Helicase, preparation method thereof and application of helicase in sequencing

Abstract

The invention provides DNA helicase BCH516 and a mutant thereof, the amino acid sequence of the helicase is as shown in SEQ ID NO: 1, and the nucleotide sequence for coding the helicase is as shown in SEQ ID NO: 2. The helicase has salt tolerance and stability, has better soluble expression quantity, and can be applied to control and characterization of nucleic acid and nanopore sequencing.

Description

A helicase, its preparation method and its application in sequencing Technical Field

[0001] The present invention belongs to the technical fields of gene sequencing, molecular detection and clinical detection, and particularly relates to a helicase, a preparation method thereof and an application thereof in sequencing. 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 length, rapid speed, in situ detection, and label-free operation. This technology is convenient and portable, adapting to diverse sequencing scenarios. Furthermore, due to its non-amplified direct sequencing nature, there is no length limit on the sequenceable DNA, allowing for real-time base calling and direct sequencing of modifications such as RNA and methylation, as well as other single molecules. Nanopore sequencing technology has broad applications in many fields, including molecular biology, medicine, epidemiology, and ecology. These include genome mapping, monitoring infectious diseases such as epidemics, detecting rare species, identifying hidden intermediates, monitoring the dynamics of non-covalent interactions, facilitating the characterization of epigenetic and post-translational modifications, and enabling rapid and inexpensive 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) serves as a signal sensor to separate two electrolyte chambers. When voltage is applied between the two electrolyte chambers, a stable perforation current is generated. When the molecule to be tested enters the nanopore, the flow of ions is hindered, resulting in fluctuations in the current signal. Different bases have different effects on the current. By detecting the current fluctuation signal of the nanopore in real time and using machine learning to analyze and decode the current signal, the 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 utilizes the principle of helicase unwinding to control the perforation motion of nucleic acid molecules, improve detection accuracy, and maintain sequencing speed and uniformity.

[0005] The helicase used in current commercial nanopore sequencers is the Dda helicase derived from the bacterial phage T4. Its yield, stability, salt tolerance, and unwinding speed all have room for improvement. It has its own limitations, and more new and different types of helicases are needed to fill the gap in the small number of commercial helicase categories.

[0006] Summary of the Invention

[0007] To address the problems of poor helicase yield, stability, and salt tolerance in the prior art, the present invention provides a helicase, a method for preparing the same, and its application in sequencing. The helicase has high salt tolerance and stability, good soluble expression, high protein yield, and DNA unwinding activity, and can be used for the control and characterization of nucleic acids and for nanopore sequencing. Specifically, the present invention provides:

[0008] In order to solve the above technical problems, the first aspect of the present invention provides a helicase, the amino acid sequence of the helicase is as shown in SEQ ID NO:1, or has at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO:1.

[0009] The gene encoding helicase comes from the deep-sea metagenome. The protein encoded by this gene has extremely high thermal stability and salt tolerance, has the ability to unwind DNA, can be used for the control and characterization of nucleic acids, and is applied to nanopore sequencing.

[0010] In some technical embodiments, the surface of the helicase has at least one mutation, wherein the mutation is a substitution of cysteine ​​by alanine, glutamine, glycine, histidine, isoleucine, leucine, valine, serine, threonine or methionine.

[0011] In some preferred technical solutions, the mutated amino acid site includes C318.

[0012] In some more preferred technical embodiments, the mutations include C318A, C318G, C318I and / or C318L.

[0013] In some technical solutions, the helicase has at least one mutation in the pin domain and / or the tower domain, wherein the mutation is a substitution of the original amino acid with cysteine ​​or a non-natural amino acid.

[0014] The tower domain and the pin domain can be cross-linked to form a DNA binding region, which facilitates rate-controlled sequencing, increases sequencing continuity and stability, and prevents fluctuations or interruptions in sequencing signals caused by DNA slipping or fluctuating during the sequencing process.

[0015] In some preferred technical solutions, the mutation site is selected from one or more of D95 to P115 and S388 to W435.

[0016] In some more preferred technical embodiments, the unnatural amino acids include but are not limited to 4-azido-L-phenylalanine, 4-azido-L-phenylalanine, 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-phenylalanine 4-Isopropyl-L-phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthylamino)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- one or more of (2-nitrobenzyl)-L-serine, (2-nitrobenzyl)-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, (2-nitrobenzyl)-L-tyrosine, and (2-nitrophenylalanine).

