Nucleic acid-polypeptide complex and preparation method therefor

Abstract

Provided is a nucleic acid-polypeptide complex which comprises a single-stranded nucleic acid and a polypeptide fragment, wherein both ends of the polypeptide fragment are covalently linked to both ends of the single-stranded nucleic acid, respectively. The nucleic acid-polypeptide complex has a high coupling efficiency, a high yield, and a simple preparation method.

Description

Nucleic acid-peptide complexes and their preparation methods Technical Field

[0001] This invention relates to the field of protein sequencing, and more specifically, to nucleic acid-peptide complexes and methods for their preparation. Further, it relates to peptide libraries, kits, applications, and methods for peptide detection. Background Technology

[0002] Peptides are compounds formed by amino acids linked by peptide bonds and are intermediate products of protein hydrolysis. They play crucial roles in human growth, development, immune regulation, and metabolism, and are also the main active ingredients in some medicinal plants. Currently, there are various methods for interpreting peptide amino acid sequences, including Adams degradation, mass spectrometry, and nanopore sequencing. However, existing peptide sequencing methods have relatively low accuracy.

[0003] Therefore, there is an urgent need to develop a new peptide sequencing method to improve the accuracy of peptide sequencing.

[0004] Summary of the Invention

[0005] The present invention aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a nucleic acid-peptide complex.

[0006] This invention is based on the following discoveries of the inventors:

[0007] Nanopore sequencing technology has seen continuous development in recent years, demonstrating great potential in peptide sequencing. However, using nanopore sequencing for peptide sequencing presents challenges such as low coupling efficiency for large molecules, cumbersome procedures, high byproduct yields, and difficulties in product purification due to reliance on special modifications. To overcome these problems, the inventors constructed a nucleic acid-peptide complex. This nucleic acid-peptide complex exhibits high coupling efficiency, high yield, and a simple preparation method.

[0008] In a first aspect, the present invention provides a nucleic acid-peptide complex. According to embodiments of the invention, the nucleic acid-peptide complex comprises a single-stranded nucleic acid and a peptide fragment; wherein the two ends of the peptide fragment are covalently linked to the two ends of the single-stranded nucleic acid, respectively. In the nucleic acid-peptide complex according to embodiments of the invention, the nucleic acid fragment in the leader region can guide peptides with different electrical charges into the pore, and the nucleic acid fragment in the rate-controlling region provides a motor protein binding region, thereby controlling the rate of peptide passage through the pore in nanopore detection.

[0009] In a second aspect, the present invention provides a method for preparing the nucleic acid-peptide complex described in the first aspect. According to an embodiment of the present invention, the method includes: annealing a single-stranded bridging oligonucleotide fragment with a single-stranded nucleic acid, and covalently linking the annealed product with a peptide fragment to obtain the nucleic acid-peptide complex. The method of the present invention is simple to prepare, has high coupling efficiency, yields a high amount of the obtained nucleic acid-peptide complex, and produces few byproducts.

[0010] In a third aspect, the present invention provides a polypeptide library. The polypeptide library according to embodiments of the present invention comprises linearized nucleic acid-peptide molecules obtained by the method described in the second aspect of the present invention. During nanopore sequencing, the polypeptide library of the present invention allows for controllable passage speed of polypeptides of different lengths and charges through the nanopore, thereby obtaining a good sequencing signal.

[0011] In a fourth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises (i) a single-stranded nucleic acid, the single-stranded nucleic acid including a leader region and a rate-controlling region, the leader region and the rate-controlling region being linked by a modified molecule or a breakable chemical bond; and (ii) a single-stranded bridging oligonucleotide fragment, the two ends of the single-stranded bridging oligonucleotide fragment being capable of being linked to the two ends of the single-stranded nucleic acid respectively through base complementary pairing. The kit of the present invention can be used to prepare peptide libraries with high coupling efficiency and high yield.

[0012] In a fifth aspect, the present invention proposes the use of the nucleic acid-peptide complex described in the first aspect of the present invention, the peptide library described in the third aspect of the present invention, and the kit described in the fourth aspect of the present invention in high-throughput sequencing.

[0013] In a sixth aspect, the present invention provides a method for detecting peptides. According to an embodiment of the present invention, the method includes: adding the peptide library described in the third aspect of the present invention into a detection solution chamber; under the action of an electric field, controlling the peptide library to pass through a nanopore via a motor protein, thereby acquiring an electrical signal corresponding to the peptide; and decoding the electrical signal to determine the amino acid information of the peptide. The method according to the embodiment of the present invention can accurately obtain the amino acid sequence information and amino acid modification information of the peptide.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0016] Figure 1 is a schematic diagram of single-stranded nucleic acid based on template reaction in Example 1.

[0017] Figure 2 is a schematic diagram of the peptide fragment based on template reaction in Example 1.

[0018] Figure 3 is a schematic diagram of the target product and byproducts in the nucleic acid-peptide complex based on template reaction in Example 1.

[0019] Figure 4 is a schematic diagram of the enrichment of the linearized nucleic acid-peptide molecules using trapping magnetic beads based on template reaction in Example 1.

[0020] Figure 5 is a schematic diagram of the nucleic acid-peptide complex sequencing library based on template reaction in Example 1.

[0021] Figure 6 is a schematic diagram of the nucleic acid-peptide complex sequencing library based on non-template reaction in Example 2.

[0022] Figure 7 shows the modified gel images of the coupling products in Examples 1 and 2.

[0023] Figure 8 shows the nanopore sequencing results of the nucleic acid-peptide complex based on template reaction in Example 1.

[0024] Figure 9 shows the nanopore sequencing results of the nucleic acid-peptide complex based on non-template reaction in Example 2.

[0025] Figure 10 shows the proportion of the target product in nanopore sequencing in Examples 1 and 2. Detailed Implementation

[0026] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0028] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this invention, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] In this invention, the terms "comprising" or "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0030] In this invention, the terms “optionally,” “optionally,” or “optionally” generally refer to events or conditions described subsequently that may but may not occur, and the description includes both cases in which such events or conditions occur and cases in which such events or conditions do not occur.

