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

Abstract

The invention provides helicase, a preparation method thereof and application of the helicase in sequencing. The amino acid sequence of the helicase is shown as SEQ ID NO: 1 or has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of identity with the amino acid sequence shown as SEQ ID NO: 1, the helicase has high thermal stability, ATP hydrolysis activity and DNA unwinding activity, and the DNA unwinding activity of the helicase is increased along with increase of salt concentration. The method can be used for control and characterization of nucleic acid and applied to nanopore sequencing.

Description

A helicase and its preparation method and 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 and an application thereof. Background Art

[0002] Nanopore sequencing, an emerging single-molecule sequencing technology, has revolutionized the genome sequencing industry with its unique advantages, including high throughput, long read lengths, rapid speed, in situ detection, and label-free operation. Devices based on this technology are lightweight and portable, adapting to diverse sequencing scenarios. Furthermore, due to its non-amplified direct sequencing nature, it has no length limit for sequenceable DNA, enabling real-time base calling and direct sequencing of RNA, modified molecules such as methylated molecules, and other single molecules. This technology has the potential to overcome the limitations of second-generation sequencing and complement other sequencing needs. Nanopore sequencing technology has broad applications in many fields, including molecular biology, medicine, epidemiology, and ecology. Applications include genome mapping, epidemic monitoring, rare species detection, identification of hidden intermediates, monitoring the dynamics of non-covalent interactions, characterizing epigenetic and post-translational modifications, and rapid and cost-effective protein sequencing.

[0003] The principle of nanopore sequencing technology is based on changes in electrical signals. A nanopore inserted into a membrane (protein or solid) as a signal sensor separates two electrolyte chambers. When voltage is applied between the two electrolyte chambers, a stable current will be generated through the nanopore. When the nucleic acid molecule to be tested enters the nanopore, it will hinder the flow of ions, thereby causing current signal fluctuations. The effects of different base nucleotides on the current are different. Therefore, by detecting the current fluctuation signal of the nanopore in real time and using machine learning to analyze and decode the current signal, the sequence information of the nucleic acid molecule to be tested can be sequenced in real time.

[0004] During this sequencing process, the extremely high speed at which nucleic acids pass through the nanopore channel makes it 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 nanopore sequencing. Currently, the most common and effective method is to utilize the unwinding mechanism of helicases to control the perforation motion of nucleic acid molecules, thereby improving detection accuracy while maintaining sequencing speed and uniformity.

[0005] The helicase used in current commercial nanopore sequencers is the DDA helicase derived from the bacterial phage T4. Its thermal stability and unwinding speed have room for improvement. It has its own limitations, and there are few similar alternative products. Therefore, the market needs more new and different types of helicases to fill the gap.

[0006] Summary of the Invention

[0007] In order to solve the problem of lack of helicase with better performance in the prior art, the present invention provides a new helicase with high salt tolerance and thermal stability and its application.

[0008] Specifically, the first aspect of the present invention provides a helicase, whose amino acid sequence is as shown in SEQ ID NO: 1 or has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO: 1. Its gene is derived from the deep-sea metagenome, so its protein has strong thermal stability. The helicase has good ATP hydrolysis activity and dsDNA unwinding activity, and its unwinding activity tends to increase with increasing salt concentration. It can be used for the control and characterization of nucleic acids, and is applied to single-molecule nanopore sequencing to output a stable sequencing current signal.

[0009] At least one cysteine ​​on the surface of the three-dimensional structure of the helicase is mutated, and the mutation is that the cysteine ​​is replaced by alanine, glutamine, glycine, histidine, isoleucine, leucine, valine, serine, threonine or methionine.

[0010] Preferably, the mutated site includes C334.

[0011] At least one long-chain amino acid on the surface of the three-dimensional structure of the helicase undergoes a mutation; the mutation is that the original amino acid is replaced by a short-chain amino acid; the short-chain amino acid is preferably alanine or serine.

[0012] Preferably, the mutated sites include at least one of 17, 23, 27, 30, 39, 41, 42, 43, 69, 176, 215, 217, 224, 231, 232, 234, 235, 263 and 270.

