CN120366293B

CN120366293B - Sequencing adapter for nanopore sequencing and its use

Info

Publication number: CN120366293B
Application number: CN202510881103.1A
Authority: CN
Inventors: 傅迪宏; 侯军军; 杨锦涛
Original assignee: Hangzhou Huada Xufeng Technology Co ltd
Current assignee: Hangzhou Huada Xufeng Technology Co ltd
Priority date: 2025-06-27
Filing date: 2025-06-27
Publication date: 2025-10-24
Anticipated expiration: 2045-06-27
Also published as: CN120366293A

Abstract

The present invention relates to the field of sequencing technology, and more particularly to sequencing adapters for nanopore sequencing and their uses. The sequencing adapters provided by the present invention incorporate different non-B-form structures into the sequencing adapters to prevent motor proteins from unwinding nucleic acids in a non-sequencing state. This allows the leading strand to pass through the secondary structure region for sequencing only when the leading strand enters the nanopore and is subjected to the electric field force. The sequencing adapters provided by the present invention for nanopore sequencing have lower synthesis costs than existing sequencing adapters.

Description

Sequencing adapter for nanopore sequencing and application thereof

Technical Field

The invention relates to the technical field of sequencing, in particular to a sequencing joint for nanopore sequencing and application thereof.

Background

Nanopore sequencing is an emerging sequencing technology in recent years, and is used as a high-throughput single-molecule sequencing method, and the principle of nanopore sequencing is to detect current changes in a pore canal caused by single-stranded nucleic acid molecules passing through a nanopore, so as to identify the base type of a perforation through signal recognition. In nanopore sequencing, the nucleic acid to be tested (DNA/RNA) is ligated to a sequencing linker loaded with a motor protein, resulting in a sequencing library that can be captured by the nanopore.

The existing nanopore sequencing connector mainly has two functions of 1 loading motor protein and 2 capturing the nanopore, so that the DNA to be tested can be subjected to nanopore sequencing. The function of capturing the nanopore is realized through a guide chain and an anchoring region, and the function of loading the motor protein needs two regions, namely 1, the loading region, namely a site to which the motor protein can be bound, is usually a section of single-stranded DNA, 2, the modification region does not function under the non-sequencing condition, does not shift, only when the guide chain part of a sequencing joint enters the nanopore, the motor protein is positioned above the nanopore, and under the action of electric field force, the motor protein moves across the modification region to perform unwinding/polymerization/enzyme cutting, starts to move relative to the direction of the inlet of the DNA to be tested, and the inlet of the DNA to be tested is sequenced.

However, the existing linker nucleic acid sequences are costly to synthesize and do not meet market demands. Thus, there is a need to develop a lower cost sequencing adapter for nanopore sequencing to reduce sequencing costs.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, it is an object of the present invention to provide a sequencing adapter for nanopore sequencing, which is designed by the inventors to prevent motor protein from unwinding nucleic acid in a non-sequencing state by designing a different non-B-form structure in the sequencing adapter so that it can pass through a secondary structure region only when a guide strand enters the nanopore under the action of an electric field force.

To this end, a first aspect of the invention provides a sequencing adapter. In some embodiments of the invention, the sequencing adapter is used for nanopore sequencing, wherein it comprises a first single strand comprising:

a first section for guiding a nucleic acid to be tested into a single-stranded nucleotide region of the nanopore;

A second segment for binding to a single-stranded nucleotide region of a motor protein;

A third segment of single stranded nucleotide region for preventing motor protein from functioning as a helic nucleic acid in a non-sequenced state;

A fourth section for connecting an analyte to be detected;

Wherein the third segment comprises a G-rich region and/or a C-rich region;

The G-rich region forms a G quadruplex structure;

The C-rich region forms an i-Motif structure.

The inventors found that a non-B-form structure (G-rich region and/or C-rich region) was designed in the sequencing linker of nanopore sequencing to prevent motor protein from unwinding nucleic acid in a non-sequencing state so that it could pass through the secondary structure region for sequencing only when the guide strand enters the nanopore under the action of electric field force. The sequencing joint synthesis cost for nanopore sequencing provided by the invention is lower than that of the existing sequencing joint.

In some embodiments of the invention, the sequencing adapter contains at least 10-30 consecutive bases T.

In some embodiments of the invention, the nucleic acid sequence of the third segment is as set forth in any one of SEQ ID NOS.22-25.

