A DNA assembly molecular machine and its application

Abstract

The present invention discloses a DNA assembly molecular machine and its applications, relating to the field of molecular biotechnology. The DNA assembly molecular machine comprises a palm domain, two terminal insertion domains, a thumb domain, a finger domain, and an exonuclease activity domain. The DNA assembly molecule exhibits excellent stability and is suitable for isothermal amplification. Furthermore, the DNA assembly molecule and its mutants possess DNA assembly capabilities in the presence of various metal ions and at different temperatures, and can be applied to nanopore sequencing, single-molecule sequencing, RCA library construction, environmental testing, and in vitro diagnostics.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, in particular to a DNA assembly molecular machine and applications thereof. Background Art

[0002] The development of DNA sequencing technology has been crucial for modern biological exploration, disease diagnosis, and human ancestry tracing. Advances in sequencing technology have led to a shift from indirect post-amplification sequencing to in situ single-molecule sequencing. Whether using Pacbio's tSMS single-molecule fluorescence sequencing technology or ONT's electrophysiological sequencing technology, sequencing length, accuracy, and efficiency are critically dependent on the molecular motors that control DNA movement and assembly. Both Pacbio and MGI's DNB-SEQ technology rely on rolling circle amplification using the phi29 polymerase, which requires high thermal stability and activity. However, in nanopore sequencing, the high activity of existing polymerases results in excessively fast DNA translocation through the pore, making it difficult to increase the DNA translocation permeation time from 10µs to 100µs. Furthermore, the DNA assembly molecular machinery requires high salt tolerance, requiring it to withstand salt concentrations exceeding 300mM.

[0003] Existing patents for phi29 polymerases have modified their heat and salt tolerance. For example, patent WO2021248757A1 improves the thermostability of commercial phi29 polymerase through 73 point mutations. The combination of mutations M97T, Y224K, and E515S increases the thermostable Tm value from 48.5°C to 50.8°C. This demonstrates that modifying commercial phi29 polymerases is not only difficult but also difficult to significantly alter the enzyme's properties. Patent CN201910778360 discloses a novel salt-tolerant polymerase. Compared to phi29 polymerase and Taq DNA polymerase, the polymerase in this patent can tolerate sodium chloride with an inflection point of 300 mM and loses activity above 500 mM. Furthermore, the polymerase is susceptible to inactivation in the presence of calcium, zinc, and lithium ions. Therefore, further designing novel DNA assembly molecular machines to improve salt tolerance, thermostability, and tolerance to heavy metal ions is a technical bottleneck that needs to be addressed in this field.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a DNA assembly molecular machine and its application to solve the above technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a DNA assembly molecular machine composed of 500-1000 amino acids, which includes a palm domain, two terminal insertion domains, a thumb domain, a finger domain and an exonuclease activity domain; wherein the palm domain is composed of 150-220 amino acids, the thumb domain is composed of 40-70 amino acids, the finger domain is composed of 30-45 amino acids forming an α-helical structure, and the exonuclease activity domain is composed of 150-220 amino acids; the two terminal insertion domains include terminal insertion domain 1 and terminal insertion domain 2, terminal insertion domain 1 is composed of 50-150 amino acids, and terminal insertion domain 2 is composed of 20-70 amino acids;

[0008] DNA assembly molecular machines have the following functions:

[0009] The ability to interact with nucleotides through amino acid side chains; and the DNA assembly molecular machine has the ability to load and / or connect DNA molecules.

[0010] The DNA assembly molecule can interact with the bases of free nucleotides through the amino acid side chains, or interact with the phosphate backbone. In an optional embodiment, the DNA assembly molecule has the function of loading and / or connecting the primer strand and the template strand.

[0011] The DNA assembly molecular machine, consisting of a palm domain, two terminal insertion domains, a thumb domain, a finger domain, and an exonuclease activity domain, has good stability and can be used for isothermal amplification. The DNA assembly molecules and their mutants provided by the present invention have DNA assembly functions under various metal ions and different temperatures, and can be applied to nanopore sequencing, single-molecule sequencing, RCA library construction, environmental testing, and in vitro diagnosis. In particular, under high salt concentrations, it has amplification activity far superior to that of existing DNA polymerases. By increasing the salt concentration, the time of DNA translocation perforation can be extended, thereby improving the accuracy of single-molecule sequencing and greatly reducing the loss of key sequencing information.