[0031] This invention proposes nucleic acid-peptide complexes, methods for preparing nucleic acid-peptide complexes, peptide libraries, reagent kits, applications, and methods for detecting peptides, which will be described in detail below.

[0032] Nucleic acid-peptide complex

[0033] In a first aspect, the present invention provides a nucleic acid-peptide complex. According to an embodiment of the invention, the nucleic acid-peptide complex comprises a single-stranded nucleic acid and a peptide fragment; wherein the two ends of the peptide fragment are covalently linked to the two ends of the single-stranded nucleic acid, respectively. According to one embodiment of the invention, a schematic diagram of the single-stranded nucleic acid is shown in FIG1, and a schematic diagram of the peptide fragment is shown in FIG2. In the nucleic acid-peptide complex according to an embodiment of the invention, the nucleic acid fragment in the leader region can guide peptides with different electrical charges into the pore, and the nucleic acid fragment in the rate control region provides a motor protein binding region, thereby controlling the rate of peptide permeation in nanopore sequencing.

[0034] According to an embodiment of the present invention, the complex further includes a single-stranded bridging oligonucleotide fragment, the two ends of which are connected to the two ends of the single-stranded nucleic acid by complementary base pairing.

[0035] According to an embodiment of the present invention, at least a portion of the 5' end sequence of the single-stranded bridging oligonucleotide fragment is adapted to be complementary to at least a portion of the 3' end sequence of the single-stranded nucleic acid, and at least a portion of the 3' end sequence of the single-stranded bridging oligonucleotide fragment is adapted to be complementary to at least a portion of the 5' end sequence of the single-stranded nucleic acid.

[0036] According to an embodiment of the present invention, the single-stranded bridging oligonucleotide fragment includes bases that are not complementary to the single-stranded nucleic acid, and the number of non-complementary bases is N, where N is an integer ≥1, such as 1, 2, 5, or 10. The number of non-complementary bases is used to adjust the spatial distance between modifying groups A and B on the single-stranded nucleic acid, thereby promoting the reaction efficiency between peptides of different lengths and single-stranded nucleic acids.

[0037] According to an embodiment of the present invention, the single-stranded nucleic acid includes a leader region and a rate-controlling region, wherein the leader region and the rate-controlling region are connected by a modified molecule or a breakable chemical bond.

[0038] According to embodiments of the present invention, the modified molecule includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition sequences.

[0039] According to embodiments of the present invention, the breakable chemical bond includes at least one of disulfide bond, diselenide bond, imine bond, acylhydrazone bond, disulfide bond, ester bond, and borate ester bond.

[0040] [Corrected according to Rule 91, 20.12.2024] According to embodiments of the present invention, the covalent connection is carried out by any one or more of the following methods: thiol-olefin connection, thiol-maleimide connection, amino-N-hydroxysuccinimide connection, carbonyl-hydroxylamine-containing oxime connection, carbonyl-hydrazone-containing compound hydrazone connection, tetrazolium-alkynyl compound connection, tetrazolium-olefin compound connection, carbonyl-urea-containing compound urea-forming connection, halogen-nucleophile substitution connection, 1,3-dipolar cycloaddition reaction connection, copper-catalyzed azide-alkynyl cycloaddition reaction connection, ruthenium-catalyzed azide-alkynyl cycloaddition reaction connection, Staudinger connection of azide-phosphorus compound, click chemistry connection of azide compound-alkynyl compound, or natural chemical connection.

[0041] According to embodiments of the present invention, the click chemistry link of the azide compound-alkynyl compound includes any one or more of the following: azide-DBCO click chemistry link, azide-OCT click chemistry link, azide-DIBO click chemistry link, azide-BARAC click chemistry link, azide-ALO click chemistry link, azide-DIFO click chemistry link, azide-MOFO click chemistry link, azide-DIBAC click chemistry link, azide-DIMAC click chemistry link, or azide-cyclooctene click chemistry link.

[0042] According to an embodiment of the present invention, the N-terminus of the polypeptide fragment is covalently linked to the 3' end of the single-stranded nucleic acid, and the C-terminus of the polypeptide fragment is covalently linked to the 5' end of the single-stranded nucleic acid; or the N-terminus of the polypeptide fragment is covalently linked to the 5' end of the single-stranded nucleic acid, and the C-terminus of the polypeptide fragment is covalently linked to the 3' end of the single-stranded nucleic acid.

[0043] According to an embodiment of the present invention, the modifying group A' of the polypeptide fragment is covalently linked to the modifying group A of the single-stranded nucleic acid.

[0044] According to an embodiment of the present invention, the modifying group B' of the polypeptide fragment is covalently linked to the modifying group B of the single-stranded nucleic acid.

[0045] According to an embodiment of the present invention, the modifying group A of the single-stranded nucleic acid and the modifying group B of the single-stranded nucleic acid may be the same or different.

[0046] According to embodiments of the present invention, the modifying group A' of the polypeptide fragment and the modifying group B' of the polypeptide fragment may be the same or different.

[0047] [Corrected according to Rule 91, 20.12.2024] According to embodiments of the present invention, the modifying group A and the modifying group A' are selected from any one of the following groups: (a) mercapto-olefin group; (b) mercapto-maleimide group; (c) amino-N-hydroxysuccinimide group; (d) carbonyl-hydroxylamine group; (e) carbonyl-hydrazine group; (f) carbonyl-urea group; (g) azide-phosphorus group; (h) azide-alkynyl group; (i) tetrazolyl-alkynyl group; (j) tetrazolyl-olefin group; (k) halogen-hydroxyl group; (l) halogen-cyano group; (m) halogen-amino group.

[0048] [Corrected according to Rule 91, 20.12.2024] According to embodiments of the present invention, when the modifying group A is a mercapto group, the modifying group A' is an olefinic group or a maleimide group; when the modifying group A is an amino group, the modifying group A' is an N-hydroxysuccinimide group; when the modifying group A is a carbonyl group, the modifying group A' is a hydroxylamine group, a hydrazine group, or a urea group; when the modifying group A is an azide group, the modifying group A' is a phosphorus group or an alkynyl group; when the modifying group A is a tetrazolium group, the modifying group A' is an alkynyl group or an olefinic group; when the modifying group A is a halogen, the modifying group A' is a hydroxyl group, a cyano group, or an amino group.

