WO2025010736A1 - Nucleic acid polypeptide complex and use thereof in peptide sequencing - Google Patents

Abstract

Provided are a nucleic acid polypeptide complex and the use thereof in peptide sequencing. The nucleic acid polypeptide complex comprises a first nucleic acid fragment, a polypeptide to be tested and a composite nucleic acid fragment which are connected in sequence, wherein the composite nucleic acid fragment comprises a nucleic acid sequence capable of forming a hairpin structure; after the hairpin structure is formed, the composite nucleic acid fragment comprises a double-stranded nucleic acid and a single-stranded nucleic acid, one end of the single-stranded nucleic acid is connected to the double-stranded nucleic acid, and the other end of the single-stranded nucleic acid is connected to the polypeptide to be tested. The detection of any polypeptide sequence can be achieved by means of guiding the polypeptide with different charges by the nucleic acid sequence to pass through the nanopore.

Description

Nucleic acid-peptide complex and its application in peptide sequencing Technical Field

The present invention belongs to the technical field of sequencing, and in particular relates to a nucleic acid-polypeptide complex and its application in peptide sequencing.

Background Art

Proteomics is one of the core areas of life science research after the completion of human genome sequencing. Fully analyzing the composition of various proteins can enable humans to have a deeper understanding of the cell regulatory mechanisms related to proteins, thereby understanding the relationship between proteins and human diseases. Therefore, in order to accurately obtain various protein sequence information, the development of single-molecule protein sequencing technology is particularly valuable.

At present, the technology of DNA sequencing using nanopores has become increasingly mature. Its principle is that under the action of electric field force, the DNA molecule chain bound to the helicase will pass through the nanopore at a relatively constant speed. Since different bases have different blocking effects on the current when passing through the nanopore, the purpose of sequencing can be achieved by analyzing the corresponding relationship between the current change and the base in this process. Similarly, measuring the change of the current signal when the polypeptide chain passes through the nanopore and analyzing its corresponding relationship with different amino acids can also achieve the purpose of polypeptide sequence reading.

In 1994, Sutherland, Todd C. et al. used nanopore detection technology to explore the detection of peptide structure (Sutherland, Todd C., et al. "Structure of peptides investigated by nanopore analysis." Nano letters 4.7 (2004): 1273-1277.). In 2018, Ji, Zhouxiang et al. constructed a nanopore sensor based on the bacteriophage motor protein channel to detect single amino acid accuracy differences (Ji, Zhouxiang, et al. "Nano-channel of viral DNA packaging motor as single pore to differentiate peptides with single amino acid difference." Biomaterials 182 (2018): 227-233.).

Although the speed of some peptides passing through the nanopore can be reduced by modifying the pore protein or optimizing the biochemical experimental conditions, thereby improving the accuracy of peptide sequencing, it is crucial to control the uniformity of the peptides passing through the nanopore channel for the precision and accuracy of peptide sequencing.

In 2021, Dekker and Cees' laboratory published an article on protein sequencing based on Hel308 rate control (Brinkerhoff, Henry, et al. "Multiple rereads of single proteins at single-amino acid resolution using nanopores." Science 374.6574 (2021): 1509-1513.). As shown in Figure 1, the polypeptide and nucleic acid are first coupled by chemical coupling to form a polypeptide-nucleic acid complex. Incubated with Hel308 to form a peptide-DNA-hel308 complex; after the complex is captured by the nanopore under the action of the electric field force, the complementary DNA chains are separated; the DNA moves from the bottom to the top of the membrane under the action of Hel308; when the polypeptide is located in the sensor area of the pore, the sequence is read. The key point of this method is to use the translocation activity of Hel308 to achieve the purpose of rate control.

In 2021, Huang Shuo's team from Nanjing University reported an article on peptide sequencing using Phi29 DNA polymerase to control the rate (Yan, Shuanghong, et al. "Single molecule ratcheting motion of peptides in a Mycobacterium smegmatis Porin A (MspA) nanopore." Nano letters, 21.15 (2021): 6703-6710.). As shown in Figure 2, Phi29 DNA polymerase is incubated with the synthesized ssDNA-peptide complex to form a complex. The polymerase is used to amplify DNA to indirectly control the speed of polypeptide movement.

In 2021, Oxford Nanopore Technologies Limited (WO2021/111125A1) disclosed a protein sequencing solution based on oligonucleotide-controlled protein speed control. The design is to first synthesize the adaptor-peptide-dsDNA tail complex, then partially anneal the single-stranded DNA to form double-stranded DNA, and then combine it with the oligonucleotide control protein to read the electrical signal through the nanopore (as shown in Figure 3). This method can solve the problem that neutral and positively charged peptides cannot penetrate the pore.

Chen, Zhijie, et al. (Chen, Zhijie, et al. "Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application." Chemical science 12.47 (2021): 15750-15756.) disclosed a similar protein sequencing scheme based on MTA helicase rate control. Its design is also to first synthesize a ssDNA-peptide-ssDNA (polyT) complex, bind to the MTA helicase without annealing to form a double strand, and drive the polypeptide fragment through the MspA-M2 nanopore protein through the regular sliding of the MTA helicase on the ssDNA chain to measure the sequencing electrical signal (as shown in Figure 4). This method can solve the problem that neutral polypeptides cannot penetrate holes.

The protein sequencing method published by Dekker and Cees uses peptides composed of amino acids with all negatively charged side chains (aspartic acid and glutamic acid) because the peptides need to be captured by the nanopore first. Therefore, this scheme cannot detect neutral and positively charged peptides. The method reported by Huang Shuo's team also has similar problems.

Although the technical solutions disclosed in Oxford Nanopore Technologies Limited patent document WO2021/111125A1 and the technical solutions disclosed in Chen, Zhijie's article can solve the problem of neutral peptide perforation, most natural wild helicases have large differences in activity during unwinding and translocation, and the speeds of unwinding and perforation are difficult to keep constant. Therefore, in order to obtain high-resolution peptide sequencing signals, the ideal situation is to move forward/translocate one base for every base unwound. Therefore, in order to obtain a truly effective helicase that can be used for peptide sequencing, it is necessary to use protein engineering methods to screen a large number of mutants, which is difficult.

Summary of the invention

The technical problem to be solved by the present invention is the defect that the prior art lacks a method capable of realizing perforation sequencing of any polypeptide, and provides a nucleic acid polypeptide complex and its application in peptide sequencing. The nucleic acid polypeptide of the present invention solves the problem that only negatively charged polypeptides are allowed to be perforated in the polymerase-based protein sequencing route, and realizes perforation sequencing of polypeptides of any charge (positive, negative, neutral).

The present invention solves the above technical problems through the following technical solutions.