[0017] In some technical embodiments, the tower domain and the pin domain of the helicase are covalently linked to one or more ends of one or more linkers, and the linkers are selected from cross-linkers, polypeptides and small molecules.

[0018] In some preferred technical schemes, the cross-linking agent includes a chemical cross-linking agent containing the following functional groups: maleimide, active ester, succinimide, azide, alkyne, phosphine, halogenated acetyl, phosgene-type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and photosensitive reagent; the alkyne includes dibenzocyclooctyne, difluorocycloalkyne and linear alkyne, the phosphine includes the phosphine used in traceless and non-traceless Staudinger combination, the halogenated acetyl includes iodoacetamide, and the photosensitive reagent includes aromatic azide and diaziridine.

[0019] In some technical solutions, one or more amino acid sites in the helicase's DNA binding region, near the ATP catalytic active center, and potentially interacting with the nanopore binding region undergo mutations; the mutations include replacing the original amino acid with an amino acid with a larger side chain. These mutations increase (i) electrostatic interactions, (ii) hydrogen bonds, and / or (iii) cation-π interactions between the at least one amino acid and one or more nucleotides in the ssDNA; substitutions increase positively charged amino acids to reduce repulsion between the motor protein and the pore, etc.

[0020] In the present invention, the amino acid sites near the ATP catalytic active center are amino acid sites that are spatially located around the ATP catalytic active center and have an impact on the ATP catalytic activity, as can be understood by those skilled in the art.

[0021] In some preferred technical embodiments, the mutated amino acid sites include one or more of M1, E2, N3, V4, K5, K75, P98, R106, F108, K160, E161, D162, F163, F167, K168, Y169, F170, E171, E172, Q173, K215, K219, D220, F416, W435, Y438 and Y439.

[0022] In some technical schemes, the long side chain amino acids on the surface of the helicase are mutated into short side chain amino acids, and the mutation sites include at least one of the following: E2A, N3A, K5A, K75A, R106A, D162A, K168A, Y169A, K215A, K219A, D220A, Y438A, Y439A, K5S, K75S, K168S, K215S, K219S, D202S, Y438S and Y439S.

[0023] The helicase has good ATP hydrolysis activity and dsDNA unwinding activity.

[0024] The helicase can be used for the control and characterization of nucleic acids and applied to single-molecule nanopore sequencing to output a stable sequencing current signal.

[0025] In order to solve the above technical problems, the second aspect of the present invention provides an isolated nucleic acid, which encodes the helicase as described in the first aspect of the present invention.

[0026] In some preferred technical embodiments, the nucleotide sequence of the helicase is as shown in SEQ ID NO: 2, or has at least 55%, at least 60%, at least 70%, 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the nucleotide sequence shown in SEQ ID NO: 2.

[0027] In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector, which comprises a promoter and the nucleic acid as described in the second aspect of the present invention.

[0028] In some preferred technical solutions, the promoter is T7; and / or the backbone plasmid of the recombinant expression vector is PET.28a(+), PET.21a(+) or PET.32a(+).

[0029] In order to solve the above technical problems, the fourth aspect of the present invention provides a transformant, which comprises the nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention.

[0030] In some preferred technical solutions, the host cell of the transformant is Escherichia coli.

[0031] This type of helicase can be successfully expressed in the Escherichia coli recombinant protein expression system, with high protein yield, high uniformity and high purity.

[0032] In some more preferred technical solutions, the host cell of the transformant is BL21(DE3), BL21Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

[0033] In order to solve the above technical problems, the fifth aspect of the present invention provides a method for preparing the helicase 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.

[0034] In order to solve the above technical problems, the sixth aspect of the present invention provides a helicase-linker complex, wherein the helicase-linker complex comprises the helicase as described in the first aspect of the present invention and a sequencing linker, and the sequencing linker comprises two DNA chains with complementary partial regions, for example, as shown in SEQ ID NO: 9 and 10, respectively.

[0035] In order to solve the above technical problems, the seventh aspect of the present invention provides a kit, which includes the helicase as described in the first aspect of the present invention and / or the helicase-linker complex as described in the sixth aspect of the present invention.