[0013] More preferably, the mutated sites include at least one of K17, N23, N27, Y30, K39, K41, K42, K43, R69, R176, R215, K217, E224, Q231, Y232, N234, K235, K263 and D270.

[0014] The helicase has at least one amino acid mutation in the pin domain and / or the tower domain, where the original amino acid is replaced by cysteine ​​or an unnatural amino acid. This allows the two domains to connect, preventing DNA from swinging out of the helicase region during nanopore sequencing. The electric field and helicase forces allow DNA to penetrate the pore, improving sequencing continuity and stability.

[0015] Preferably, the mutated sites include at least one of positions 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, and 143 of the pin domain, and / or positions 365, At least one of 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400.

[0016] More preferably, the mutated sites include at least one of L117, K118, L119, D120, Y121, G122, L123, D124, S125, D126, N127, A128, S129, E130, S131, T132, K133, P134, K135, L136, V137, K138, N139, T140, D141, K142, and F143 of the pin domain, and / or Y365, E366, , E367, Y368, N369, D370, L371, I372, D373, K374, R375, L376, Q377, F378, A379, K380, Q381, S382, V383, G384, K385, D386, R387, R388, N389, A390, W391, K392, E393, Y394, F395, K396, L397, K398, N399 and R400.

[0017] Such unnatural amino acids include, but are not limited to, 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}propanoic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropionyl)amino]phenyl}propanoic 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-(naphthyl-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- At least one of (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.

[0018] The pin domain and the tower domain are connected; wherein the connection includes but is not limited to covalent connection, covalent and non-covalent binding connection or non-covalent connection; the covalent connection includes but is not limited to commercial cross-linking agent connection, protein fusion connection, polypeptide molecule connection and / or synthetic small molecule connection; the commercial cross-linking agent includes but is not limited to chemical cross-linking agents including the following functional groups: maleimide, active ester, succinimide, azide, alkyne, difluorocycloalkyne and linear alkyne), phosphine, haloacetyl, phosgene-type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and / or photosensitizer.

[0019] The tower domain and pin domain of the helicase are covalently linked to one or more ends of one or more linkers.

[0020] The second aspect of the present invention provides an isolated nucleic acid encoding the helicase as described in the first aspect of the present invention.

[0021] Preferably, the nucleotide sequence of the helicase is as shown in SEQ ID NO:2 or has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:2.

[0022] The third aspect of the present invention provides a recombinant expression vector comprising a promoter and the nucleic acid as described in the second aspect.

[0023] Preferably, the promoter is T7; and / or the backbone plasmid of the recombinant expression vector is PET.28a(+), PET.21a(+) or PET.32a(+).

[0024] The fourth aspect of the present invention provides a transformant comprising a host cell and the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect.

[0025] Preferably, the host cell is Escherichia coli, more preferably BL21(DE3), BL21Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

[0026] The fifth aspect of the present invention provides a method for preparing the helicase as described in the first aspect, wherein the transformant as described in the fourth aspect is cultured in a culture medium and fermented to produce the helicase with high homogeneity and protein purity.

[0027] The sixth aspect of the present invention provides a helicase-sequencing adapter complex, which includes the helicase as described in the first aspect, and a sequencing adapter.

[0028] In a seventh aspect, the present invention provides a kit comprising the helicase according to the first aspect and / or the helicase-sequencing adapter complex according to the sixth aspect; preferably, it further comprises a single-stranded DNA containing an anchor molecule at the 5' end, a nanopore protein, an electrical signal detector, a membrane and / or a buffer; more preferably, the anchor 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 membrane is an amphiphilic membrane (such as a phospholipid bilayer), a high molecular polymer membrane (such as a diblock copolymer, a triblock copolymer) or any combination thereof; the nanopore is a transmembrane protein pore or a solid-state pore; 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; 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.

[0029] The eighth aspect of the present invention provides the use of the helicase described in the first aspect, the helicase-sequencing adapter complex described in the sixth aspect, or the kit described in the seventh aspect in high-throughput sequencing or the preparation of sequencing-related reagents.

[0030] Preferably, the high-throughput sequencing is nanopore sequencing.

[0031] The ninth aspect of the present invention provides a DNA unwinding method, which comprises unwinding double-stranded DNA using the helicase described in the first aspect, the helicase-sequencing adapter complex described in the sixth aspect, or the kit described in the seventh aspect.