[0049] [Corrected according to Rule 91, 20.12.2024] According to embodiments of the present invention, the modifying group B and the modifying group B' are selected from any one of the following groups: (a) mercapto-olefin group; (b) mercapto-maleimide group; (c) amino-N-hydroxysuccinimide group; (d) carbonyl-hydroxylamine group; (e) carbonyl-hydrazine group; (f) carbonyl-urea group; (g) azide-phosphorus group; (h) azide-alkynyl group; (i) tetrazolyl-alkynyl group; (j) tetrazolyl-olefin group; (k) halogen-hydroxyl group; (l) halogen-cyano group; (m) halogen-amino group.

[0050] [Corrected according to Rule 91, 20.12.2024] According to embodiments of the present invention, when the modifying group B is a mercapto group, the modifying group B' is an olefinic group or a maleimide group; when the modifying group B is an amino group, the modifying group B' is an N-hydroxysuccinimide group; when the modifying group B is a carbonyl group, the modifying group B' is a hydroxylamine group, a hydrazine group, or a urea group; when the modifying group B is an azide group, the modifying group B' is a phosphorus group or an alkynyl group; when the modifying group B is a tetrazolium group, the modifying group B' is an alkynyl group or an olefinic group; when the modifying group B is a halogen, the modifying group B' is a hydroxyl group, a cyano group, or an amino group.

[0051] [Corrected according to Article 91, December 2024] Those skilled in the art will understand that if modifying group A' can react with modifying group A to form a covalent link, it means that modifying group A' and modifying group A are paired reactive groups. Similarly, if modifying group B' can react with modifying group B to form a covalent link, it means that modifying group B' and modifying group B are also paired reactive groups. This invention does not particularly limit the specific reactive groups used in pairs; any group capable of forming a covalent link (such as covalent coupling) within the scope of the art can be used. Furthermore, in specific groups, A and A' can be used interchangeably, and B and B' can also be used interchangeably. For example, groups used in pairs include: thiol and olefin group, thiol and maleimide group, amino and N and hydroxysuccinimide group, carbonyl and hydroxylamine group, carbonyl and hydrazine group, carbonyl and urea group, azido and phosphorus group, azido and alkynyl group, tetrazolium and alkynyl group, tetrazolium and olefin group, halogen and hydroxyl group, halogen and cyano group, halogen and amino group, etc.

[0052] Methods for preparing nucleic acid-peptide complexes

[0053] In a second aspect, the present invention provides a method for preparing the nucleic acid-peptide complex described in the first aspect. According to an embodiment of the present invention, the method includes: annealing a single-stranded bridging oligonucleotide fragment with a single-stranded nucleic acid, and covalently linking the annealed product with a peptide fragment to obtain the nucleic acid-peptide complex. The method of the present invention is simple to prepare, has high coupling efficiency, yields a high amount of the obtained nucleic acid-peptide complex, and produces few byproducts.

[0054] According to an embodiment of the present invention, the method further includes: digesting the nucleic acid-peptide complex with an exonuclease to remove byproducts, thereby obtaining a cyclic nucleic acid-peptide complex.

[0055] According to an embodiment of the present invention, the byproducts include a template nucleic acid with one polypeptide molecule coupled to one end, two polypeptide molecules simultaneously coupled to both ends of the template nucleic acid, and an unreacted template nucleic acid without polypeptide molecules coupled to it. A schematic diagram of the byproducts is shown in Figure 3.

[0056] According to embodiments of the present invention, the exonuclease includes at least one of exonuclease VII, exonuclease I, exonuclease V, exonuclease T, and T5 exonuclease. The exonuclease can remove coupling byproducts, thereby improving the purity of the nucleic acid-peptide complex.

[0057] According to an embodiment of the present invention, the method further includes: the single-stranded nucleic acid includes a leader region and a rate-controlling region, the leader region and the rate-controlling region are connected by a modifying molecule, and the cyclic nucleic acid-peptide complex contains the modifying molecule; the cyclic nucleic acid-peptide complex is cleaved using a nuclease capable of cleaving the modifying molecule to obtain a linearized nucleic acid-peptide molecule.

[0058] According to embodiments of the present invention, when the modified molecule is deoxyribose phosphate, the nuclease is apurine endonuclease 1; when the modified molecule is hypoxanthine nucleotide, the nuclease is acid endonuclease V; when the modified molecule is ribonucleotide, the nuclease is an RNA endonuclease; when the modified molecule is deoxyuridine nucleotide, the nuclease is a User enzyme; and when the modified molecule is a restriction endonuclease recognition sequence, the nuclease is a restriction endonuclease that recognizes the sequence.

[0059] According to an embodiment of the present invention, the method further includes:

[0060] The single-stranded nucleic acid includes a leader region and a rate-controlling region, the leader region and the rate-controlling region are connected by a breakable chemical bond, and the cyclic nucleic acid-peptide complex contains the breakable chemical bond;

[0061] The cyclic nucleic acid-peptide complex is treated under conditions that allow the chemical bonds to be broken, thereby obtaining a linearized nucleic acid-peptide molecule.

[0062] According to embodiments of the present invention, the cyclic nucleic acid-peptide complex is treated with light, heat, electromagnetic radiation, reducing agents, acidic reagents, or alkaline reagents to break the chemical bonds.

[0063] According to embodiments of the present invention, the reducing agent is selected from at least one of glutathione and dithiothreitol. When the leader region and rate-controlling region of a single-stranded nucleic acid are connected by a disulfide bond or a diselenylene bond, the chemical bond can be broken by the action of reducing agents such as glutathione and dithiothreitol.

[0064] According to embodiments of the present invention, the acidic reagent is selected from at least one of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, carbonic acid, lactic acid, citric acid, malic acid, succinic acid, benzoic acid, tartaric acid, and maleic acid.

[0065] According to an embodiment of the present invention, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, hexamethylenetetramine, ethylenediamine, ethanolamine, dibutylamine, and hexylamine.

[0066] According to an embodiment of the present invention, the enrichment of the linearized nucleic acid-peptide molecules using capturing magnetic beads is carried out in the following manner: the surface of the capturing magnetic beads is connected to a nucleic acid capturing sequence, which is at least partially complementary to the nucleic acid sequence in the linearized nucleic acid-peptide molecule, thereby enriching the linearized nucleic acid-peptide molecule.