[0036] In some preferred technical solutions, the kit further 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 sequencing buffer.

[0037] In some preferred technical solutions, 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 membrane is an amphiphilic membrane (e.g., a phospholipid bilayer), a polymer membrane (e.g., 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, InvG, GspD, or any combination thereof; and the sequencing buffer is a dihydrogen phosphate-dihydrogen 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.

[0038] In order to solve the above technical problems, the eighth aspect of the present invention provides the use of the helicase described in the first aspect of the present invention or the helicase-linker complex described in the sixth aspect of the present invention in sequencing or preparing sequencing-related reagents.

[0039] In order to solve the above technical problems, the ninth aspect of the present invention provides a DNA unwinding method, which comprises unwinding double-stranded DNA using the helicase as described in the first aspect of the present invention or the helicase-linker complex as described in the sixth aspect of the invention or the kit as described in the seventh aspect of the present invention.

[0040] In order to solve the above technical problems, the tenth aspect of the present invention provides a sequencing method, which includes the following steps: using the DNA unwinding method as described in the ninth aspect of the present invention to sequence the double-stranded DNA while unwinding it.

[0041] The beneficial effects brought about by the technical solution of the present invention are:

[0042] The present invention provides a helicase, a preparation method thereof, and its application in sequencing. The gene of the helicase is derived from the deep-sea metagenome, so that the protein itself has high salt tolerance and stability. The protein has good soluble expression, high protein yield and DNA unwinding activity, can be used for the control and characterization of nucleic acids, and is applied to nanopore sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 shows the purification results of BCH516 using molecular sieve Superdex 200. (A) Elution profile of BCH516 using molecular sieve Superdex 200; (B) Elution profile of BCH516 using molecular sieve gel.

[0044] FIG2 is the ATPase activity detection of BCH516 protein.

[0045] FIG3 is a dsDNA melting activity assay of the BCH516 protein (low salt reaction buffer 1).

[0046] FIG4 is a dsDNA melting activity assay of the BCH516 protein (high salt reaction buffer 2).

[0047] FIG5 is a detection of the restriction sequence blocking the BCH516 protein depolymerization activity (low salt reaction buffer).

[0048] FIG6 is a detection of the restriction sequence blocking the BCH516 protein melting activity (high salt reaction buffer).

[0049] FIG7 is a schematic diagram of the connector (a: upper chain; b: lower chain).

[0050] 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).

[0051] FIG9 is a schematic diagram of a nanopore sequencing patch clamp amplifier.

[0052] FIG10 is a diagram of the BCH516 sequencing current signal. DETAILED DESCRIPTION

[0053] Example 1 Cloning, expression and purification of BCH516

[0054] 1. Cloning and Expression of BCH516

[0055] The BCH516 DNA sequence was ligated into the PET.28a(+) plasmid to synthesize the full-length DNA sequence (Liuhe BGI). Using dual restriction enzyme sites Nde1 and Xho1, the resulting plasmid was labeled PET.28a(+)-BCH516. The BCH516 protein expressed from this plasmid has a thrombin cleavage site at its N-terminus and a 6×His tag at its N-terminus.

[0056] Transform the cloned PET.28a(+)-BCH516 plasmid into E. coli expression strain BL21(DE3) or its derivatives. Pick a single colony and inoculate it into 20 mL of LB medium containing kanamycin resistance and culture it at 37°C with shaking overnight. Then transfer it into 2 L of LB medium containing kanamycin resistance and culture it at 37°C with shaking until the OD 600 =0.6-0.8, cooled to 16°C, added with IPTG at a final concentration of 500 μM to induce expression overnight, and obtained BCH516 bacteria.