[0012] In an optional embodiment, the palm domain consists of 150-200 amino acids, or 160-220 amino acids, or 180-220 amino acids.

[0013] In an optional embodiment, the thumb domain is composed of 40, 45, 50, 55, 60, 65 or 70 amino acids, forming a domain with a length of 30-45 angstroms between the exonuclease active domain and the palm domain to prevent the dissociation of the enzyme and DNA during DNA assembly.

[0014] In an optional embodiment, the finger domain is composed of 30, 32, 36, 38, 40 or 45 amino acids to form an α-helical structure, and the main function of this structure is to bind DNA and assist the exonuclease activity.

[0015] In an optional embodiment, the exonuclease activity domain consists of 150, 160, 170, 180, 190, 200, 210 or 220 amino acids.

[0016] The terminal insertion domain includes terminal insertion domain 1 and terminal insertion domain 2. Terminal insertion domain 1 consists of 50-150 amino acids, and terminal insertion domain 2 consists of 20-70 amino acids, which realizes the effective separation of template chain and non-template chain and contributes to effective chain displacement and continuity.

[0017] In a preferred embodiment of the present invention, the amino acid type of each domain of the DNA assembly molecular machine is selected from at least one of the following: natural amino acids, unnatural amino acids, modified natural amino acids, and modified unnatural amino acids.

[0018] The natural amino acid is selected from glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, histidine, tryptophan, cysteine, asparagine, aspartic acid, glutamic acid, glutamine, lysine, tyrosine, methionine, arginine, cystine, or C1-C4 alkyl esters of the above amino acids.

[0019] The unnatural amino acid is selected from p-acetyl-phenylalanine, p-ethynyl-phenylalanine, p-propargyloxyphenylalanine, p-azido-phenylalanine, β-alanine (βAla), 6-aminohexanoic acid (Aca), and tetraethylene glycol (Teg, NH2-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-COOH) with an amino group at one end and a carboxyl group at the other end, NAEK, Lys-azido, or other groups containing one, two or three of a diaziridine or azide structure.

[0020] The modification is selected from at least one of glycosylation, phosphorylation, ubiquitination, S-nitrosylation, methylation, N-acetylation, succinylation, hydroxylation, lipidation and polyisoprenylation.

[0021] In an alternative embodiment, the methylation is selected from N-methylation or O-methylation;

[0022] In an alternative embodiment, the glycosylation is selected from ADP-ribosylation.

[0023] In a preferred embodiment of the present invention, there are two amino acid sequences on the palm domain that form a β-pleated sheet.

[0024] The two amino acid sequences forming the β-pleated sheet are: EWKFKMV and ATAT;

[0025] Deletion (βDel) of the amino acid sequence (EWKFKMV) of the β-pleated sheet results in decreased assembly activity.

[0026] In an optional embodiment, the thumb domain has an alpha-helix-forming amino acid sequence: DPNDFTEEEIKRKNI.

[0027] In some embodiments, the DNA assembly molecules provided herein, compared to existing DNA polymerases, include a sequence in the thumb domain that can form an α-helical structure. This α-helix is ​​crucial for DNA assembly. Deletion of the α-helical sequence in the thumb domain results in a loss of DNA amplification activity in the DNA assembly molecules. This structure is a key sequence in DNA assembly molecules.

[0028] The inventors discovered that the formation of an α-helical structure in the thumb domain plays a key role in enhancing the DNA amplification activity of DNA assembly molecules. The specific mechanism is that this structure enhances the binding ability of DNA and enzymes. The amino acid sequence set forth in any one of SEQ ID NOs: 1-7 forms an α-helical structure in the thumb domain, while the amino acid sequence set forth in any one of SEQ ID NOs: 8-9 does not form an α-helical structure in the thumb domain.