[0067] According to an embodiment of the present invention, the nucleic acid capture sequence includes a release region nucleic acid sequence and a complementary region nucleic acid sequence in the direction from the end near the capture magnetic bead to the end away from the capture magnetic bead.

[0068] According to an embodiment of the present invention, the complementary region nucleic acid sequence is adapted to at least partially complement the nucleic acid sequence in the linearized nucleic acid-peptide molecule.

[0069] According to an embodiment of the present invention, the nucleic acid sequence of the release region includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition site.

[0070] According to an embodiment of the present invention, the linearized nucleic acid-peptide molecule is released from the trapping magnetic bead by cleaving the nucleic acid sequence in the release region using a nuclease.

[0071] The specific process of enriching linearized nucleic acid-peptide molecules using capture magnetic beads is illustrated in Figure 4.

[0072] peptide library

[0073] In a third aspect, the present invention provides a polypeptide library. The polypeptide library according to embodiments of the present invention comprises linearized nucleic acid-peptide molecules obtained by the method of the second aspect of the present invention. During nanopore sequencing, the polypeptide library of the present invention allows for controlled passage speeds of polypeptides of different lengths and electrical charges through the nanopore, thereby obtaining good sequencing signals.

[0074] According to an embodiment of the present invention, the polypeptide library further includes a adapter that is linked to the nucleic acid-polypeptide molecule.

[0075] According to an embodiment of the present invention, the connector is a connector-motor protein complex containing motor proteins.

[0076] Reagent test kit

[0077] In a fourth aspect, the present invention provides a kit. According to embodiments of the present invention, the kit comprises (i) a single-stranded nucleic acid, the single-stranded nucleic acid including a leader region and a rate-controlling region, the leader region and the rate-controlling region being linked by a modified molecularly breakable chemical bond; and (ii) a single-stranded bridging oligonucleotide fragment, the two ends of the single-stranded bridging oligonucleotide fragment being capable of being linked to the two ends of the single-stranded nucleic acid respectively through base complementary pairing. The kit of the present invention can be used to prepare peptide libraries with high coupling efficiency and high yield.

[0078] According to an embodiment of the present invention, one end of the lead region is connected to the modified molecule, and the other end is connected to the modifying group A; and one end of the rate control region is connected to the modified molecule or a breakable chemical bond, and the other end is connected to the modifying group B; wherein the modifying group A and the modifying group B are the same or different, and the single-stranded nucleic acid can be covalently linked to the polypeptide fragment through the modifying group A and the modifying group B.

[0079] According to embodiments of the present invention, the modified molecule includes at least one of: deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition sequence.

[0080] According to embodiments of the present invention, the breakable chemical bond includes at least one of disulfide bond, diselenide bond, imine bond, acylhydrazone bond, disulfide bond, ester bond, and borate ester bond.

[0081] [Corrected according to Rule 91 20.12.2024] According to an embodiment of the present invention, the modifying group A and the modifying group B are the same and are selected from any one of the following: mercapto, olefinic group, maleimide group, amino, N-hydroxysuccinimide group, carbonyl, hydroxylamine group, hydrazine, urea group, azide group, phosphorus group, alkynyl, tetrazolium group, halogen, hydroxyl and cyano.

[0082] [Corrected according to Rule 91 20.12.2024] According to an embodiment of the present invention, the modifying group A and the modifying group B are different, and the modifying group A and the modifying group B are independently selected from: mercapto, olefinic group, maleimide group, amino, N-hydroxysuccinimide group, carbonyl, hydroxylamine group, hydrazine, urea group, azide group, phosphorus group, alkynyl, tetrazolium group, halogen, hydroxyl and cyano.

[0083] According to embodiments of the present invention, the kit further comprises at least one of nuclease, ligase, capture magnetic beads, adapter, reducing agent, acidic reagent, and basic reagent.

[0084] According to an embodiment of the present invention, the nuclease is selected from: exonuclease, endonuclease V, apurine-free endonuclease I, restriction endonuclease and User enzyme, and the nuclease is capable of cleaving the modified molecule.

[0085] According to an embodiment of the present invention, the reducing agent is selected from at least one of glutathione and dithiothreitol.

[0086] According to embodiments of the present invention, the acidic reagent is selected from at least one of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, carbonic acid, lactic acid, citric acid, malic acid, succinic acid, benzoic acid, tartaric acid, and maleic acid.

[0087] According to an embodiment of the present invention, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, hexamethylenetetramine, ethylenediamine, ethanolamine, dibutylamine, and hexylamine.

[0088] According to an embodiment of the present invention, the surface of the capturing magnetic beads is connected to a nucleic acid capturing sequence.

[0089] According to an embodiment of the present invention, the nucleic acid capture sequence includes a release region nucleic acid sequence and a complementary region nucleic acid sequence in the direction from the end near the capture magnetic bead to the end away from the capture magnetic bead.

[0090] According to an embodiment of the present invention, the complementary region nucleic acid sequence is adapted to at least partially complement the single-stranded nucleic acid.

[0091] According to an embodiment of the present invention, the nucleic acid sequence of the release region includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition site.

[0092] use

[0093] In a fifth aspect, the present invention proposes the use of the nucleic acid-peptide complex described in the first aspect of the present invention, the peptide library described in the third aspect of the present invention, and the kit described in the fourth aspect of the present invention in high-throughput sequencing.

[0094] According to an embodiment of the present invention, the high-throughput sequencing includes single-molecule nanopore sequencing.

[0095] Methods for detecting peptides

[0096] In a sixth aspect, the present invention provides a method for detecting peptides. According to an embodiment of the present invention, the method includes: adding a peptide library as described in the third aspect of the present invention into a detection solution chamber; under the action of an electric field, controlling the peptide library to pass through a nanopore via a motor protein, thereby acquiring an electrical signal corresponding to the peptide; and decoding the electrical signal to determine the amino acid information of the peptide. The method according to the embodiment of the present invention can accurately obtain the amino acid information of the peptide, such as the amino acid sequence, amino acid side chain modifications, or the presence of a target peptide.