[0057] 2. Purification of BCH516

[0058] Buffer A: 20mM Tris-HCl pH 7.5, 250mM NaCl, 20mM Imidazole

[0059] Buffer B: 20mM Tris-HCl pH 7.5, 250mM NaCl, 300mM Imidazole

[0060] Buffer C: 20mM Tris-HCl pH 7.5, 80mM NaCl

[0061] Buffer D: 20mM Tris-HCl pH 7.5, 1000mM NaCl

[0062] Buffer E: 20mM Tris-HCl pH 7.5, 200mM NaCl

[0063] Collect the expressed BCH516 cells, resuspend them in Buffer A, disrupt them using a cell disruptor, and centrifuge to collect the supernatant. Mix the supernatant with Ni-NTA filler pre-equilibrated with Buffer A and allow to bind for 1 hour. Collect the filler and wash it extensively with Buffer A until all contaminants are washed out. Next, add Buffer B to the filler to elute the protein. Pass the eluted protein through a desalting column (Cytiva, Sephadex G-25) equilibrated with Buffer C, switching the buffer from Buffer B to Buffer C. The protein solution from the desalting column is then added to ssDNA cellulose filler equilibrated with Buffer C. An appropriate amount of coagulation protease is 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 is performed at 4°C and incubated overnight on a rotary shaker.

[0064] 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 using 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.

[0065] As shown in Figure 1, the purification yielded a relatively high amount of pure BGH516 protein with a uniform peak shape. On average, 1 L of fermentation yielded approximately 1.39 mg of the target protein, significantly higher than the yield of helicase DDA (0.23 mg per 1 L).

[0066] Example 2 ATPase activity detection of BCH516 protein

[0067] 1. Preparation of double-stranded DNA (ovDNA) and single-stranded DNA (ssDNA): SEQ ID NO: 3 and SEQ ID NO: 4 were annealed to form ovDNA with 20 T residues overhanging the 5' end. The annealing process was incubation at 95°C for 5 minutes, followed by a cooling rate of 0.1°C / s to 25°C, and continued incubation for 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.

[0068] Table 1 ovDNA annealing formula

[0069] Solution volume 100 μM SEQ ID NO: 3 5 μL 100 μM SEQ ID NO: 4 5 μL TE buffer (pH=8) 40 μL

[0070] 2. Prepare high salt reaction buffer (2×): 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl2, 1.0 M KCl.

[0071] 3. Dilute the protein: Dilute the BCH516 protein to 10 μM with 1× PBS.

[0072] 4. Perform ATP hydrolysis reaction: Add the corresponding reagents according to the reaction system in Table 2, incubate at 30°C for 30 minutes, and inactivate at 80°C for 5 minutes. ①② are experimental groups, and ③④⑤⑥ are corresponding control groups, with three replicates per group.

[0073] Table 2 ATP hydrolysis reaction system

[0074] No. Reaction buffer (2×) DNA Protein H2O ① 10μL 1μL (ovDNA) 1μL 8μL ② 10μL 1μL (ssDNA) 1μL 8μL ③ 10μL——1μL9μL ④ 10μL 1μL (ovDNA)——9μL

[0075] ⑤10μL1μL(ssDNA)——9μL⑥10μL————10μL

[0076] 5. Detection of the remaining ATP in the reaction: Use the ATP detection kit (Biyuntian, S0026B) to determine the remaining ATP concentration in the reaction according to the manufacturer's instructions.

[0077] 6. Experimental results: As shown in Figure 2, BCH516 has the activity of hydrolyzing ATP under high salt conditions.

[0078] Example 3 Detection of dsDNA Melting Activity of BCH516 Protein

[0079] 1. Preparation of double-stranded DNA (ovDNA): Sequence ID NO: 5 and Sequence ID NO: 6 were annealed to form ovDNA with 20 T residues overhanging the 5' end. The annealing process was as follows: incubate at 95°C for 5 minutes, cool to 25°C at a rate of 0.1°C / s, and incubate for 30 minutes. The annealing recipe is shown in Table 3.

[0080] Table 3 ovDNA annealing formula

[0081] Solution volume 100 μM SEQ ID NO: 5 5 μL 100 μM SEQ ID NO: 6 5 μL TE buffer (pH = 8) 40 μL

[0082] 2. Prepare reaction buffer: low salt reaction buffer 1 is 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl; high salt reaction buffer 2 is 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 500 mM KCl.

[0083] 3. Prepare the reaction solution

[0084] Experimental reaction solution: Take 3 μL 10 μM annealed ovDNA, 6 μL 100 μM SEQ ID NO: 7 (20x competitor DNA, this strand can anneal with complementary DNA to prevent re-annealing of the initial substrate and loss of fluorescence), and 6 μL 100 mM ATP to 585 μL low salt reaction buffer or high salt reaction buffer

[0085] Positive control solution: Take 1 μL 10 μM SEQ ID NO: 6, 2 μL 100 μM SEQ ID NO: 7 (20-fold competitor DNA, this strand can anneal with complementary DNA to prevent re-annealing of the initial substrate and loss of fluorescence), and 2 μL 100 mM ATP to 195 μL low salt reaction buffer or high salt reaction buffer

[0086] 4. Dilute the protein: Dilute the BCH516 protein to 4.8 μM with 1× PBS.

