WO2024138380A1 - Helicase, preparation method therefor, and use thereof in high-throughput sequencing - Google Patents

Abstract

The present invention provides a helicase, a preparation method therefor, and the use thereof in high-throughput sequencing. The amino acid sequence of the helicase is as shown in SEQ ID NO: 1, or has 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 in SEQ ID NO: 1. The helicase still has high unwinding activity in a high-salt environment, can be used for control and characterization of a nucleic acid, and is 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. In addition, real-time base calling can also achieve direct sequencing of modified molecules such as RNA, methylation, and other single molecules. Nanopore sequencing technology has wide application value in many fields such as molecular biology, medicine, epidemiology, and ecology, such as genome mapping, epidemic and other infectious diseases, detection of rare species, and rapid and economical 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 voltage is applied to 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. Since different nucleotides have different effects on the current, the sequence information of the nucleic acid molecule to be tested can be identified by detecting the current fluctuation signal of the nanopore in real time and analyzing and decoding the current signal with the help of machine learning.

In the sequencing process, since the nucleic acid molecules pass through the nanopore channel at an extremely high speed, the sequence information of the nucleic acid molecules cannot be accurately obtained. Therefore, effectively reducing and controlling the perforation motion of nucleic acid molecules is a key technical problem in realizing nanopore sequencing. At present, the most common effective method is to use helicase to unwind to control the perforation motion of nucleic acid molecules, improve detection accuracy, and maintain sequencing speed and sequencing uniformity. In addition to the highly conserved regions (mainly II and VI regions) that bind to ATP, play ATP hydrolase activity, and provide power for unwinding, the helicase also has two key domains: Pin (pin structure) and Tower (tower structure). These two structures interact to form an "arch" structure that allows single-stranded DNA to pass through. In addition, Pin also plays a role in unwinding double-stranded DNA into single-stranded DNA and assisting in promoting the directional movement of nucleic acid single strands.

The helicase in the currently commercialized nanopore sequencer is the DDA helicase derived from the bacteriophage T4, which has its own limitations, such as its stability, salt tolerance and unwinding speed. In particular, in terms of salt tolerance, high salt will inhibit the unwinding activity of the DDA helicase, causing its unwinding speed to decrease, and it cannot fully exert its unwinding ability, thereby weakening its sequencing speed in nanopore sequencing applications and reducing sequencing efficiency. Therefore, more new helicases with better performance are needed on the market.

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 novel helicase with high salt tolerance and stability, a preparation method thereof, and an application in high-throughput sequencing. Specifically, the present invention provides:

a) A helicase, whose amino acid sequence is as shown in SEQ ID NO:1 or has 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 can be successfully expressed in the Escherichia coli recombinant protein expression system, the protein itself has high homogeneity and purity, and has good ATP hydrolysis activity and dsDNA unwinding activity. Since its gene is derived from the deep-sea metagenome, the proteins derived from this genome have extremely high thermal stability and salt tolerance. Therefore, the helicase shows better unwinding activity in a high-salt environment than in a low-salt environment, and can bind well to single-stranded DNA and unwind double-stranded DNA. The helicase 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.

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, so as to improve the uniformity of the protein, thereby improving indicators such as sequencing uniformity.

Preferably, the mutated sites include at least one of C21, C50, C56, C91, C156, C279, C367 and C379.

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 mutation site of the pin domain is at least one of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 and 103; and/or the mutation site of the tower domain is at least one of 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367 and 368.

More preferably, the mutation site of the pin domain is at least one of G85, I86, S87, P88, T89, V90, D91, K92, K93, E94, L95, E96, F97, E98, H99, V97, N98, I99, P100, S101, L102 and W103; and/or the mutation site of the tower domain is at least one of L337, Y338, At least one of E339, V340, A341, N342, Y343, Y344, D345, Y346, Q347, Q348, 1347, A348, D349, Y350, Y351, E352, H353, 1354, A355, W356, N357, M358, K359, T360, P361, Q362, A363, K364, A365, K366, A367 and Y368.

