Sequencing adapters for nanopore sequencing and uses thereof

Abstract

The present application relates to the field of sequencing technology, and particularly relates to a sequencing adaptor for nanopore sequencing and purposes thereof. The sequencing adaptor provided by the present application is designed with different non-B-form structures in the sequencing adaptor, which is used to prevent the motor protein from unwinding the nucleic acid in a non-sequencing state, so that the motor protein can only pass through the secondary structure region under the action of the electric field force when the leading strand enters the nanopore for sequencing. The sequencing adaptor for nanopore sequencing provided by the present application has a lower synthesis cost than the existing sequencing adaptor.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of sequencing technology, in particular to a sequencing adaptor for nanopore sequencing and use thereof. BACKGROUND

[0002] Nanopore sequencing is a newly emerging sequencing technology in recent years. As a high-throughput single molecule sequencing method, the principle of nanopore sequencing is to detect the current change in the pore when a single-stranded nucleic acid molecule passes through the nanopore, so as to identify the signal and then determine the type of base passing through the pore. In nanopore sequencing, the nucleic acid (DNA / RNA) to be tested is connected with a sequencing adaptor loaded with a motor protein to obtain a sequencing library that can be captured by a nanopore.

[0003] The existing nanopore sequencing adaptor mainly performs two functions: 1. loading a motor protein; and 2. nanopore capture, so that the DNA to be tested can be subjected to nanopore sequencing. The function of nanopore capture is achieved through a primer strand and an anchoring region, and the function of loading a motor protein requires two regions: 1. a loading region, i.e. a site where the motor protein can bind, which is usually a single-stranded DNA; and 2. a modification region, in which the motor protein does not function and does not displace under non-sequencing conditions. Only when the primer strand of the sequencing adaptor enters the nanopore, the motor protein is located above the nanopore, and the movement direction of the DNA to be tested and the motor protein is opposite, under the action of an electric field force, the motor protein crosses the modification region to unwind / polymerize / enzymatically cut, and starts to move in the opposite direction of the DNA to be tested entering the pore for sequencing.

[0004] However, the synthesis cost of the nucleic acid sequence of the existing adaptor is high, which does not meet the market demand. Therefore, there is an urgent need to develop a sequencing adaptor for nanopore sequencing with lower cost to reduce the sequencing cost. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a sequencing adaptor for nanopore sequencing. The inventors designed different non-B-form structures in the sequencing adaptor to prevent the motor protein from unwinding the nucleic acid in the non-sequencing state, so that it can pass through the secondary structure region only under the action of the electric field force when the primer strand enters the nanopore for sequencing.

[0006] To this end, the present application provides a sequencing adaptor in the first aspect. In some embodiments of the present application, the sequencing adaptor is used for nanopore sequencing, wherein it comprises a first single strand, which comprises:

[0007] a first section, which is a single-stranded nucleotide region for guiding the nucleic acid to be tested to enter the nanopore;

[0008] a second segment for binding a single-stranded nucleotide region of a motor protein;

[0009] a third segment for preventing the motor protein from performing a function of unwinding nucleic acid in a non-sequencing state;

[0010] a fourth segment for connecting an analyte to be detected;

[0011] wherein the third segment contains a G-rich region and / or a C-rich region;

[0012] the G-rich region forms a G-quadruplex structure;

[0013] the C-rich region forms an i-Motif structure.

[0014] The inventors have found that designing a non-B-form structure (G-rich region and / or C-rich region) in a sequencing adaptor for nanopore sequencing for preventing the motor protein from unwinding nucleic acid in a non-sequencing state, so that the motor protein can only pass through the secondary structure region under the action of an electric field force when the leading strand enters the nanopore for sequencing. The sequencing adaptor for nanopore sequencing provided by the present application has a lower synthesis cost than the existing sequencing adaptor.

[0015] In some embodiments of the present application, the sequencing adaptor contains at least 10-30 consecutive bases T.

[0016] In some embodiments of the present application, the nucleic acid sequence of the third segment is as shown in any one of SEQ ID NOs: 22-25.

