Helicase mutant as well as preparation method and application thereof in high-throughput sequencing

Abstract

The invention provides a helicase mutant as well as a preparation method and application thereof in high-throughput sequencing. According to the helicase mutant, at least one cysteine on the surface of a three-dimensional structure of an amino acid sequence as shown in SEQ ID NO: 1 is substituted by alanine. The cysteine is C < 133 >, C < 164 >, C < 292 >, C < 323 > and / or C < 347 >; preferably, the amino acid sequence of the helicase mutant is as shown in SEQ NO: 6. The helicase mutant has good DNA unwinding activity and good protein homogeneity, reduces cross-linking interaction between protein molecules, improves the suitability of the helicase mutant with perforin, and improves the stability of sequencing and the integrity and continuity of an electric signal, so that the helicase mutant has a better sequencing effect, can be used for control and characterization of nucleic acid, and can be applied to the field of biochemistry. And the method is applied to single-molecule nanopore sequencing.

Description

A helicase mutant, its preparation method, and its application in high-throughput sequencing Technical Field

[0001] The present invention belongs to the field of biotechnology or sequencing, and particularly relates to a helicase mutant, a preparation method thereof, and an application thereof in high-throughput sequencing. Background Art

[0002] Nanopore sequencing, an emerging single-molecule sequencing technology, has revolutionized the genetic sequencing industry with its unique advantages, including high throughput, long read lengths, rapid speed, in situ detection, and label-free operation. This technology eliminates the need for imaging equipment, enabling portable systems to meet diverse sequencing scenarios. Furthermore, due to its non-amplified direct sequencing nature, there is no length limit for sequenceable DNA, enabling real-time base calling and direct sequencing of RNA, modifications such as methylation, and other single molecules. Nanopore sequencing technology has broad applications in fundamental life science research and biomedical clinical practice.

[0003] Nanopore sequencing is a sequencing technology based on electrical signals. dsDNA is unwound into ssDNA by DNA helicase. Under the influence of an electric field, ssDNA passes through porins anchored to biological or artificial membranes. As ssDNA passes through the nanopore, the amount of current obstruction varies depending on the bases. By detecting the current fluctuation signal of the nanopore and analyzing it through machine learning, the DNA sequence of the perforated pore is determined.

[0004] During this sequencing process, due to the extremely high speed at which nucleic acid molecules pass through the nanopore channel, it is difficult to accurately obtain polynucleotide sequence information. Therefore, effectively reducing and controlling the perforation motion of nucleic acid molecules is a key technical challenge in achieving nanopore sequencing. Currently, the most common and effective method is to use the unwinding principle of helicases to control the perforation motion of nucleic acid molecules and improve detection accuracy. In order to better maintain sequencing speed and sequencing uniformity, it is urgent to specifically modify and improve the helicase enzyme to enhance its control over the movement of polynucleotides and enhance the stability of nanopore sequencing applications.

[0005] Summary of the Invention

[0006] The helicase in the current commercialized nanopore sequencer is the DDA helicase derived from the bacterial phage T4, which has extremely low yield and limited application range. We discovered a helicase BCH105 (patent number PCT / CN2021 / 143662), whose gene is derived from the deep-sea metagenome and has extremely high thermal stability and salt tolerance. BCH105 has a very high expression level in the Escherichia coli recombinant protein expression system and a very high yield. In addition, BCH105 has a special pin structure, which gives it good single-stranded DNA binding and double-stranded DNA unwinding activity, so that it can be used for the control and characterization of nucleic acids, and applied to single-molecule nanopore sequencing. However, BCH105 itself has some intermolecular cross-links. By mutating and modifying certain key sites and changing the cysteine ​​on its surface, the problem of intermolecular cross-linking interactions of its protein can be reduced. In addition, BCH105 will interact with perforin during application, which will have a certain impact on the sequencing signal and sequencing speed. By mutating the amino acids at certain key sites and modifying the interface where it may interact with the porin, the helicase can be increased to interact with DNA or porin, promoting its formation of a stable complex with DNA, making it better compatible with the perforin, and thus having a better sequencing effect. In addition, by modifying the helicase pin (Pin) structure and tower (Tower) domain, the integrity of the electrical signal can be improved. In summary, the protein homogeneity of BCH105 itself, as well as sequencing persistence, stability and sequencing integrity, all have great room for improvement. Therefore, the present invention provides a helicase mutant, a preparation method thereof, and its application in high-throughput sequencing.

[0007] Specifically, the first aspect of the present invention provides a helicase mutant in which at least one cysteine ​​on the surface of the three-dimensional structure of the amino acid sequence shown in SEQ ID NO: 1 is replaced by alanine, thereby reducing the cross-linking phenomenon between protein molecules.

[0008] The cysteine ​​is C133, C164, C292, C323 and / or C347; preferably, the amino acid sequence of the helicase mutant is as shown in SEQ ID NO: 6.

[0009] The helicase mutant further comprises at least one amino acid mutation in the pin domain, the tower domain, the fragment V66-N84, and / or the fragment K294-A321, wherein the amino acid mutation is a substitution of the original amino acid with cysteine ​​or a non-natural amino acid, and / or is cross-linked with at least one chemical cross-linker, thereby improving cross-linking efficiency and thus improving sequencing performance.