[0029] In a preferred embodiment of the present invention, the DNA assembly molecular machine comprises:

[0030] (a): A mutant obtained by mutating the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid at the site where the mutant interacts with DNA is substituted or modified;

[0031] and / or, one or more amino acids are inserted into the amino acid sequence shown in SEQ ID NO: 1;

[0032] and / or, one or more amino acids are deleted from the amino acid sequence shown in SEQ ID NO: 1;

[0033] and / or, the amino acid sequence shown in SEQ ID NO: 1 is side-chain modified;

[0034] Or (b): a mutant obtained by mutating the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, wherein the mutation position of the mutant is determined as follows: aligning the mutant with the sequence of SEQ ID NO: 1, the mutation position of the mutant corresponds to the functional site of the sequence of SEQ ID NO: 1; at least one of the amino acids at the mutation position of the mutant is substituted, deleted, inserted or modified.

[0035] The sequence alignment method can be performed using the Blast website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for Protein Blast alignment; the same results can be obtained using other sequence alignment methods or tools well known in the art.

[0036] It should be noted that the mutation position of the mutant obtained by mutating the sequence shown in any one of SEQ ID NOs: 2-9 may be the same as or different from that of SEQ ID NO: 1 (for example, SEQ ID NOs: 2, 3, and 5). The specific position of the mutation is determined based on the aforementioned sequence alignment. As long as it corresponds to the site in SEQ ID NO: 1 after alignment with the reference sequence (SEQ ID NO: 1), it is the mutation site described in the present invention.

[0037] The amino acid sequence shown in SEQ ID NO: 1 is derived from Bacillus phage, has a length of 608 aa, and has a sequence similarity of 58% with phi29. The UniProt database number is A0A1X9SGT8.

[0038] The amino acid sequence information of SEQ ID NOs: 2-9 is shown in Table 1.

[0039] Table 1 Gene list

[0040]

[0041] In a preferred embodiment of the present invention, (a): the substituted or modified amino acid in the amino acid sequence of SEQ ID NO: 1 is at least one of D12, E14, T15, Y60, H62, N63, F66, D67, F70, V94, S123, L124, D146, Y149, D170, D257, V258, S260, Y262, T365, I372, W374, K378, K379, R338, K392, L393, N396, S397, Y399, G400, F402, K428, E429, T443, D465, D467, K507, Y509 and K547;

[0042] (b) The functional site is at least one of D12, E14, T15, Y60, H62, N63, F66, D67, F70, V94, S123, L124, D146, Y149, D170, D257, V258, S260, Y262, T365, I372, W374, K378, K379, R338, K392, L393, N396, S397, Y399, G400, F402, K428, E429, T443, D465, D467, K507, Y509 and K547 of the sequence shown in SEQ ID NO: 1.

[0043] The functions of the functional sites of the sequence shown in SEQ ID NO: 1 are shown in Table 2 below:

[0044] Table 2 SEQ ID NO: 1 Key functional sites (A0A1X9SGT8)

[0045]

[0046] The inventors discovered that mutations in the D12 protein reduced its assembly activity, while simultaneous mutations in D12A and D67A resulted in a loss of assembly activity. This contrasts with the existing phi29 protein, which exhibits enhanced DNA amplification activity following mutations in the D12 and D67 sites.

[0047] In an alternative embodiment, at least one of the following positions shown in SEQ ID NO: 2 is mutated: D27, E29, T30, Y77, H79, F83, D84, F87, L113, S138, L139, D161, Y164, D185, D265, V266, S268, Y270, S372, I379, K381, V385, K386, R394, K398, L399, N402, A403, Y405, G406, F408, E435, E436, T450, R455, D472, D474, K512, Y514 and K550;

[0048] In an alternative embodiment, at least one of the following positions shown in SEQ ID NO: 3 is mutated: D9, E11, T12, Y56, H58, F62, D63, F66, T93, S122, L123, E145, Y148, D169, D249, V250, S252, Y254, V357, I364, R366, I370, K371, V381, K385, L386, N389, S390, Y392, G393, F395, K421, E423, T436, R440, D458, D460, K500, Y502 and K532;

[0049] In an alternative embodiment, at least one of the following positions shown in SEQ ID NO:5 is mutated: D272, E274, T275, Y322, H324, F328, D329, F332, S371, S395, L396, D418, Y421, D442, D532, V533, S535, Y537, V667, K676, I680, K681, R689, K693, L694, N697, N698, Y700, G701, M703, R728, A729, T744, R748, D771, D773, K812, Y814 and K834.