[0087] 5. Prepare the melt reaction: Divide into experimental group ①, negative group ②, and positive group ③. Add the corresponding reagents according to Table 4. Use a microplate reader to detect the kinetic changes of fluorescence intensity within 30 minutes at 30°C. Repeat three times for each group.

[0088] 6. Data analysis: Calculate the percentage of the fluorescence value of the experimental group and the negative control group relative to the fluorescence value of the positive control group.

[0089] Table 4 Melting reaction formula

[0090] No. Solution 1 Solution 2 ① 58.5 μL experimental reaction solution 1.5 μL protein ② 58.5 μL experimental reaction solution 1.5 μL reaction buffer ③ 58.5 μL positive control solution 1.5 μL reaction buffer

[0091] 7. Experimental results:

[0092] Within the tolerance range and instrument fluctuations, we calculated the ratio of the measured fluorescence value to the fluorescence value of the positive control and plotted the experimental results (due to instrument sensitivity, the negative control group also had fluorescence absorption readings). The experimental results show 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 in reaction time, indicating that the activity of the sample was in unwinding double-stranded DNA, and the unwinding direction was 5'-3'.

[0093] As shown in Figures 3 and 4, BCH516 exhibits dsDNA unwinding activity under both low-salt (150 mM KCl final concentration in the reaction solution) and high-salt (500 mM KCl final concentration in the reaction solution) conditions. Within the 0.15–0.5 M KCl range, BCH516 exhibited minimal dsDNA unwinding activity.

[0094] Example 4 Detection of the melting activity of BCH516 by limiting sequence blockade

[0095] 1. Preparation of double-stranded DNA (ovDNA) containing a restriction sequence: SEQ ID NO: 5 and SEQ ID NO: 8 were annealed to form ovDNA (containing a restriction sequence) with 20 T residues overhanging the 5' end. The annealing process was incubation at 95°C for 5 minutes, followed by a cooling rate of 0.1°C / s to 25°C, and incubation for 30 minutes. The annealing recipe is shown in Table 5.

[0096] Table 5 ovDNA (containing restriction sequences) annealing formula

[0097] Solution volume 100 μM SEQ ID NO: 5 5 μL 100 μM SEQ ID NO: 8 5 μL TE buffer (pH=8) 40 μL

[0098] 2. Prepare reaction buffer: low salt reaction buffer is 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 150 mM KCl; high salt reaction buffer is 100 mM HEPES (pH = 8.0), 1 mg / mL BSA, 10 mM MgCl2, 500 mM KCl.

[0099] 3. Prepare the reaction solution:

[0100] Experimental reaction solution: Take 3 μL 10 μM annealed ovDNA (containing the restriction sequence), 6 μL 100 μM SEQ ID NO: 7 (20x competitor DNA), and 6 μL 100 mM ATP to 585 μL low salt reaction buffer or high salt reaction buffer

[0101] Positive control solution: add 1 μL 10 μM SEQ ID NO: 11, 2 μL 100 μM SEQ ID NO: 7 (20x competitor DNA), and 2 μL 100 mM ATP to 195 μL low salt reaction buffer or high salt reaction buffer

[0102] 4. Dilute the protein: Dilute the BCH516 protein to 4.8 μM with 1× PBS.

[0103] 5. Prepare the melt reaction: Divide into experimental group ①, negative group ②, and positive group ③. Add the corresponding reagents according to Table 6. Use a microplate reader to detect the kinetic changes of fluorescence intensity within 30 minutes at 30°C. Repeat three times for each group.

[0104] 6. Data analysis: Calculate the percentage of the fluorescence value of the experimental group and the negative control group relative to the fluorescence value of the positive control group.