In some embodiments of the invention, the sequencing adapter further comprises a second single strand comprising a ligation segment complementarily paired with a fourth segment of the first single strand for ligation of an analyte to be detected.

In a second aspect the invention provides the use of a sequencing adapter according to the first aspect in the preparation of a kit for nanopore sequencing.

In a third aspect, the invention provides a kit for nanopore sequencing. In some embodiments of the invention, the kit comprises the sequencing adapter of the first aspect, further comprising a polynucleotide binding protein selected from at least one of a polymerase, helicase, or exonuclease.

In a fourth aspect the invention provides a construct. In some embodiments of the invention, the construct comprises a nucleic acid to be sequenced and a sequencing adapter of the first aspect, wherein the sequencing adapter is attached to either or both ends of the nucleic acid to be sequenced.

In a fifth aspect, the invention provides a complex for sequencing. In some embodiments of the invention, the complex comprises a polynucleotide binding protein, and

The sequencing adapter of the first aspect or the construct of the fourth aspect,

The polynucleotide binding protein is selected from at least one of a polymerase, a helicase, or an exonuclease.

In a sixth aspect, the invention provides a method of constructing a sequencing complex. In some embodiments of the invention, the method comprises:

(1) Constructing the sequencing adapter of the first aspect;

(2) Assembling the sequencing adapter with a nucleic acid to be sequenced to form a construct;

(3) Contacting the construct with a polynucleotide binding protein so as to obtain a sequencing complex;

The seventh aspect of the invention provides the use of a sequencing adapter according to the first aspect, a construct according to the fourth aspect, a complex for sequencing according to the fifth aspect in nanopore sequencing.

The eighth aspect of the present invention provides a nanopore sequencing method. In some embodiments of the invention, the nanopore sequencing method comprises nanopore sequencing of a nucleic acid to be sequenced using at least one of the sequencing adapter of the first aspect, the kit of the third aspect, the construct of the fourth aspect, the complex for sequencing of the fifth aspect.

The inventor designs different non-B-form structures in the sequencing joint by optimizing the sequencing joint for sequencing the nanopore, and the sequencing joint is used for preventing motor protein from unwinding nucleic acid in a non-sequencing state, so that the motor protein can pass through a secondary structure region only when a guide chain enters the nanopore under the action of electric field force, and sequencing is performed. By introducing a natural nucleotide sequence with a secondary structure on the linker, a novel nanopore sequencing linker can be constructed and sequencing can be successfully performed. The sequencing joint has low synthesis cost.

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.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a schematic representation of a sequencing adapter according to one embodiment of the present invention;

FIG. 2 shows the results of non-denaturing PAGE electrophoresis of DNA samples prepared in example 1, wherein lanes 1-9 are 50 bp DNA ladder (TIANGEN), G4-1, G4-1_neg, G4-2, G4-2_neg, ds-1, G4-2_neg, ds-2, ds-2_neg, respectively;

FIG. 3 shows the inhibition of helicase Dda by a fluorescence quenching assay to detect DNA samples;

FIG. 4 shows nanopore sequencing current signals, wherein A-E are the current signals of neg-Adp, a1-Adp, a2-Adp, a3-Adp, a4-Adp linker and the DNA to be tested to which it is attached, respectively.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.

In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.

As used herein, the term "B-form structure" refers to the most stable and common double helix conformation of DNA under physiological conditions, and its structural features include right-hand double helix, base pairing, specific helix parameters, and the presence of major and minor grooves. The stability, functional adaptability and dynamic nature of the B-form DNA enable it to efficiently perform important biological processes such as replication, transcription, etc. in an organism.

In this context, the term "motor protein" refers to a class of proteins capable of converting chemical energy (typically from ATP hydrolysis) into mechanical energy. In nanopore sequencing, motor Protein (motorprotein) plays a vital role, which is able to control the speed of DNA or RNA molecules through the nanopore, keeping it at a suitable rate. Such speed control is critical for accurate detection of the current change caused by each base. By stably controlling the movement of DNA or RNA molecules, motor proteins can reduce noise of current signals, improving sequencing accuracy and resolution. During nanopore sequencing, a motor protein binds to a linker of a library molecule, helping DNA or RNA molecules to stably enter the nanopore. In addition, motor proteins (e.g., phi29 DNA polymerase) are capable of binding to DNA double strands during nanopore sequencing and performing the function of unwinding nucleic acids under the drive of an electric field to unwind the double strands into single stranded nucleic acids. This process ensures that single-stranded nucleic acids can pass smoothly through the nanopore, thereby achieving accurate detection of each base.