[0050] The corresponding sites of four typical polymerase key functional sites are shown in Table 3:

[0051]

[0052]

[0053] In a preferred embodiment of the present invention, the DNA assembly molecular machine comprises:

[0054] A polypeptide having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% homology to the amino acid sequence shown in any one of SEQ ID NOs: 1-9.

[0055] The DNA assembly molecular machine of the present invention is regulated by an agonist, and the agonists that can be used include: 0.01-100mM Co 2+ , Ni 2+ , Cu 2+ , Mn 2+ , Zn 2+ , Ca 2+ Especially under conditions of calcium ion concentration, the DNA assembly molecule provided by the present invention exhibits higher DNA amplification activity than other proteins (phi29).

[0056] In a second aspect, the present invention further provides a nucleic acid molecule encoding the above-mentioned DNA assembly molecular machine.

[0057] In a third aspect, the present invention also provides a vector or recombinant cell containing the above-mentioned nucleic acid molecule.

[0058] The term "vector" is used herein in its most general sense and includes any intermediate for nucleic acid that enables the nucleic acid to be, for example, introduced into a prokaryotic and / or eukaryotic cell and, where appropriate, integrated into the genome. Vectors of this type are preferably replicated and / or expressed in the cell. Vectors include plasmids, phagemids, phages, or viral genomes. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, typically a circular DNA double strand that can replicate independently of chromosomal DNA.

[0059] The term "recombinant cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid. The term "recombinant cell" according to the present invention comprises prokaryotes (e.g., Escherichia coli) or eukaryotic cells (e.g., mammalian cells, particularly human cells, yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats or primates. Cells can be derived from multiple tissue types and include primary cells and cell lines. Nucleic acid can be present in a host cell in a single copy form or in two or more copy forms, and in one embodiment, is expressed in a recombinant cell.

[0060] In an alternative embodiment, the recombinant cell is a eukaryotic cell;

[0061] In an alternative embodiment, the recombinant cell is a mammalian cell;

[0062] In an alternative embodiment, the recombinant cell is HEK293.

[0063] In a fourth aspect, the present invention further provides a method for DNA replication or amplification, which comprises using the above-mentioned DNA assembly molecular machine as a DNA polymerase to replicate or amplify DNA.

[0064] In a fifth aspect, the present invention further provides use of the DNA assembly molecular machine in any of the following applications:

[0065] 1) Acts as a DNA polymerase;

[0066] 2) Acts as RNA polymerase;

[0067] 3) Control the movement of DNA or RNA;

[0068] 4) preparing single-molecule sequencing reagents or kits;

[0069] 5) preparing nanopore sequencing reagents or kits;

[0070] 6) Assembling molecular machines chain reactions;

[0071] 7) catalyze DNA replication and / or catalyze DNA amplification;

[0072] 8) catalyzing rolling circle amplification and / or catalyzing multiple strand displacement amplification;

[0073] 9) Perform DNA sequencing, RNA sequencing, or whole genome sequencing;

[0074] 10) RCA library construction;

[0075] 11) Genome amplification coverage detection;

[0076] 12) Preparing a kit product for catalyzing DNA replication and / or catalyzing DNA amplification;

[0077] 13) preparing products for catalyzing rolling circle amplification and / or catalyzing multiple strand displacement amplification;

[0078] 14) preparing products for performing DNA sequencing, RNA sequencing, or whole genome sequencing;

[0079] 15) Preparation of products for RCA library construction;

[0080] 16) Prepare products for genome amplification coverage detection.