[0010] Preferably, the mutation sites of the pin domain are 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 28, 129, 130, 131, 132, 133 and 134; the mutation sites of the tower domain are 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415 at least one of 79, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, and 393; the mutation sites of the fragment V66-N84 are at least one of 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, and 84. Less one; and / or the mutation position of fragment K294-A321 is at least one of 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320 and 321.

[0011] More preferably, the mutation sites of the pin domain are G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119, T120, T121, K122, A123, K124, L125, V126, V127, at least one of N128, K129, F130, N131, E132, A133 and L134; the mutation sites of the tower domain are P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, at least one of D379, R380, S381, K382, A383, W384, K385, K386, Y387, F388, K389, L390, K391, E392 and K39; the mutation sites of the fragment V66-N84 are V66, T67, S68, P69, T70, H71, K72, A73, V74, R75, V76, S78, L79, N80, M81, L82, K83 and N84; at least one; and / or the mutation site of fragment K294-A321 is at least one of K294, D295, E296, L297, V298, F299, Q300, E301, T302, Y303, T304, D305, S306, K307, G308, N309, I310, I311, V312, S313, N314, G315, E316, I317, I318, E319, V320 and A321.

[0012] The unnatural amino acid is selected from 4-azido-L-phenylalanine (PAZF), 4-azido-L-phenylalanine (PAZF-HCl), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenoyl)-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3-{4-[(propan-2-ylsulfanyl)carbonyl]-L-phenylalanine ]phenyl}propionic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropionyl)amino]phenyl}propionic acid, O-methyl-L-tyrosine, 4-amino-L-phenylalanine, 4-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3-iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)- L-Alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthalen-2-ylamino)propionic acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl)propionic acid, (2R)-2-aminooctanoate 3-(2,2′-bipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolinyl)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl] propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine and 2-nitrophenylalanine; and / or, the chemical cross-linking agent is selected from maleimide, active ester, succinimide, azide, alkyne, phosgene-type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and photosensitizer.

[0013] The helicase mutant has one or more amino acid residues at the N-terminus truncated on the amino acid sequence shown in SEQ ID NO: 6, for example, amino acids M1-A7 are truncated. The helicase mutant can still be stable in a monomeric state.

[0014] The helicase mutant has at least one amino acid mutation in K2, D4, L5, K82, N83, N84, G85, I86, and D87 of the amino acid sequence shown in SEQ ID NO: 6, wherein the amino acid mutation is a substitution of the original amino acid with another amino acid; preferably, the other amino acid is alanine. The helicase mutant is still stable in a monomeric state.

[0015] The helicase mutant has at least one mutation in the porin interface interaction region, wherein the mutation includes substitution of the original amino acid with alanine, serine, glutamine, leucine, threonine, histidine, or glycine. The helicase mutant still retains the ability to control the movement of polynucleotides and is applicable to nanopore sequencing technology.

[0016] Preferably, the mutation site in the region interacting with the porin interface is at least one of 1, 2, 3, 4, 5, 12, 14, 16, 18, 21, 28, 63, 82, 124, 129, 158, 165, 189, 194, 196, 199, 202, 203, 205, 206, 207, 212, 214, 215, 218, 223, 244, 247, 261, 263, 271, 281, 289, 294, 307, 324, 331, 333, 336, 340, 341, 349, 367, 376, 382, ​​385, 386, 389 and 393.

[0017] More preferably, the mutation sites in the region interacting with the porin interface are M1, K2, H3, D4, L5, Q12, Y14, F16, D18, K21, K28, K63, K82, K124, K129, K158, K165, K189, H194, K196, K199, R202, Q203, E205, D206, N207, K212, At least one of Q214, E215, K218, K223, K244, K247, K261, K263, K271, R281, K289, K294, K307, K324, K331, K333, K336, K340, K341, R349, K367, K376, K382, K385, K386, K389 and K393.

[0018] The helicase mutant has at least one mutation in the ATP hydrolysis domain, wherein the original amino acid is replaced by another charged amino acid or an amino acid with a relatively small side chain; preferably, the other charged amino acid or amino acid with a relatively small side chain is alanine, aspartic acid, glutamine, or glycine. The helicase mutant has enhanced unwinding activity, thereby improving sequencing performance and is applicable to nanopore sequencing technology.

[0019] Preferably, the mutation sites of the ATP hydrolysis region are 40, 41, 42, 43, 44, 45, 46, 143, 144, 145, 146, 147, 148, 149, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 , 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444 and 445.

[0020] More preferably, the mutation sites in the ATP hydrolysis region are G40, F41, A42, G43, S44, G45, K46, E144, A145, S146, M147, V148, S149, G171, D172, S173, Y174, Q175, L176, L177, P178, V179, D180, D181, E182, D183, S184, S185, I186, L197, T198, K199, V200, V201, R202, Q203, A204, E205, D206, N207 , I208, I209, I210, A211, S213, Q214, E215, L216, I217, K218, A219, M220, D221, Q222, K223, I405, H406, K407, L408, Q409, G410, S411, T412, Y413, Q414, N430, R431, D432, N433, V434, L435, R436, L437, V438, Y439, V440, G441, I442, T443, R444 and A445.