The first aspect of the present invention provides a nucleic acid-polypeptide complex, wherein the nucleic acid-polypeptide complex comprises a first nucleic acid fragment, a polypeptide to be detected, and a composite nucleic acid fragment connected in sequence;

Wherein, the composite nucleic acid fragment comprises a nucleic acid sequence that can form a hairpin structure, and after forming the hairpin structure, the composite nucleic acid fragment comprises a double-stranded nucleic acid and a single-stranded nucleic acid, one end of the single-stranded nucleic acid is connected to the double-stranded nucleic acid, and the other end of the single-stranded nucleic acid is connected to the polypeptide to be detected;

In some preferred embodiments of the present invention, the length of the single-stranded nucleic acid is greater than or equal to the length of the polypeptide to be detected.

In some embodiments of the present invention, the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, and the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure. After the hairpin structure is formed, the third nucleic acid fragment is complementary to a part of the second nucleic acid fragment to form a double-stranded nucleic acid, and the other part of the second nucleic acid fragment is a single-stranded nucleic acid.

In the above scheme, in the composite nucleic acid fragment, the connection between the end of the second nucleic acid fragment and the end of the third nucleic acid fragment can form a covalent connection by chemical connection, enzyme connection, etc., or no covalent connection reaction is required. Due to the partial double-stranded structure between the third nucleic acid fragment and the second nucleic acid fragment, the two form a stable composite nucleic acid fragment.

In other embodiments of the present invention, the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, after the hairpin structure is formed, the two ends of the third nucleic acid fragment are complementary to each other to form a double-stranded nucleic acid, the second nucleic acid fragment is a single-stranded nucleic acid, and one end of the second nucleic acid fragment is connected to the double-stranded nucleic acid, and the other end of the second nucleic acid fragment is connected to the polypeptide to be tested.

In the above scheme, in the composite nucleic acid fragment, the junction between the second nucleic acid fragment end and the third nucleic acid fragment end needs to be connected by chemical, biological, enzymatic or other connection methods to form a covalent bond to form a stable composite nucleic acid fragment.

In some embodiments of the present invention, the nucleic acid-polypeptide complex further includes a fourth nucleic acid fragment, the 5' end nucleic acid sequence of the fourth nucleic acid fragment is complementary to at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment to form a double-stranded chain, and the 3' end of the fourth nucleic acid fragment is optionally connected to an anchoring portion.

In some preferred embodiments of the present invention, after the hairpin structure is formed, the stability of the double-stranded nucleic acid in the hairpin structure of the composite nucleic acid fragment is higher than the stability of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment.

In other preferred embodiments of the present invention, the Tm value of the double-stranded nucleic acid in the hairpin structure of the composite nucleic acid fragment is higher than the Tm value of the double-stranded nucleic acid formed by the complementarity of the 5' end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment.

In some embodiments of the present invention, the length of the first nucleic acid fragment is 5 to 200 nt, preferably 10 to 50 nt; the length of the composite nucleic acid fragment is 80 to 2000 nt, preferably 50 to 200 nt.

In some embodiments of the present invention, the length of the second nucleic acid fragment is 40 to 1000 nt, preferably 50 to 200 nt.

In some embodiments of the present invention, the length of the third nucleic acid fragment is 40 to 1000 nt, preferably 50 to 200 nt.

In some embodiments of the present invention, the length of the fourth nucleic acid fragment is 20-500 nt, preferably 25-100 nt.

In some embodiments of the present invention, the first nucleic acid fragment, the composite nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment is DNA or a nucleic acid analog.

In some preferred embodiments of the present invention, the nucleic acid analog is LNA or PNA.

In other preferred embodiments of the present invention, the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are single-stranded DNA.

In some embodiments of the present invention, the anchoring moiety is selected from any one or a combination of lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and/or amino acids; preferably cholesterol, palmitate or tocopherol.

In some specific embodiments of the present invention, the sequence of the first nucleic acid fragment is shown as SEQ ID NO:2.

In some specific embodiments of the present invention, the sequence of the second nucleic acid fragment is as shown in positions 2 to 55 of SEQ ID NO:1.

In some specific embodiments of the present invention, the sequence of the third nucleic acid fragment is as shown in SEQ ID NO:4.

In some specific embodiments of the present invention, the sequence of the fourth nucleic acid fragment is as shown in SEQ ID NO:5.

The second aspect of the present invention provides a method for preparing the nucleic acid-polypeptide complex as described in the first aspect, the method comprising the steps of subjecting the composite nucleic acid fragment or the second nucleic acid fragment modified with maleamide at the 5' end to a test polypeptide through a thiol-maleamide addition reaction to obtain the nucleic acid-polypeptide complex;

And/or, the method includes the step of obtaining the nucleic acid-polypeptide complex by reacting the first nucleic acid fragment whose 3' end is modified with DBCO with the polypeptide to be tested through an azide-DBCO click chemistry reaction.

The third aspect of the present invention provides a nucleic acid-protein complex, wherein the nucleic acid-protein complex comprises a polymerase and the nucleic acid-polypeptide complex as described in the first aspect;

Wherein, the polymerase binds to the single-stranded nucleic acid at the interface between the single-stranded nucleic acid and the double-stranded nucleic acid in the nucleic acid-polypeptide complex.

In some embodiments of the present invention, the polymerase is a polymerase capable of synthesizing double-stranded DNA using the amplified single-stranded DNA as a template.

In some preferred embodiments of the present invention, the polymerase is a DNA polymerase.

In the present invention, the polymerase is, for example, Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, enzyme, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, or any combination thereof.

When the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid fragment, the fourth nucleic acid fragment and/or the composite nucleic acid fragment is not DNA, the polymerase should be adaptively adjusted to a polymerase corresponding to nucleic acid.

The fourth aspect of the present invention provides a sequencing complex, the sequencing complex comprising a nanopore and the nucleic acid polypeptide complex as described in the first aspect or the nucleic acid protein complex as described in the third aspect; the nucleic acid polypeptide complex or the nucleic acid protein complex can move axially relative to the nanopore;

Wherein, in the nucleic acid-polypeptide complex or the nucleic acid-protein complex, the first nucleic acid fragment and the polypeptide sequence to be detected pass through the nanopore and move axially relative to the nanopore.

In some embodiments of the present invention, the nanopore is embedded in an electrically insulating film.

In some embodiments of the present invention, the nanopore is a transmembrane protein pore or a solid-state pore.

In some preferred embodiments of the present invention, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector protein, InvG, GspD or any combination thereof.

In some embodiments of the present invention, the nanopore is a modified nanopore.

In some preferred embodiments of the present invention, the modified nanopore is a polypeptide-modified nanopore.

In some embodiments of the present invention, the polypeptide is a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker or any combination thereof.

In some embodiments of the present invention, the electrically insulating film is an amphiphilic film, a high molecular polymer film or any combination thereof.

In some preferred embodiments of the present invention, the electrical insulating membrane is a phospholipid bilayer, a diblock copolymer or a triblock copolymer.

