WO2024138635A1 - Helicase and preparation method therefor and use thereof in high-throughput sequencing - Google Patents

Abstract

Provided are a helicase and a preparation method therefor and a use thereof in sequencing. The amino acid sequence of the helicase is as shown by 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 to the amino acid sequence as shown by SEQ ID NO:1. The helicase has high thermal stability, ATP hydrolysis activity and DNA unwinding activity, the DNA unwinding activity increases with increasing salt concentration, and the helicase can be used for control and characterization of nucleic acids, and can be applied to nanopore sequencing.

Description

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

The invention belongs to the field of biotechnology or sequencing, and specifically relates to a helicase and an application thereof.

Background technique

As an emerging single-molecule sequencing technology, nanopore sequencing technology has brought disruptive changes to the gene sequencing industry with its unique advantages such as high throughput, long read length, fast speed, in situ detection and label-free operation. The equipment based on this technology is light and portable, and can meet different sequencing scenarios. And because of its non-amplified direct sequencing nature, there is no length limit on the sequenceable DNA, allowing real-time base calling, and can also achieve direct sequencing of RNA, methylation and other modified molecules, as well as other single molecules, which is expected to break through the limitations of second-generation sequencing and serve as a supplement to meet different sequencing needs. Nanopore sequencing technology has a wide range of application values in many fields such as molecular biology, medicine, epidemiology and ecology, such as genome mapping, epidemic and other infectious diseases, detection of rare species, identification of hidden intermediates, dynamic monitoring of biological non-covalent interactions, characterization of epigenetic and post-translational modifications, and fast and inexpensive protein sequencing.

The principle of nanopore sequencing technology is based on changes in electrical signals. Two electrolyte chambers filled with electrolyte are separated by a nanopore inserted into a membrane (protein or solid) as a signal sensor. When a 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, the flow of ions will be hindered, resulting in current signal fluctuations. Different base nucleotides have different effects on the current. 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.

In the sequencing process, the nucleic acid molecules pass through the nanopore channel at an extremely high speed, so it is impossible to accurately obtain the polynucleotide sequence information. Therefore, effectively reducing and controlling the perforation movement of nucleic acid molecules is a key technical issue in achieving nanopore sequencing. At present, the most common and effective method is to use the idea of helicase unwinding to control the perforation movement of nucleic acid molecules, improve detection accuracy, and maintain sequencing speed and sequencing uniformity.

The helicase in the 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.

Summary of the invention

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

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 genes are derived from deep-sea metagenomes, so its proteins have strong thermal stability. The helicase has good ATP hydrolysis activity and dsDNA unwinding activity, and its unwinding activity tends to increase with the increase of salt concentration. It can be used for the control and characterization of nucleic acids, and applied to single-molecule nanopore sequencing to output stable sequencing current signals.

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.

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.

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 by cysteine or a non-natural amino acid. The purpose is to connect the two domains so that the DNA will not leave the helicase region due to swinging during nanopore sequencing, but will be perforated under the action of the electric field force and the helicase, thereby improving the continuity and stability of sequencing.

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 the tower domain at least one of 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.

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.

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.

The tower domain and the pin domain of the helicase are covalently linked to one or more ends of one or more connectors.

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.

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.

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.

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

The fourth aspect of the present invention provides a transformant, which comprises a host cell and the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect.

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

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 to ferment and produce the helicase with high uniformity and protein purity.

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.

The seventh aspect of the present invention provides a kit, which includes the helicase as described in the first aspect and/or the helicase-sequencing adapter complex as described in the sixth aspect; preferably, it also includes 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, a copolymer tri-block) 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.

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 preparation of sequencing-related reagents.

Preferably, the high-throughput sequencing is nanopore sequencing.

The ninth aspect of the present invention provides a method for unwinding a DNA strand, comprising unwinding a 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.

The tenth aspect of the present invention provides a sequencing method, which comprises the following steps:

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

Beneficial effects brought by the technical solution of the present invention:

The present invention provides a new type of helicase, named BCH866, whose gene is derived from deep-sea metagenome, which can be soluble expressed and purified through the Escherichia coli system, has high thermal stability, ATP hydrolysis activity and DNA unwinding activity, and its DNA unwinding activity increases with increasing salt concentration. The helicase can be used for the control and characterization of nucleic acids and applied to nanopore sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the molecular sieve Superdex 200 purification results of BCH866. (A) is the molecular sieve Superdex 200 elution graph of BCH866; (B) is the molecular sieve gel elution graph of BCH866.