[0017] In some embodiments of the present application, the sequencing adaptor further comprises a second single strand, the second single strand comprising a connecting segment for connecting an analyte to be detected, which is complementary to the fourth segment of the first single strand.

[0018] The second aspect of the present application provides use of the sequencing adaptor of the first aspect in preparing a kit for nanopore sequencing.

[0019] The third aspect of the present application provides a kit for nanopore sequencing. In some embodiments of the present application, the kit comprises the sequencing adaptor of the first aspect, and further comprises a polynucleotide binding protein; the polynucleotide binding protein is selected from at least one of a polymerase, a helicase or an exonuclease.

[0020] The fourth aspect of the present application provides a construct. In some embodiments of the present application, the construct comprises a nucleic acid to be sequenced and the sequencing adaptor of the first aspect, wherein the sequencing adaptor is connected to either end or both ends of the nucleic acid to be sequenced.

[0021] The fifth aspect of the present application provides a complex for sequencing. In some embodiments of the present application, the complex comprises a polynucleotide binding protein, and

[0022] the sequencing adaptor of the first aspect or the construct of the fourth aspect,

[0023] The polynucleotide binding protein is selected from at least one of a polymerase, a helicase or an exonuclease.

[0024] The sixth aspect of the present application provides a method of constructing a sequencing complex. In some embodiments of the present application, the method comprises:

[0025] (1) constructing the sequencing adaptor of the first aspect;

[0026] (2) assembling the sequencing adaptor with a nucleic acid to be sequenced to form a construct;

[0027] (3) contacting the construct with a polynucleotide binding protein to obtain a sequencing complex;

[0028] The polynucleotide binding protein is selected from at least one of a polymerase, a helicase or an exonuclease.

[0029] The seventh aspect of the present application provides the use of the sequencing adaptor of the first aspect, the construct of the fourth aspect, the complex for sequencing of the fifth aspect in nanopore sequencing.

[0030] The eighth aspect of the present application provides a method of nanopore sequencing. In some embodiments of the present application, the method of nanopore sequencing comprises nanopore sequencing a nucleic acid to be sequenced using at least one of the sequencing adaptor of the first aspect, the kit of the third aspect, the construct of the fourth aspect, the complex for sequencing of the fifth aspect.

[0031] The inventors have optimized the sequencing adaptor for nanopore sequencing, designed different non-B-form structures in the sequencing adaptor to prevent the motor protein from unwinding the nucleic acid in the non-sequencing state, so that it can only pass through the secondary structure region under the action of the electric field force when the leading strand enters the nanopore for sequencing. By introducing the natural nucleotide sequence of the secondary structure on the adaptor, a new nanopore sequencing adaptor can be constructed, and sequencing can be successfully performed. The sequencing adaptor has low synthesis cost.

[0032] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0033] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings, wherein:

[0034] Figure 1 A schematic diagram showing the sequencing adaptor of one embodiment of the present application is shown;

[0035] Figure 2 A non-denaturing PAGE electrophoresis result of the DNA sample prepared in Example 1 is shown, 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;

[0036] Figure 3 A fluorescence quenching experiment for detecting the inhibition of DNA sample on helicase Dda is shown;

[0037] Figure 4 A nanopore sequencing current signal is shown, wherein A-E are the current signals of neg-Adp, a1-Adp, a2-Adp, a3-Adp, a4-Adp adaptor and the DNA to be detected connected thereto, respectively. DETAILED DESCRIPTION

[0038] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0039] It should be noted that the terms "first", "second" are used only for the purpose of description, and should not be understood as indicating or implying relative importance or implying the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0040] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be approximations that allow for significant variation. Within each range, other values can exist that are not expressly identified. Other values within the scope of the ranges and values disclosed herein should be considered as potentially inscribed in the ranges and values.

[0041] In order that the present application can be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in this document concur in meaning with the general use of their respective fields.

[0042] In the present text, the terms "comprising" or "including" are of an open-ended nature, i.e. meaning including the indicated features but not excluding others.