A fifth aspect of the present invention provides a method for polypeptide sequencing, the method comprising the following steps:

(1) applying an electric field force to the sequencing complex as described in the fourth aspect, so that the first nucleic acid fragment is captured by the nanopore, and the first nucleic acid fragment pulls the polypeptide to be tested so that the polypeptide to be tested at least partially passes through the nanopore from the first electric field end to the second electric field end;

(2) The polymerase synthesizes a double-stranded nucleic acid using the composite nucleic acid fragment or the single-stranded nucleic acid of the second nucleic acid fragment from the end of the double-stranded nucleic acid as a template, and pulls the polypeptide to be tested from the second electric field end through the nanopore to the first electric field end, generating and recording the current change in the nanopore.

In the present invention, the first nucleic acid fragment is used to provide negative charge, and provides a pulling force to pass through the nanopore under the action of the electric field force.

In the present invention, the hairpin structure of the composite nucleic acid fragment provides a double-stranded region and an exposed single-stranded region for the nucleic acid-polypeptide complex, which is conducive to the polymerase correctly identifying the starting site of double-stranded synthesis for amplification and sequencing.

In the present invention, the fourth nucleic acid fragment can be used as a switch for automated sequencing. Prevent the polymerase from starting to synthesize double-stranded DNA in advance. When the fourth nucleic acid fragment is separated from the composite nucleic acid fragment or the second nucleic acid fragment, the polymerase recognizes the exposed start site of amplification and amplification begins; when the fourth nucleic acid fragment is in a binding state with the composite nucleic acid fragment or the second nucleic acid fragment, the polymerase cannot recognize the start site of amplification and amplification cannot be performed.

Furthermore, the anchoring portion of the fourth nucleic acid fragment may be used to aggregate the complex to the periphery of the pore.

In the present invention, the third nucleic acid fragment is not directly connected to the second nucleic acid fragment by chemical bonds or enzymes, but is only connected to the second nucleic acid fragment by a hairpin structure formed by annealing. The third nucleic acid fragment provides an annealing site, thereby combining the second nucleic acid fragment and the third nucleic acid fragment without using chemical/enzymatic connection.

In the present invention, the spacer is used to present a more obvious peak on the current curve with a smaller volume, so as to facilitate the determination of the starting position of the polypeptide signal. The spacer is, for example, abasic deoxynucleoside, isp6, isp18, polyT, etc.

Since polymerase amplification is slower and the amplification and translocation speeds are less volatile, it has a greater advantage over helicase.

In some embodiments of the present invention, the first electric field end is the nanopore end in a negative electric field, and the second electric field end is the nanopore end in a positive electric field.

In some embodiments of the present invention, after the first nucleic acid fragment is captured by the nanopore, the fourth nucleic acid fragment is separated from the composite nucleic acid fragment or the second nucleic acid fragment under the action of an electric field force.

In some embodiments of the present invention, the electric field force is formed by applying a voltage, and the voltage is above 50 mV.

In some preferred embodiments of the present invention, the voltage is 100 mV-300 mV.

In some embodiments of the present invention, the method further comprises step (3): analyzing the current change to identify the amino acid sequence of the polypeptide to be detected.

A sixth aspect of the present invention provides a polypeptide sequencing device, the polypeptide sequencing device comprising: a sample processing module, a nanopore sequencing module, a power module and a detection and analysis module;

Wherein, the sample processing module processes the polypeptide to be tested to form a nucleic acid-protein complex as described in the third aspect, and transfers it to the nanopore sequencing module; the nanopore sequencing module sequences the polypeptide to be tested under the action of the power module, and the detection and analysis module detects and analyzes the current changes during the sequencing process;

Alternatively, the sample processing module processes the polypeptide to be tested to form a nucleic acid-polypeptide complex as described in the first aspect, and transfers it to a nanopore sequencing module; the nanopore sequencing module includes a polymerase, and sequences the polypeptide to be tested under the action of the power module, and the detection and analysis module detects and analyzes the current changes during the sequencing process.

A seventh aspect of the present invention provides a kit, the kit comprising a first nucleic acid fragment and a composite nucleic acid fragment, Wherein, the composite nucleic acid fragment comprises a nucleic acid sequence that can form a hairpin structure, and after forming the hairpin structure, the composite nucleic acid fragment comprises a double-stranded nucleic acid and a single-stranded nucleic acid, one end of the single-stranded nucleic acid is connected to the double-stranded nucleic acid, and the other end of the single-stranded nucleic acid is connected to the polypeptide to be detected;

After modification, the first nucleic acid fragment can be linked to the polypeptide to be detected and the composite nucleic acid fragment in sequence.

In some preferred embodiments of the present invention, the length of the single-stranded nucleic acid is greater than or equal to the length of the polypeptide to be detected.

In some embodiments of the present invention, the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, and the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure. After the hairpin structure is formed, the third nucleic acid fragment is complementary to a part of the second nucleic acid fragment to form a double-stranded nucleic acid, and the other part of the second nucleic acid fragment is a single-stranded nucleic acid.

In other embodiments of the present invention, the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, after the hairpin structure is formed, the two ends of the third nucleic acid fragment are complementary to each other to form a double-stranded nucleic acid, the second nucleic acid fragment is a single-stranded nucleic acid, and one end of the second nucleic acid fragment is connected to the double-stranded nucleic acid, and the other end of the second nucleic acid fragment is connected to the polypeptide to be tested.

In some embodiments of the present invention, the kit further comprises a fourth nucleic acid fragment, the 5' end nucleic acid sequence of the fourth nucleic acid fragment is complementary to at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment to form a double strand, and the 3' end of the fourth nucleic acid fragment is optionally connected to an anchoring portion.

In some preferred embodiments of the present invention, after the hairpin structure is formed, the stability of the double-stranded nucleic acid in the hairpin structure of the composite nucleic acid fragment is higher than the stability of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment.

In some preferred embodiments of the present invention, the Tm value of the double-stranded nucleic acid of the hairpin structure of the composite nucleic acid fragment is higher than the Tm value of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment.

In some preferred embodiments of the present invention, the anchoring moiety is selected from any one or a combination of lipids, fatty acids, sterols, polymer materials, carbon nanotubes, polypeptides, proteins and/or amino acids.

In some specific embodiments of the present invention, the anchoring moiety is cholesterol, palmitate or tocopherol.

In some embodiments of the present invention, the first nucleic acid fragment, the composite nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment is DNA or a nucleic acid analog.

In some preferred embodiments of the present invention, the nucleic acid analog is LNA or PNA.

In some other preferred embodiments of the present invention, the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are preferably single-stranded DNA.

In some specific embodiments of the present invention, the sequence of the first nucleic acid fragment is shown as SEQ ID NO:2.

In some specific embodiments of the present invention, the sequence of the second nucleic acid fragment is as shown in positions 2 to 55 of SEQ ID NO:1.

In some specific embodiments of the present invention, the sequence of the third nucleic acid fragment is as shown in SEQ ID NO:4.