[0081] In a sixth aspect, the present invention further provides a method for DNA molecular assembly using the above-mentioned DNA assembly molecular machine, which comprises carrying out a reaction in a reaction system; the reaction system comprises 0.3M-1.5M salt ions.

[0082] The DNA assembly molecular machine provided by the present invention has higher activity than phi29 when the salt ion concentration is higher than 300 nM, indicating that the DNA assembly molecular machine provided by the present invention can be applied to high salt concentration environments, such as nanopore sequencing.

[0083] In an alternative embodiment, the salt ion is sodium, potassium, calcium, magnesium, manganese, nickel, cobalt, copper or zinc.

[0084] In an optional embodiment, the reaction system further includes calcium ions.

[0085] The present invention has the following beneficial effects:

[0086] Compared to existing DNA polymerases, the DNA assembly molecule provided by the present invention has a sequence in the thumb domain that can form an α-helical structure, which is particularly critical for improving the DNA amplification activity of the DNA assembly molecule. The DNA assembly molecule has good stability and is suitable for isothermal amplification.

[0087] Furthermore, the DNA assembly molecules and their mutants provided by the present invention possess DNA assembly capabilities in the presence of various metal ions and at different temperatures, and can be applied to nanopore sequencing, single-molecule sequencing, RCA library construction, environmental testing, and in vitro diagnostics. In particular, at high salt concentrations, they exhibit amplification activity far superior to that of existing DNA polymerases. By increasing the salt concentration, the DNA translocation perforation time can be prolonged, thereby improving the accuracy of single-molecule sequencing and significantly reducing the loss of critical sequencing information. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0089] Figure 1 The predicted structure of the protein having the amino acid sequence shown in SEQ ID NO.1;

[0090] Figure 2 The predicted structure of the protein having the amino acid sequence shown in SEQ ID NO.2;

[0091] Figure 3The predicted structure of the protein having the amino acid sequence shown in SEQ ID NO.3;

[0092] Figure 4 The predicted structure of the protein having the amino acid sequence shown in SEQ ID NO.4;

[0093] Figure 5 A diagram comparing the structure of the protein having the amino acid sequence shown in SEQ ID NO. 1 with the existing DNA assembly molecular machine phi29;

[0094] Figure 6 This is the result of SDS-PAGE protein electrophoresis;

[0095] Figure 7 Figure 2 shows the detection results after rolling circle amplification of each DNA assembly molecular machine;

[0096] Figure 8 This is the result diagram of the effect of heavy metal ions on amplification;

[0097] Figure 9 This is the result diagram of the effect of NaCl concentration on amplification;

[0098] Figure 10 The graph shows the activity test results of each mutant. DETAILED DESCRIPTION

[0099] Reference will now be made in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0100] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a person skilled in the art. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0101] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0102] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0103] Example 1

[0104] This embodiment provides a DNA assembly molecular machine, and its structure is analyzed.

[0105] The amino acid sequence of the DNA assembly molecular machine of this embodiment is shown in SEQ ID NO.1. The structure is analyzed by structure prediction software and the following is obtained: Figure 1 The structure shown in FIG. 1 shows that amino acids 1-192 constitute the exonuclease active domain, amino acids 192-268 and 437-549 constitute the palm domain, amino acids 269-367 constitute the terminal insertion domain 1, amino acids 368-404 constitute the finger domain, amino acids 405-436 constitute the terminal insertion domain 2, and amino acids 550-606 constitute the thumb structure.

[0106] This structure is compared with the existing structure of the DNA assembly molecular machine phi29 (PDB ID: 1xhx) (ref. Figure 5 The thumb structure has an extra α-helix, while the palm structure has a longer β-sheet structure.

[0107] Example 2

[0108] This embodiment provides a DNA assembly molecular machine, and its structure is analyzed.

[0109] The amino acid sequence of the DNA assembly molecular machine of this embodiment is shown in SEQ ID NO. 2. The structure is analyzed by structure prediction software and the following is obtained: Figure 2 The structure shown in FIG. 1 shows an exonuclease activity domain composed of amino acids 1-205, a palm domain composed of amino acids 206-276 and 443-551, a terminal insertion domain 1 composed of amino acids 277-375, a finger domain composed of amino acids 376-409, a terminal insertion domain 2 composed of amino acids 410-442, and a thumb domain composed of amino acids 552-598.