[0097] The sequence of the present invention is shown in the table below:

[0098] sequence list

[0099] It should be noted that EMCS refers to 6-maleimide hexanoic acid N-hydroxysuccinimide ester.

[0100] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0101] Example 1:

[0102] 1. Obtaining nucleic acid-peptide complexes

[0103] (1) A template DNA (specific nucleotide sequence as shown in SEQ ID NO: 1) was formed by introducing deoxyxanthine nucleotides between DNA1 (specific nucleotide sequence as shown in SEQ ID NO: 2) and DNA2 (specific nucleotide sequence as shown in SEQ ID NO: 3). The template DNA was synthesized by Changzhou Xinyisheng Life Technology Co., Ltd.

[0104] (2) Dissolve the template DNA powder from step (1) in 1×PBS solution to prepare a 100 μM working solution A, dissolve the bridging oligonucleotide fragment (DNA3) powder (specific nucleotide sequence as shown in SEQ ID NO: 4) in 1×PBS solution to prepare a 100 μM working solution B, and dissolve the polypeptide fragment powder (specific amino acid sequence as shown in SEQ ID NO: 8) in 1×PBS solution to prepare a 1 mM working solution C;

[0105] (3) Take 5 μL each of working solution A and B from step (2), mix them evenly, and anneal them using a PCR instrument. After annealing, they can be stored at 4℃ for later use.

[0106] (4) Take 5 μL of working solution C from step (2), add 5 μL of 1 mM tris(2-carbonylethyl)phosphohydrochloride (TCEP), and incubate at room temperature for 30 min;

[0107] (5) Add the mixed solution from step (4) to the mixed solution from step (3) after annealing, and react overnight at room temperature to obtain the crude product of nucleic acid-peptide complex.

[0108] 2. Remove byproducts

[0109] (1) Add 2 μL of exonuclease VII (NEB, M0379S) to the crude product solution of nucleic acid-peptide complex and incubate at 37°C for 30 min to remove template DNA with one peptide molecule coupled at one end, two peptide molecules coupled at both ends, and no peptide molecules coupled.

[0110] (2) After incubation, heat at 95°C for 10 min to inactivate exonuclease VII;

[0111] (3) Add 3.5 μL of NEB buffer 4 (NEB#B7004) and 1 μL of endonuclease V (NEB, M0305) to the solution in step (2), and incubate at 37°C for 1 h to linearize the template DNA in the nucleic acid-peptide complex and obtain the target product after removing by-products, which is used for subsequent magnetic bead capture.

[0112] 3. Preparation and enrichment of nucleic acid-peptide complex sequencing libraries

[0113] (1) Take 100 μL of streptavidin (SA) magnetic beads and 200 pmol of capture sequence (Capture DNA, the specific nucleotide sequence is shown in SEQ ID NO: 5) and incubate with shaking at room temperature for 1 h. After discarding the supernatant, wash twice with 1×PBS (0.1% Tween) to remove excess free capture sequence (Capture DNA) to obtain magnetic beads with capture strands. Store at 4℃ for later use.

[0114] (2) Anneal the top chain Y-top (specific nucleotide sequence as shown in SEQ ID NO: 6) and bottom chain Y-bottom (specific nucleotide sequence as shown in SEQ ID NO: 7) of the adapter in a 1:1 ratio to obtain the annealed product, and then incubate it with DNA helicase to obtain the adapter containing DNA helicase, which is the Y-type adapter, and store it at 4℃ for later use.

[0115] (3) Take 10 μL of the target product obtained in the above steps after removing byproducts and add it to the magnetic beads with trapping chains obtained in step (1). Shake and incubate at 30°C for 1 h. After incubation, discard the supernatant and wash twice with 1×PBS.

[0116] (4) Add the Y-type adapter obtained in step (2) to the magnetic beads with the capture chain in step (3) at a ratio of 2:1, and perform the ligation reaction using the T4 ligase (NEB, #M2200L) kit at room temperature for 30 min; after the reaction, discard the supernatant and wash twice with 1×PBS.

[0117] (5) Add 10 μL of 1×PBS to resuspend the magnetic beads obtained in (4), and add 0.5 μL of ribonuclease A (RNase A) (NEB, #M0305). Digest at 30°C for 40 min.

[0118] (6) Collect the supernatant to obtain a nucleic acid-peptide complex sequencing library based on template reaction, as shown in Figure 5.

[0119] Example 2

[0120] The construction process of this embodiment is basically the same as that of embodiment 1, except that:

[0121] (1) Take 5 μL of polypeptide solution (specific amino acid sequence as shown in SEQ ID NO: 8) (1 mM, 1×PBS), add 5 μL of 1 mM tris(2-carbonylethyl)phosphohydrochloride (TCEP), and incubate at room temperature for 30 min;

[0122] (2) Add 5 μL of DNA2 solution (specific nucleotide sequence as shown in SEQ ID NO: 2) (100 μM, 1×PBS) to the solution in step (1) and react at room temperature for 6 h. Then add 5 μL of DNA1 solution (specific nucleotide sequence as shown in SEQ ID NO: 1) (100 μM, 1×PBS) and continue to react at room temperature overnight.

[0123] (3) Anneal the top linker Y-Top (specific nucleotide sequence as shown in SEQ ID NO: 6) and the bottom linker Y-boTTom (specific nucleotide sequence as shown in SEQ ID NO: 7) in a 1:1 ratio to obtain the annealed product. Then, co-incubate it with DNA helicase to obtain the linker containing DNA helicase, which is the Y-type linker. Store it at 4℃ for later use.

[0124] (4) Add the Y-type adapter obtained in step (3) to the product solution in step (2) at a ratio of 2:1, and perform the ligation reaction using a T4 ligase (NEB, #M2200L) kit at room temperature for 30 min. After the reaction, obtain a nucleic acid-peptide complex sequencing library based on non-template reaction, as shown in Figure 6.

[0125] DNA1, DNA2, the template-based nucleic acid-peptide complex sequencing library constructed in Example 1, and the non-template-based nucleic acid-peptide complex sequencing library constructed in Example 2 were subjected to electrophoresis at 150V for 90 minutes using 12% polyacrylamide denaturing gel. The results are shown in Figure 7.