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 helicase has at least one mutation in the DNA binding region and/or near the ATP catalytic active center, wherein the mutation includes replacement of the original amino acid with an amino acid with a larger side chain, such as increasing the number of carbon atoms, increasing the length and/or increasing the molecular volume, thereby increasing (i) electrostatic interaction; (ii) hydrogen bonding and/or (iii) cation-pi (cation-π) interaction between at least one amino acid and one or more nucleotides in ssDNA.

Preferably, the mutation site in the DNA binding region is at least one of 63, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, 95 and 96; and/or the mutation site near the ATP catalytic active center is at least one of 154, 155, 156, 158, 159, 160, 161, 174, 175, 177, 178, 179 and 181.

More preferably, the mutation site in the DNA binding region is at least one of H63, E73, T79, V80, H81, S82, A83, L84, G85, I86, S87, L95 and E96; and/or the mutation site near the ATP catalytic active center is at least one of D154, D155, P156, I158, S159, P160, V161, P174, M175, N177, T178, G179 and L181.

The helicase has at least one mutation in the DNA binding region and the nanopore binding region, and the mutation includes replacing the original amino acid with an amino acid with a non-positive surface charge and/or an amino acid with a side chain length shorter than the original amino acid, thereby reducing the repulsion between the motor protein and the pore, etc.

Preferably, the mutation occurs in at least one of positions 1, 2, 3, 5, 7, 8, 9, 10, 36, 42, 45, 67, 74, 97, 207, 208, 209, 220, 221, 222, 374, 408 and 411 of the DNA binding region and the nanopore binding region.

More preferably, the mutation occurs in at least one of M1, N2, S3, N5, D7, Q8, Q9, K10, K36, K42, K45, D67, K74, F97, R207, K208, D209, K220, K221, D222, H374, D408 and K411 in the DNA binding region and the nanopore binding region.

b) An isolated nucleic acid encoding a helicase as described in a).

Preferably, the nucleotide sequence of the helicase is as shown in SEQ ID NO:2 or has 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.

c) A recombinant expression vector comprising a promoter and the nucleic acid as described in b).

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

d) A transformant comprising a host cell and the nucleic acid as described in b).

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

e) A method for preparing the helicase described in a), comprising culturing the transformant described in d) in a culture medium to ferment and produce the helicase.

f) A helicase-sequencing adapter complex, comprising the helicase as described in a) and a sequencing adapter.

g) A kit comprising the helicase as described in a) and/or the helicase-sequencing adapter complex as described in f); preferably also 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 buffer.

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

The nanopore is a transmembrane protein pore or a solid-state pore; preferably, the transmembrane protein pore is selected from hemolysin, MspA, MspB, MspC, MspD, FraC, ClyA, PA63, CsgG, CsgD, XcpQ, SP1, phi29 connector protein (phi29connector), InvG, GspD or any combination thereof.

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

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.

h) Use of the helicase as described in a), the helicase-sequencing adapter complex as described in f), or the kit as described in g) in high-throughput sequencing.

Preferably, the high-throughput sequencing is nanopore sequencing.

i) A method for unwinding a DNA strand, comprising unwinding a double-stranded DNA using the helicase described in a), the helicase-sequencing adapter complex described in f), or the kit described in g).

j) A sequencing method comprising the following steps:

The DNA is unwound using the DNA unwinding method described in i), and the obtained single-stranded DNA is sequenced simultaneously.

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

The present invention provides a new type of helicase, named BCH666, whose gene is derived from the deep-sea metagenome. The protein itself has good salt tolerance and stability, as well as DNA unwinding activity. It can have high unwinding activity in a high-salt environment, and 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 results of molecular sieve Superdex 200 purification of BCH666 protein; Figure 1A shows the elution diagram of molecular sieve Superdex 200 purification of BCH666; Figure 1B shows the SDS-PAGE electrophoresis results after molecular sieve elution of BCH666.

Figure 2 is the structural diagram of the BCH666 protein predicted using Alphafold 2 software.

FIG3 shows the ATPase activity detection result of BCH666 protein.

FIG. 4 shows the dsDNA melting activity detection results of BCH666 protein (low salt reaction buffer 1).

FIG5 shows the dsDNA melting activity detection result of BCH666 protein (high salt reaction buffer 2).