[0110] Example 3

[0111] This embodiment provides a DNA assembly molecular machine, and its structure is analyzed.

[0112] The amino acid sequence of the DNA assembly molecular machine of this embodiment is shown in SEQ ID NO.3. The structure is analyzed by structure prediction software and the following is obtained: Figure 3 The structure shown in FIG. 1 shows an exonuclease activity domain composed of amino acids 1-189, a palm domain composed of amino acids 190-260 and 429-537, a terminal insertion domain 1 composed of amino acids 261-360, a finger domain composed of amino acids 361-396, a terminal insertion domain 2 composed of amino acids 397-428, and a thumb structure composed of amino acids 538-578.

[0113] Example 4

[0114] This embodiment provides a DNA assembly molecular machine, and its structure is analyzed.

[0115] The amino acid sequence of the DNA assembly molecular machine of this embodiment is shown in SEQ ID NO.5. The structure is analyzed by structure prediction software and the following is obtained: Figure 4 The structure shown in FIG. 265-462 amino acids constitute the exonuclease active domain, amino acids 463-545 and 737-839 constitute the palm domain, amino acids 546-670 constitute the terminal insertion domain 1, amino acids 671-703 constitute the finger domain, amino acids 704-736 constitute the terminal insertion domain 2, and amino acids 840-915 constitute the thumb structure.

[0116] Example 5

[0117] In this example, protein expression was performed.

[0118] Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize the nucleic acid sequences corresponding to the amino acid sequences represented by SEQ ID Nos. 1, 2, 3, 5, and 6. The resulting sequences were transferred into the pET30a vector to generate an expression vector for the DNA assembly molecular machine. The expression vector was then transformed into competent Escherichia coli BL21(DE3) cells (Beijing Quanshijin Biotechnology Co., Ltd.), cultured in ampicillin-resistant medium, and verified by sequencing. After successful transformation, a single colony was selected and inoculated into 5 mL of LB liquid medium for overnight culture. The colony was then transferred to 1 L of LB liquid medium and cultured with shaking for 4 hours until the OD value reached 0.6. IPTG was then added to a final concentration of 500 μM and continued to be shaken overnight. The culture was centrifuged, the cells were harvested, and the cells were lysed using an ultrasonic cell disruptor at 80 W for 1 second with 2 second intervals for a total of 20 minutes. The protein (i.e., the DNA assembly molecular machine) was then purified using affinity chromatography on a His-tag column.

[0119] The obtained DNA assembly molecular machine was subjected to SDS-PAGE protein electrophoresis to verify the size of the DNA assembly molecular machine protein. The molecular weight of the DNA assembly molecular machine was 76 kD.

[0120] The results of SDS-PAGE protein electrophoresis are as follows Figure 6 shown. Figure 6 Lane 1 is phi29 protein, lane 2 is protein SEQ ID NO: 1, lane 3 is SEQ ID NO: 2, lane 4 is SEQ ID NO: 3, lane 5 is SEQ ID NO: 5, and lane 6 is SEQ ID NO: 6. The protein size is about 70 kDa.

[0121] Example 6

[0122] This example uses rolling circle amplification to detect the function of DNA assembly molecular machines.

[0123] Each histone prepared in Example 5, phi29, and a control group were used as DNA assembly molecular machines for rolling circle amplification. Specifically, 200 nM of the DNA assembly molecular machine, 500 nM of the hairpin template, and reaction buffer were mixed to obtain a reaction system. The reaction buffer consisted of 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 200 μM dNTPs. The pH of the reaction buffer was 7.5 at 25°C. The nucleotide sequence of the hairpin template was as follows:

[0124] A sequence:

[0125] 5'-p-TTGGCATATCGTACGATATGCCACCACCACCACCACAACCACCACCACCAAGCGATACGCGTATCGCTTA-3';

[0126] B sequence:

[0127] 5-AAAAAAACCTTCCTTTT-ATATGCCAATAAGCGATACG-3'.