[0032] A tenth aspect of the present invention provides a sequencing method comprising the following steps:

[0033] The DNA is sequenced while being unwound using the DNA unwinding method as described in the ninth aspect.

[0034] The beneficial effects brought about by the technical solution of the present invention are:

[0035] This invention provides a novel helicase, named BCH866, derived from a deep-sea metagenome. It can be soluble expressed and purified in Escherichia coli. It exhibits high thermal stability, ATP hydrolysis activity, and DNA unwinding activity, with the DNA unwinding activity increasing with increasing salt concentration. This helicase can be used for nucleic acid manipulation and characterization, and has applications in nanopore sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows the purification results of BCH866 using molecular sieve Superdex 200. (A) shows the elution profile of BCH866 using molecular sieve Superdex 200; (B) shows the elution profile of BCH866 using molecular sieve gel.

[0037] Figure 2 shows the Alphafold2 predicted structure of BCH866.

[0038] Figure 3 shows the thermal stability test results of BCH866. (A) Thermal stability test of BCH866 after heating at different temperatures for 1 hour, with arrows indicating the target protein; (B) Thermal stability test of the control DDA mutant after heating at different temperatures for 1 hour, with arrows indicating the target protein.

[0039] FIG4 is the ATPase activity detection of BCH866 protein.

[0040] FIG5 is a dsDNA melting activity assay of the BCH866 protein (low salt reaction buffer).

[0041] FIG6 is a dsDNA melting activity assay of the BCH866 protein (high salt reaction buffer).

[0042] FIG7 is a schematic diagram of the connector (a: upper chain; b: lower chain).

[0043] Figure 8 is a schematic diagram of a sequencing library containing helicase (a: upper strand; b: lower strand; c: double-stranded target fragment; d: helicase; e: cholesterol-labeled double-stranded DNA).

[0044] FIG9 is a schematic diagram of a nanopore sequencing patch clamp amplifier.

[0045] FIG10 is a diagram of the BCH866 sequencing current signal. DETAILED DESCRIPTION

[0046] Example 1 Cloning, expression and purification of BCH866

[0047] 1. Cloning and Expression of BCH866

[0048] The amino acid sequence of BCH866 is shown in SEQ ID NO: 1, and its full-length DNA sequence is shown in SEQ ID NO: 2. The full-length DNA sequence was synthesized (Liuhe BGI) and ligated into the PET.28a(+) plasmid using the double restriction enzyme sites Nde1 and Xho1. The resulting plasmid was labeled PET.28a(+)-BCH866. The BCH866 protein expressed from this plasmid has a thrombin restriction site and a 6×His tag at its N-terminus.

[0049] Transform the PET.28a(+)-BCH866 plasmid into E. coli BL21(DE3) or its derivatives. Pick a single colony and inoculate it into 20 mL of LB medium containing kanamycin resistance. Cultivate with shaking at 37°C overnight. Then, inoculate it into 2 L of LB medium containing kanamycin resistance and incubate with shaking at 37°C until OD600 = 0.6-0.8. Cool the culture to 16°C and induce expression overnight with IPTG at a final concentration of 500 μM to obtain BCH866 cells.

[0050] 2. Purification of BCH866 Protein

[0051] The buffer solution used is as follows:

[0052] 1) Buffer A: 20mM Tris-HCl pH 7.5, 250mM NaCl, 20mM imidazole;

[0053] 2) Buffer B: 20 ​​mM Tris-HCl pH 7.5, 250 mM NaCl, 300 mM imidazole;

[0054] 3) Buffer C: 20mM Tris-HCl pH 7.5, 80mM NaCl;

[0055] 4) Buffer D: 20mM Tris-HCl pH 7.5, 1000mM NaCl;

[0056] 5) Buffer E: 20mM Tris-HCl pH 7.5, 200mM NaCl.