FIG6 shows the detection result of the restriction sequence blocking the melting activity of BCH666 protein (low salt reaction buffer).

FIG. 7 shows the detection result of the restriction sequence blocking the BCH666 protein depolymerization activity (high salt reaction buffer).

FIG8 is a schematic diagram of the connector structure (a: upper chain; b: lower chain).

FIG9 is a schematic diagram of the structure of a sequencing library containing a helicase (a: upper chain; b: lower chain; c: double-stranded target fragment; d: helicase; e: cholesterol-labeled double-stranded DNA).

FIG. 10 is a schematic diagram of an electrical signal amplifier.

FIG. 11 is a diagram showing the current signal obtained when the BCH666 protein is used for sequencing.

Detailed ways

Example 1 Cloning, expression and purification of BCH666 protein

1. Cloning and expression of BCH666 protein

The amino acid sequence of BCH666 protein 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(+)-BCH666. The BCH666 protein expressed by the plasmid has a thrombin restriction site and a 6×His tag at its N-terminus.

The PET.28a(+)-BCH666 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 BCH666 bacteria.

2. Purification of BCH666 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: 20 mM Tris-HCl pH 7.5, 200 mM NaCl.

Collect BCH666 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 bind 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. The eluted protein is passed through a HiTrap desalting column (Sephadex G-25, product number 29048684, Cytiva) equilibrated with 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 thrombin, digest and bind overnight at 4℃. The enzyme 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. Collect the ssDNA cellulose filler, at which time the target protein is specifically adsorbed to the ssDNA filler. Wash the ssDNA cellulose filler 3-4 times with Buffer C to remove the impurities that are not adsorbed to the ssDNA cellulose filler, then elute with buffer D to destroy the specific adsorption of the target protein and the ssDNA filler, and elute the target protein into the solution. The protein purified from ssDNA cellulose was concentrated in a 4°C precooled centrifuge through a 30K ultrafiltration concentration tube (Merck millipore), the parameters were set to a speed of 3000g, each centrifugation time was 10min, repeated several times, and the final protein volume was concentrated to 2mL. Then, the molecular sieve Superdex 200 was applied, 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 BCH666 protein. As shown in Figure 1, after purification, a large amount of BCH666 protein with good purity can be obtained. The peak shape of the protein is uniform and the purity is high.

3. Use AlphaFold 2 to predict the structure of BCH666 protein

The structure of BCH666 protein was predicted with the help of AlphaFold 2 software, and the results are shown in Figure 2. The root mean square error (RMSD) between the predicted value and the true value of the protein skeleton structure is

The protein includes a helix, a sheet and a loop. The structure of the BCH666 protein is similar to that of a conventional 5'-3' helicase, with a long and flexible pin domain.

Example 2 Detection of ATPase activity of BCH666 protein

Synthetic SEQ ID NO:3-8 (Liuhe BGI).

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

Use TE buffer (pH = 8) to dissolve SEQ ID NO: 3 and SEQ ID NO: 4 into a mother solution with a final concentration of 100 μM. Anneal SEQ ID NO: 3 and SEQ ID NO: 4 to ovDNA-1 with 20 Ts hanging from the 5' end, with a concentration of 10 μM. The annealing process is incubation at 95°C for 5 minutes, cooling down to 25°C at a rate of 0.1°C/s, and continuing to incubate for 30 minutes. The annealing formula is shown in Table 1. Dilute 100 μM SEQ ID NO: 4 to 10 μM 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×): 20 mM HEPES (pH 8.0), 4 mM ATP, 4 mM MgCl ₂ , 1.0 M KCl.

3. Dilution of protein

Dilute BCH666 protein to 10 μM with 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	BCH666 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 FIG3 . Under high salt conditions, the BCH666 protein has the activity of hydrolyzing ATP.