In some specific embodiments of the present invention, the sequence of the fourth nucleic acid fragment is as shown in SEQ ID NO:5.

In some embodiments of the present invention, the kit further comprises any one or a combination of a polymerase, a nanopore, an electrical insulating membrane and a reaction buffer.

An eighth aspect of the present invention provides a use of the nucleic acid-polypeptide complex as described in the first aspect, the nucleic acid-protein complex as described in the third aspect, or the sequencing complex as described in the fourth aspect in nanopore sequencing.

Beneficial effects brought by the technical solution of the present invention:

The present invention can construct a DNA-polypeptide-DNA ternary complex and use negatively charged DNA as a guide sequence to pull polypeptides of different charges into the nanopore, thereby realizing the detection of any polypeptide sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of protein sequencing technology based on Hel308 rate control.

Figure 2 is a schematic diagram of peptide sequencing technology using Phi29 DNA polymerase to control the rate.

FIG. 3 is a schematic diagram of protein sequencing technology based on oligonucleotide-controlled protein and rate control.

FIG. 4 is a schematic diagram of protein sequencing technology based on MTA helicase rate control.

FIG5 is a schematic diagram of a protein sequencing process according to an embodiment of the present invention, wherein A is a schematic diagram of a sequencing complex; and B is a schematic diagram of a sequencing process.

Figure 6 is a chemical reaction process for connecting DNA1 to a polypeptide according to Example 1: DNA1 containing a maleimide modification at the 5' end is connected to the N-terminal cysteine residue of the polypeptide via a thiol-maleimide addition reaction to obtain a DP complex, and is further connected to DNA2 containing a DBCO modification at the 3' end via a click chemistry reaction with the azide-modified lysine on the C-terminal side chain of the polypeptide to obtain a DPD complex.

FIG. 7 is a chromatogram showing the purification of (a) DNA1-Peptide1, (b) DNA1-Peptide2, and (c) DNA1-Peptide3 by high performance liquid chromatography (HPLC) according to Example 1. FIG.

8 is a chromatogram showing the purification of (a) DNA1-Peptide1-DNA2, (b) DNA1-Peptide2-DNA2, and (c) DNA1-Peptide3-DNA2 by high performance liquid chromatography (HPLC) according to Example 2.

Figure 9 is a HPLC purity analysis chromatogram and mass spectrometry characterization diagram of DNA1-Peptide-DNA2 shown in Example 2, wherein (a) and (b) correspond to DNA1-Peptide1-DNA2, (c) and (d) correspond to DNA1-Peptide2-DNA2, and (e) and (f) correspond to DNA1-Peptide3-DNA2.

FIG. 10 is a diagram showing the basic principle of detecting the DPD complex using a nanopore according to Example 3.

FIG. 11 shows the nanopore test results of DNA 3 according to Example 3.

FIG. 12 shows the nanopore test results of DNA 3 according to Example 3.

FIG. 13 shows the nanopore test results of DNA1-Peptide1-DNA2 according to Example 4.

FIG. 14 shows the nanopore test results of DNA1-Peptide1-DNA2 according to Example 4.

FIG. 15 shows the nanopore test results of DNA1-Peptide2-DNA2 according to Example 5. FIG.

FIG. 16 shows the nanopore test result of DNA1-Peptide2-DNA2 according to Example 5. FIG.

FIG. 17 shows the nanopore test results of DNA1-Peptide3-DNA2 according to Example 6.

FIG. 18 shows the nanopore test results of DNA1-Peptide3-DNA2 according to Example 6.

DETAILED DESCRIPTION

definition

The term "nucleotide sequence" or "nucleic acid sequence" as used herein refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. The term "nucleic acid" as used herein is a single-stranded nucleotide sequence in which the 3' and 5' ends on each nucleotide are connected by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized and manufactured in vitro, or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as DNA or RNA that has been methylated, or RNA that has been subjected to post-transcriptional modification, such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA) and peptide nucleic acids (PNA). The size of a nucleic acid is usually expressed as the number of nucleotides (nt) in the case of a single-stranded polynucleotide.

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. Peptides may be prepared using recombinant techniques, such as by expressing recombinant or synthetic polynucleotides.

The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. A protein may consist of a single polypeptide or may include multiple polypeptides assembled to form a multimer. A multimer may be a homo-oligomer or a hetero-oligomer. A protein may be a naturally occurring or wild-type protein or a modified or non-naturally occurring protein. A protein may differ from a wild-type protein, for example, by the addition, substitution or deletion of one or more amino acids.

The term "anchoring" refers to fixing a substance in a relatively stable manner within a structure or within a certain range on its surface by relying on polarity, intermolecular forces, chemical bonds, etc.

In some embodiments, the present invention constructs a DNA-peptide-DNA ternary complex structure through thiol-maleimide addition reaction and azide-DBCO click chemistry reaction, and purifies it by HPLC to obtain a pure product, which is used as a substrate for polypeptide nanopore sequencing. DNA1 is a DNA fragment amplified by Phi29 DNA polymerase, and DNA2 is a DNA fragment that guides the polypeptide into the pore.

The synthesized DNA-peptide-DNA complex is combined with the hairpin DNA fragment, the protective DNA fragment and the Phi29 DNA polymerase through annealing and co-incubation to form a sequencing complex. The complex is mixed with the dNTP solution and added to the solution chamber of the sequencing chip. Since there is a sterol structure at the end of DNA5, the entire annealed library will be anchored on the membrane to connect it to the nanopore. Under the action of the electric field force, the chain containing DNA-peptide-DNA will move toward the trans end under the action of the electric field force, thereby tearing apart the double-stranded interaction between DNA5 and DNA1, exposing the 3' end of DNA4 to start synthesizing the DNA double strand. Through the reaction of continuously synthesizing the DNA double strand, the polypeptide is stably pulled through the nanopore and an electrical signal is obtained (the schematic diagram of the process is shown in Figure 5).

By comparing the nanopore electrical signals of the DNA-peptide-DNA and DNA control groups, it is demonstrated that the scheme proposed in the present invention is feasible for nanopore detection of polypeptides with different electrical properties, so that Phi29 DNA polymerase can be used to control the speed of nanopore sequencing of any polypeptide.

The DNA and polypeptide sequences used in the following examples were synthesized by BGI Liuhe, and the sequence information is as follows (from left to right): Right is from 5' to 3' end):

DNA1 (SEQ ID NO: 1):

/Maleimide/NAGAACTTTAGAACTTTTCAGATCTCACTATCGCATTCTCATGCAGGTCGTAGCC, wherein /Maleimide/ can be connected to the C-terminus of the polypeptide.

DNA2 (SEQ ID NO:2):

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT/DBCO/, wherein /DBCO/ can be connected to the N-terminus of the polypeptide (i.e., LYS(N3)).