[0057] BCH866 cells were harvested, resuspended in Buffer A, disrupted using a cell disruptor, and then centrifuged to collect the supernatant. The supernatant was mixed with Ni-NTA filler previously equilibrated with Buffer A and allowed to bind for 1 hour. The filler was collected and washed extensively with Buffer A until all contaminants were removed. Next, Buffer B was added to the filler to elute the protein. The eluted protein was passed through a desalting column (Cytiva, Sephadex G-25) equilibrated with Buffer C, replacing the buffer from Buffer B to Buffer C. The protein solution from the desalting column was then added to ssDNA cellulose filler equilibrated with Buffer C. An appropriate amount of coagulation protease was added. This enzyme specifically recognizes the thrombin cleavage site amino acid sequence LVPRGS in the vector sequence PET28(a)+, thereby cleaving the affinity His tag carried by the protein. This procedure was performed at 4°C and incubated overnight on a rotary shaker.

[0058] The next day, the ssDNA cellulose filler was collected. At this time, the target protein was specifically adsorbed to the ssDNA filler. The ssDNA cellulose filler was washed 3-4 times with Buffer C to remove impurities that were not adsorbed to the ssDNA cellulose filler. Then, it was eluted with Buffer D to destroy the specific adsorption of the target protein to the ssDNA filler and elute the target protein into the solution. The protein purified from ssDNA cellulose was concentrated through a 30K ultrafiltration concentrator tube (Merck Millipore) in a centrifuge pre-cooled at 4°C. The parameters were set to 3000g speed, each centrifugation time was 10 minutes, and the final protein volume was concentrated to 2mL. Finally, it was passed through a molecular sieve Superdex 200 (Cytiva) using Buffer E as the molecular sieve buffer. The target protein peak was collected, concentrated, and frozen.

[0059] Figure 1 shows the results of the BCH866 protein purification using Superdex 200 molecular sieves. As shown in Figure 1, a relatively large amount of BGH866 protein with good purity was obtained after purification. The protein exhibited uniform peak shape and high purity.

[0060] 3. AlphaFold2 structure prediction of BCH866 protein

[0061] Using AlphaFold 2 software, we performed structural prediction on the BCH866 protein, as shown in Figure 2. The protein comprises a helix, sheet, and loop. The overall structure of the BCH866 protein is similar to that of conventional 5'-3' helicases, but its pin and tower domains are closer together, resulting in a more compact DNA binding region.

[0062] Example 2 Thermal stability test of BCH866 protein

[0063] The purified BCH866 protein was diluted to 1 mg / mL with Buffer E and aliquoted into seven tubes of equal volume. One tube was placed in a 4°C refrigerator, and the remaining tubes were incubated for 1 hour in a metal bath at different temperatures. The metal bath temperatures were set at 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C. After the reaction, the proteins incubated at different temperatures were immediately centrifuged at high speed at 4°C, 10,000 rpm, and 10 minutes. After centrifugation, equal volumes of the protein supernatant were heat-denatured in a 95°C metal bath for 10 minutes and characterized by SDS-PAGE (10%). 10 μL of sample was added to each well. The protein gel image is shown in Figure 3. The control group consisted of the DDA mutant protein (SEQ ID NO: 11), using exactly the same operating conditions.

[0064] Figure 3 shows the behavior of BCH866 and the control DDA mutant proteins at different temperatures. As shown in Figure 3, BCH866 protein was only slightly degraded after heating at 40°C for 1 hour, while only 20% of the DDA mutant protein remained stable and undegraded under the same conditions. Approximately 30% of the BCH866 protein remained stable and undegraded after heating at 45°C for 1 hour, while the DDA mutant was almost completely degraded under the same conditions.

[0065] The experimental results show that the BCH866 protein has better thermal stability than the DDA mutant, can maintain protein stability at higher temperatures, and has stronger heat resistance. This may be related to the fact that its genome comes from the deep-sea metagenome under extreme environmental conditions. This performance also gives it a wider application prospect in sequencing.

[0066] Example 3 ATPase activity detection of BCH866 protein

[0067] 1. Preparation of double-stranded DNA (ovDNA-1) and single-stranded DNA (ssDNA):

[0068] SEQ ID NO: 3 and SEQ ID NO: 4 were synthesized (Liuhe BGI) and annealed to ovDNA-1 with 20 T residues in the 5' overhang. The annealing process was incubation at 95°C for 5 minutes, cooling to 25°C at a rate of 0.1°C / s, and incubation for another 30 minutes. The annealing recipe is shown in Table 1. 100 μM of SEQ ID NO: 4 was diluted to 10 μM in TE buffer (pH = 8) to prepare ssDNA.