Example 3 Detection of dsDNA melting activity of BCH666 protein

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

TE buffer (pH = 8) was used to dissolve SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 into a mother solution with a final concentration of 100 μM. SEQ ID NO: 5 and SEQ ID NO: 6 were annealed to ovDNA-2 with 20 Ts hanging from the 5' end, with a concentration of 10 μM. The annealing process was incubated at 95°C for 5 minutes, cooled to 25°C at a rate of 0.1°C/s, and incubated 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: 100 mM HEPES (pH = 8.0), 1 mg/mL BSA, 10 mM MgCl ₂ , 150 mM KCl;

High salt reaction buffer: 100 mM HEPES (pH=8.0), 1 mg/mL BSA, 10 mM MgCl ₂ , 500 mM KCl.

3. Prepare the reaction solution

Experimental reaction solution: Add 3μL 10μM ovDNA-2, 6μL 100μM SEQ ID NO:7 (20x competitive DNA) and 6μL 100mM ATP to 582μL low salt reaction buffer. Add 3μL 10μM ovDNA-2, 6μL 100μM SEQ ID NO:7 (20x competitive DNA) and 6μL 100mM ATP to 582μL high salt reaction buffer. SEQ ID NO:7 acts as excess capture DNA, preferentially annealing with complementary DNA to prevent re-annealing of the initial substrate and loss of fluorescence.

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

Dilute BCH666 protein 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), and the results are shown in Figures 4 and 5.

From the experimental results in Figures 4 and 5, 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 BCH666 protein has the activity of unwinding double-stranded DNA, and its unwinding direction is 5'-3'.

Moreover, Figure 4 is a result diagram under low salt conditions, and Figure 5 is a result diagram under high salt conditions. Comparison of the two shows that as the salt concentration increases, the activity of BCH666 protein in unwinding dsDNA increases.

Example 4 Detection of the melting activity of BCH666 by limiting sequence blocking

1. Preparation of double-stranded DNA containing restriction sequences (ovDNA-3)

SEQ ID NO: 5 and SEQ ID NO: 8 were annealed to ovDNA-3 (containing a restriction sequence) with 20 Ts hanging from the 5' end, with a concentration of 10 μM. The annealing process was incubated at 95°C for 5 minutes, cooled to 25°C at a rate of 0.1°C/s, and incubated for 30 minutes. The annealing formula is shown in Table 5.

Table 5 ovDNA-3 (containing restriction sequences) annealing formula

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

2. Prepare reaction buffer

The low-salt reaction buffer was 100 mM HEPES (pH = 8.0), 1 mg/mL BSA, 10 mM MgCl ₂ , 150 mM KCl;

The high salt reaction buffer was 100 mM HEPES (pH=8.0), 1 mg/mL BSA, 10 mM MgCl ₂ , 500 mM KCl.

3. Prepare the reaction solution

Experimental reaction solution: Take 3μL 10μM ovDNA-3 (containing the restriction sequence), 6μL 100μM SEQ ID NO:7 (20-fold competitive DNA) and 6μL 100mM ATP and add them to 582μL low salt reaction buffer. Take 3μL 10μM ovDNA-3, 6μL 100μM SEQ ID NO:78 (20-fold competitive DNA) and 6μL 100mM ATP and add them to 585μL high salt reaction buffer.

Positive control solution: add 1μL 10μM SEQ ID NO:8, 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:8, 2μL 100μM SEQ ID NO:7 (20x competitive DNA) and 2μL 100mM ATP to 195μL high salt reaction buffer.

4. Dilution of protein

Dilute BCH666 protein to 4.8 μM with 1× PBS.

5. Prepare the melting reaction

According to Table 6, add the corresponding reagents, ①② for experimental groups, ③④ for negative control groups, ⑤⑥ for positive control groups, use an enzyme reader to detect the kinetic changes of fluorescence intensity within 30 minutes of the reaction at 30°C, and repeat 3 times for each group.

Table 6 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 1× PBS
④	Negative control group	58.5μL experimental reaction solution (high salt)	1.5 μL 1× PBS
⑤	Positive control group	58.5μL positive control solution (low salt)	1.5 μL 1× PBS

⑥

Positive control group

58.5μL positive control solution (high salt)

1.5 μL 1× 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

The experimental results were plotted in the same manner as in Example 3, as shown in Figures 6 and 7. Under low salt conditions, the restriction sequence almost completely blocked the BCH666 protein from unwinding dsDNA. As shown in Figure 7, under high salt conditions, the restriction sequence could not block the BCH666 protein from unwinding dsDNA.