[0126] A nanopore detection platform based on a patch-clamp platform was used to perform nanopore sequencing on the template-reaction-based nucleic acid-peptide complex sequencing library constructed in Example 1 and the non-template-reaction-based nucleic acid-peptide complex sequencing library constructed in Example 2. Referring to the single-channel electrophysiological detection system described by Geng Jia and Guo Peixuan (“Application of Phage phi29 DNA Packaging Motor Phospholipid Membrane Chip in Single-Molecular Detection and Nanomedicine”, Life Sciences, 2011, 23(11):1114-1129), a nanopore detection platform based on a patch-clamp platform was constructed. CsgG pore protein was inserted into the phospholipid bilayer membrane to form a single-channel nanopore. The template-reaction-based nucleic acid-peptide complex sequencing library constructed in Example 1 and the non-template-reaction-based nucleic acid-peptide complex sequencing library constructed in Example 2 were respectively added to this single-channel system. The changes in current amplitude and the current signals generated when different protein libraries passed through the nanopore were detected and recorded using the patch-clamp system, and the data were analyzed. Data analysis yielded sequencing results as shown in Figures 8 and 9, and the target product percentage as shown in Figure 10.

[0127] Compared to Example 2, the yield of nucleic acid-peptide complexes based on template reactions was higher, and the byproducts of nucleic acid-peptide complexes based on template reactions were lower. This indicates that the coupling efficiency of nucleic acid-peptide complexes based on template reactions is improved and the byproduct yield is lower, as shown in the denaturing gel results in Figure 7. The solid boxes mark the nucleic acid-peptide product bands, and the dashed boxes mark the "nucleic acid-peptide" bands. Lanes 3 and 4 are the electrophoresis results for DNA1 and DNA2, respectively. Lane 5 is the electrophoresis result after the template nucleic acid is digested with endonuclease V. The bands in lane 5 are basically consistent with the positions of DNA1 and DNA2, indicating that the template nucleic acid can be successfully digested by endonuclease V. Lane 6 is the electrophoresis result of the product from the circular template reaction in Example 1, and lane 7 is the electrophoresis result of the product from the non-template reaction in Example 2. Compared to non-template reactions, the coupling efficiency of nucleic acid-peptide pairs is significantly improved under circular DNA template conditions. This is because the template of circular DNA brings the functional groups at the ends of nucleic acids closer together, effectively increasing the coupling efficiency of the peptide's two ends. In contrast, for free coupling reactions, after a peptide is coupled to DNA2, it is difficult to continue coupling to DNA1, resulting in a low yield of nucleic acid-peptide pairs.

[0128] The obtained conjugated products were tested using sequencing. Figure 8 shows the sequencing results of the sequencing library in Example 1, and Figure 9 shows the sequencing results of the sequencing library in Example 2. In the figures, "√" indicates the sequencing results of the complete "nucleic acid-peptide" target product, while "×" indicates the sequencing results of adapters or byproducts. Figures 8-9 clearly demonstrate that after reacting with the circular DNA template and purification, the proportion of the target product in the sequencing data significantly increased.

[0129] Furthermore, the sequencing results of three repeated experiments were manually analyzed. Within ten minutes of sequencing, the number of complete "nucleic acid-peptide" target products and the total number of sequences were counted, yielding the target product percentage = (number of target product sequences / total number of sequences) * 100%. As shown in Figure 10, after template reaction and purification, the percentage of target products in the sequencing data significantly increased, by 47% compared to the non-template reaction.

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0131] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A nucleic acid-peptide complex, characterized in that, include: A single-stranded nucleic acid and a polypeptide fragment; wherein the two ends of the polypeptide fragment are covalently linked to the two ends of the single-stranded nucleic acid, respectively.

2. The complex according to claim 1, characterized in that, The complex further includes a single-stranded bridging oligonucleotide fragment, the two ends of which are connected to the two ends of the single-stranded nucleic acid through complementary base pairing.

3. The complex according to claim 2, characterized in that, At least a portion of the 5' end sequence of the single-stranded bridging oligonucleotide fragment is adapted to be complementary to at least a portion of the 3' end sequence of the single-stranded nucleic acid, and at least a portion of the 3' end sequence of the single-stranded bridging oligonucleotide fragment is adapted to be complementary to at least a portion of the 5' end sequence of the single-stranded nucleic acid.

4. The complex according to claim 2 or 3, characterized in that, The single-stranded bridging oligonucleotide fragment includes bases that are not complementary to the single-stranded nucleic acid, and the number of such non-complementary bases is N, where N is an integer ≥ 1.

5. The complex according to claim 1, characterized in that, The single-stranded nucleic acid includes a leader region and a rate-controlling region, which are connected by a modified molecule or a breakable chemical bond.

6. The complex according to claim 5, characterized in that, The modified molecule includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition sequences; Optionally, the breakable chemical bond includes at least one of disulfide bond, diselenide bond, imine bond, acylhydrazone bond, disulfide bond, ester bond, and borate ester bond.

7. [Correction 20.12.2024 according to Rule 91] The complex according to claim 1, characterized in that, The covalent linkage is performed through any one or more of the following methods: linkage of thiol-olefin compound, linkage of thiol-maleimide compound, linkage of amino group with N-hydroxysuccinimide compound, oxime linkage of carbonyl-hydroxylamine compound, hydrazone linkage of carbonyl-hydrazol compound, linkage of tetrazolium-alkynyl compound, linkage of tetrazolium-olefin compound, urea linkage of carbonyl-urea compound, halogen-nucleophilic substitution linkage, 1,3-dipolar cycloaddition reaction linkage, copper-catalyzed azide-alkynyl cycloaddition reaction linkage, ruthenium-catalyzed azide-alkynyl cycloaddition reaction linkage, Staudinger linkage of azide-phosphorus compound, click chemistry linkage of azide compound-alkynyl compound, or natural chemical linkage. Preferably, the click chemistry link of the azide compound-alkynyl compound includes any one or more of the following: azide-DBCO click chemistry link, azide-OCT click chemistry link, azide-DIBO click chemistry link, azide-BARAC click chemistry link, azide-ALO click chemistry link, azide-DIFO click chemistry link, azide-MOFO click chemistry link, azide-DIBAC click chemistry link, azide-DIMAC click chemistry link, or azide-cyclooctene click chemistry link.