As used herein, the term "G-rich region" refers to guanine-rich DNA or RNA sequence regions that form a stable G-quadruplex structure.

As used herein, the term "C-rich region" refers to a cytosine-rich region of DNA or RNA sequence. These regions have specific structures and functions in the biological molecule, in particular, are capable of forming specific secondary structures (e.g., i-motif structures).

According to a specific embodiment of the present invention, there is provided a sequencing adapter for nanopore sequencing comprising a first single strand comprising:

A fourth section for connecting an analyte to be detected;

Wherein the third segment comprises a G-rich region and/or a C-rich region;

The G-rich region forms a G quadruplex structure;

The C-rich region forms an i-Motif structure.

According to a specific embodiment of the invention, the sequencing adapter contains at least 10-30 consecutive bases T. For example, the sequencing adapter may contain 10, 15, 20, 25, 30 consecutive bases T. These consecutive bases T are typically located in the first segment of the sequencing adapter. For example, consecutive bases T in the first segment (also referred to as the leader) are shown in the sequence SEQ ID NO. 21 (5'-TTTTTTTTTTTTTTTTTTTT-3').

According to a specific embodiment of the present invention, the first section and the second section may be the same section.

According to a specific embodiment of the present invention, the nucleic acid sequence of the third segment is shown as any one of SEQ ID NOS.22-25.

SEQ ID NO: 22:5’-GGGTGGGTGGGTGGGT-3’

SEQ ID NO: 23:5’-GGGTGGGTGGGTGGGTTTGGGTGGGTGGGTGGGT-3’

SEQ ID NO: 24:5’-CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG-3’

SEQ ID NO: 25: 5’-CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG -3’。

According to a specific embodiment of the invention, the sequencing adapter further comprises a second single strand comprising a ligation segment for ligation of an analyte to be detected complementarily paired with the fourth segment of the first single strand.

According to a specific embodiment of the present invention, the sequencing linker according to the present invention comprises a first single strand and a second single strand, wherein the first single strand comprises a first segment (guide region), a second segment (loading region), a third segment (blocking region) and a fourth segment, the fourth segment comprises a double strand pairing region and a segment for binding an analyte to be detected, and the second single strand comprises a side chain region, a segment for complementarily pairing with the fourth segment and a linking segment for binding an analyte to be detected, as shown in FIG. 1.

According to a specific embodiment of the invention, the sequence of the double-stranded pairing region in the sequencing adapter according to the invention is shown as 5'-GGTTGTTTCTGTTGGTGCTGATATTGCT-3' (SEQ ID NO: 26) or 5'-GCAATATCAGCACCAACAGAAACAACC-3' (SEQ ID NO: 27). It should be noted that the double-stranded pairing region sequences in the sequencing linker structure, or any sequence capable of complementary pairing, which can be used for nanopore sequencing are encompassed within the scope of the present invention. However, the length of the double-stranded pairing region needs to satisfy the length range commonly used in the art. In addition, it should be noted that there is a sticky end T at the 3' end of the first single strand.

It should be noted that, there is no particular requirement regarding the length and sequence of the side chain region of the second single strand, and all sequences of the side chain region of the sequencing linker useful in the art for nanopore sequencing are encompassed within the scope of the present invention. For example 5'-TTTGAGGCGAGCGGTCAA-3' (SEQ ID NO: 28).

According to a specific embodiment of the present invention, the sequencing adapter described above can be used to prepare a kit for nanopore sequencing.

According to a specific embodiment of the present invention, there is provided a kit for nanopore sequencing, the kit comprising a sequencing adapter as described above, and further comprising a polynucleotide binding protein selected from at least one of a polymerase, a helicase or an exonuclease.

It should be noted that any type of polymerase, helicase or exonuclease known in the art that can be used for nanopore sequencing is within the scope of the present invention.

According to a specific embodiment of the present invention, there is provided a construct comprising a nucleic acid to be sequenced and a sequencing adapter as described above, wherein the sequencing adapter is attached to either or both ends of the nucleic acid to be sequenced. Based on the construct structure, nanopore sequencing of the nucleic acid to be sequenced is achieved.