Example 5 Application of Nanopore Sequencing of BCH666 Protein

1. Two partially complementary DNA chains (upper chain, SEQ ID NO: 9 and lower chain, SEQ ID NO: 10) were annealed to form a linker (as shown in Figure 8), and then connected to the double-stranded target fragment to be tested using a rapid T4 DNA ligase kit (NEB, E6057AVIAL) and purified to obtain a sequencing library.

The connection steps are as follows: Take out the fast T4 DNA ligase from the -20℃ refrigerator, flick the tube wall to mix, centrifuge instantly, and place on ice. Thaw the fast connection reaction buffer, mix by pipetting, centrifuge instantly, and then place on ice. Prepare the reaction mixture (120μL fast connection 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 connection 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 connection 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.

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 (Eppendorf, 0030108051) 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 (Eppendorf, 0030108051) on the magnetic rack, add 900μL of washing buffer [20mM Tris (pH＝7.5), 2500mM NaCl,], remove the DNA low adsorption tube (Eppendorf, 0030108051) from the magnetic rack, and gently tap the tube wall to mix the magnetic beads. After mixing, put it back on the magnetic rack and let it stand for 2-5 minutes until all the magnetic beads are against the wall. Carefully absorb 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 (Eppendorf, 0030108051) from the magnetic rack, add 68μL elution buffer [20mM Tris (pH＝7.5), 50mM NaCl] to elute the DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube. Incubate at room temperature for 10 minutes. Centrifuge the DNA low adsorption tube (Eppendorf, 0030108051) and place it on the magnetic rack. Let it stand for 2-5 minutes until the liquid is clear. Transfer 66μL of supernatant to a new 1.5mL DNA low adsorption tube (Eppendorf, 0030108051). 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.

2. The BCH666 protein and the sequencing library were incubated at 25°C for 1 h (molar concentration ratio 1:8) to form a sequencing library containing the helicase BCH666.

3. The sequencing library containing helicase BCH666 was incubated with single-stranded DNA containing cholesterol at the 5' end (ssDNA-chol, SEQ ID NO: 11) 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 sequencing library loaded and increase the library capture rate (the schematic diagram of the adapter is shown in Figure 9, the star represents cholesterol, and the triangle represents the helicase BCH666).

4. Use a patch clamp amplifier or other electrical signal amplifier to collect current signals (as shown in Figure 10). According to the method disclosed in the literature (Ji Z, Guo P. Channel from bacterial virus T7DNA packaging motor for the differentiation of peptides composed of a mixture of acidic and basic amino acids. Biomaterials. 2019 May 21; 214: 119-222), a single-channel nanopore detection system based on patch clamp and signal amplifier was constructed. The Teflon membrane with micron-sized pores (diameter 50-200 μm) in the middle 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 bimolecular phospholipid membrane is formed at the micropores of the two chambers, and the nanopore protein CsgG-Eco-(Y51A/F56Q/R97W/R192D-StrepII(C)) is added; after a single nanopore protein is inserted into the phospholipid membrane, electrical measurements are obtained; the sequencing library obtained in step 3 is added, 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 BCH666. The buffer used in this experiment is: 0.47M KCl, 25mM HEPES, 1mM EDTA, 5mM ATP, 25mM MgCl ₂ , pH 7.6, and the sequencing temperature is 28°C.

5. The sequencing electrical signal is shown in FIG11 . As can be seen from the figure, as the helicase BCH666 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 BCH666 can be used for nanopore sequencing.