[0069] Table 1 ovDNA-1 annealing formula

[0070] Solution volume 100 μM SEQ ID NO: 3 5 μL 100 μM SEQ ID NO: 4 5 μL TE buffer (pH = 8) 40 μL

[0071] 2. Prepare high salt reaction buffer

[0072] High salt reaction buffer (2×): 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl2, 1.0 M KCl.

[0073] 3. Dilute protein

[0074] BCH866 protein was diluted to 10 μM in 1× PBS.

[0075] 4. ATP hydrolysis reaction

[0076] Add the corresponding reagents according to the reaction system in Table 2, incubate at 30°C for 30 minutes for ATP hydrolysis reaction test, and inactivate at 80°C for 5 minutes. ①② are experimental groups, ③④⑤⑥ are corresponding control groups, and each group has 3 replicates.

[0077] Table 2 ATP hydrolysis reaction system

[0078] No. Reaction buffer (2×) DNA BCH6X Protein H2O ①10μL1μL(ovDNA-1)1μL8μL ②10μL1μL(ssDNA)1μL8μL ③10μL——1μL9μL ④10μL1Μl(ovDNA-1)——9μL ⑤10μL1μL(ssDNA)——9μL ⑥10μL————10μL

[0079] 5. Detection of Remaining ATP in the Reaction

[0080] The ATP concentration remaining in the reaction was determined using an ATP detection kit (Beyotime, S0026B) according to the manufacturer's instructions.

[0081] 6. Experimental Results

[0082] The results are shown in FIG4 . Under the condition of a final KCl concentration of 500 mM, BCH866 has the activity of hydrolyzing ATP.

[0083] Example 4 Detection of dsDNA Melting Activity of BCH866

[0084] 1. Preparation of double-stranded DNA (ovDNA-2)

[0085] SEQ ID NO: 5 and SEQ ID NO: 6 were synthesized and annealed to ovDNA-2 with 20 T residues in the 5' overhang. The annealing process was incubation at 95°C for 5 minutes, cooling at a rate of 0.1°C / s to 25°C, and incubation for 30 minutes. The annealing recipe is shown in Table 3.

[0086] Table 3 ovDNA-2 annealing formula

[0087] Solution volume 100 μM SEQ ID NO: 5 5 μL 100 μM SEQ ID NO: 6 5 μL TE buffer (pH = 8) 40 μL

[0088] 2. Prepare reaction buffer

[0089] Low salt reaction buffer: 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl;

[0090] High salt reaction buffer: 100 mM HEPES (pH=8.0), 1 mg / mL BSA, 10 mM MgCl2, 500 mM KCl.

[0091] 3. Prepare the reaction solution

[0092] Experimental reaction solution: Add 3 μL of 10 μM ovDNA-2, 6 μL of 100 μM SEQ ID NO: 7 (20x competitor DNA, this strand can anneal with complementary DNA to prevent reannealing of the initial substrate and loss of fluorescence), and 6 μL of 100 mM ATP to 585 μL of low-salt reaction buffer. Add 3 μL of 10 μM ovDNA-2, 6 μL of 100 μM SEQ ID NO: 7 (20x competitor DNA), and 6 μL of 100 mM ATP to 585 μL of high-salt reaction buffer.

[0093] Positive control: Add 1 μL of 10 μM SEQ ID NO:6, 2 μL of 100 μM SEQ ID NO:7 (20x competitor DNA), and 2 μL of 100 mM ATP to 195 μL of low-salt reaction buffer. Add 1 μL of 10 μM SEQ ID NO:6, 2 μL of 100 μM SEQ ID NO:7 (20x competitor DNA), and 2 μL of 100 mM ATP to 195 μL of high-salt reaction buffer.

[0094] 4. Dilute protein

[0095] BCH866 protein was diluted to 4.8 μM in 1× PBS.

[0096] 5. Prepare the Melting Reaction

[0097] The corresponding reagents were added according to Table 4. ①② were experimental groups, ③④ were negative control groups, and ⑤⑥ were positive control groups. The kinetic changes of fluorescence intensity within 30 min of the reaction were detected using a microplate reader at 30°C. Each group was repeated three times.