According to a specific embodiment of the present invention, there is provided a complex for sequencing, the complex comprising a polynucleotide binding protein, and

The sequencing linker as described above or the construct as described above, and the polynucleotide binding protein is at least one selected from the group consisting of a polymerase, a helicase and an exonuclease.

According to a specific embodiment of the present invention, there is provided a method of constructing a sequencing complex, the method comprising:

(1) Constructing the sequencing adapter;

The sequencing complex and the nanopore sequencing chip constructed based on the invention realize the sequencing of the nucleic acid to be sequenced. It should be noted that, the nanopore sequencing chip may contain a transmembrane pore protein, which may be inserted into the sequencing chip in advance, and the transmembrane pore may be a protein pore or a solid state pore.

According to a specific embodiment of the invention, the invention provides the use of the sequencing adapter, construct, complex for sequencing as described above in nanopore sequencing.

According to a specific embodiment of the present invention, there is provided a nanopore sequencing method comprising nanopore sequencing of a nucleic acid to be sequenced using at least one of the sequencing adaptors described above, the kit described above, the construct described above, the complex for sequencing described above.

The aspects of the present disclosure will be explained below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 preparation of DNA with non-B-form Structure

In this example, a DNA sample having a non-B-form DNA structure was prepared by annealing two chemically synthesized nucleotide chains having complementary fragments.

1. Ordering nucleotide chains (primers) shown in the following Table 1 from an organism, and dissolving the solid primers with nuclease-free water to a stock solution with a final concentration of 100. Mu.M according to the manufacturer's instructions;

TABLE 1

Note that the 3 '-end of the nucleic acid sequence shown in SEQ ID NO. 1-7 in Table 1 is linked to a fluorescence reporter group 6-FAM (6-carboxyfluorescein), and the 5' -end of the nucleic acid sequence shown in SEQ ID NO. 8 is linked to a fluorescence quenching group BHQ1.

2. And respectively taking 5 mu L of Top strand primer and 5 mu L of Bottom strand primer stock, adding into 40 mu L of an Anneal buffer solution (70 mM KCl,20 mM KH ₂PO₄, pH=7.0), mixing together, fully oscillating and uniformly mixing by using a vortex oscillator, and annealing the primer mixture by using a thermal cycler to obtain a DNA sample G4-1/G4-1_neg/G4-2/G4-2_neg/ds-1/ds-2/ds-2_neg.

The DNA samples G4-1/G4-1_neg/G4-2/G4-2_neg/ds-1/ds-2/ds-2_neg were mass-detected using 15% native PAGE, and the results are shown in FIG. 2, indicating that the target DNA samples were successfully obtained.

EXAMPLE 2 cloning, expression and purification of helicase Dda

This example prepares the helicase Dda by recombinant expression in e.coli, which is used as a sequenced motor protein. The preparation process of the helicase Dda is as follows:

1. ordering cDNA full-length sequence of full-length Dda from an organism, connecting the cDNA full-length sequence into PET.28a (+) plasmid, and using double enzyme cutting sites of Nde I and Xho I, so that the expressed Dda protein N end has 6 x His tag and thrombin (thrombin) enzyme cutting site;

2. the cloned PET.28a (+) -Dda plasmid was transformed into Arcticexpress (DE 3) competent bacteria (Tolo Biotech., 96183-02) or derivatives thereof. Single colonies were picked and inoculated into 5 mL LB medium containing kanamycin, and shake cultured overnight at 37 ℃. Then transferring into LB (containing kanamycin) of 1L, culturing at 37 ℃ in a shaking way until the OD600 = 0.6-0.8, cooling to 16 ℃, and adding IPTG with a final concentration of 500 mu M to induce Dda expression overnight;

3. Five buffers were prepared according to the following formulation:

Buffer A, 20 mM Tris-HCl pH 7.5,250 mM NaCl,20 mM imidazole;

Buffer B, 20 mM Tris-HCl pH 7.5,250 mM NaCl,300 mM imidazole;

Buffer C, 20mM Tris-HCl pH 7.5,50 mM NaCl;

buffer D, 20 mM Tris-HCl pH 7.5,1000 mM NaCl;

buffer E, 20 mM Tris-HCl pH 7.5,100 mM NaCl;