DNA3 (SEQ ID NO:3):

TTTTTTTTTTTTTTTTTTTTTTTT/NAGAACTTTAGAACTTTTCAGATCTCACTATCGCATTCTCATGCAGGTCGTAGCC

DNA4 (SEQ ID NO:4):

GCGTACGCCTACGGTTTTCCGTAGGCGTACGCGGCTACGACCTGCATGAGAATGC DNA5 (SEQ ID NO:5):

GATAGTGAGATCTGATTTCCCAAATTTAAA/cholesterol/

Peptide1(SEQ ID NO:6):CGSGDDGSG{LYS(N3)}

Peptide2(SEQ ID NO:7):CGSGYYGSG{LYS(N3)}

Peptide3(SEQ ID NO:8):CGSGRRRGSG{LYS(N3)}

Among them, the structure of Maleimide is as follows:

N (abasic spacer) is a spacer of abasic deoxynucleoside, and its structure is shown below:

LYS(N3) is an azidation modification of the Lys side chain (the azide group replaces the original amino group), and its structural formula is as follows:

DNA3 is the control sequence (Control) used to verify the present invention, which simulates the combined sequence of DNA2+DNA1. Specifically, 25-mer polyT is used to simulate DNA2, and then the complete DNA1 sequence except the maleimide modification group is added to its 3' end.

DNA4 is used to anneal with DNA3 or DNA1 in DNA1-Peptide-DNA2 to form a hairpin-shaped double-stranded structure.

DNA5 is used to anneal with DNA3 or DNA1 in DNA1-Peptide-DNA2 to form a protective fragment of a 15bp double-stranded structure, which prevents the polymerase from starting to synthesize the DNA double strand prematurely.

Example 1: Construction of nucleic acid-polypeptide complex (DP ligation product)

(1) Experimental methods

Three DP-linked products were prepared: (a) DNA1-Peptide1, (b) DNA1-Peptide2, and (c) DNA1-Peptide3. DNA1 is the amplified DNA sequence shown in FIG5, and its sequence is SEQ ID NO: 1. Peptide1, Peptide2, and Peptide 3 are the peptides to be tested shown in FIG5, and their sequences are SEQ ID NO: 6-8, respectively. The preparation method of each DP-linked product is as follows:

DNA1 powder was fully dissolved in pure water, and the concentration was quantified by Qubit ssDNA detection kit (ThermoFisher). Peptide powder was fully dissolved in pure water to 20 mg/mL. 1 μL 10× ligation reaction buffer (1M HEPES (pH=7.2), 50 mM EDTA) was added to the EP tube, and 80 nmol of peptide and 200 mM TCEP solution were added, with a molar ratio of peptide to TCEP of 1:1. Pure water was then added to dilute the reaction solution, vortexed and mixed, and the metal bath was 25°C for 10 minutes; 5 nmol of DNA1 was added after 10 minutes, and the final reaction volume was 10 μL. After vortexing and mixing, the metal bath was 25°C for 4 hours. After the reaction, the DNA1-Peptide (DP) ligation product fraction was purified and collected using a 1260 Infinity II high performance liquid chromatograph (Agilent), and the fraction was lyophilized overnight for subsequent ligation reactions.

(2) Experimental results

Figure 6 shows the chemical reaction process of connecting DNA1 to peptides. Figure 7 shows the HPLC purification chromatograms of (a) DNA1-Peptide1, (b) DNA1-Peptide2, and (c) DNA1-Peptide3, where the highest peak between 10 and 17 minutes corresponds to the DP complex fractions taken.

Example 2: Preparation of DPD linker

(1) Experimental methods

Three DPD conjugates were prepared: (a) DNA1-Peptide1-DNA2, (b) DNA1-Peptide2-DNA2, and (c) DNA1-Peptide3-DNA2. DNA2 is the guide DNA sequence shown in FIG5 , and its sequence is SEQ ID NO: 2. The preparation method of each is as follows:

DNA2 powder was fully dissolved in pure water, and its concentration was quantitatively detected by Qubit ssDNA detection kit (ThermoFisher). The DP connection product and DNA2 were in a molar ratio of 1:7.5. The DNA2 solution was added to the lyophilized powder of the DP connection product obtained in Example 2, and 10 μL 10× connection reaction buffer (1M HEPES (pH＝7.2), 50mM EDTA) was added. The final reaction system was adjusted to 100 μL with pure water, vortexed and mixed, and the metal bath was 25°C for 16 hours. After the reaction, the 1260 Infinity II high performance liquid chromatograph (Agilent) was used for purification and the DNA1-Peptide-DNA2 (DPD) connection product fraction was collected. The concentration of the fractions was quantitatively detected using the Qubit ssDNA detection kit (ThermoFisher). A small amount of the purified product was retained in an EP tube for HPLC purity identification and mass spectrometry characterization. The remaining sample was packaged, freeze-dried overnight, and stored at -80°C.

(2) Experimental results

Figure 8 shows (a) DNA1-Peptide1-DNA2, (b) DNA1-Peptide2-DNA2, (c) DNA1- HPLC purification chromatogram of Peptide3-DNA2, in which the two highest peaks between 14 and 18 minutes correspond to the DPD complex fractions taken. Figure 9 shows (a) HPLC purity analysis chromatogram and (b) mass spectrum of DNA1-Peptide1-DNA2; (c) HPLC purity analysis chromatogram and (d) mass spectrum of DNA1-Peptide2-DNA2; (e) HPLC purity analysis chromatogram and (f) mass spectrum of DNA1-Peptide3-DNA2. HPLC purity analysis shows that the purity of the three DPD complexes is above 85%, and the highest can exceed 95%. The experimental molecular weight shown in the mass spectrum also matches the theoretical molecular weight of the three DPD complexes, respectively, proving that three DPD conjugates were obtained in this embodiment.

Example 3: Nanopore sequencing of DNA 3-5 annealing complex

(1) Experimental methods

The sequence of DNA3 is SEQ ID NO: 3; DNA4 is the hairpin fragment shown in Figure 5, and its sequence is SEQ ID NO: 4; DNA5 is the protection fragment shown in Figure 5, and its sequence is SEQ ID NO: 5.

DNA3, DNA4 and DNA5 powders were fully dissolved in pure water, and their concentrations were quantitatively detected by Qubit ssDNA detection kit (ThermoFisher). DNA3, DNA4 and DNA5 aqueous solutions were added to EP tubes at a molar ratio of 1:1:2, and annealed at room temperature for 20 minutes. The concentration of the annealed complex was quantitatively detected using Qubit dsDNA HS detection kit (ThermoFisher), and then an appropriate amount of annealed complex was taken and incubated with 1μL Phi29 DNA polymerase (NEB) in a total volume of 8μL 1×PBS for 30 minutes, wherein the final concentration of the annealed complex in the incubation mixture was 75nM. As shown in Figure 10, a patch clamp amplifier was used to collect current signals. The electrolytic cell was divided into two chambers by a Teflon membrane with micron-sized pores (diameter 50-200μm) in the middle: a cis chamber and a trans chamber; a pair of Ag/AgCl electrodes were placed in each chamber; after a layer of bimolecular phospholipid membrane was formed at the micropores of the two chambers, the nanopore protein MspA was added; after a single nanopore protein MspA was inserted into the phospholipid membrane, the above-mentioned co-incubation mixture and 30μL 10Mm dNTP (NEB) were added, 180mV was applied, and the current data was recorded.