Figure 2 shows the Alphafold2 predicted structure of BCH866.

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

FIG. 4 is the detection of ATPase activity of BCH866 protein.

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

FIG. 6 is a dsDNA melting activity assay of the BCH866 protein (high salt reaction buffer).

FIG. 7 is a schematic diagram of a connector (a: upper chain; b: lower chain).

FIG8 is a schematic diagram of a sequencing library containing a helicase (a: upper strand; b: lower strand; c: double-stranded target fragment; d: helicase; e: cholesterol-labeled double-stranded DNA).

FIG. 9 is a schematic diagram of a nanopore sequencing patch clamp amplifier.

FIG. 10 is a diagram of the BCH866 sequencing current signal.

Detailed ways

Example 1 Cloning, expression and purification of BCH866

1. Cloning and expression of BCH866

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 sites Nde1 and Xho1, and the obtained plasmid was labeled PET.28a(+)-BCH866. The BCH866 protein expressed by the plasmid has a thrombin restriction site and a 6×His tag at its N-terminus.

The PET.28a(+)-BCH866 plasmid was transformed into the E. coli expression bacteria BL21 (DE3) or its derivatives. A single colony was picked and inoculated into 20 mL of LB medium containing kanamycin resistance, and cultured at 37°C overnight with shaking. Then, it was transferred into 2 L of LB medium containing kanamycin resistance, cultured at 37°C with shaking until OD600 = 0.6-0.8, cooled to 16°C, and IPTG was added at a final concentration of 500 μM to induce expression overnight to obtain BCH866 bacteria.

2. Purification of BCH866 Protein

The buffer formulation used is as follows:

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

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

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

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

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

Collect BCH866 bacteria, resuspend the bacteria in Buffer A, break the bacteria with a cell disruptor, and then centrifuge to obtain the supernatant. Mix the supernatant with the Ni-NTA filler that has been equilibrated with Buffer A in advance and combine for 1 hour. Collect the filler and wash the filler with Buffer A in large quantities until no impurities 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, and change the protein buffer from Buffer B to Buffer C. Then, add the protein solution that has passed through the desalting column to the ssDNA cellulose filler equilibrated with Buffer C, and add an appropriate amount of coagulation protease, which can specifically recognize the thrombin cleavage site amino acid sequence LVPRGS in the vector sequence PET28(a)+, thereby removing the affinity His tag carried by the protein. The operation is performed at 4°C and incubated overnight on a rotary shaker.

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 the impurities that were not adsorbed to the ssDNA cellulose filler, and then 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. The protein purified from ssDNA cellulose was concentrated in a 4°C precooled centrifuge through a 30K ultrafiltration concentrator tube (Merck millipore). The parameters were set to a speed of 3000g, and the centrifugation time was 10min each time. Repeated several times to concentrate the final protein volume to 2mL. Finally, it was passed through the molecular sieve Superdex 200 (Cytiva), and the molecular sieve buffer used was Buffer E. The target protein peak was collected, concentrated and frozen.

Figure 1 shows the results of the molecular sieve Superdex 200 purification of BCH866 protein. As shown in Figure 1, after purification, a large amount of BGH866 protein with good purity can be obtained. The peak shape of the protein is uniform and the purity is high.

3. AlphaFold2 structure prediction of BCH866 protein

With the help of AlphaFold 2 software, the structure of BCH866 protein was predicted, and the results are shown in Figure 2. The protein includes a helix structure, a sheet structure, and a loop structure. The structure of BCH866 protein is similar to the overall structure of conventional 5'-3' helicases, but its pin domain and tower domain are closer, and the DNA binding region is more compact.