The sequences used in the present invention are as follows:

Amino acid sequence of BCH666 protein (SEQ ID NO: 1)

DNA sequence corresponding to BCH666 protein (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'-TTTTTTTTTTTTTTTTTTTTTTTYYYY-AATGCCTAAGTACTGCTTTTCGACGC-FAM-3'(Y＝iSp18)

SEQ ID NO:9:

5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTYYYY-GGTTGTTTCTGTTGGTGCTGATATTGCT-3'(Y＝iSp18)

SEQ ID NO: 10:

5’-GCAATATCAGCACCAACAGAAACAACCTTTGAGGCGAGCGGTCAA-3’

SEQ ID NO: 11:

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 (15)

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 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 three-dimensional structure surface 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 sites include at least one of C21, C50, C56, C91, C156, C279, C367 and C379.
3. 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 mutation site of the pin domain is at least one of 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102 and 103; and/or the mutation site of the tower domain is at least one of 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367 and 368;

   More preferably, the mutation site of the pin domain is at least one of G85, I86, S87, P88, T89, V90, D91, K92, K93, E94, L95, E96, F97, E98, H99, V97, N98, I99, P100, S101, L102 and W103; and/or the mutation site of the tower domain is at least one of L337, Y338, E339, V340, A34 1. At least one of N342, Y343, Y344, D345, Y346, Q347, Q348, I347, A348, D349, Y350, Y351, E352, H353, I354, A355, W356, N357, M358, K359, T360, P361, Q362, A363, K364, A365, K366, A367 and Y368.
4. The helicase of claim 3, 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-(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-quinolyl)propionic acid, 4 -benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl]propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-({[(2-nitrobenzyl)oxy]carbonyl}amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine and 2-nitrophenylalanine.
5. The helicase according to claim 1, characterized in that the helicase has at least one mutation in the DNA binding region and/or near the ATP catalytic active center, wherein the mutation includes replacement of the original amino acid with an amino acid with a larger side chain;

   Preferably, the mutation site of the DNA binding region is at least one of 63, 73, 79, 80, 81, 82, 83, 84, 85, 86, 87, 95 and 96; and/or the mutation site near the ATP catalytic active center is at least one of 154, 155, 156, 158, 159, 160, 161, 174, 175, 177, 178, 179 and 181;

   More preferably, the mutation site in the DNA binding region is at least one of H63, E73, T79, V80, H81, S82, A83, L84, G85, I86, S87, L95 and E96; and/or the mutation site near the ATP catalytic active center is at least one of D154, D155, P156, I158, S159, P160, V161, P174, M175, N177, T178, G179 and L181.
6. The helicase according to claim 1, characterized in that the helicase has at least one mutation in the DNA binding region and the nanopore binding region, wherein the mutation comprises substitution of the original amino acid with an amino acid having a non-positive surface charge and/or an amino acid having a side chain length shorter than the original amino acid;

   Preferably, the mutation occurs in at least one of positions 1, 2, 3, 5, 7, 8, 9, 10, 36, 42, 45, 67, 74, 97, 207, 208, 209, 220, 221, 222, 374, 408 and 411 of the DNA binding region and the nanopore binding region;

   More preferably, the mutation occurs in at least one of M1, N2, S3, N5, D7, Q8, Q9, K10, K36, K42, K45, D67, K74, F97, R207, K208, D209, K220, K221, D222, H374, D408 and K411 in the DNA binding region and the nanopore binding region.
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 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.
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 a host cell and the nucleic acid according to claim 7 or the recombinant expression vector according to claim 8;

   Preferably, the host cell is Escherichia coli, more preferably BL21(DE3), BL21 Star(DE3)pLyss, Rossata(DE3) or Lemo21(DE3).
10. A method for preparing the helicase as described in any one of claims 1 to 6, characterized in that the transformant as described in claim 9 is cultured in a culture medium to ferment and produce the helicase.
11. A helicase-sequencing adapter complex, characterized in that it comprises the helicase according to any one of claims 1 to 6, and a sequencing adapter.
12. A kit, characterized in that the kit comprises the helicase according to any one of claims 1 to 6 and/or the helicase-sequencing adapter complex according to claim 11; preferably also 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 buffer;

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

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

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

    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.
13. Use of the helicase according to any one of claims 1 to 6, the helicase-sequencing adapter complex according to claim 11, or the kit according to claim 12 in high-throughput sequencing;

    Preferably, the high-throughput sequencing is nanopore sequencing.
14. A method for DNA unwinding, characterized in that it comprises unwinding double-stranded DNA using the helicase according to any one of claims 1 to 6, the helicase-sequencing adapter complex according to claim 11, or the kit according to claim 12.
15. 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 14.

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