[0043] In the present text, the terms "optionally", "optional" or "optional" generally mean that the event or circumstance subsequently described can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] In the present text, the term "B-form structure" refers to the most stable and most common double helix conformation of DNA under physiological conditions, the structural features of which include right-handed double helix, base pairing, specific helical parameters, and the presence of major and minor grooves. The stability, functional adaptability and dynamics of B-form DNA enable it to efficiently carry out important biological processes such as replication, transcription, etc. in living organisms.

[0045] In the present text, the term "motor protein" refers to a class of proteins that can convert chemical energy (usually from ATP hydrolysis) into mechanical energy. In nanopore sequencing, motor proteins play a crucial role in controlling the speed of DNA or RNA molecules passing through the nanopore, keeping them at a suitable rate. This speed control is crucial for accurately detecting the current changes caused by each base. By stably controlling the movement of DNA or RNA molecules, motor proteins can reduce the noise of the current signal, improving the accuracy and resolution of sequencing. During nanopore sequencing, motor proteins bind to the linker of the library molecule, helping the DNA or RNA molecule to enter the nanopore stably. In addition, motor proteins (such as phi29 DNA polymerase) can bind to the DNA double strand during nanopore sequencing and, under the drive of the electric field, perform the function of unwinding nucleic acids to unwind the double strand into a single strand of nucleic acid. This process ensures that the single strand of nucleic acid can pass through the nanopore smoothly, thereby achieving accurate detection of each base.

[0046] In the present text, the term "G-rich region" refers to a region of DNA or RNA sequence rich in guanine, which can form stable G-quadruplex structures.

[0047] In the present text, the term "C-rich region" refers to a region of DNA or RNA sequence rich in cytosine. These regions have special structures and functions in biomolecules, especially the ability to form specific secondary structures (such as i-motif structures).

[0048] According to a specific embodiment of the present application, the present application provides a sequencing adapter for nanopore sequencing, comprising a first single strand, the first single strand comprising:

[0049] a first segment for guiding the single-stranded nucleotides of the nucleic acid to be tested into a single-stranded nucleotide region of the nanopore;

[0050] a second segment for binding the motor protein;

[0051] a third segment for preventing the motor protein from performing the function of unwinding the nucleic acid in a non-sequencing state;

[0052] a fourth segment for connecting the analyte to be tested;

[0053] wherein the third segment contains a G-rich region and / or a C-rich region;

[0054] the G-rich region forms a G-quadruplex structure;

[0055] the C-rich region forms an i-Motif structure.

[0056] According to a specific embodiment of the present application, the sequencing adaptor contains at least 10-30 consecutive bases T. For example, the sequencing adaptor can contain 10, 15, 20, 25, 30 consecutive bases T. These consecutive bases T are usually located in the first segment of the sequencing adaptor. For example, the consecutive bases T in the first segment (also referred to as the guide region) are shown in the sequence SEQ ID NO: 21 (5'-TTTTTTTTTTTTTTTTTTTT-3').

[0057] According to a specific embodiment of the present application, the first segment and the second segment can be the same segment.

[0058] According to a specific embodiment of the present application, the nucleic acid sequence of the third segment is shown in any one of SEQ ID NO: 22-25.

[0059] SEQ ID NO: 22: 5'-GGGTGGGTGGGTGGGT-3'

[0060] SEQ ID NO: 23: 5'-GGGTGGGTGGGTGGGTTTGGGTGGGTGGGTGGGT-3'

[0061] SEQ ID NO: 24: 5'-CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG-3'

[0062] SEQ ID NO: 25: 5'-CGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGG -3'.

[0063] According to a specific embodiment of the present invention, 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 the analyte to be detected.

[0064] According to a specific embodiment of the present invention, the sequencing adapter of the present invention is as follows Figure 1 As shown, the sequencing adapter includes a first single strand and a second single strand, wherein the first single strand includes a first segment (guide region), a second segment (loading region), a third segment (blocking region) and a fourth segment, and the fourth segment includes a double-stranded pairing region and a segment for connecting to the analyte to be tested; the second single strand includes a side chain region, a segment complementary to the fourth segment and a connecting segment for connecting to the analyte to be tested.