Example 2 Thermal stability test of BCH866 protein

The purified BCH866 protein was diluted to 1 mg/mL with protein storage buffer Buffer E and divided into 7 tubes of equal volume, one of which was placed in a 4°C refrigerator, and the remaining tubes were placed in metal bath instruments at different temperatures for incubation for 1 hour. The temperatures of the metal baths were set to 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively. After the reaction, the proteins incubated at different temperatures were immediately centrifuged at high speed, and the centrifugation conditions were: 4°C, 10000rpm, 10min. After the centrifugation, the same volume of protein supernatant was taken and heated in a 95°C metal bath for denaturation for 10min, and characterized by SDS-PAGE (10%). 10μL of sample was added to each loading well, and the gel map of the protein is shown in Figure 3. The control group was the DDA mutant (SEQ ID NO: 11) protein, and the same operating conditions were used.

Figure 3 shows the status of BCH866 and the control group DDA mutant proteins at different temperatures. As shown in Figure 3, the BCH866 protein was only slightly degraded after being heated at 40°C for 1 hour, while under the same conditions, only 20% of the DDA mutant protein remained stable without precipitation and degradation. About 30% of the BCH866 protein remained stable without precipitation and degradation after being heated at 45°C for 1 hour, while the DDA mutant was almost completely degraded under the same conditions.

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.

Example 3 Detection of ATPase activity of BCH866 protein

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

SEQ ID NO:3 and SEQ ID NO:4 were synthesized (Liuhe BGI), and SEQ ID NO:3 and SEQ ID NO:4 were annealed to ovDNA-1 with 20 Ts hanging from the 5'. The annealing process was incubated at 95℃ for 5 minutes, cooled to 25℃ at a rate of 0.1℃/s, and incubated for 30 minutes. The annealing formula is shown in Table 1. 100μM SEQ ID NO:4 was diluted to 10μM with TE buffer (pH=8) as ssDNA.

Table 1 ovDNA-1 annealing formula

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

2. Prepare high salt reaction buffer

High salt reaction buffer (2×): 20mM HEPES (pH8.0), 4mM ATP, 4mM MgCl2, 1.0M KCl.

3. Dilution of protein

Dilute BCH866 protein to 10 μM in 1× PBS.

4. ATP hydrolysis reaction

According to the reaction system in Table 2, add the corresponding reagents, incubate at 30℃ for 30min to test the ATP hydrolysis reaction, and inactivate at 80℃ for 5min. ①② are experimental groups, ③④⑤⑥ are corresponding control groups, and each group has 3 replicates.

Table 2 ATP hydrolysis reaction system

serial number Reaction buffer (2×) DNA BCH6X protein _H2O ① 10μL 1μL (ovDNA-1) 1μL 8μL ② 10μL 1μL (ssDNA) 1μL 8μL ③ 10μL —— 1μL 9μL ④ 10μL 1μl(ovDNA-1) —— 9μL ⑤ 10μL 1μL (ssDNA) —— 9μL ⑥ 10μL —— —— 10μL

5. Detection of Remaining ATP in the Reaction

The ATP detection kit (Biyuntian, S0026B) was used to determine the residual ATP concentration in the reaction according to the manufacturer's instructions.

6. Experimental Results

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

Example 4 Detection of dsDNA melting activity of BCH866

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

Synthesize SEQ ID NO:5 and SEQ ID NO:6, and anneal SEQ ID NO:5 and SEQ ID NO:6 to ovDNA-2 with 20 Ts hanging at 5'. The annealing process is incubating at 95℃ for 5 minutes, cooling at a rate of 0.1℃/s to 25℃, and incubating for 30 minutes. The annealing formula is shown in Table 3.

Table 3 ovDNA-2 annealing formula

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

2. Prepare reaction buffer

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

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

3. Prepare the reaction solution

Experimental reaction solution: Take 3μL 10μM ovDNA-2, 6μL 100μM SEQ ID NO:7 (20x competitive DNA, this chain can anneal with complementary DNA to prevent re-annealing of the initial substrate and loss of fluorescence), 6μL 100mM ATP to 585μL low salt reaction buffer. Take 3μL 10μM ovDNA-2, 6μL 100μM SEQ ID NO:7 (20x competitive DNA), 6μL 100mM ATP to 585μL high salt reaction buffer.

Positive control solution: add 1μL 10μM SEQ ID NO:6, 2μL 100μM SEQ ID NO:7 (20x competitive DNA), and 2μL 100mM ATP to 195μL low salt reaction buffer. Add 1μL 10μM SEQ ID NO:6, 2μL 100μM SEQ ID NO:7 (20x competitive DNA), and 2μL 100mM ATP to 195μL high salt reaction buffer.