[0021] The helicase mutant has a double substitution selected from the following on the amino acid sequence shown in SEQ ID NO: 6:

[0022] L369C and D117C;

[0023] L369C and V119C;

[0024] L369C and D113C;

[0025] K373C and G114C;

[0026] K373C and D112C;

[0027] W384C and F110C;

[0028] W384C and D112C;

[0029] W384C and G114C;

[0030] L369C and T115C;

[0031] L369C and N118C;

[0032] K373C and T115C;

[0033] K373C and A116C;

[0034] K373C and D117C;

[0035] K373C and N118C;

[0036] K373C and V119C;

[0037] H108C and F388C;

[0038] V119C and W384C;

[0039] R380C and A116C;

[0040] K373C and T120C;

[0041] L369C and T121C;

[0042] A116C and 381C;

[0043] T121C and K373C;

[0044] T120C and L369C;

[0045] T121C and 388C.

[0046] Preferably, when it has L369C and V119C double substitutions, the helicase mutant further has a mutation selected from the following:

[0047] (1)K21S, K28S, K189S, H194S, K196S, K199S, Q203S, N207S, K212S, Q214S, K21 8S, K223S, R202K, E215A, K2A, H3A, D4A, L5A, Y14A, F16A, D18A, K63A, K82A, K1 65A, K244A, K247A, K261A, R281A, K307A, K324A, K331A, K333A, K336A, K341A, R349A, K124A, K129A, K158A, K271A, K376A, K382A, K385A, K393A, E205D monosubstituted; or

[0048] (2) K72C and G315C, or E205A and D206A disubstituted; or

[0049] (3) truncation of amino acids 1-7; or

[0050] (4) M1G and an H before the amino acid sequence are truncated.

[0051] The second aspect of the present invention provides an isolated nucleic acid encoding the helicase mutant as described in the first aspect of the present invention.

[0052] Preferably, the nucleotide sequence of the helicase mutant is shown in SEQ ID NO:8.

[0053] The third aspect of the present invention provides a recombinant expression vector comprising a promoter and the nucleic acid according to the second aspect of the present invention.

[0054] Preferably, the promoter is T7; and / or the backbone plasmid of the recombinant expression vector is PET.28a(+), PET.21a(+), or PET.32a(+).

[0055] The fourth aspect of the present invention provides a transformant comprising a host cell and the nucleic acid according to the second aspect of the present invention.

[0056] Preferably, the host cell is Escherichia coli, more preferably BL21(DE3), BL21Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

[0057] The fifth aspect of the present invention provides a method for preparing the helicase mutant as described in the first aspect of the present invention, which comprises culturing the transformant as described in the fourth aspect of the present invention in a culture medium and fermenting it to produce the helicase mutant.

[0058] The sixth aspect of the present invention provides a helicase mutant-sequencing adapter complex, which includes the helicase mutant described in the first aspect of the present invention and a sequencing adapter.

[0059] The seventh aspect of the present invention provides a kit, which includes the helicase mutant described in the first aspect of the present invention and / or the helicase mutant-sequencing adapter complex described in the sixth aspect of the present invention; preferably also includes a single-stranded DNA containing an anchor molecule at the 5' end, a nanopore, a nanopore protein, an electrical signal detector, a membrane and / or a buffer.

[0060] Preferably, the anchoring molecule is a hydrophobic molecule, preferably selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and / or amino acids, such as cholesterol, palmitate or tocopherol.

[0061] The nanopore is a transmembrane protein pore or a solid-state pore; preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein (phi29 connector), InvG, GspD or any combination thereof.

[0062] The membrane is an amphiphilic membrane (such as a phospholipid bilayer), a high molecular polymer membrane (such as a di-block copolymer, a tri-block copolymer) or any combination thereof.

[0063] The buffer is a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, a MOPS buffer system or any combination thereof.

[0064] The eighth aspect of the present invention provides the use of the helicase mutant described in the first aspect of the present invention, the helicase mutant-sequencing adapter complex described in the sixth aspect of the present invention, or the kit described in the seventh aspect of the present invention in high-throughput sequencing.

[0065] Preferably, the high-throughput sequencing is nanopore sequencing.

[0066] The ninth aspect of the present invention provides a DNA unwinding method, which comprises unwinding double-stranded DNA using the helicase mutant described in the first aspect of the present invention, the helicase mutant-sequencing adapter complex described in the sixth aspect of the present invention, or the kit described in the seventh aspect of the present invention.

[0067] A tenth aspect of the present invention provides a sequencing method comprising the following steps:

[0068] The DNA is sequenced while being unwound using the DNA unwinding method described in the ninth aspect of the present invention.

[0069] The beneficial effects brought about by the technical solution of the present invention are:

[0070] The present invention provides helicase mutants based on the helicase BCH105, designated BCH105-1, BCH105-2, and BCH105-3. These helicase mutants possess excellent DNA unwinding activity and good protein homogeneity, enabling the control and characterization of nucleic acids and their application in single-molecule nanopore sequencing. Furthermore, compared to BCH105, these helicase mutants exhibit reduced cross-linking interactions between protein molecules, improving their compatibility with perforin, enhancing sequencing stability, and improving the integrity and continuity of electrical signals, resulting in superior sequencing performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 is an SDS-PAGE image of BCH105 (left) and BCH105-1 (right) after protein purification.

[0072] FIG2 is an SDS-PAGE image of BCH105 (left) and BCH105-1 (right) after incubation with oxidants (10%).

[0073] Figure 3 is an SDS-PAGE image of BCH105-2 protein after purification (10%)

[0074] FIG4 is a graph showing the purity of the constructed BCH105-2 complex (DNA Native PAGE / 8%).