[0065] According to a specific embodiment of the present invention, the double-stranded pairing region sequence in the sequencing adapter described herein is represented by 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 sequence in the sequencing adapter structure used for nanopore sequencing, or any sequence capable of complementary pairing, is covered by the scope of protection of the present invention. However, the length of the double-stranded pairing region must meet the length range commonly used in the art. Furthermore, it should be noted that the 3' end of the first single strand contains a sticky end T.

[0066] It should be noted that there are no specific requirements for the length and sequence of the side chain region of the second single-stranded sequence. All sequences of the side chain region of sequencing adapters used in nanopore sequencing that are available in the art are covered by the scope of protection of the present invention. For example, it can be 5'-TTTGAGGCGAGCGGTCAA-3' (SEQ ID NO: 28).

[0067] According to a specific embodiment of the present invention, the above-mentioned sequencing adapter of the present invention can be used to prepare a kit for nanopore sequencing.

[0068] According to a specific embodiment of the present invention, the present invention provides a kit for nanopore sequencing, which includes the sequencing adapter described above and a polynucleotide binding protein; the polynucleotide binding protein is selected from at least one of a polymerase, a helicase or a nuclease exonuclease.

[0069] It is to be understood that any of the polymerase, helicase or exonuclease types known in the art that can be used for nanopore sequencing are encompassed by the present application.

[0070] According to an embodiment of the present application, the present application provides a construct comprising a nucleic acid to be sequenced and the sequencing adaptor as described above, wherein the sequencing adaptor is linked to either end or both ends of the nucleic acid to be sequenced. Based on the construct, nanopore sequencing of the nucleic acid to be sequenced is achieved.

[0071] According to an embodiment of the present application, the present application provides a complex for sequencing, the complex comprising a polynucleotide binding protein, and

[0072] the sequencing adaptor as described above or the construct as described above; the polynucleotide binding protein is selected from at least one of a polymerase, a helicase or an exonuclease.

[0073] According to an embodiment of the present application, the present application provides a method of constructing a sequencing complex, the method comprising:

[0074] (1) constructing the sequencing adaptor as described above;

[0075] (2) assembling the sequencing adaptor with a nucleic acid to be sequenced to form a construct;

[0076] (3) contacting the construct with a polynucleotide binding protein to obtain a sequencing complex;

[0077] the polynucleotide binding protein is selected from at least one of a polymerase, a helicase or an exonuclease.

[0078] Based on the sequencing complex and the nanopore sequencing chip constructed according to the present application, sequencing of the nucleic acid to be sequenced is achieved. It is to be understood that the nanopore sequencing chip can contain a transmembrane pore protein, which can be inserted into the sequencing chip in advance, and the transmembrane pore can be a protein pore or a solid pore.

[0079] According to an embodiment of the present application, the present application provides the use of the sequencing adaptor, the construct, the complex for sequencing as described above in nanopore sequencing.

[0080] According to an embodiment of the present application, the present application provides a nanopore sequencing method, the nanopore sequencing method comprising nanopore sequencing of a nucleic acid to be sequenced by using at least one of the sequencing adaptor as described above, the kit as described above, the construct as described above, the complex for sequencing as described above.

[0081] The scheme of the present disclosure will be explained below in connection with examples. Those skilled in the art will understand that the examples below are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If a specific technique or condition is not specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained commercially.

[0082] Example 1 Preparation of DNA with non-B-form structure

[0083] In this example, DNA samples with non-B-form DNA structure were prepared by annealing two chemically synthesized nucleotide chains with complementary fragments.

[0084] 1. The nucleotide chains (primers) shown in Table 1 below were ordered from Shengwo Biological, and the solid primers were dissolved in nuclease-free water to a final concentration of 100 μM stock solution according to the manufacturer's instructions;

[0085] Table 1

[0086]

[0087] Note: The 3' end of the nucleic acid sequence shown in SEQ ID NO: 1-7 in Table 1 is connected to the fluorescent reporter group 6-FAM (6-carboxyfluorescein); the 5' end of the nucleic acid sequence shown in SEQ ID NO: 8 is connected to the fluorescent quencher group BHQ1.