[0105] Table 6 Melting reaction formula

[0106] No. Solution 1 Solution 2 ① 58.5 μL experimental reaction solution 1.5 μL protein ② 58.5 μL experimental reaction solution 1.5 μL 1× PBS

[0107] ③58.5μL positive control solution 1.5μL 1×PBS

[0108] 7. Experimental results:

[0109] As shown in Figures 5 and 6, under low-salt (final KCl concentration in the reaction solution was 150 mM) and high-salt (final KCl concentration in the reaction solution was 500 mM) conditions, the limiter sequence can block BCH516 from unwinding dsDNA. This indicates that the limiter sequence can block its unwinding activity, preventing it from unwinding when not in contact with the nanopore. However, under the action of current, it can cross the limiter sequence and initiate unwinding sequencing.

[0110] Example 5 Nanopore Sequencing Application of BCH516 Protein

[0111] 1. Two partially complementary DNA strands (top strand, SEQ ID NO: 9 and bottom strand, SEQ ID NO: 10) were annealed to form a linker (as shown in Figure 7). The linker was then ligated to the double-stranded target fragment using T4 DNA ligase at room temperature and purified to prepare a sequencing library.

[0112] 2. BCH516 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. The helicase-containing sequencing library was incubated with single-stranded DNA (ssDNA-chol, SEQ ID NO: 11) containing cholesterol at its 5' end at room temperature for 10 minutes. As shown in Figure 8, the ssDNA-chol sequence is complementary to a portion of the bottom strand of the adaptor. Cholesterol binding to the phospholipid membrane reduces library loading and improves capture efficiency.

[0114] 4. Use a patch clamp amplifier (as shown in Figure 9) 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 bimolecular phospholipid membrane is formed at the micropores of both chambers, and the nanopore protein is added. Electrical measurements are obtained after a single nanopore protein, MspA, 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 BCH516 yielded the sequencing signal shown in Figure 10. As the helicase guides the DNA single strand into the nanopore, the current is partially blocked, decreasing. Because different nucleotides have different sizes, the amount of current blocked also varies, resulting in the visible fluctuations in the current signal.

[0116] The sequences involved in the present invention are as follows:

[0117] Amino acid sequence of BCH516 (SEQ ID NO:1): MENVKLTEDQLKIIKTVEEDIKNNQFINQKVLLTGSAGTGKTTTLIELIKLIESKTKKNIQVLTPTHQSSIVIHKILQGYNLKRTEIGTIHSYFDIKPDIDDNGNRVFKPSKSDPEIMHDIFIIDESSMIDSVLYEIITKYLSPFPVIFVGDEYQLPPVKEDFSPVFKYFEEQENLGNTKYIFKLEKIIRTQNKNSLIFEKFRELIKRYKDENYKAPLKDINNLINEFQTFSESQFPDFFNKYVEERFQNKKNIKIGTFTNNFTDYYNLHFRRFDNEVKTPDNKYSEGDKLLLNGPYNYWNLAMNKEKLSTRDFMSVCNYLKNGEEIKVNEVIESTLEIRNLSSLETAVISFNQEIPEFPNIKEIAKKGISFEVYNIQGELFNSSKPSKKSLIIDKNGKAKDFLRKFLNFAKIYNFTNKGGHGRRITKKSKKELWKLYYTLSDYFADVTYTYSSTIHKLQGQTLDEIFIDTRDFNHLYNTDYNLFLRLLYVGITRTSNEVFILK

[0118]

[0119] SEQ ID NO:3: 5'-GCGTCGAAAAGCAGTACTTAGGCATT-3'

[0120] SEQ ID NO:4': 5'-TTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-3'

[0121] SEQ ID NO:5:5'-BHQ-1-GCGTCGAAAAGCAGTACTTAGGCATT-3'

[0122] SEQ ID NO:6: 5'-TTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-FAM-3'

[0123] SEQ ID NO:7: 5'-AATGCCTAAGTACTGCTTTTTCGACGCT-3'

[0124] SEQ ID NO: 8: 5'-TTTTTTTTTTTTTTTTTTTTTYYYYAATGCCTAAGTACTGCTTTTCGACGC-FAM-3' (Y=iSP18)

[0125] SEQ ID NO:9: 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTYYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3'(Y=iSP18)

[0126] SEQ ID NO:10: 5'-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3'

[0127] SEQ ID NO: 11: 5'-cholesterol-TTGACCGCTCGCCTC-3'

[0128] 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 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology to the amino acid sequence shown in SEQ ID NO:

1.