4. The Dda-expressing cells were collected, resuspended in buffer a, disrupted by a cell disrupter, and the supernatant was centrifuged. The supernatant was mixed with Ni-NTA filler equilibrated with buffer A in advance and combined for 1h. The packing was collected and washed extensively with buffer a until no contaminating proteins were washed out. The Dda was then eluted by adding buffer B to the packing. And (3) passing the obtained Dda through a desalting column with well balanced buffer solution C, and replacing the buffer solution. Thrombin (thrombin) (assist organism, 20402ES 05) was then added, followed by 4 ℃ cleavage and overnight binding to a well-equilibrated ssDNA cellulose (Sigma, D8273-10G) pad in buffer C. The ssDNA cellulose wad was collected, washed 3-4 times with buffer C, and then eluted with buffer D. The ssDNA cellulose purified protein was concentrated and then passed over molecular sieve Superdex 200 (Sigma, GE 28-9909-44) using molecular sieve buffer E. Collecting target protein peak, concentrating, and freezing. The concentration of the purified protein was quantified using Nanodrop.

Nucleotide sequence of helicase Dda (SEQ ID NO: 9):

ATGACATTTGATGATTTGACCGAAGGCCAGAAAAATGCCTTTAACATTGTTATGAAGGCTATTAAAGAAAAGAAACATCATGTAACTATTAATGGACCTGCTGGTACCGGTAAGACTACTCTTACTAAGTTCATCATTGAAGCTTTAATATCTACGGGTGGAACTGGTATTATTTTAGCAGCTCCTACACATGCAGCTAAAAAGATTCTTTCAAAACTATCAGGGAAAGAAGCGAGTACTATTCATAGTATTCTTAAAATTAACCCAGTAACATATGAAGAAAATGTTCTTTTTGAACAAAAAGAAGTACCTGATTTAGCCAAATGCAGAGTATTAATCTGCGACGAAGTGTCAATGTATGATAGAAAGCTATTTAAAATTCTGCTTTCAACTATTCCACCTTGGTGTACTATAATTGGAATAGGGGATAATAAGCAAATCAGACCTGTTGAACCAGGAGAAAATACTGCTTATATCAGTCCATTCTTTACACATAAAGATTTTTATCAGTGTGAACTCACTGAAGTTAAACGCAGTAATGCTCCTATTATTGATGTAGCTACTGACGTTCGCAACGGTAAGTGGAATTATGATAAAGTTGTTGACGGGCATGGAGTACGTGGATTTACTGGTGATACCGCTTTACGCGATTTTATGGTAAATTATTTTTCAATCGTCAAATCACTAGATGATTTGTTTGAAAATCGCGTAATGGCATTTACGAATAAATCTGTTGACAAGTTAAATAGCATTATTCGTAAAAAGATTTTTGAAACTGATAAAGATTTTATTGTCGGTGAAATTATTGTAATGCAGGAACCATTATTTAAAACATATAAAATTGATGGAAAGCCTGTGTCAGAAATTATTTTTAATAACGGACAATTAGTTCGTATTATAGAAGCAGAGTATACATCAACGTTTGTTAAAGCCCGTGGTGTTCCTGGAGAATATCTAATTCGTCATTGGGATTTAACAGTAGAAACTTATGGCGATGATGAATATTATCGTGAAAAGATTAAAATAATTTCATCTGACGAAGAATTGTATAAGTTTAACCTATTTTTAGCTAAAACAGCAGAAACTTATAAAAATTGGAACAAAGGCGGAAAAGCTCCGTGGAGTGATTTTTGGGATGCTAAATCACAGTTTAGTAAAGTGAAAGCACTTCCTGCATCAACATTCCATAAAGCGCAGGGTATGTCTGTAGACCGTGCTTTCATTTATACGCCTTGTATTCATTATGCAGATGTTGAATTAGCTCAACAACTTCTTTATGTTGGTGTCACCCGTGGTCGTTATGATGTGTTTTATGTATGA

Amino acid sequence of helicase Dda protein (SEQ ID NO: 10):

MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGGTGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCRVLICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVEPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWNYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLAKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV* （* Indicating termination

Example 3 fluorescence quenching experiments to test the effect of non-B-form structures on helicase

In this example, the helicase Dda was tested for its ability to helicase Dda in a DNA with a non-B-form structure using fluorescence quenching experiments. The FAM group and BHQ1 group of the DNA double strand obtained in example 1 were close to each other when the DNA double strand was formed, fluorescence quenching was performed, and when the helicase released the DNA double strand, fluorescence was recovered.