[0088] 2. Respectively, 5 μL of Top strand primer and 5 μL of Bottom strand primer stock solution were added to 40 μL of Anneal buffer (70 mM KCl, 20 mM KH2PO4, pH = 7.0), mixed together, and fully mixed using a vortex shaker. The primer mixture was annealed using a thermal cycler to obtain DNA samples G4-1 / G4-1_neg / G4-2 / G4-2_neg / ds-1 / ds-2 / ds-2_neg.

[0089] The DNA samples G4-1 / G4-1_neg / G4-2 / G4-2_neg / ds-1 / ds-2 / ds-2_neg were quality tested using 15% non-denaturing PAGE, and the results are shown in Figure 2 , indicating that the target DNA samples were successfully obtained.

[0090] Example 2 Cloning, expression and purification of helicase Dda

[0091] This example prepared helicase Dda by recombinant expression in E. coli, which was used as the motor protein for sequencing. The preparation of helicase Dda was as follows:

[0092] 1. The full-length sequence of cDNA of Dda was ordered from Shenguo Bio, which was ligated into the PET.28a(+) plasmid using double enzyme cutting sites Nde I and Xho I, so that the N-terminal of the expressed Dda protein had a 6*His tag and a thrombin enzyme cutting site;

[0093] 2. The cloned PET.28a(+)-Dda plasmid was transformed into ArcticExpress (DE3) competent bacteria (Tolo Biotech., 96183-02) or its derivative bacteria. A single colony was picked and inoculated into 5 mL of LB medium containing kanamycin and incubated at 37°C overnight. Then, it was transferred into 1 L of LB (containing kanamycin) and incubated at 37°C until the OD600=0.6-0.8, then cooled to 16°C and induced Dda expression by adding IPTG at a final concentration of 500 μM overnight;

[0094] 3. Five kinds of buffers were prepared according to the following formula:

[0095] Buffer A: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM imidazole;

[0096] Buffer B: 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0097] Buffer C: 20 mM Tris-HCl pH 7.5, 50 mM NaCl;

[0098] Buffer D: 20 mM Tris-HCl pH 7.5, 1000 mM NaCl;

[0099] Buffer E: 20 mM Tris-HCl pH 7.5, 100 mM NaCl;

[0100] 4. Collect the bacteria expressing Dda, resuspend the bacteria with buffer A, break the bacteria with a cell disruptor, then centrifuge to collect the supernatant. Mix the supernatant with Ni-NTA filler which has been equilibrated with buffer A, and combine for 1 hour. Collect the filler, and wash the filler with buffer A until no impurities are washed out. Then add buffer B to the filler to elute Dda. The eluted Dda is passed through a desalting column equilibrated with buffer C to change the buffer. Then add thrombin (Yixing Bio, 20402ES05), and then add to ssDNA cellulose (Sigma, D8273-10G) filler equilibrated with buffer C, and cut and combine overnight at 4°C. Collect the ssDNA cellulose filler, wash with buffer C for 3-4 times, and then elute with buffer D. Concentrate the protein purified by ssDNA cellulose, and pass through a Superdex 200 (Sigma, GE28-9909-44) molecular sieve, with the molecular sieve buffer being buffer E. Collect the protein peak, concentrate, and freeze. Use a Nanodrop to quantify the concentration of the purified protein.

[0101] Nucleotide sequence of helicase Dda (SEQ ID NO: 9):

[0102]

[0103] Amino acid sequence of helicase Dda protein (SEQ ID NO: 10):

[0104] MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGGTGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCR VLICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVEPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWNYDKVVDGHGVRGFTGDTALRDFMV NYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETY GDDEYYREKIKIKIISSDEELYKFNLFLAKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV* (* indicates termination)

[0105] Example 3 Fluorescence quenching experiment to test the effect of non-B-form structure on helicase

[0106] In this example, a fluorescence quenching experiment was used to examine the ability of helicase Dda to unwind DNA with a non-B-form structure. The FAM and BHQ1 groups of the DNA duplex obtained in Example 1 approached each other during duplex formation, resulting in fluorescence quenching. Fluorescence was restored after the helicase unwound the DNA duplex.