[0128] The reaction system was placed at a temperature of 30° C. for 2 h to obtain a reaction product.

[0129] Test results reference Figure 7 As shown in the test results, it can be seen that the DNA assembly molecular machine prepared in Example 6 has DNA assembly activity and can extend the template chain to form a product chain, but its activity is not as good as that of phi29 polymerase under the test conditions.

[0130] Experimental Example 1

[0131] The influencing factor experiment was carried out according to Example 6.

[0132] The reaction system for heavy metal ion detection is: 50mM Tris-HCl, 100uM metal ions, 10mM (NH4)2SO4, 4mM DTT, 0.25mM dNTP, 1uM DNA, 0.8uM protein.

[0133] Reference Figure 8 As shown, heavy metal ion effect analysis showed that A0A1X9SGT8 exhibited higher activity than other proteins under calcium ion concentration conditions.

[0134] Furthermore, the activity of A0A1X9SGT8 was detected by different salt concentrations as in Example 6. Figure 9 As shown, at concentrations below 300 mM NaCl, the DNA amplification activity was inferior to that of commercial phi29.

[0135] When the salt ion concentration is higher than 300 mM, the activity is stronger than phi29 (ie, Phi29 shown in the figure).

[0136] This indicates that the DNA assembly molecular machine of the present invention can be used in a high salt concentration environment.

[0137] Experimental Example 2

[0138] The activity of the mutants of SEQ ID NO: 1 was verified.

[0139] The activity of each mutant of A0A1X9SGT8 was tested using the detection method of Example 6. Figure 10 As shown:

[0140] When amino acids 571-585 (DPNDFTEEEIKRKNI) are deleted, i.e., αdel, the DNA assembly molecular machine loses its DNA amplification activity, indicating that this domain is essential for DNA assembly in the DNA assembly molecular machine, and this structure is also the key sequence provided by the present invention.

[0141] Deletion (βDel) of amino acids 525-532 (EWKFKMV) reduced assembly activity, confirming the importance of this domain. Mutations at positions D12 and D67 also resulted in a loss of activity. However, mutations at these two sites in the patented phi29 protein enhanced activity. This demonstrates the significant differences between the present invention's DNA molecular assembly machinery and the patented protein.

[0142] Figure 10 In the data, DD refers to the simultaneous mutation of D12 and D67. The control group is the WT group with 100 mM EDTA added.

[0143] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of a DNA assembly molecular machine in any of the following applications, characterized in that: The amino acid sequence of the DNA assembly molecular machine is shown in SEQ ID NO:

1. The application is in a high-salt reaction system, wherein the reaction system includes 0.3M-1M salt ions: 1) Acts as a DNA polymerase; 2) Control the movement of DNA; 3) Preparation of single-molecule sequencing reagents or kits; 4) Preparation of nanopore sequencing reagents or kits; 5) Catalyze DNA amplification; 6) catalyzing rolling circle amplification and / or catalyzing multiple strand displacement amplification; 7) Perform DNA sequencing or whole genome sequencing; 8) RCA library construction; 9) Genome amplification coverage detection; 10) Preparing a kit product for catalyzing DNA replication; 11) preparing products for catalyzing rolling circle amplification and / or catalyzing multiple strand displacement amplification; 12) Preparation of products for DNA sequencing or whole genome sequencing; 13) Preparation of products for RCA library construction; 14) Preparing products for genome amplification coverage detection; The use is not for the purpose of diagnosing a disease.

2. A method for assembling DNA molecules using a DNA assembly molecular machine, characterized in that: The method is not intended for disease diagnosis, and includes performing a reaction in a high-salt reaction system; the reaction system includes 0.3M-1M salt ions, and the amino acid sequence of the DNA assembly molecular machine is shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that The salt ion is sodium, potassium, calcium, magnesium, manganese, nickel, cobalt, copper or zinc.

4. The method according to claim 2, characterized in that The reaction system also includes calcium ions.

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