4. Dilution of protein

BCH866 protein was diluted to 4.8 μM with 1× PBS.

5. Prepare the melting reaction

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 an ELISA reader at 30°C, with 3 replicates per group.

Table 4 Melting reaction formula

serial number category Solution 1 Solution 2 ① test group 58.5μL experimental reaction solution (low salt) 1.5 μL protein ② test group 58.5μL experimental reaction solution (high salt) 1.5 μL protein ③ Negative control group 58.5μL experimental reaction solution (low salt) 1.5 μL PBS ④ Negative control group 58.5μL experimental reaction solution (high salt) 1.5 μL PBS ⑤ Positive control group 58.5μL positive control solution (low salt) 1.5 μL PBS ⑥ Positive control group 58.5μL positive reaction solution (high salt) 1.5 μL PBS

6. Data Analysis

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.

7. Experimental Results

Within the error range and the allowable instrument fluctuation, the experimental results were plotted by calculating the ratio of the fluorescence value of the experimental group to the fluorescence value of the positive control group, and the ratio of the fluorescence value of the negative control group to the fluorescence value of the positive control group (due to the sensitivity of the instrument, the negative control group had fluorescence absorption readings), as shown in Figures 5 and 6.

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'.

Moreover, Figure 5 is a result diagram under low salt conditions, and Figure 6 is a result diagram under high salt conditions. Comparison of the two shows that as the salt concentration increases, the activity of BCH866 protein in unwinding dsDNA increases.

Example 5 Nanopore sequencing application of BCH866 protein

1. Two partially complementary DNA chains (upper chain, SEQ ID NO: 8 and lower chain, SEQ ID NO: 9) were annealed to form a linker (as shown in Figure 7), which was then connected to the double-stranded target fragment using rapid T4 DNA ligase (NEB, E6057AVIAL) and purified to obtain a sequencing library.

2. The connection steps are as follows: Take out T4 DNA ligase from the -20℃ refrigerator, flick the tube wall to mix, centrifuge instantly, and place on ice. Thaw the quick ligation reaction buffer, mix by pipetting, centrifuge instantly, and then place on ice. Prepare the reaction mixture (120μL quick ligation reaction buffer, 60μL T4 DNA ligase, 30μL 10μM adapter). Then, add 390μL of the purified end-repaired, "A"-added and purified products of the double-stranded target fragment to the ligation reaction mixture. Use the flared pipette tip to gently pipette and mix 6 times, centrifuge instantly to collect the reaction solution at the bottom of the tube, and then place it in a metal bath preheated at 25℃ to carry out the ligation reaction, and the timer counts for 30 minutes. After the reaction is completed, centrifuge the reaction tube instantly and collect the reaction solution at the bottom of the tube.

3. The purification steps are as follows: 30 minutes in advance, take out Ampure XP magnetic beads (Beckman Coulter, A63882) from the 4-degree refrigerator, shake and mix, and place at room temperature. Shake and mix thoroughly before use. Pipette 240 μL of magnetic beads into the DNA low-adsorption tube (Eppendorf, 0030108051) containing the sample connection product after the reaction, flick the tube wall by hand to mix, or gently blow at least 6 times with a flared gun tip until completely mixed. The last time should ensure that all the liquid and magnetic beads in the pipette tip are injected into the tube. Incubate at room temperature for 5 minutes on a rotating mixer. Centrifuge the DNA low-adsorption tube instantly and place it on a magnetic rack. Let it stand for 2 to 5 minutes until the liquid is clear. Use a pipette to carefully aspirate the supernatant and discard it. Keep the DNA low adsorption tube on the magnetic rack, add 900μL of washing buffer [20mM Tris (pH＝7.5), 2500mM NaCl], remove the DNA low adsorption tube from the magnetic rack, and flick the tube wall to mix the magnetic beads. After mixing, place it back on the magnetic rack and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully aspirate the supernatant and discard it. After removing the centrifuge tube from the magnetic rack, centrifuge it instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube. Remove the DNA low adsorption tube from the magnetic rack, add 68μL of elution buffer [20mM Tris (pH＝7.5), 50mM NaCl] to elute the DNA, and flick the tube wall to mix. Centrifuge instantly for 3 seconds and collect the liquid in the tube to the bottom of the tube. Incubate at room temperature for 10 minutes. After instant centrifugation, place the DNA low adsorption tube on a magnetic rack and let it stand for 2 to 5 minutes until the liquid is clear. Transfer 66 μL of supernatant to a new 1.5 mL DNA low adsorption tube. The remaining sample can be used for concentration determination. It is recommended to use Qubit-dsDNA HS Assay Kit (Thermofisher, Q32854) to determine the concentration.