[0107] 1. Prepare buffer S according to Table 2 below:

[0108] Table 2

[0109]

[0110] After the buffer S was prepared, it was filtered through a 0.2 μm filter membrane.

[0111] 2. Prepare Dda solution of 0.75 μΜ helicase: Prepare 0.5x buffer S with buffer S and NF Water at a ratio of 1:1, dilute the helicase Dda solution purified in Example 2 to a solution of 0.75 μΜ with 0.5x buffer S. Prepare 100 mM ATP solution with 0.5x buffer S.

[0112] 3. Prepare the fluorescence quenching experiment system according to Table 3:

[0113] Table 3

[0114]

[0115] SEQ ID NO: 11: AGCAATATCAGCACCAACAGAAACAACC

[0116] 4. Prepare the single-strand control system of the fluorescence quenching experiment according to Table 4:

[0117] Table 4

[0118]

[0119] 5. Add the above experimental system to the 96-well plate, and for the experimental system of G4-1 sequence, add 50 μL to six wells respectively. Before the reaction, add 10 μL of 0.75 μΜ Dda solution to three wells respectively, and add 10 μL of 0.5x buffer S to the other three wells as a control reaction without helicase. Add 50 μL of SEQ ID NO. 1 single-strand to three wells respectively as a control reaction, and before the reaction, add 10 μL of 0.75 μΜ Dda solution to the three wells respectively.

[0120] For the experimental system of G4-1 / G4-1_neg / G4-2 / G4-2_neg / ds-1 / ds-2 / ds-2_neg, refer to the G4-1 sequence.

[0121] 6. After adding the Dda helicase, use the microplate reader (TECAN, SPARK) to detect the fluorescence recovery of the experimental system in each well within 2 h, and analyze the inhibition efficiency of different sequences on the helicase according to the experimental results as Figure 3 By comparing the negative control sequences G4-1_neg / G4-2_neg / ds-2_neg that do not contain non-B-form structure sequences, the G4-1 / G4-2 / ds-1 / ds-2 sequences that can form G4 strands or i-motif structures have a higher inhibition rate on the helicase, which can hinder the helicase from unwinding the nucleic acid double strand.

[0122] Example 4 Preparation of linker DNA

[0123] In this embodiment, the linker DNA with non-B-form DNA structure is prepared by annealing two chemically synthesized nucleotide chains with complementary fragments.

[0124] 1. Order the nucleotide chains in Table 5 from Shengwo Biological, and dissolve the solid primers into a stock solution with a final concentration of 100 μM using nuclease-free water according to the manufacturer's instructions.

[0125] Table 5

[0126]

[0127] 2. Take 5 μL of the Top strand primer stock solution and 5 μL of the Bottom strand primer stock solution, add them to 40 μL of Anneal buffer (70 mM KCl, 20 mM KH2PO4, pH = 7.0), mix them together, and use a vortex shaker to mix them thoroughly. Use a thermal cycler to anneal the primer mixture to obtain the linker a1-Adp / a2-Adp / a3-Adp / a4-Adp / neg-Adp.

[0128] Example 5 Preparation of DNA to be tested

[0129] In this embodiment, the Lambda phage genome is amplified to obtain an amplicon as the DNA to be tested.

[0130] In this embodiment, a sequence of about 500 bp in length from the Lambda phage genome is amplified by polymerase chain reaction (PCR) to serve as the insert substrate for the subsequent ligation reaction (i.e., the target nucleotide to be tested). The specific process is as follows:

[0131] 1. Order the sequences shown in SEQ ID NO: 18 and SEQ ID NO: 19 from Shengwo Biological, and dissolve the SEQ ID NO: 18 primer (Primer #1) and the SEQ ID NO: 19 primer (Primer #2) into a stock solution with a final concentration of 100 μM using TE buffer (pH = 8) according to the manufacturer's instructions. Then take 10 μL of the stock solution and dilute it into a working solution with a final concentration of 10 μM by adding 90 μL of TE buffer (pH = 8).