8. The complex according to claim 1 or 7, characterized in that, The N-terminus of the polypeptide fragment is covalently linked to the 3' end of the single-stranded nucleic acid, and the C-terminus of the polypeptide fragment is covalently linked to the 5' end of the single-stranded nucleic acid; or The N-terminus of the polypeptide fragment is covalently linked to the 5' end of the single-stranded nucleic acid, and the C-terminus of the polypeptide fragment is covalently linked to the 3' end of the single-stranded nucleic acid.

9. The complex according to claim 8, characterized in that, The modifying group A' of the polypeptide fragment is covalently linked to the modifying group A of the single-stranded nucleic acid; Optionally, the modifying group B' of the polypeptide fragment is covalently linked to the modifying group B of the single-stranded nucleic acid; Optionally, the modifying group A of the single-stranded nucleic acid and the modifying group B of the single-stranded nucleic acid may be the same or different; Optionally, the modifying group A' of the polypeptide fragment and the modifying group B' of the polypeptide fragment may be the same or different.

10. [Correction 20.12.2024 according to Rule 91] The complex according to claim 9, characterized in that, The modifying group A and the modifying group A' are selected from any one of the following groups: (a) thiol-olefin group; (b) Thio-maleimide group; (c) Amino-N-hydroxysuccinimide group; (d) Carbonyl-hydroxylamine group; (e) Carbonyl-hydrazine; (f) Carbonyl-urea group; (g) Azide-phosphorus group; (h) Azide-alkynyl; (i) Tetrazolyl-alkynyl; (j) Tetrazoazolyl-olefin group; (k) Halogen-hydroxyl group; (l) Halogen-cyano; (m) Halogen-amino.

11. [Correction 20.12.2024 according to Rule 91] The complex according to claim 10, characterized in that, When the modifying group A is a thiol group, the modifying group A' is an olefin group or a maleimide group; When the modifying group A is an amino group, the modifying group A' is an N-hydroxysuccinimide group; When the modifying group A is a carbonyl group, the modifying group A' is a hydroxylamine group, a hydrazine group, or a urea group; When the modifying group A is an azide group, the modifying group A' is a phosphorus group or an alkynyl group; When the modifying group A is a tetrazolium group, the modifying group A' is an alkynyl group or an olefinic group; When the modifying group A is a halogen, the modifying group A' is a hydroxyl, cyano, or amino group.

12. [Correction 20.12.2024 according to Rule 91] The complex according to claim 9, characterized in that, The modifying group B and the modifying group B' are selected from any one of the following groups: (a) thiol-olefin group; (b) Thio-maleimide group; (c) Amino-N-hydroxysuccinimide group; (d) Carbonyl-hydroxylamine group; (e) Carbonyl-hydrazine; (f) Carbonyl-urea group; (g) Azide-phosphorus group; (h) Azide-alkynyl; (i) Tetrazolyl-alkynyl; (j) Tetrazoazolyl-olefin group; (k) Halogen-hydroxyl group; (l) Halogen-cyano; (m) Halogen-amino.

13. [Correction 20.12.2024 according to Rule 91] The complex according to claim 12, characterized in that, When the modifying group B is a thiol group, the modifying group B' is an olefin group or a maleimide group; When the modifying group B is an amino group, the modifying group B' is an N-hydroxysuccinimide group; When the modifying group B is a carbonyl group, the modifying group B' is a hydroxylamine group, a hydrazine group, or a urea group; When the modifying group B is an azide group, the modifying group B' is a phosphorus group or an alkynyl group; When the modifying group B is a tetrazolium group, the modifying group B' is an alkynyl group or an olefinic group; When the modifying group B is a halogen, the modifying group B' is a hydroxyl, cyano, or amino group.

14. A method for preparing the nucleic acid-peptide complex according to any one of claims 1 to 13, characterized in that, include: The single-stranded bridging oligonucleotide fragment is annealed to a single-stranded nucleic acid, and the annealed product is covalently linked to a polypeptide fragment to obtain the nucleic acid-polypeptide complex.

15. The method according to claim 14, characterized in that, Further includes: The nucleic acid-peptide complex is obtained by digesting it with an exonuclease to remove byproducts.

16. The method according to claim 15, characterized in that, The byproducts include template nucleic acid with one polypeptide molecule coupled to one end, template nucleic acid with two polypeptide molecules coupled to both ends simultaneously, and template nucleic acid without reacting and without polypeptide molecules coupled. Optionally, the exonuclease includes at least one of exonuclease VII, exonuclease I, exonuclease V, exonuclease T, and T5 exonuclease.

17. The method according to claim 15, characterized in that, Further includes: The single-stranded nucleic acid includes a leader region and a rate-controlling region, the leader region and the rate-controlling region are connected by a modifying molecule, and the cyclic nucleic acid-peptide complex contains the modifying molecule; The cyclic nucleic acid-peptide complex is cleaved using a nuclease capable of cleaving the modified molecule to obtain a linearized nucleic acid-peptide molecule.

18. The method according to claim 17, characterized in that, When the modified molecule is deoxyribose phosphate, the nuclease is apurine endonuclease 1; when the modified molecule is hypoxanthine nucleotide, the nuclease is acid endonuclease V; when the modified molecule is ribonucleotide, the nuclease is an RNA endonuclease; when the modified molecule is deoxyuridine nucleotide, the nuclease is User enzyme; when the modified molecule is a restriction endonuclease recognition sequence, the nuclease is a restriction endonuclease that recognizes the sequence.

19. The method according to claim 15, characterized in that, Further includes: The single-stranded nucleic acid includes a leader region and a rate-controlling region, the leader region and the rate-controlling region are connected by a breakable chemical bond, and the cyclic nucleic acid-peptide complex contains the breakable chemical bond; The cyclic nucleic acid-peptide complex is treated under conditions that allow the chemical bonds to be broken, thereby obtaining a linearized nucleic acid-peptide molecule.

