is embedded into the solid-state nanopores of the holding device.

[0037] 2. Detection of the read length of the peptide chain:

[0038] Step 4, under the action of the functional protein with the ability to control the translocation speed, the DNA molecule modified with the peptide chain passes through the functional protein with the ability to read embedded in the solid-state nanopores of the chip thin film to achieve the complete reading of the peptide chain information.

[0039] The following takes MspA with the ability to read, phi29-DNAP with the ability to control the translocation speed, and glass nanotubes as examples for detailed description.

[0040] Example

[0041] A protein sequencing method based on a nano-motion platform and nanopores includes the following steps:

[0042] 1. Construct a protein sequencing system (as Figure 1 shown):

[0043] (1) Fabricate nanopores on the chip thin film and glass nanotube: Use the focused helium ion beam method to fabricate solid-state nanopores in the chip thin film, use the laser pulling method to fabricate glass nanopores, and fabricate a solid-state nanopore on the chip thin film and the glass nanotube respectively;

[0044] (2) Detection platform construction: Combine the glass nanotube with nanopores prepared with the sub-nanometer single-molecule manipulation system, place it together with the chip film with processed nanopores in a KCl solution, and apply electrodes at both ends of the solution. Adjust the distance between the glass nanopore and the nanopore of the chip film through the sub-nanometer single-molecule manipulation system;

[0045] (3) Electrically driven embedding of functional proteins into solid-state nanopores: Add the solution containing MspA protein to the chip film end, and apply a certain bias voltage to embed MspA into the solid-state nanopore of the chip film; Inject the solution containing phi29-DNAP into the glass nanotube. Under the action of the electric field force of the bias voltage, this protein moves to the tip position of the glass nanotube and embeds into the solid-state nanopore at this position.

[0046] 2. Detection of the reading length of the peptide chain (the detection process is as Figure 2 shown):

[0047] (4) Detection of the reading length of the part of the DNA molecule modified by the peptide chain: During the process of phi29-DNAP synthesizing a double-stranded DNA molecule using a single-stranded DNA molecule as a template, the region of the DNA modified by the peptide drives through MspA embedded in the solid-state nanopore at the chip film end, and starts to read the peptide chain sequence. The distance between the chip film position and the glass nanotube is adjusted through the sub-nanometer single-molecule manipulation system to avoid the phenomenon of reading termination caused by the movement of the peptide-modified end on the DNA molecule to the phi29-DNAP position.

[0048] In the present invention, a solid-state nanopore channel is processed on the chip film, and proteins with high-resolution reading ability for various molecules including proteins and DNA are embedded into this solid-state nanopore to form a reading part for peptide chain sequence information; A holding device is fixed at the upper end of the chip film through the sub-nanometer single-molecule manipulation system. The holding device can be connected or combined with proteins having the function of a biomolecular motor. The sub-nanometer single-molecule manipulation system is used to precisely control the up and down movement of the holding device at the nanometer level, 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 reading length caused by space limitation in protein sequencing.

[0049] Therefore, the present invention adopts the above-mentioned protein sequencing method based on a nano-motion platform and nanopores, and uses the 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, overcoming the problem of difficult reading length due to the limitation of the nano space during the current detection of protein molecules.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements do not enable the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A protein sequencing method based on a nano-motion platform and a nanopore, characterized in that, It includes the following steps: 1) Construct a protein sequencing system: Step 1, fabricate nano-through holes: Fabricate solid-state nanopores on the chip thin film and the holding device respectively; Step 2, build a detection platform: Combine the holding device connected with the functional protein and the sub-nanometer single-molecule manipulation system, and assemble them with the chip thin film with nanopores processed; Step 3, electro-drive the functional proteins into the solid-state nanopores: Respectively embed two functional proteins with the ability to read proteins and DNA and the ability to control the translocation speed into the solid-state nanopores to obtain a protein sequencing system based on the nano-motion 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 DNA molecule modified with a peptide chain passes through the functional protein with the ability to read, realizing the complete reading of the peptide chain information. The distance between the chip thin film and the holding device is adjusted by the sub-nanometer single-molecule manipulation system.

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

3. A protein sequencing method based on a nano-motion 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 nanopore of the chip thin film is adjusted through the sub-nanometer single-molecule manipulation system.

4. A protein sequencing method based on a nano-motion platform and a nanopore according to claim 1, characterized in that: In Step 3, the functional protein with the ability to read is embedded in the solid-state nanopore of the chip thin film, and the functional protein with the ability to control the translocation speed is embedded in the solid-state nanopore of the holding device.

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