4. The BCH866 protein and the sequencing library were incubated at 25°C for 1 h (molar concentration ratio 1:8) to form a sequencing library containing helicase.

5. The sequencing library containing the helicase was incubated with single-stranded DNA containing cholesterol at the 5' end (ssDNA-chol, SEQ ID NO: 10) at room temperature for 10 minutes. The ssDNA-chol sequence is complementary to a part of the lower chain of the adapter. After cholesterol binds to the phospholipid membrane, it can reduce the amount of library loading and increase the capture rate (the schematic diagram of the adapter is shown in Figure 8, the star represents cholesterol, and the triangle represents the helicase BCH866).

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 micron-sized hole in the middle (diameter 50-200μm) divides the electrolytic cell into two chambers, the cis chamber and the trans chamber; a pair of Ag/AgCl electrodes are placed in each of the cis chamber and the trans chamber; a layer of bimolecular phospholipid membrane is formed at the micropores of the two chambers and then the nanopore protein is added; electrical measurements are obtained after a single nanopore protein is inserted into the phospholipid membrane; the reaction product of step 3 is added, 180mV 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: 0.47M KCl, 25mM HEPES, 1mM EDTA, 30mM ATP, 25mM MgCl ₂ , pH 8, and the sequencing temperature is 30℃.

7. The sequencing electrical signal is shown in FIG10 . As can be seen from the figure, as the helicase BCH866 controls the DNA single strand to enter the nanopore, part of the current is blocked and the current becomes smaller. Since the sizes of different nucleotides are different, the size of the blocked current is also different, so a fluctuating current signal can be seen. This example proves that the helicase BCH866 can be used for nanopore sequencing.

The sequences used in the present invention are as follows:

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

DNA sequence of BCH866 (SEQ ID NO: 2)

SEQ ID NO:3:

5’-GCGTCGAAAAGCAGTACTTAGGCATT-3’

SEQ ID NO:4:

5’-TTTTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-3’

SEQ ID NO:5:

5’-BHQ-1-GCGTCGAAAAGCAGTACTTAGGCATT-3’

SEQ ID NO:6:

5’-TTTTTTTTTTTTTTTTTTTTTTTAATGCCTAAGTACTGCTTTTCGACGC-FAM-3’

SEQ ID NO:7:

5’-AATGCCTAAGTACTGCTTTTCGACGCT-3’

SEQ ID NO:8:

5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-YYYYGGTTGTTTCTGTTGGTGCTGATATTGCT-3'(Y＝iSP18)

SEQ ID NO:9:

5’-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3’

SEQ ID NO: 10:

5’-cholesterol-TTGACCGCTCGCCTC-3’

Wherein, iSP18 is shown in the following formula I:

Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims (16)

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. 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. 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. 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. The helicase of claim 4, wherein the unnatural amino acid includes but is 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-( 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. 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. The helicase of claim 1, wherein the tower domain and the pin domain of the helicase are covalently linked to one or more ends of one or more connectors. 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. 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(+). 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). A method for preparing the helicase as described in any one of claims 1 to 7, characterized in that the transformant as described in claim 10 is cultured in a culture medium to ferment and produce the helicase. A helicase-sequencing adapter complex, characterized in that it comprises the helicase according to any one of claims 1 to 7, and a sequencing adapter. 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 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 (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. 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. A DNA unwinding method, characterized in that it comprises unwinding double-stranded DNA 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. A sequencing method, characterized in that it comprises the following steps: The DNA is sequenced while being unwound using the DNA unwinding method as described in claim 15.

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