[0075] FIG5 is a schematic diagram of the connector (a: upper chain; b: lower chain).

[0076] Figure 6 is a schematic diagram of the sequencing library containing BCH105 and its mutants (a: upper strand; b: lower strand; c: double-stranded target fragment; d: BCH105 and its mutants; e: chol-ssDNA).

[0077] FIG7 is a translocation diagram of the changes in the perforation current of the nucleic acid fragment to be tested in BCH105-2.

[0078] FIG8 is a protein SDS-PAGE image of the BCH105-3 mutant (10%).

[0079] FIG9 is a graph showing the purity of the constructed mutant BCH105-3 complex (DNA Native PAGE / 8%).

[0080] FIG10 is a translocation diagram showing the changes in the perforation current of the nucleic acid fragment to be tested in BCH105-3.

[0081] FIG11 shows the electrical signal integrity rate of BCH105 mutants (tested at 28° C.).

[0082] Figure 12 is an SDS-PAGE image of BCH105-33 protein after purification (10%)

[0083] FIG13 is a graph showing the purity of the constructed mutant BCH105-33 complex (DNA Native PAGE / 8%).

[0084] FIG14 is a translocation diagram showing the changes in the perforation current of the nucleic acid fragment to be tested, BCH105-33.

[0085] FIG15 is a distribution diagram of sequencing speeds of BCH105-3, BCH105-33, and BCH105-72 (tested at 30° C.).

[0086] Figure 16 is a diagram of the Alphafold2 predicted structure of BCH105. DETAILED DESCRIPTION

[0087] Example 1 Cloning, expression and purification of BCH105 and its mutant BCH105-1

[0088] 1. Vector Construction and Expression of BCH105 Helicase and Its Mutant BCH105-1

[0089] The amino acid sequence of BCH105 is shown in SEQ ID NO:1, and its full-length DNA sequence is shown in SEQ ID NO:5. This full-length DNA sequence was synthesized (Liuhe BGI) and ligated into the PET-28a(+) plasmid. Using dual restriction enzyme sites, Nde1 and Xho1, the expressed BCH105 protein has a His tag and a thrombin restriction site at its N-terminus. Mutants were constructed by site-directed mutagenesis based on this plasmid.

[0090] The cloned PET.28a(+)-BCH105 plasmid and its mutant BCH105-1 were transformed into E. coli expression strain BL21(DE3) or its derivatives. A single colony was picked and inoculated into 5 mL of LB medium containing kanamycin resistance. The culture was shaken at 37°C overnight. The culture was then transferred to 1 L of LB medium containing kanamycin resistance and shaken at 37°C until OD600 = 0.6-0.8. The culture was cooled to 16°C and IPTG was added to a final concentration of 500 μM to induce expression overnight.

[0091] 2. Purification of BCH105 and its mutant BCH105-1

[0092] The buffer solution used is as follows:

[0093] Buffer A: 20mM Tris-HCl pH 8.0, 200mM NaCl, 20mM imidazole

[0094] Buffer B: 20 ​​mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM imidazole

[0095] Buffer C: 20mM Tris-HCl pH 8.0

[0096] Buffer D: 20mM Tris-HCl pH 8.0, 50mM NaCl

[0097] Buffer E: 20mM Tris-HCl pH 8.0, 1000mM NaCl

[0098] Buffer F: 20mM Tris-HCl pH 8.0, 80mM NaCl

[0099] The expressing cells were collected and centrifuged at 6500 rpm to collect the expressed BCH105 and its mutant BCH105-1. The cells were resuspended in buffer A, disrupted using a cell pressure disruptor, and then centrifuged at 18000 rpm for 1 hour. The supernatant was mixed with Ni-NTA medium equilibrated in buffer A and allowed to bind for 1 hour. The medium was collected and washed extensively with buffer B until all contaminants were washed out. Thrombin was then added to the medium and incubated overnight at 4°C for 12-16 hours. The flow-through was collected and diluted to 50 mM NaCl with buffer C. The flow-through, i.e., the target protein, was then passed through a QFF column with buffer D and gradient eluted with buffer E. The target protein peak was collected and concentrated. After protein concentration, the protein was applied to a Superdex 200 column using Buffer F. The target protein peak was collected, concentrated, and frozen. As shown in Figure 1, the purified mutant protein was obtained in large quantities and of high purity.

[0100] Example 2 Test of the intermolecular aggregation state of BCH105 and its mutant BCH105-1

[0101] 1. Determination of the intermolecular or intramolecular aggregation state of BCH105

[0102] BCH105 and oxidant were incubated at a constant temperature of 30°C for 0.5 h. The protein concentration was fixed at 5 μM, and the oxidant concentrations were 0 and 150 μM, respectively. After the incubation, 10 μL of the sample was taken and run on SDS-PAGE gel (10%). The results are shown in Figure 2.

[0103] As shown in Figure 2 (left), in the absence of an oxidant, only one target protein band is observed on SDS-PAGE. However, after incubation with an oxidant, the protein band shifts upward and multiple bands appear, indicating cross-linking between BCH105 proteins. It is speculated that in the presence of an oxidant, cross-linking occurs between or within the protein molecules, leading to protein aggregation and upward shifting of the band. This can lead to heterogeneity in this type of protein, posing a potential risk in sequencing applications.