1. Buffer S was prepared as follows in table 2:

TABLE 2

After completion of the preparation of buffer S, it was filtered through a 0.2 μm filter.

2. A solution of helicase (0.75. Mu.M) was prepared by preparing 0.5 Xbuffer S from buffer S and NF Water in a 1:1 ratio, and diluting the solution of helicase (Dda) purified in example 2 with 0.5 Xbuffer S to a solution of 0.75. Mu.M. 100 mM ATP solution was prepared using 0.5 Xbuffer S.

3. The fluorescence quenching experiment system was prepared as follows in table 3:

TABLE 3 Table 3

SEQ ID NO: 11:AGCAATATCAGCACCAACAGAAACAACC

4. A single-stranded control system for fluorescence quenching experiments was formulated as follows in table 4:

TABLE 4 Table 4

5. The above experiment system was added to a 96-well plate, and 50. Mu.L of each of the six wells was added to the G4-1 sequence experiment system. Before the reaction, 10. Mu.L of 0.75. Mu.M Dda solution was added to each of the three wells, and 10. Mu.L of 0.5 Xbuffer S was added to the other three wells as a control reaction without helicase. As a control reaction, 50. Mu.L of single strand of SEQ ID NO.1 was added to each of the three wells, and 10. Mu.L of 0.75. Mu.M Dda solution was added to each of the three wells before the reaction.

The reference G4-1 sequence was entered for the experimental system of G4-1/G4-1_neg/G4-2/G4-2_neg/ds-1/ds-2/ds-2_neg.

6. After adding Dda helicase, detecting the fluorescence recovery condition of each experimental system in 2 h by using an enzyme-labeled instrument (TECAN, SPARK), analyzing the inhibition efficiency of different sequences on the helicase according to the experimental result as shown in figure 3, and comparing the negative control sequences G4-1_neg/G4-2_neg/ds-2_neg which do not contain the non-B-form structural sequence, wherein the inhibition rate of the G4 chain body or the G4-1/G4-2/ds-1/ds-2 sequence of the i-motif structure on the helicase is higher, and the helicase can be prevented from unwinding the nucleic acid double chain.

Example 4 preparation of linker DNA

In this example, a linker DNA having a non-B-form DNA structure was prepared by annealing two chemically synthesized nucleotide chains having complementary fragments.

1. The nucleotide chains of Table 5 below were ordered from the organisms and the primers in solid form were dissolved in nuclease-free water to give a stock solution with a final concentration of 100. Mu.M as per the manufacturer's instructions.

TABLE 5

2. Respectively taking 5 mu L of stock solution of Top strand primer and 5 mu L of stock solution of Bottom strand primer, adding into 40 mu L of Anneal buffer (70 mM KCl,20 mM KH ₂PO₄, pH=7.0), mixing, fully oscillating and uniformly mixing by using a vortex oscillator, and annealing the primer mixture by using a thermal cycler to obtain the adaptor a1-Adp/a2-Adp/a3-Adp/a4-Adp/neg-Adp.

Example 5 preparation of DNA to be tested

In this example, the Lambda phage genome was amplified to obtain an amplicon as the DNA to be tested.

In this example, a sequence of about 500 bp a in the Lambda phage genome was amplified by Polymerase Chain Reaction (PCR) and used as an insert substrate for subsequent ligation reactions (i.e., the target nucleotide to be tested). The specific process is as follows:

1. The sequences shown as SEQ ID NO. 18 and SEQ ID NO. 19 were ordered from the organisms and the SEQ ID NO. 18 Primer (Primer # 1) and the SEQ ID NO. 19 Primer (Primer # 2) were dissolved in TE buffer (pH=8) to a final concentration of 100. Mu.M according to the manufacturer's instructions. Subsequently, 10. Mu.L of stock solution was taken and 90. Mu.L of TE buffer (pH=8) was added to dilute the stock solution to a final concentration of 10. Mu.M.

SEQ ID NO: 18:5’-GCCATCAGATTGTGTTTGTTAGT-3’

SEQ ID NO: 19:5’-AAGCTTCGAGTCAGTACCGATGT-3’

2. PCR reactions were performed using Lambda phage genomic DNA (NEB, N3011L) as template. The PCR mixture was prepared on ice according to the formulation shown in Table 6.