[0132] SEQ ID NO: 18: 5'-GCCATCAGATTGTGTTTGTTAGT-3'

[0133] SEQ ID NO: 19: 5'-AAGCTTCGAGTCAGTACCGATGT-3'

[0134] 2. PCR reaction was performed with Lambda phage genomic DNA (NEB, N3011L) as template. PCR mix was prepared according to the recipe in Table 6 on ice.

[0135] Table 6: PCR mix recipe

[0136]

[0137] After the PCR mix was well mixed by vortex, it was put into a PCR machine and run according to the program in Table 7.

[0138] Table 7: PCR program

[0139]

[0140] 3. Ampure XP beads (Beckman Coulter, A63882) were taken out from the fridge and equilibrated at room temperature for 30 minutes by vortex. 100 μL equilibrated beads were added into the PCR mix and vortexed well. After a short spin, the tube was placed at room temperature for 10 minutes.

[0141] 4. The tube was placed on a magnetic stand for 10 minutes until the beads were completely absorbed to the side of the stand and the solution was clear. The supernatant was carefully removed.

[0142] 5. The beads were resuspended with 200 μL 80% ethanol solution and washed by pipetting. The tube was placed on a magnetic stand for 10 minutes until the beads were completely absorbed to the side of the stand and the solution was clear. The supernatant was carefully removed.

[0143] 6. The ethanol solution washing step was repeated once. After the supernatant was removed, the tube was placed on a magnetic stand until the beads were dry. 100 μL TE buffer (pH = 8) was added to resuspend the beads and the tube was placed at room temperature for 10 minutes.

[0144] 7. The tube was placed on a magnetic stand until the beads were completely absorbed to the side of the stand. The supernatant was transferred to a new tube. The sequence of the amplified insert was SEQ ID NO: 20.

[0145] SEQ ID NO: 20:

[0146] GCCATCAGATTGTGTTTGTTAGTCGCTTTTTTTTTTTGGAATTTTTTTTTTGGAATTTTTTTTTTGCGCTAACAACCTCCTGCCGTTTTGCCCGTGCATATCGGTCACGAACAAATCTGATTACTAAACACAGTAGCCTGGATTTGTTCTATCAGTAATCGACCTTATTCCTAATTAAATAGAGCAAATCCCCTTATTGGGGGTAAGACATGAAGATGCCAGAAAAACATGACCTGTTGGCCGCCATTCTCGCGGCAAAGGAACAAGGCATCGGGGCAATCCTTGCGTTTGCAATGGCGTACCTTCGCGGCAGATATAATGGCGGTGCGTTTACAAAAACAGTAATCGACGCAACGATGTGCGCCATTATCGCCTAGTTCATTCGTGACCTTCTCGACTTCGCCGGACTAAGTAGCAATCTCGCTTATATAACGAGCGTGTTTATCGGCTACATCGGTACTGACTCGAAGCTT

[0147] Example 6 Library ligation and nanopore sequencing

[0148] This example constructs a nanopore detection platform based on a patch clamp platform, and performs nanopore sequencing on the target sequencing library prepared in Example 5 to verify that the linker constructed in this application can limit the motor protein and be passed by the motor protein under the action of the sequencing voltage, so as to achieve the purpose of sequencing the sequencing library in the nanopore.

[0149] 1. The Lambda0.5k in Example 5 was end-repaired, 5' phosphorylated and 3' A-added using a library preparation kit (Hangzhou Huadaixun Technology Co., Ltd.). The total reaction system was 60 μL, including: 6 μL end-repair enzyme 1 buffer, 3 μL end-repair enzyme 1, 3 μL end-repair enzyme 2 buffer, 4 μL end-repair enzyme 2, 1 μg DCS500 fragment (SEQ ID NO: 20) obtained in Example 5, and supplemented with nuclease-free water to 60 μL. The reaction solution was prepared on ice, and after thorough shaking and mixing, it was placed in a thermal cycler, incubated at 20°C for 10 minutes, and incubated at 65°C for 10 minutes. The reaction product was purified using 1x volume magnetic beads, and quantified using a Qubit DNA HS kit (Yixing Biotechnology).