[0104] 2. Determination of the intermolecular or intramolecular aggregation state of BCH105 and BCH105-1

[0105] BCH105-1 is based on the Alphafold2 predicted structure of the BCH105 protein ( Figure 16 ), in which all five cysteines located on the surface of the protein's three-dimensional structure were mutated to alanine. The specific mutation site information is shown in Table 1. After protein expression and purification, the protein was mixed with an oxidant in appropriate proportions and reacted using the same method as above.

[0106] The results are shown in Figure 2 (right). As can be seen from the figure, the BCH105-1 protein has only one target protein band both before and after adding an oxidant. This indicates that after the cysteine ​​on the protein surface is mutated to alanine, the phenomenon of intermolecular or intramolecular cross-linking can be significantly reduced, which can increase the uniformity of the protein and make it better suitable for nanopore sequencing.

[0107] Example 3 Construction of BCH105 series mutant complexes

[0108] Helicases that form relatively pure complexes with DNA are better suited for nanopore sequencing, improving the stability and sustainability of nanopore sequencing. Therefore, constructing protein-DNA complexes is a crucial step in nanopore sequencing. The purity of the complex (i.e., a 1:1 protein to adapter ratio) directly impacts sequencing results. A free adapter to protein ratio of N:1 can lead to pore blockage or other poor sequencing results. Higher-purity complexes improve sequencing efficiency, making it crucial to improve the purity of the complex. Constructing the helicase-DNA adapter complex requires cross-linking the protein to the protein-binding base region of the adapter to prevent the target DNA from detaching from the sequencing region during sequencing, which could lead to unstable and unsustainable sequencing. By analyzing the helicase structure, we mutated amino acids in the pin and tower regions of BCH105-1, introducing cysteine ​​residues to generate the mutant BCH105-2 (SEQ ID NO:6). The specific mutation sites are shown in Table 1, and the protein results are shown in Figure 3. The BCH105-2 protein and DNA linker were mixed in appropriate proportions and incubated at 28°C for 1 hour. The protein and crosslinker were then added to an oxidative crosslinker in appropriate proportions and incubated at 28°C for 1.5 hours. Finally, ATP was added to a final concentration of 4 μM and incubated at 30°C for 1.5 hours. 10 μL of the sample was used as the pre-purification gel sample. The remaining sample was purified using magnetic beads to obtain the desired complex, which was then stored in 20% glycerol and frozen at -80°C. A small amount of sample (equal weight to the pre-purification sample) was taken as the post-purification sample and run on a DNA Native PAGE gel. The results are shown in Figure 4.

[0109] As can be seen from Figure 4, protein BCH105-2 can form a complex with DNA under the action of oxidants, but the protein and DNA form multiple bands. The second brightest band was identified as a 1:1 stable complex. The bands above this band are all N:1 bands, and the band below this band is a free DNA linker band.

[0110] Example 4 Characterization of BCH105-2 and its mutants and control nucleic acids

[0111] 1. Two partially complementary DNA strands (upper strand, SEQ ID NO: 2 and lower strand, SEQ ID NO: 3) were annealed to form a linker, which was then ligated to the double-stranded target fragment using T4 DNA ligase (NEB, E6057AVIAL) at room temperature and purified to prepare a sequencing library.

[0112] 2. BCH105-2 protein was incubated with the sequencing library at 25°C for 1 h (molar concentration ratio 1:8) to form a sequencing library containing helicase.

[0113] 3. Incubate the helicase-containing sequencing library with single-stranded DNA (ssDNA-chol, SEQ ID NO: 4) containing cholesterol at its 5' end at room temperature for 10 minutes. The ssDNA-chol sequence is complementary to a portion of the lower strand of the adapter. Cholesterol binds to the phospholipid membrane, reducing library loading and improving capture efficiency.

[0114] 4. Use a patch clamp amplifier or other electrical signal amplifier to collect current signals. A Teflon membrane with a micrometer-sized pore (50-200 μm in diameter) in the center divides the electrolytic cell into two chambers: the cis chamber and the trans chamber. A pair of Ag / AgCl electrodes is placed in each chamber. A bilayer of phospholipid membranes is formed at the micropores of each chamber, and the nanopore protein CsgG-Eco-(Y51A / F56Q / R97W / R192D-StrepII(C)) is added. Electrical measurements are obtained after a single nanopore protein is inserted into the phospholipid membrane. The reaction product from step 3 is added, and 180 mV is applied. The sequencing library is captured by the nanopore, and the nucleic acids pass through the nanopore under the control of the helicase. The buffer used in this experiment is: 0.47 M KCl, 25 mM HEPES, 1 mM EDTA, 5 mM ATP, 25 mM MgCl2, pH 7.6, and the sequencing temperature is 28°C.

[0115] 5. Sequencing experiments using BCH105-2 yielded the sequencing signal shown in Figure 7. As the helicase guides the DNA single strand into the nanopore, the current is partially blocked, decreasing. Because the size of different nucleotides varies, the amount of current blocked also varies, resulting in the visible fluctuations in the current signal.