TABLE 6 PCR mixture formulation

After the PCR mixture was thoroughly mixed by shaking, the mixture was placed in a PCR apparatus, and the procedure shown in Table 7 was performed.

TABLE 7 PCR procedure

3. Ampure XP beads (Beckman Coulter, A63882) were removed from the refrigerator in advance, mixed by shaking and allowed to equilibrate at room temperature for half an hour. 100 mu L of balanced magnetic beads are added into the PCR system, and the mixture is stirred and mixed uniformly, centrifuged briefly and then kept stand at room temperature for 10 minutes.

4. The centrifuge tube was placed on a magnetic rack for 10 minutes and after the beads were fully adsorbed to the side of the magnetic rack and the solution was fully clarified, the supernatant was carefully removed.

5. The beads were resuspended using 200 μl of 80% ethanol solution and washed with a pipette blow, the centrifuge tube was placed on a magnetic rack for 10 minutes, and after the beads were fully adsorbed to the magnetic rack side and the solution was fully clarified, the supernatant was carefully removed.

6. Repeating the above ethanol solution cleaning step once, removing the supernatant, placing the centrifuge tube on a magnetic rack, standing, adding 100 μl TE buffer (pH=8) to resuspend the magnetic beads after the surfaces of the magnetic beads become dry, and standing at room temperature for 10 minutes.

7. And placing the centrifuge tube on a magnetic rack, transferring the supernatant to a new centrifuge tube after the magnetic beads are all adsorbed to the side of the magnetic rack, and obtaining the amplified insert sequence SEQ ID NO. 20.

SEQ ID NO: 20:

GCCATCAGATTGTGTTTGTTAGTCGCTTTTTTTTTTTGGAATTTTTTTTTTGGAATTTTTTTTTTGCGCTAACAACCTCCTGCCGTTTTGCCCGTGCATATCGGTCACGAACAAATCTGATTACTAAACACAGTAGCCTGGATTTGTTCTATCAGTAATCGACCTTATTCCTAATTAAATAGAGCAAATCCCCTTATTGGGGGTAAGACATGAAGATGCCAGAAAAACATGACCTGTTGGCCGCCATTCTCGCGGCAAAGGAACAAGGCATCGGGGCAATCCTTGCGTTTGCAATGGCGTACCTTCGCGGCAGATATAATGGCGGTGCGTTTACAAAAACAGTAATCGACGCAACGATGTGCGCCATTATCGCCTAGTTCATTCGTGACCTTCTCGACTTCGCCGGACTAAGTAGCAATCTCGCTTATATAACGAGCGTGTTTATCGGCTACATCGGTACTGACTCGAAGCTT

EXAMPLE 6 library ligation and nanopore sequencing

In the embodiment, a nanopore detection platform based on a patch clamp platform is constructed, and nanopore sequencing is carried out on the target sequencing library prepared in the embodiment 5, so that the joint constructed by the application is verified to be capable of limiting motor protein, and the motor protein passes through under the action of sequencing voltage, so that the aim that the sequencing library is sequenced in the nanopore is fulfilled.

1. Lambda 0.5k in example 5 was end repaired, 5 'phosphorylated and 3' plus A using library preparation kit (Hangzhou Hua order wind technologies Co., ltd.). The total amount of the reaction system was 60. Mu.L, which consisted of 6. Mu.L of the end-repairing enzyme 1 buffer, 3. Mu.L of the end-repairing enzyme 1, 3. Mu.L of the end-repairing enzyme 2 buffer, 4. Mu.L of the end-repairing enzyme 2, 1. Mu.g of the DCS500 fragment (SEQ ID NO: 20) obtained in example 5, and was supplemented with nuclease-free water to 60. Mu.L. The reaction solution was prepared on ice, and after thoroughly shaking and mixing, it was placed on a thermal cycler and incubated at 20℃for 10 minutes and 65℃for 10 minutes. The reaction products were purified using 1 Xvolume magnetic beads and quantitated using the Qubit DNA HS kit (next holy biotechnology).