(2) Experimental results

Figures 11 and 12 show the signal data of two parallel DNA3 nanopore experiments, where (a) represents the overall through-pore signal from the opening current to the 25-mer polyT signal at the 5' end of DNA3. It can be seen that under a fixed voltage of 180mV, the nanopore current first drops from about 150pA to about 50pA from the opening current, and then rises back to about 70-80pA and oscillates for a period of time. This signal corresponds to the pore entry signal of the annealing complex, but at this time the protective fragment DNA5 has not yet detached from the complex. When DNA5 is torn apart under the action of the electric field force, the electrical signal immediately presents three 40-50pA signal valleys as shown in (b), followed by a signal peak of about 100pA. This characteristic signal interval corresponds to the abasic spacer in the DNA3 sequence and a relatively continuous repeating sequence at its 3' end. After the characteristic signal is read, the nanopore current drops to a stable platform of about 60pA, corresponding to the 25-mer polyT at the 5' end of DNA3.

The above results prove that the complex formed by DNA3-5 can pass through the nanopore, and further confirm that the annealing complex constructed using DNA4 and DNA5 can realize signal reading according to the scheme designed by the present invention.

Example 4: Nanopore sequencing of DNA1-Peptide1-DNA2

(1) Experimental methods

DNA4 is the hairpin fragment shown in Figure 5, and its sequence is SEQ ID NO: 4; DNA5 is the protection fragment shown in Figure 5, and its sequence is SEQ ID NO: 5.

Take the freeze-dried DNA1-Peptide1-DNA2 obtained in Example 2, and mix the DNA1-Peptide1-DNA2 dry powder and DNA4 and DNA5 solutions in a molar ratio of 1:1:2 in an EP tube according to the amount of packaging, and anneal at room temperature for 20 minutes to form the annealing complex shown in Figure 5. The concentration of the annealing complex was quantitatively detected using the Qubit dsDNA HS detection kit (ThermoFisher), and then an appropriate amount of the annealing complex was taken and incubated with 1 μL of Phi29 DNA polymerase (NEB) in a total volume of 8 μL of 1×PBS for 30 minutes, wherein the final concentration of the annealing complex in the incubation mixture was 75 nM. A patch clamp amplifier was used to collect current signals. The electrolytic cell was divided into two chambers by a Teflon membrane with micron-sized pores (diameter 50-200μm) in the middle: a cis chamber and a trans chamber; a pair of Ag/AgCl electrodes were placed in each chamber; after a layer of bimolecular phospholipid membrane was formed at the micropores of the two chambers, nanopore protein was added; after a single nanopore protein was inserted into the phospholipid membrane, the above-mentioned co-incubation mixture and 30μL 10Mm dNTP (NEB) were added, 180mV was applied, and the current data was recorded.

(2) Experimental results

Figures 13 and 14 show two parallel DNA1-Peptide1-DNA2 nanopore experimental signal data, respectively, where (a) represents the nanopore current data from the opening current to the current signal fluctuating steadily in the 80-100pA interval. It can be seen that, similar to Figures 11 and 12, the pore entry signal of the annealing complex and the characteristic signal intervals of the three current signal valleys are successfully identified. Since the sequence of DNA1 in DNA1-Peptide1-DNA2 is consistent with the 3' end sequence of DNA3 in Example 3, a characteristic signal the same as the "three consecutive signal valleys" presented when the corresponding DNA3 in Example 3 passes through the nanopore will be generated, which is called the characteristic signal interval, and the third signal valley in the characteristic interval can be roughly estimated as the starting end of the polypeptide signal. Next, since in DNA1-Peptide1-DNA2, the peptide Peptide1 is sequenced immediately after DNA1, while in Example 3, the 25-mer polyT is sequenced in DNA3, so the current fluctuation trends after the characteristic signal interval are obviously different: DNA1-Peptide1-DNA2 generates a current of "40pA→70pA→80pA→70pA→80pA", as shown in Figure 13 (b) and Figure 14 (b); the polyT of DNA3 generates a relatively stable current of about 60pA. The characteristic signal interval with three signal valleys similar to that shown in Example 3 generated by DNA1-Peptide1-DNA2 after passing through the nanopore, and the fluctuation signal generated by the above-mentioned peptide Peptide1 after passing through the nanopore is different from that when 25-mer polyT is pierced, which proves that negatively charged polypeptides can be captured by nanopores and detected under the speed control of Phi29 DNA polymerase.

Example 5: Nanopore sequencing of DNA1-Peptide2-DNA2

(1) Experimental methods

Referring to the experimental method of Example 4, DNA1-Peptide1-DNA2 was replaced by DNA1-Peptide2-DNA2.

(2) Experimental results

Figures 15 and 16 show two parallel DNA1-Peptide2-DNA2 nanopore experimental signal data, where (a) represents the nanopore current data from the opening current to the current signal fluctuating steadily in the 80-100pA interval. It can be seen that, similar to Figures 11 and 12, the pore entry signal of the annealing complex and the characteristic signal intervals of the three current signal valleys are successfully identified. Similar to Figures 13 and 14, since the sequence of DNA1 in DNA1-Peptide2-DNA2 is consistent with the 3' end sequence of DNA3, a characteristic signal interval of "three consecutive signal valleys" will be generated when the corresponding DNA3 in Example 3 passes through the nanopore, and the third signal valley of the characteristic interval can be roughly estimated as the starting end of the polypeptide signal. Next, since in DNA1-Peptide2-DNA2, the peptide Peptide2 is sequenced immediately after DNA1, while in Example 3, the 25-mer polyT is sequenced in DNA3, so the current fluctuation trends after the characteristic signal interval are obviously different: DNA1-Peptide2-DNA2 generates a current of "40pA→60pA→40pA→70pA→85pA" as shown in Figures 15(b) and 16(b); the polyT of DNA3 generates a relatively stable current of about 60pA. The characteristic interval with three signal valleys similar to that shown in Example 3 generated by DNA1-Peptide2-DNA2 after passing through the nanopore, as well as the fluctuation signal generated by the above-mentioned peptide Peptide2 after passing through the nanopore, which is different from that when 25-mer polyT is pierced, proves that electrically neutral peptides can be captured by nanopores and detected under the speed control of Phi29 DNA polymerase.

Example 6: Nanopore sequencing of DNA1-Peptide3-DNA2

(1) Experimental methods

Referring to the experimental method of Example 4, DNA1-Peptide1-DNA2 was replaced by DNA1-Peptide3-DNA2.