[0116] Example 5 Amino acid modification and optimization of the Pin and Tower domains of the BCH105 series helicases

[0117] To better apply helicases like BCH105 to nanopore sequencing, a key sequencing metric must be considered: the completeness of the sequencing signal. This refers to the degree to which the signal generated by sequencing a known sequence with a unique signal is fully recognized and learned by the algorithm. A higher completeness of the signal, the better the sequencing performance. In-depth analysis of helicases in nanopore sequencing has revealed that the Pin and Tower domains contribute to this performance. Because BCH105-1, resulting from surface cysteine ​​mutations to alanine, lacks intermolecular crosslinking and is therefore well-suited for nanopore sequencing, the researchers are modifying the Pin and Tower domains to introduce one or more cysteines for crosslinking. By modifying these sites, they hope to further enhance the performance of BCH105 helicases in nanopore sequencing.

[0118] The constructed plasmid was site-directed mutagenesis, and the mutants were BCH105-3 to BCH105-26. The mutation information is shown in Table 1. The subsequent mutant protein expression, purification and sequencing were carried out in the same manner as above.

[0119] The experimental results are shown in Figures 8-10. As can be seen from Figure 8, the protein purity is high. As can be seen from the comparison between Figure 9 and Figure 4, the purity of the complex of the BCH105-3 mutant (i.e., the protein to DNA ratio is 1:1) is higher than that of BCH105-2. After the complex was constructed and tested on the machine, the perforation current translocation diagram of the BCH105-3 nucleic acid fragment to be tested is shown in Figure 10. The sequencing electrical signal integrity rate has been greatly improved, indicating that the modification of the amino acids in the Pin and Tower domains can greatly improve the electrical signal integrity rate of helicases such as BCH105 and optimize their performance in nanopore sequencing technology.

[0120] Example 6 Modification and optimization of amino acids at the possible interaction interface between BCH105 series helicases and porins

[0121] To better apply helicases like BCH105 in nanopore sequencing, it's important to evaluate BCH105's performance in nanopore sequencing using more metrics, such as signal capture rate and sequencing speed. Higher speeds and signal capture rates indicate greater sequencing throughput. Helicases function by interacting with porins and DNA during sequencing. Improving the helicase's compatibility with porins or DNA can improve sequencing performance. By optimizing the Pin and Tower domains, the efficiency and purity of the 1:1 complex formation during complex construction were improved, thereby enhancing sequencing performance. After analyzing the sequencing results, BCH105-3 was screened out for its improved performance in capturing complete electrical signals. Therefore, based on BCH105-3, certain key sites that may interact with the perforin interface were modified, mainly targeting amino acid mutations in the 1A / 2A / 2B regions. The mutation direction mainly replaced positively charged amino acids with neutral or negatively charged amino acids, and / or replaced long side chain amino acids with short side chain amino acids, thereby increasing the compatibility of helicases such as BCH105 with porins and completing the optimization of certain key sequencing indicators.

[0122] The constructed plasmid was site-directed mutagenesis, and the mutants were BCH105-27 to BCH105-73. The mutation site information is shown in Table 1. The subsequent mutant protein expression, purification and sequencing were carried out in the same manner as above.

[0123] The sequencing results are shown in Figures 12-14.

[0124] As shown in Figure 15 , the speeds of BCH105-33 and BCH105-72 are both higher than that of BCH105-3, indicating that the 1A / 2A / 2B domains of helicases such as BCH105 interact with porins during sequencing, and the direction of amino acid mutation is also relatively clear.

[0125] Table 1 Mutation site information of BCH105 mutants

[0126]

[0127]

[0128]

[0129]

[0130] Table 2. Various indicators of BCH105 mutants in nanopore sequencing technology

[0131] Name Speed ​​(nt / s) Single-hole channel capture number Completeness rate BCH105-22104436% BCH105-324525566% BCH105-42371838% BCH105-523821810% BCH105-61998820% BCH105-723216949% BCH105-824613522% BCH105-91321937% BCH105-102304411% BCH105-1123214339% BCH105-122409225% BCH105-1324519639%

[0132] BCH105-142381230%BCH105-1524024410%BCH105-1623633221%BCH105-1734076 50%BCH105-1832713532%BCH105-192953251%BCH105-2031610811%BCH105-2137 53019%BCH105-2236510423%BCH105-233202330%BCH105-2435129853%BCH105-2 53311646%BCH105-2633818446%BCH105-2721631062%BCH105-283424158%BCH105 -2934534067%BCH105-3030431575%BCH105-3130031673%BCH105-3231331468%B CH105-33358270.563%BCH105-3423624369%BCH105-3534319963%BCH105-362707 5.564%BCH105-372077168%BCH105-38336115.563%BCH105-3934320863%BCH105 -4017917869%BCH105-4133620763%BCH105-42363169.562%BCH105-4332716662%

[0133] BCH105-4433091.567%BCH105-453327263%BCH105-4630011557%BCH105-4731811 2.668%BCH105-482504466%BCH105-4932414365%BCH105-5032376.564%BCH105-51 36015751%BCH105-52345138.545%BCH105-533495373%BCH105-543321364%BCH10 5-5536082.537%BCH105-5636088.570%BCH105-573494063%BCH105-5834856.539% BCH105-5934810744%BCH105-603502676%BCH105-6136282.568%BCH105-6236230 72%BCH105-6334067.567%BCH105-643446863%BCH105-6536066.558%BCH105-6633 062.561%BCH105-673558952%BCH105-683555169%BCH105-6935098.562%BCH105-7 03532772%BCH105-7136082.571%BCH105-7236576.556%BCH105-73355nt / s15362%

[0134] The sequences used in the present invention are as follows:

[0135] Amino acid sequence of BCH105 (SEQ ID NO: 1):

[0136]

[0137] SEQ ID NO: 2:

[0138] 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-

[0139] YYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3'(Y=iSp18)

[0140] Wherein, iSp18 is represented by the following formula I:

[0141]

[0142] SEQ ID NO: 3:

[0143] 5'-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'

[0144] SEQ ID NO: 4:

[0145] 5'-cholesterol-TTGACCGCTCGCCTC-3'

[0146] DNA sequence corresponding to BCH105 protein (SEQ ID NO: 5)

[0147]

[0148]

[0149] Amino acid sequence of BCH105-1 (SEQ ID NO: 6)

[0150]

[0151] BCH105-2 amino acid sequence (SEQ ID NO: 7)

[0152]

[0153] DNA sequence corresponding to BCH105-1 protein (SEQ ID NO: 8)

[0154]

[0155]

[0156] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A helicase mutant, characterized in that In the helicase mutant, at least one cysteine ​​on the surface of the three-dimensional structure of the amino acid sequence shown in SEQ ID NO: 1 is substituted by alanine.

2. The helicase mutant according to claim 1, characterized in that The cysteine ​​is C133, C164, C292, C323 and / or C347; preferably, the amino acid sequence of the helicase mutant is as shown in SEQ ID NO:

6.

3. The helicase mutant according to claim 2, characterized in that The helicase mutant further has at least one amino acid mutation in the pin domain, the tower domain, the fragment V66-N84 and / or the fragment K294-A321, wherein the amino acid mutation is a substitution of the original amino acid with cysteine ​​or an unnatural amino acid, and / or is cross-linked with at least one chemical cross-linking agent; Preferably, the mutation sites of the pin domain are 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 28, 129, 130, 131, 132, 133 and 134; the mutation sites of the tower domain are 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 3 at least one of 79, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392 and 393; the mutation site of the fragment V66-N84 is at least one of 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 and 84 less than one; and / or the mutation position of fragment K294-A321 is at least one of 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320 and 321; More preferably, the mutation sites of the pin domain are G96, T97, I98, H99, H100, F101, L102, N103, L104, K105, L106, D107, H108, G109, F110, A111, D112, D113, G114, T115, A116, D117, N118, V119, T120, T121, K122, A123, K124, L125, V126, V127, 7, at least one of N128, K129, F130, N131, E132, A133 and L134; the mutation sites of the tower domain are P355, S356, S357, Y358, N359, E360, F361, N362, D363, L364, L365, D366, K367, Y368, L369, A370, D371, A372, K373, I374, A375, K376, G377, Y378, at least one of V76, T67, S68, P69, T70, H71, K72, A73, V74, R75, V76, S78, L79, N80, M81, L82, K83 and N84; at least one of; and / or the mutation site of fragment K294-A321 is at least one of K294, D295, E296, L297, V298, F299, Q300, E301, T302, Y303, T304, D305, S306, K307, G308, N309, I310, I311, V312, S313, N314, G315, E316, I317, I318, E319, V320 and A321.

4. The helicase mutant according to claim 3, characterized in that The non-natural amino acid is selected from 4-azido-L-phenylalanine (PAZF), 4-azido-L-phenylalanine (PAZF-Hcl), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylselenoyl)-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3-{4-[(propan-2-ylsulfanyl)carbonyl]- ]phenyl}propionic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropionyl)amino]phenyl}propionic acid, O-methyl-L-tyrosine, 4-amino-L-phenylalanine, 4-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3-iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)- L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthalen-2-ylamino)propionic acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl)propionic acid, (2R)-2-aminooctanoate 3-(2,2′-bipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolyl)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl] propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine and 2-nitrophenylalanine; and / or, the chemical cross-linking agent is selected from maleimide, active ester, succinimide, azide, alkyne, phosgene-type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and photosensitizer.

5. The helicase mutant according to claim 2, characterized in that The helicase mutant has one or more amino acid residues at the N-terminus truncated in the amino acid sequence shown in SEQ ID NO: 6, for example, the amino acids M1-A7 are truncated.

6. The helicase mutant according to claim 2, characterized in that The helicase mutant has at least one amino acid mutation in K2, D4, L5, K82, N83, N84, G85, I86 and D87 in the amino acid sequence as shown in SEQ ID NO:6, and the amino acid mutation is that the original amino acid is replaced by other amino acids; preferably, the other amino acid is alanine.

7. The helicase mutant according to claim 2, characterized in that The helicase mutant has at least one mutation in the region interacting with the porin interface, wherein the mutation includes replacement of the original amino acid with alanine, serine, glutamine, leucine, threonine, histidine or glycine; Preferably, the mutation site in the region interacting with the porin interface is at least one of 1, 2, 3, 4, 5, 12, 14, 16, 18, 21, 28, 63, 82, 124, 129, 158, 165, 189, 194, 196, 199, 202, 203, 205, 206, 207, 212, 214, 215, 218, 223, 244, 247, 261, 263, 271, 281, 289, 294, 307, 324, 331, 333, 336, 340, 341, 349, 367, 376, 382, ​​385, 386, 389 and 393; More preferably, the mutation sites in the region interacting with the porin interface are M1, K2, H3, D4, L5, Q12, Y14, F16, D18, K21, K28, K63, K82, K124, K129, K158, K165, K189, H194, K196, K199, R202, Q203, E205, D206, N207, K212, At least one of Q214, E215, K218, K223, K244, K247, K261, K263, K271, R281, K289, K294, K307, K324, K331, K333, K336, K340, K341, R349, K367, K376, K382, K385, K386, K389 and K393.