[0098] Table 4 Melting reaction formula

[0099] No. Category Solution 1 Solution 2 ① Experimental group 58.5μL experimental reaction solution (low salt) 1.5μL protein ② Experimental group 58.5μL experimental reaction solution (high salt) 1.5μL protein ③ Negative control group 58.5μL experimental reaction solution (low salt) 1.5μL PBS ④ Negative control group 58.5μL experimental reaction solution (high salt) 1.5μL PBS ⑤ Positive control group 58.5μL positive control solution (low salt) 1.5μL PBS ⑥ Positive control group 58.5μL positive reaction solution (high salt) 1.5μL PBS

[0100] 6. Data Analysis

[0101] The percentages of the fluorescence values ​​of the experimental group and the negative control group relative to the fluorescence value of the positive control group were calculated.

[0102] 7. Experimental Results

[0103] Within the error range and the allowable instrument fluctuation, the ratio of the fluorescence value of the experimental group to the fluorescence value of the positive control group, as well as the ratio of the fluorescence value of the negative control group to the fluorescence value of the positive control group, were calculated to plot the experimental results (due to the sensitivity of the instrument, the negative control group had fluorescence absorption readings), as shown in Figures 5 and 6.

[0104] From the experimental results in Figures 5 and 6, it can be seen that the negative control group in each experiment remained unchanged during the measurement process, while the fluorescence value of the experimental group gradually increased with the increase of reaction time, indicating that the BCH866 protein has the activity of unwinding double-stranded DNA, and its unwinding direction is 5'-3'.

[0105] Furthermore, Figure 5 shows the results under low salt conditions, and Figure 6 shows the results under high salt conditions. Comparison of the two shows that the activity of BCH866 protein in unwinding dsDNA increases with increasing salt concentration.

[0106] Example 5 Nanopore Sequencing Application of BCH866 Protein

[0107] 1. Two partially complementary DNA strands (upper strand, SEQ ID NO: 8 and lower strand, SEQ ID NO: 9) were annealed to form a linker (as shown in Figure 7). The linker was then ligated to the double-stranded target fragment using Rapid T4 DNA Ligase (NEB, E6057AVIAL) and purified to obtain a sequencing library.

[0108] 2. The ligation steps are as follows: Take the T4 DNA ligase out of the -20°C freezer, gently tap the tube wall to mix, centrifuge briefly, and place on ice. Thaw the quick ligation reaction buffer, pipette to mix, centrifuge briefly, and then place on ice. Prepare the reaction mixture (120 μL quick ligation reaction buffer, 60 μL T4 DNA ligase, 30 μL 10 μM adapter). Then, add 390 μL of the purified end-repaired, "A"-added, and purified product of the double-stranded target fragment to the ligation reaction mixture. Use a flared pipette tip to gently pipette and mix 6 times, centrifuge briefly to collect the reaction solution at the bottom of the tube, and then place it in a metal bath preheated at 25°C for the ligation reaction, with a timer for 30 minutes. After the reaction is completed, centrifuge the reaction tube briefly to collect the reaction solution at the bottom of the tube.

[0109] 3. Purification steps are as follows: 30 minutes in advance, remove Ampure XP magnetic beads (Beckman Coulter, A63882) from a 4°C refrigerator, shake well, and bring to room temperature. Shake thoroughly before use. Pipette 240 μL of magnetic beads into a DNA low-binding tube (Eppendorf, 0030108051) containing the sample ligation product. Mix thoroughly by gently tapping the tube or pipetting gently with a flared pipette tip at least six times. Ensure all liquid and beads in the pipette tip are pipetted into the tube. Incubate on a rotary mixer at room temperature for 5 minutes. Centrifuge the DNA low-binding tube briefly, place on a magnetic rack, and let stand for 2-5 minutes until the liquid clears. Carefully aspirate and discard the supernatant. Keep the DNA low-adsorption tube on the magnetic stand, add 900μL of washing buffer [20mM Tris (pH=7.5), 2500mM NaCl], remove the DNA low-adsorption tube from the magnetic stand, and gently tap the tube wall to mix the magnetic beads. After mixing, place it back on the magnetic stand and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully aspirate the supernatant and discard it. Remove the centrifuge tube from the magnetic stand and centrifuge it briefly. After separation on the magnetic stand, use a small-scale pipette to absorb the remaining liquid at the bottom of the tube. Remove the DNA low-adsorption tube from the magnetic stand and add 68μL of elution buffer [20mM Tris (pH=7.5), 50mM NaCl] to elute the DNA. Gently tap the tube wall by hand to mix. Centrifuge briefly for 3 seconds and collect the liquid in the tube to the bottom of the tube. Incubate at room temperature for 10 minutes. After brief centrifugation, place the DNA low-binding tube on a magnetic stand and let it stand for 2–5 minutes until the liquid clears. Transfer 66 μL of the supernatant to a new 1.5 mL DNA low-binding tube. The remaining sample can be used for concentration determination. The Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) is recommended for concentration determination.