(2) Experimental results

Figures 17 and 18 show two parallel DNA1-Peptide3-DNA2 nanopore experimental signal data, respectively, where (a) represents the nanopore current data from the opening current to the current signal fluctuating steadily in the 30pA interval. It can be seen that, similar to Figures 11 and 12, the pore entry signal of the annealing complex and the characteristic signal intervals of the three current signal valleys were successfully identified. Similar to Figures 13-16, since the sequence of DNA1 in DNA1-Peptide3-DNA2 is consistent with the 3' end sequence of DNA3 in Example 3, a characteristic signal interval of "three consecutive signal valleys" will be generated when the corresponding DNA3 in Example 3 passes through the nanopore, and the third signal valley of the characteristic signal interval can be roughly estimated as the starting end of the polypeptide signal. Next, since in DNA1-Peptide3-DNA2, the peptide Peptide3 is sequenced immediately after DNA1, while in Example 3, the 25-mer polyT is sequenced in DNA3, the current fluctuation trends after the characteristic signal interval are obviously different: DNA1-Peptide3-DNA2 generates a current of "50pA→85pA→95pA→120pA→30pA", as shown in Figure 17 (b) and Figure 18 (b); DNA3's polyT generates a relatively stable current of about 60pA. The current generated by DNA1-Peptide3-DNA2 after passing through the nanopore is similar to that in Example 3. The characteristic signal interval with three signal valleys shown in Example 3, as well as the fluctuation signal generated after the above-mentioned peptide Peptide3 passes through the nanopore, which is different from that when 25-mer polyT is pierced, proves that positively charged peptides can be captured by the nanopore and detected under the control of the Phi29 DNA polymerase.

Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims (31)