20. The method according to claim 19, characterized in that, The cyclic nucleic acid-peptide complex is treated with light, heat, electromagnetic radiation, reducing agents, acidic reagents, or alkaline reagents to break the chemical bonds. Optionally, the reducing agent is selected from at least one of glutathione and dithiothreitol; Optionally, the acidic reagent is selected from at least one of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, carbonic acid, lactic acid, citric acid, malic acid, succinic acid, benzoic acid, tartaric acid, and maleic acid; Optionally, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, hexamethylenetetramine, ethylenediamine, ethanolamine, dibutylamine, and hexylamine.

21. The method according to any one of claims 17-20, characterized in that, The method further includes: enriching the linearized nucleic acid-peptide molecules using capturing magnetic beads.

22. The method according to claim 21, characterized in that, The enrichment of the linearized nucleic acid-peptide molecules using trapping magnetic beads is carried out in the following manner: The surface of the capturing magnetic beads is connected to a nucleic acid capturing sequence, which is at least partially complementary to the nucleic acid sequence in the linearized nucleic acid-peptide molecule, thereby enriching the linearized nucleic acid-peptide molecule; Optionally, the nucleic acid capture sequence includes a release region nucleic acid sequence and a complementary region nucleic acid sequence from the end near the capture magnetic bead to the end away from the capture magnetic bead; Optionally, the complementary region nucleic acid sequence is adapted to be at least partially complementary to the nucleic acid sequence in the linearized nucleic acid-peptide molecule; Optionally, the nucleic acid sequence of the release region includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition site; Optionally, the linearized nucleic acid-peptide molecule is released from the trapping magnetic bead by cleaving the nucleic acid sequence in the release region using a nuclease.

23. A polypeptide library, characterized in that, Includes linearized nucleic acid-peptide molecules obtained by the method of any one of claims 17-22.

24. The library according to claim 23, characterized in that, It further includes a connector that links to the nucleic acid-peptide molecule; Preferably, the connector is a connector-motor protein complex containing motor proteins.

25. A reagent kit, characterized in that, The invention includes (i) a single-stranded nucleic acid, the single-stranded nucleic acid including a leader region and a rate-controlling region, the leader region and the rate-controlling region being connected by a modified molecule or a breakable chemical bond; and (ii) a single-stranded bridging oligonucleotide fragment, the two ends of the single-stranded bridging oligonucleotide fragment being able to be connected to the two ends of the single-stranded nucleic acid respectively through complementary base pairing.

26. The reagent kit according to claim 25, characterized in that, One end of the pilot region is connected to the modified molecule, and the other end is connected to the modifying group A; and one end of the rate control region is connected to the modified molecule or a breakable chemical bond, and the other end is connected to the modifying group B; wherein the modifying group A and the modifying group B may be the same or different, and the single-stranded nucleic acid can be covalently linked to the polypeptide fragment through the modifying group A and the modifying group B.

27. The reagent kit according to claim 26, characterized in that, The modified molecule includes at least one of: deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition sequence; Optionally, the breakable chemical bond includes at least one of disulfide bond, diselenide bond, imine bond, acylhydrazone bond, disulfide bond, ester bond, and borate ester bond.

28. [Correction 20.12.2024 according to Rule 91] The kit according to claim 26, characterized in that, The modifying group A and modifying group B are the same and are selected from any one of the following: mercapto, olefinic group, maleimide group, amino, N-hydroxysuccinimide group, carbonyl, hydroxylamine group, hydrazine, urea group, azide group, phosphorus group, alkynyl, tetrazolium group, halogen, hydroxyl and cyano.

29. [Correction 20.12.2024 according to Rule 91] The kit according to claim 26, characterized in that, The modifying group A and the modifying group B are different, and the modifying group A and the modifying group B are each independently selected from: mercapto, olefinic group, maleimide group, amino, N-hydroxysuccinimide group, carbonyl, hydroxylamine group, hydrazine, urea group, azide group, phosphorus group, alkynyl, tetrazolium group, halogen, hydroxyl, cyano.

30. The reagent kit according to any one of claims 25-29, characterized in that, Further including at least one of nuclease, ligase, trapping magnetic beads, adapter, reducing agent, acidic reagent and basic reagent.

31. The reagent kit according to claim 30, characterized in that, The nuclease is selected from: exonuclease, endonuclease V, apurine-free endonuclease I, restriction endonuclease, and User enzyme, and the nuclease is capable of cleaving the modified molecule.

32. The reagent kit according to claim 30, characterized in that, The reducing agent is selected from at least one of glutathione and dithiothreitol; Optionally, the acidic reagent is selected from at least one of hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, carbonic acid, lactic acid, citric acid, malic acid, succinic acid, benzoic acid, tartaric acid, and maleic acid; Optionally, the alkaline reagent is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, triethylamine, pyridine, hexamethylenetetramine, ethylenediamine, ethanolamine, dibutylamine, and hexylamine.

33. The reagent kit according to claim 30, characterized in that, The surface of the capture magnetic beads is linked to a nucleic acid capture sequence; Optionally, the nucleic acid capture sequence includes a release region nucleic acid sequence and a complementary region nucleic acid sequence from the end near the capture magnetic bead to the end away from the capture magnetic bead; Optionally, the complementary region nucleic acid sequence is adapted to be complementary to at least a portion of the sequence of the single-stranded nucleic acid; Optionally, the nucleic acid sequence of the release region includes at least one of deoxyribose phosphate, hypoxanthine nucleotide, ribonucleotide, deoxyuridine nucleotide, and restriction endonuclease recognition site.

34. Use of the nucleic acid-peptide complex according to any one of claims 1-13, the peptide library according to claim 23 or 24, and the kit according to any one of claims 25-33 in high-throughput sequencing.

35. The use according to claim 34, characterized in that, The high-throughput sequencing includes single-molecule nanopore sequencing.

36. A method for detecting polypeptides, characterized in that, The method includes: The polypeptide library as described in claim 23 or 24 is added to the detection solution chamber. Under the action of an electric field, the polypeptide library is controlled by a motor protein to pass through a nanopore, thereby obtaining the electrical signal corresponding to the polypeptide. The electrical signal is decoded to determine the amino acid information of the polypeptide. Optionally, the amino acid information of the polypeptide includes amino acid sequence information and amino acid modification information.

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