[0110] 4. Incubate the BCH866 protein and sequencing library at 25°C for 1 hour (molar concentration ratio 1:8) to form a sequencing library containing helicase.

[0111] 5. The helicase-containing sequencing library was incubated with single-stranded DNA (ssDNA-chol, SEQ ID NO: 10) 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 binding to the phospholipid membrane reduces library loading and improves capture efficiency (a schematic of the adapter is shown in Figure 8, where the star represents cholesterol and the triangle represents the helicase BCH866).

[0112] 6. Use a patch clamp amplifier or other electrical signal amplifier to collect current signals (as shown in Figure 9). 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 bimolecular phospholipid membrane is formed at the micropores of the two chambers, and the nanopore protein 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 acid passes through the nanopore under the control of the helicase BCH866. The buffer used in this experiment is: 0.47 M KCl, 25 mM HEPES, 1 mM EDTA, 30 mM ATP, 25 mM MgCl2, pH 8, and the sequencing temperature is 30°C.

[0113] 7. The sequencing electrical signal is shown in Figure 10. As can be seen from the figure, as the helicase BCH866 guides a single DNA strand into the nanopore, some of the current is blocked, causing the current to decrease. Because different nucleotides have different sizes, the amount of current blocked also varies, resulting in the visible fluctuations in the current signal. This example demonstrates the potential of the helicase BCH866 for nanopore sequencing.

[0114] The sequences used in the present invention are as follows:

[0115] Amino acid sequence of BCH866 (SEQ ID NO: 1)

[0116]

[0117] DNA sequence of BCH866 (SEQ ID NO: 2)

[0118]

[0119]

[0120] SEQ ID NO: 3:

[0121] 5'-GCGTCGAAAAGCAGTACTTAGGCATT-3'

[0122] SEQ ID NO: 4:

[0123] 5'-TTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-3'

[0124] SEQ ID NO: 5:

[0125] 5'-BHQ-1-GCGTCGAAAAGCAGTACTTAGGCATT-3'

[0126] SEQ ID NO: 6:

[0127] 5'-TTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-FAM-3'

[0128] SEQ ID NO: 7:

[0129] 5'-AATGCCTAAGTACTGCTTTTTCGACGCT-3'

[0130] SEQ ID NO:8:

[0131] 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-YYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3'(Y=iSP18)

[0132] SEQ ID NO:9:

[0133] 5'-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'

[0134] SEQ ID NO: 10:

[0135] 5'-cholesterol-TTGACCGCTCGCCTC-3'

[0136] Wherein, iSP18 is represented by the following formula I:

[0137]

[0138]

[0139] 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, characterized in that The amino acid sequence of the helicase is as shown in SEQ ID NO:1 or has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The helicase according to claim 1, characterized in that At least one cysteine ​​on the surface of the three-dimensional structure of the helicase is mutated, and the mutation is that the cysteine ​​is replaced by alanine, glutamine, glycine, histidine, isoleucine, leucine, valine, serine, threonine or methionine; Preferably, the mutated site includes C334.

3. The helicase according to claim 1, characterized in that At least one long-chain amino acid on the surface of the three-dimensional structure of the helicase is mutated; the mutation is that the original amino acid is replaced by a short-chain amino acid; the short-chain amino acid is preferably alanine or serine; Preferably, the mutated sites include at least one of 17, 23, 27, 30, 39, 41, 42, 43, 69, 176, 215, 217, 224, 231, 232, 234, 235, 263 and 270; More preferably, the mutated sites include at least one of K17, N23, N27, Y30, K39, K41, K42, K43, R69, R176, R215, K217, E224, Q231, Y232, N234, K235, K263 and D270.