A nucleic acid-polypeptide complex, characterized in that the nucleic acid-polypeptide complex comprises a first nucleic acid fragment, a polypeptide to be detected and a composite nucleic acid fragment connected in sequence; Wherein, the composite nucleic acid fragment comprises a nucleic acid sequence that can form a hairpin structure, and after forming the hairpin structure, the composite nucleic acid fragment comprises a double-stranded nucleic acid and a single-stranded nucleic acid, one end of the single-stranded nucleic acid is connected to the double-stranded nucleic acid, and the other end of the single-stranded nucleic acid is connected to the polypeptide to be detected; Preferably, the length of the single-stranded nucleic acid is greater than or equal to the length of the polypeptide to be detected. The nucleic acid-polypeptide complex as described in claim 1 is characterized in that the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, and after the hairpin structure is formed, the third nucleic acid fragment is complementary to a part of the second nucleic acid fragment to form a double-stranded nucleic acid, and the other part of the second nucleic acid fragment is a single-stranded nucleic acid. The nucleic acid-polypeptide complex as described in claim 1 is characterized in that the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, and after the hairpin structure is formed, the two ends of the third nucleic acid fragment are complementary to each other to form a double-stranded nucleic acid, the second nucleic acid fragment is a single-stranded nucleic acid, and one end of the second nucleic acid fragment is connected to the double-stranded nucleic acid, and the other end of the second nucleic acid fragment is connected to the polypeptide to be tested. The nucleic acid-polypeptide complex according to any one of claims 1 to 3, characterized in that the nucleic acid-polypeptide complex further comprises a fourth nucleic acid fragment, the 5' end nucleic acid sequence of the fourth nucleic acid fragment is complementary to at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment to form a double strand, and the 3' end of the fourth nucleic acid fragment is optionally connected to an anchoring portion; Preferably, after the hairpin structure is formed, the stability of the double-stranded nucleic acid in the hairpin structure of the composite nucleic acid fragment is higher than the stability of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment; More preferably, the Tm value of the double-stranded nucleic acid of the hairpin structure of the composite nucleic acid fragment is higher than the Tm value of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment. The nucleic acid-polypeptide complex according to any one of claims 1 to 4 is characterized in that the length of the first nucleic acid fragment is 5 to 200 nt, preferably 10 to 50 nt; the length of the composite nucleic acid fragment is 80 to 2000 nt, preferably 50 to 200 nt; the length of the second nucleic acid fragment is 40 to 1000 nt, preferably 50 to 200 nt; the length of the third nucleic acid fragment is 40 to 1000 nt, preferably 50 to 200 nt; the length of the fourth nucleic acid fragment is 20-500 nt, preferably 25-100 nt. The nucleic acid-polypeptide complex according to any one of claims 1 to 5, characterized in that the first nucleic acid fragment, the composite nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are DNA or nucleic acid analogs; the nucleic acid analogs are preferably LNA or PNA; the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are preferably single-stranded DNA. The nucleic acid-polypeptide complex according to claim 4, characterized in that the anchoring moiety is selected from any one or a combination of lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and/or amino acids; preferably cholesterol, palmitate or tocopherol. The nucleic acid-polypeptide complex according to any one of claims 1 to 7, wherein the sequence of the first nucleic acid fragment is as shown in SEQ ID NO: 2; And/or, the sequence of the second nucleic acid fragment is as shown in positions 2 to 55 of SEQ ID NO:1; And/or, the sequence of the third nucleic acid fragment is shown in SEQ ID NO:4; And/or, the sequence of the fourth nucleic acid fragment is as shown in SEQ ID NO:5. A method for preparing a nucleic acid-polypeptide complex according to any one of claims 1 to 8, characterized in that the method comprises the step of subjecting the composite nucleic acid fragment or the second nucleic acid fragment modified with maleamide at the 5' end to a test polypeptide through a thiol-maleamide addition reaction to obtain the nucleic acid-polypeptide complex; And/or, the method includes the step of obtaining the nucleic acid-polypeptide complex by reacting the first nucleic acid fragment whose 3' end is modified with DBCO with the polypeptide to be tested through an azide-DBCO click chemistry reaction. A nucleic acid-protein complex, characterized in that the nucleic acid-protein complex comprises a polymerase and the nucleic acid-polypeptide complex according to any one of claims 1 to 8; Wherein, the polymerase is bound to the junction of the single-stranded nucleic acid and the double-stranded nucleic acid in the nucleic acid-polypeptide complex. The nucleic acid-protein complex according to claim 10, characterized in that the polymerase is a polymerase capable of synthesizing double-stranded DNA using the amplified single-stranded DNA as a template; Preferably, the polymerase is a DNA polymerase; More preferably, the polymerase is: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase or any combination thereof. A sequencing complex, characterized in that the sequencing complex comprises a nanopore and the nucleic acid-polypeptide complex according to any one of claims 1 to 8 or the nucleic acid-protein complex according to claim 10 or 11; the nucleic acid-polypeptide complex or the nucleic acid-protein complex can move axially relative to the nanopore; Wherein, in the nucleic acid-polypeptide complex or the nucleic acid-protein complex, the first nucleic acid fragment and the polypeptide sequence to be detected pass through the nanopore and move axially relative to the nanopore. The sequencing complex of claim 12, wherein the nanopore is embedded in an electrically insulating membrane. The sequencing complex according to claim 13, wherein the nanopore is a transmembrane protein pore or a solid Preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, Phi29 connector protein, InvG, GspD or any combination thereof. The sequencing complex as described in claim 13 or 14 is characterized in that the nanopore is a modified nanopore; the modified nanopore is preferably a polypeptide-modified nanopore; preferably, the polypeptide is a tag, an enzyme cleavage site, a signal peptide or a leader peptide, a detectable marker or any combination thereof. The sequencing complex as described in claim 13 is characterized in that the electrically insulating membrane is an amphiphilic membrane, a high molecular polymer membrane or any combination thereof; preferably, the electrically insulating membrane is a phospholipid bilayer, a diblock copolymer or a triblock copolymer. A method for polypeptide sequencing, characterized in that the method comprises the following steps: (1) applying an electric field force to the sequencing complex as described in any one of claims 12 to 16, so that the first nucleic acid fragment is captured by the nanopore, and the first nucleic acid fragment pulls the polypeptide to be tested so that the polypeptide to be tested at least partially passes through the nanopore from the first electric field end to the second electric field end; (2) The polymerase synthesizes a double-stranded nucleic acid using the composite nucleic acid fragment or the single-stranded nucleic acid of the second nucleic acid fragment from the end of the double-stranded nucleic acid as a template, and pulls the polypeptide to be tested from the second electric field end through the nanopore to the first electric field end, generating and recording the current change in the nanopore. The method of claim 17, wherein the first electric field end is the nanopore end in a negative electric field, and the second electric field end is the nanopore end in a positive electric field. The method according to claim 17 or 18 is characterized in that after the first nucleic acid fragment is captured by the nanopore, the fourth nucleic acid fragment is separated from the composite nucleic acid fragment or the second nucleic acid fragment under the action of the electric field force. The method according to any one of claims 17 to 19 is characterized in that the electric field force is formed by applying a voltage, and the voltage is above 50 mV; preferably, the voltage is 100 mV-300 mV. The method according to any one of claims 17 to 20, characterized in that the method further comprises the steps of (3): Analyze the current changes to identify the amino acid sequence of the polypeptide to be tested. A polypeptide sequencing device, characterized in that the polypeptide sequencing device comprises: a sample processing module, a nanopore sequencing module, a power module and a detection and analysis module; Wherein, the sample processing module processes the polypeptide to be tested to form the nucleic acid-protein complex as claimed in claim 10 or 11, and transfers it to the nanopore sequencing module; the nanopore sequencing module sequences the polypeptide to be tested under the action of the power module, and the detection and analysis module detects and analyzes the current changes during the sequencing process; Alternatively, the sample processing module processes the polypeptide to be tested to form the nucleic acid-polypeptide complex as described in any one of claims 1 to 8, and transfers it to the nanopore sequencing module; the nanopore sequencing module includes a polymerase, and sequences the polypeptide to be tested under the action of the power module, and the detection and analysis module processes the current during the sequencing process. Detect and analyze changes. A kit, characterized in that the kit comprises a first nucleic acid fragment and a composite nucleic acid fragment, Wherein, the composite nucleic acid fragment comprises a nucleic acid sequence that can form a hairpin structure, and after forming the hairpin structure, the composite nucleic acid fragment comprises a double-stranded nucleic acid and a single-stranded nucleic acid, one end of the single-stranded nucleic acid is connected to the double-stranded nucleic acid, and the other end of the single-stranded nucleic acid is connected to the polypeptide to be detected; The first nucleic acid fragment can be sequentially connected to the polypeptide to be tested and the composite nucleic acid fragment after modification; Preferably, the length of the single-stranded nucleic acid is greater than or equal to the length of the polypeptide to be detected. The kit as described in claim 23 is characterized in that the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, and after the hairpin structure is formed, the third nucleic acid fragment is complementary to a part of the second nucleic acid fragment to form a double-stranded nucleic acid, and the other part of the second nucleic acid fragment is a single-stranded nucleic acid. The kit as described in claim 23 is characterized in that the composite nucleic acid fragment is composed of a second nucleic acid fragment and a third nucleic acid fragment connected in sequence, the third nucleic acid fragment contains a nucleic acid sequence that can form a hairpin structure, after the hairpin structure is formed, the two ends of the third nucleic acid fragment are complementary to each other to form a double-stranded nucleic acid, the second nucleic acid fragment is a single-stranded nucleic acid, and one end of the second nucleic acid fragment is connected to the double-stranded nucleic acid, and the other end of the second nucleic acid fragment is connected to the polypeptide to be tested. The kit according to claim 24 or 25, characterized in that the kit further comprises a fourth nucleic acid fragment, the 5' end nucleic acid sequence of the fourth nucleic acid fragment is complementary to at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment to form a double strand, and the 3' end of the fourth nucleic acid fragment is optionally connected to an anchoring portion; Preferably, after the hairpin structure is formed, the stability of the double-stranded nucleic acid in the hairpin structure of the composite nucleic acid fragment is higher than the stability of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment; More preferably, the Tm value of the double-stranded nucleic acid of the hairpin structure of the composite nucleic acid fragment is higher than the Tm value of the double-stranded nucleic acid formed by the complementarity of the 5'-end nucleic acid sequence of the fourth nucleic acid fragment and at least a portion of the single-stranded nucleic acid in the composite nucleic acid fragment or the second nucleic acid fragment. The kit according to claim 26, characterized in that the anchoring moiety is selected from any one or a combination of lipids, fatty acids, sterols, polymer materials, carbon nanotubes, polypeptides, proteins and/or amino acids; preferably cholesterol, palmitate or tocopherol. The kit according to any one of claims 23 to 27, characterized in that the first nucleic acid fragment, the composite nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are DNA or nucleic acid analogs; the nucleic acid analogs are preferably LNA or PNA; the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid and/or the fourth nucleic acid fragment are preferably single-stranded DNA. The kit according to any one of claims 23 to 28, wherein the sequence of the first nucleic acid fragment is as shown in SEQ ID NO: 2; And/or, the sequence of the second nucleic acid fragment is as shown in positions 2 to 55 of SEQ ID NO:1; And/or, the sequence of the third nucleic acid fragment is shown in SEQ ID NO:4; And/or, the sequence of the fourth nucleic acid fragment is as shown in SEQ ID NO:5. The kit according to any one of claims 23 to 29, characterized in that the kit further comprises any one or a combination of a polymerase, a nanopore, an electrically insulating membrane and a reaction buffer. A use of the nucleic acid-polypeptide complex according to any one of claims 1 to 8, the nucleic acid-protein complex according to claim 10 or 11, or the sequencing complex according to any one of claims 12 to 16 in nanopore sequencing.