8. The helicase mutant according to claim 2, characterized in that The helicase mutant has at least one mutation in the ATP hydrolysis region, wherein the mutation includes replacement of the original amino acid by other charged amino acids or amino acids with relatively small side chains; preferably, the other charged amino acids or amino acids with relatively small side chains are alanine, aspartic acid, glutamine or glycine; Preferably, the mutation sites in the ATP hydrolysis region are 40, 41, 42, 43, 44, 45, 46, 143, 144, 145, 146, 147, 148, 149, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207 , 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, and 445; More preferably, the mutation sites in the ATP hydrolysis region are G40, F41, A42, G43, S44, G45, K46, E144, A145, S146, M147, V148, S149, G171, D172, S173, Y174, Q175, L176, L177, P178, V179, D180, D181, E182, D183, S184, S185, I186, L197, T198, K199, V200, V201, R202, Q203, A204, E205, D206, N207 , I208, I209, I210, A211, S213, Q214, E215, L216, I217, K218, A219, M220, D221, Q222, K223, I405, H406, K407, L408, Q409, G410, S411, T412, Y413, Q414, N430, R431, D432, N433, V434, L435, R436, L437, V438, Y439, V440, G441, I442, T443, R444 and A445.

9. The helicase mutant according to any one of claims 2 to 8, characterized in that The helicase mutant has a double substitution selected from the following on the amino acid sequence shown in SEQ ID NO:6: L369C and D117C; L369C and V119C; L369C and D113C; K373C and G114C; K373C and D112C; W384C and F110C; W384C and D112C; W384C and G114C; L369C and T115C; L369C and N118C; K373C and T115C; K373C and A116C; K373C and D117C; K373C and N118C; K373C and V119C; H108C and F388C; V119C and W384C; R380C and A116C; K373C and T120C; L369C and T121C; A116C and 381C; T121C and K373C; T120C and L369C; T121C and 388C; Preferably, when it has L369C and V119C double substitutions, the helicase mutant further has a mutation selected from the following: (1)K21S, K28S, K189S, H194S, K196S, K199S, Q203S, N207S, K212S, Q214S, K21 8S, K223S, R202K, E215A, K2A, H3A, D4A, L5A, Y14A, F16A, D18A, K63A, K82A, K1 65A, K244A, K247A, K261A, R281A, K307A, K324A, K331A, K333A, K336A, K341A, R349A, K124A, K129A, K158A, K271A, K376A, K382A, K385A, K393A, E205D single substitution; or (2) K72C and G315C, or E205A and D206A disubstituted; or (3) truncation of amino acids 1 to 7; or (4) M1G and an H before the amino acid sequence are truncated.

10. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the helicase mutant according to any one of claims 2 to 9; Preferably, the nucleotide sequence of the helicase mutant is as shown in SEQ ID NO:

8.

11. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a promoter and the nucleic acid according to claim 10; Preferably, the promoter is T7; and / or the backbone plasmid of the recombinant expression vector is PET.28a(+), PET.21a(+), or PET.32a(+).

12. A transformant, characterized in that: The transformant comprises a host cell and the nucleic acid according to claim 10; Preferably, the host cell is Escherichia coli, more preferably BL21(DE3), BL21 Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

13. A method for preparing the helicase mutant according to any one of claims 2 to 9, characterized in that: The transformant according to claim 12 is cultured in a culture medium to ferment and produce the helicase mutant.

14. A helicase mutant-sequencing adapter complex, characterized in that It comprises the helicase mutant according to any one of claims 2 to 9, and a sequencing adapter.

15. A kit, characterized in that: The kit comprises the helicase mutant according to any one of claims 2 to 9 and / or the helicase mutant-sequencing adapter complex according to claim 14; preferably also comprises a single-stranded DNA containing an anchor molecule at the 5' end, a nanopore, a nanopore protein, an electrical signal detector, a membrane and / or a buffer; Preferably, the anchoring molecule is a hydrophobic molecule, preferably selected from any one or more of the following: lipids, fatty acids, sterols, carbon nanotubes, polypeptides, proteins and / or amino acids, such as cholesterol, palmitate or tocopherol; The nanopore is a transmembrane protein pore or a solid-state pore; 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 membrane is an amphiphilic membrane, a high molecular polymer membrane or any combination thereof; The buffer is a dihydrogen phosphate-hydrogen phosphate buffer system, a carbonic acid-sodium bicarbonate buffer system, a Tris-HCl buffer system, a HEPES buffer system, a MOPS buffer system or any combination thereof.

16. Use of the helicase mutant according to any one of claims 2 to 9, the helicase mutant-sequencing adapter complex according to claim 14, or the kit according to claim 15 in high-throughput sequencing; Preferably, the high-throughput sequencing is nanopore sequencing.

17. A DNA denaturing method, characterized in that: The method comprises using the helicase mutant according to any one of claims 2 to 9, the helicase mutant-sequencing adapter complex according to claim 14 or the kit according to claim 15 to unwind the double-stranded DNA.

18. A sequencing method, characterized in that: It includes the following steps: The DNA is sequenced while being unwound using the DNA unwinding method as described in claim 17.