[0150] 2. The end-repaired test DNA and the adapters obtained in Example 4 were connected respectively. The total volume of the connection reaction system was 100 μL, including: 60 μL of the end-repaired test DNA (1 μg) in Example 5, 25 μL of 4x connection buffer (library preparation kit), 10 μL of T4 DNA ligase (library preparation kit), adapters a1-Adp / a2-Adp / a3-Adp / a4-Adp / neg-Adp (Example 4), and adding nuclease-free water to 100 μL. After thorough mixing and centrifugation, the reaction system was placed in a 25°C constant temperature metal bath for 30 min. The product was purified using 1x volume of magnetic beads, and quantified using the Qubit DNA HS kit to obtain the connected library.

[0151] 3. Helicase Dda binding and cross-linking. Helicase Dda was introduced to the test library with adapters obtained in step 2, and the following reaction system was prepared: 200 ng of the library obtained in step 2 was added to 10 μL, 25 μL of binding buffer (HEPES-Na 50 mM, KCl 100 mM, EDTA 1 mM, MgCl2 5 mM), 1 μM helicase Dda 5 μL, and NF water 10 μL. After mixing the reaction system, a PCR instrument was used for 25°C incubation for 60 min. After incubation, 1 mM TMAD 1 μL was added.

[0152] 4. The target sequencing library prepared in the example was sequenced using a nanopore detection platform. The neg-Adp adapter did not have a sequencing switch structure, and before the library entered the nanopore, the motor protein unwound the library and fell off, so that under the action of the electric field force, the transient passage of the single-stranded nucleic acid through the nanopore could not generate a sequencing signal, and the transient puncture signal was as shown in Figure 4 A. The non-B-form structure in the a1-Adp, a2-Adp, a3-Adp, and a4-Adp adapters could achieve the effect of limiting helicase Dda, so that the single-stranded nucleic acid could pass through the nanopore at a steady speed under the action of the electric field force, as shown in Figure 4 B-E, the current signal for sequencing was collected, and the poly dT or polydA characteristic signal of the test sequence SEQ ID NO: 20 was detected in the current signal of each new adapter.

[0153] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", "some implementations" or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples, without contradiction.

[0154] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A sequencing adaptor for nanopore sequencing, wherein, comprises a first single strand comprising: a first segment for guiding a single-stranded nucleotide region of a nucleic acid to be sequenced into a nanopore; a second segment for binding a motor protein; a third segment for preventing the motor protein from performing a function of unwinding nucleic acid in a non-sequencing state; a fourth segment for connecting an analyte to be sequenced; wherein the third segment comprises a C-rich region; the C-rich region forms an i-Motif structure, the nucleic acid sequence of the third segment is as set forth in any one of SEQ ID NOs: 24-25.

2. The sequencing adaptor of claim 1, wherein, the sequencing adaptor comprises at least 10-30 consecutive bases of T.

3. The sequencing adaptor of claim 1, wherein, the sequencing adaptor further comprises a second single strand comprising a connecting segment for connecting an analyte to be sequenced that is complementary to the fourth segment of the first single strand.

4. Use of the sequencing adaptor of any one of claims 1-3 in the preparation of a kit for nanopore sequencing.

5. A kit for nanopore sequencing, wherein, comprising the sequencing adaptor of any one of claims 1-3, further comprising a polynucleotide binding protein; the polynucleotide binding protein is a helicase.

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

7. A complex for sequencing, wherein, comprising a polynucleotide binding protein, and the sequencing adaptor of any one of claims 1-3 or the construct of claim 6; the polynucleotide binding protein is a helicase.

8. A method of constructing a sequencing complex, wherein, the method comprises: (1) constructing the sequencing adaptor of any one of claims 1-3; (2) assembling the sequencing adaptor with a 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 adaptor of any one of claims 1-3, the construct of claim 6, or the complex for sequencing of claim 7 in nanopore sequencing.

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

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