2. The helicase according to claim 1, wherein The surface of the helicase has at least one mutation, wherein the mutation is a substitution of cysteine ​​by alanine, glutamine, glycine, histidine, isoleucine, leucine, valine, serine, threonine or methionine; Preferably, the mutated amino acid site includes C318; More preferably, the mutations include C318A, C318G, C318I and / or C318L.

3. The helicase according to claim 1 or 2, wherein It has at least one mutation in the pin domain and / or the tower domain, wherein the mutation is a substitution of the original amino acid with cysteine ​​or a non-natural amino acid; Preferably, the mutated sites are selected from one or more of D95 to P115 and S388 to W435; More preferably, the unnatural amino acids include 4-azido-L-phenylalanine, 4-azido-L-phenylalanine, 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, Acid, 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 One or more of aminooctanoate 3-(2,2′-dipyridin-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.

4. The helicase according to claim 3, wherein The tower domain and the pin domain of the helicase are covalently linked to one or more ends of one or more linkers selected from the group consisting of a cross-linker, a polypeptide, and a small molecule; Preferably, the cross-linking agent includes a chemical cross-linking agent containing the following functional groups: maleimide, active ester, succinimide, azide, alkyne, phosphine, haloacetyl, phosgene-type reagent, sulfonyl chloride reagent, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and photosensitive reagent; the alkyne includes dibenzocyclooctyne, difluorocycloalkyne and linear alkyne, the phosphine includes phosphine used in traceless and non-traceless Staudinger bonding, the haloacetyl includes iodoacetamide, and the photosensitive reagent includes aryl azide and diaziridine.

5. The helicase according to any one of claims 1 to 4, wherein One or more of the amino acid sites in the DNA binding region, near the ATP catalytic active center, and the amino acid site that may interact with the nanopore binding region of the helicase undergoes a mutation; the mutation includes replacing the original amino acid with an amino acid with a larger side chain; Preferably, the mutated amino acid sites include one or more of M1, E2, N3, V4, K5, K75, P98, R106, F108, K160, E161, D162, F163, F167, K168, Y169, F170, E171, E172, Q173, K215, K219, D220, F416, W435, Y438 and Y439.

6. The helicase according to any one of claims 1 to 5, wherein The long side chain amino acids on the surface of the helicase are mutated into short side chain amino acids, and the mutation sites include at least one of the following: E2A, N3A, K5A, K75A, R106A, D162A, K168A, Y169A, K215A, K219A, D220A, Y438A, Y439A, K5S, K75S, K168S, K215S, K219S, D202S, Y438S and Y439S.

7. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the helicase according to any one of claims 1 to 6; Preferably, the nucleotide sequence of the helicase is as shown in SEQ ID NO:2, or has at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the nucleotide sequence shown in SEQ ID NO:

2.

8. 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(+).

9. A transformant, characterized in that The transformant comprises the nucleic acid according to claim 7 or the recombinant expression vector according to claim 8; Preferably, the host cell of the transformant is Escherichia coli, more preferably BL21(DE3), BL21 Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).

10. A method for preparing the helicase according to any one of claims 1 to 6, characterized in that: The transformant according to claim 9 is cultured in a culture medium to ferment and produce the helicase.

11. A helicase-linker complex, characterized in that The helicase-linker complex comprises the helicase according to any one of claims 1 to 6 and a sequencing linker, wherein the sequencing linker comprises two DNA chains with complementary partial regions.

12. A kit, characterized in that The kit comprises the helicase according to any one of claims 1 to 6 and / or the helicase-adapter complex according to claim 11; preferably further comprising 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 sequencing 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 membrane is an amphiphilic membrane, a polymer membrane 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, InvG, GspD or any combination thereof; the sequencing buffer is a dihydrogen phosphate-dihydrogen 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.

13. Use of the helicase according to any one of claims 1 to 6 or the helicase-linker complex according to claim 11 in sequencing or preparing sequencing-related reagents.

14. A DNA denaturing method, characterized in that: The method comprises unwinding double-stranded DNA using the helicase according to any one of claims 1 to 6, the helicase-linker complex according to claim 11, or the kit according to claim 12.

15. A sequencing method, characterized in that: It includes the following steps: The double-stranded DNA is sequenced while being unwound using the DNA unwinding method as described in claim 14.

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