2. The DNA to be tested obtained by end repair was ligated to the adaptor obtained in example 4, respectively. The total amount of ligation reaction was 100. Mu.L, which consisted of 60. Mu.L of DNA to be tested after end repair (1. Mu.g) in example 5, 25. Mu.L of 4 Xligation buffer (library preparation kit), 10. Mu. L T4 of 4 DNA ligase (library preparation kit), linker a 1-Adp/a 2-Adp/a 3-Adp/a 4-Adp/neg-Adp (example 4), and nuclease-free water was added to make up to 100. Mu.L. After fully mixing and centrifuging, placing the mixture in a metal bath with constant temperature of 25 ℃ for reaction for 30min. The product was purified using 1 Xvolume magnetic beads and quantitated using the Qubit DNA HS kit to give a ligated library.

3. The helicase Dda binds and crosslinks. And (3) introducing helicase Dda into the library to be tested, which is obtained in the step (2), wherein the library is obtained in the step (2), water is supplemented to 10 mu L by 200 ng, 25 mu L of binding buffer (HEPES-Na 50 mM,KCl 100 mM,EDTA 1mM,MgCl ₂ 5 mM), 5 mu L of 1 mu M helicase Dda and 10 mu L of NF water are prepared. After the reaction system was mixed, the mixture was incubated at 25℃with a PCR instrument at 60 min. After the incubation was completed, 1mM TMAD 1. Mu.L was added.

4. Nanopore sequencing was performed on the target sequencing library prepared in the examples using a nanopore detection platform. The neg-Adp linker has no sequencing switch structure, and the motor protein unwinds the library and falls off before the library enters the nanopore, so that the nucleic acid single strand can not generate a sequencing signal through the nanopore instantaneously under the action of electric field force, and the instantaneous perforation signal is shown as A in FIG. 4. The non-B-form structure in the a1-Adp, a2-Adp, a3-Adp and a4-Adp joints can realize the effect of limiting the helicase Dda, so that a nucleic acid single chain can stably pass through a nanopore under the action of electric field force, as B-E in figure 4, current signals for sequencing are acquired, and each current signal measured by the new joint has a characteristic signal of poly dT or poly dA of a sequence SEQ ID NO: 20 to be sequenced.

In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A sequencing adapter for nanopore sequencing, comprising a first single strand comprising:

The first section is used to guide the nucleic acid to be tested into the single-stranded nucleotide region of the nanopore;

The second segment is a single-stranded nucleotide region for binding to the motor protein;

The third segment is a single-stranded nucleotide region used to prevent the motor protein from performing the function of unwinding nucleic acid in the non-sequencing state;

The fourth section is used for connecting the analyte to be tested;

wherein the third segment contains a C-rich region;

The C-rich region forms an i-Motif structure,

The nucleic acid sequence of the third segment is shown in any one of SEQ ID NOs: 24-25.

2. The sequencing adapter according to claim 1, wherein the sequencing adapter contains at least 10-30 consecutive bases T.

3 . The sequencing adapter according to claim 1 , wherein the sequencing adapter further comprises a second single strand, wherein the second single strand comprises a connecting segment complementary to the fourth segment of the first single strand and used for connecting to an analyte to be detected.

4. Use of the sequencing adapter according to any one of claims 1 to 3 in preparing a kit for nanopore sequencing.

5. A kit for nanopore sequencing, comprising the sequencing adapter according to any one of claims 1 to 3, and a polynucleotide binding protein; the polynucleotide binding protein is a helicase.

6. A construct comprising a nucleic acid to be sequenced and a sequencing adapter according to any one of claims 1 to 3, wherein the sequencing adapter is connected to either or both ends of the nucleic acid to be sequenced.

7. A complex for sequencing, comprising a polynucleotide binding protein and

The sequencing adapter according to any one of claims 1 to 3 or the construct according to claim 6;

The polynucleotide binding protein is a helicase.

8. A method for constructing a sequencing complex, wherein the method comprises:

(1) constructing the sequencing adapter according to any one of claims 1 to 3;

(2) assembling the sequencing adapter and the nucleic acid to be sequenced to form a construct;

(3) contacting the construct with a polynucleotide binding protein to obtain a sequencing complex;

The polynucleotide binding protein is a helicase.

9. Use of the sequencing adapter according to any one of claims 1 to 3, the construct according to claim 6, or the complex for sequencing according to claim 7 in nanopore sequencing.

10. A nanopore sequencing method, comprising performing nanopore sequencing on a nucleic acid to be sequenced using at least one of the sequencing adapter according to any one of claims 1 to 3, the kit according to claim 5, the construct according to claim 6, or the complex for sequencing according to claim 7.