4. The helicase according to claim 1, characterized in that The helicase has at least one amino acid mutation in the pin domain and / or the tower domain, wherein the amino acid mutation is a substitution of the original amino acid with cysteine ​​or a non-natural amino acid; Preferably, the mutated sites include at least one of positions 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 and 143 of the pin domain, and / or positions 365, at least one of 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, and 400; More preferably, the mutated sites include at least one of L117, K118, L119, D120, Y121, G122, L123, D124, S125, D126, N127, A128, S129, E130, S131, T132, K133, P134, K135, L136, V137, K138, N139, T140, D141, K142, and F143 of the pin domain, and / or Y365, E366, , E367, Y368, N369, D370, L371, I372, D373, K374, R375, L376, Q377, F378, A379, K380, Q381, S382, V383, G384, K385, D386, R387, R388, N389, A390, W391, K392, E393, Y394, F395, K396, L397, K398, N399 and R400.

5. The helicase according to claim 4, characterized in that The unnatural amino acids include, but are not limited to, 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}propanoic acid, (2S)-2-amino-3-{4-[(2-amino-3-sulfanylpropionyl)amino]phenyl}propanoic 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-(naphth-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- At least one of (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.

6. The helicase according to claim 4 or 5, characterized in that The pin domain and the tower domain are connected; Wherein, the connection includes but is not limited to covalent connection, covalent and non-covalent binding connection or non-covalent connection; the covalent connection includes but is not limited to commercial cross-linking agent connection, protein fusion connection, polypeptide molecule connection and / or synthetic small molecule connection; the commercial cross-linking agent includes but is not limited to chemical cross-linking agents including the following functional groups: maleimide, active ester, succinimide, azide, alkyne, difluorocyclic alkyne and linear alkyne), phosphine, haloacetyl, phosgene type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and / or photosensitive reagent.

7. The helicase according to claim 1, characterized in that The tower domain and the pin domain of the helicase are covalently linked to one or more ends of one or more connectors.

8. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the helicase according to any one of claims 1 to 7; Preferably, the nucleotide sequence of the helicase is as shown in SEQ ID NO:2 or has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:

2.

9. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a promoter and the nucleic acid according to claim 7; Preferably, the promoter is T7; and / or the backbone plasmid of the recombinant expression vector is PET.28a(+), PET.21a(+) or PET.32a(+).

10. A transformant, characterized in that: The transformant comprises a host cell and the nucleic acid according to claim 8 or the recombinant expression vector according to claim 9; Preferably, the host cell is Escherichia coli, more preferably BL21(DE3), BL21 Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

11. A method for preparing the helicase according to any one of claims 1 to 7, characterized in that: The transformant according to claim 10 is cultured in a culture medium to ferment and produce the helicase.

12. A helicase-sequencing adapter complex, characterized in that The method comprises the helicase according to any one of claims 1 to 7, and a sequencing adapter.

13. A kit, characterized in that: The kit comprises the helicase according to any one of claims 1 to 7 and / or the helicase-sequencing adapter complex according to claim 12; preferably, it also comprises a single-stranded DNA containing an anchor molecule at the 5' end, a nanopore protein, an electrical signal detector, a membrane and / or a buffer; more preferably, the anchor 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 membrane is an amphiphilic membrane, a high molecular polymer membrane or any combination thereof; the nanopore is a transmembrane protein pore or a solid pore; 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; 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.

14. Use of the helicase according to any one of claims 1 to 7, the helicase-sequencing adapter complex according to claim 12, or the kit according to claim 13 in high-throughput sequencing or preparation of sequencing-related reagents; Preferably, the high-throughput sequencing is nanopore sequencing.

15. A DNA denaturing method, characterized in that: The method comprises using the helicase according to any one of claims 1 to 7, the helicase-sequencing adapter complex according to claim 12 or the kit according to claim 13 to unwind the double-stranded DNA.

16. 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 15.

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