Improved T7 endonuclease for specific recognition of cleavage mismatched DNA

Abstract

The invention relates to the technical field of molecular biology, in particular to improved T7 endonuclease for specifically recognizing and cutting mismatched DNA. The improved T7 endonuclease is obtained by carrying out site-directed mutagenesis modification on the wild T7 endonuclease, has excellent capability of recognizing and cutting mismatched DNA (Deoxyribose Nucleic Acid) and can better reserve a correct sequence, and the recognizing and cutting capability of the improved T7 endonuclease is superior to that of the wild T7 endonuclease and that of commercial T7 endonuclease. Therefore, the improved T7 endonuclease has important application value in the technical field of genome packaging, and has important application potential in the fields of molecular biology and synthetic biology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to improved T7 endonuclease which can specifically recognize and cleave mismatched DNA. BACKGROUND

[0002] Oligonucleotide assembly which relies on DNA polymerase and error correction enzyme is an important technology for artificially synthesizing spliced genes or DNA sequences. Currently, the error rate of this technology is as high as 1-10 errors per 1000 base pairs. The causes of errors include low purity of oligonucleotides, mismatching during annealing and hybridization, base mutations, and fragment insertion or deletion, etc.

[0003] T7 endonuclease is derived from T7 bacteriophage and is an enzyme with nucleic acid structure selectivity. It can recognize mismatched regions in double-stranded DNA, including imperfectly paired DNA, cross-shaped structure DNA, Holliday-type structure DNA, and heteroduplex DNA, etc. T7E1 enzyme eliminates DNA containing mismatches by cleaving the first, second or third phosphodiester bond at the 5' end of the mismatched base. It is applied to DNA sequence assembly error correction, gene editing efficiency verification, and gene typing, etc.

[0004] Wild-type T7 endonuclease can effectively recognize more than 1 base mismatch and is not sensitive to single base point mutations. The current technical demand is to improve its ability to recognize and cleave mismatched sequences, especially single base mismatches, so as to achieve efficient error correction in DNA synthesis assembly and improve the accuracy of the target sequence. It can also be better applied to gene mutation detection and gene typing. SUMMARY

[0005] To solve the above technical problems, the present application first mutates the wild-type T7 endonuclease at a single point to improve its recognition and cleavage ability for mismatched DNA. For example, the present application attempts to mutate I9K, S24A, W39L, F63I, I92F, S96N, F112W, I118F, A127E, G146K, etc. The results show that the six mutant T7 endonucleases I9K, F63I, I92F, S96N, F112W, and G146K have better mismatch cleavage activity than the wild-type T7 endonuclease, but I9K, I92F, and G146K have different degrees of cleavage effect on the correct sequence. Further, the present application attempts to mutate multiple sites to obtain an improved T7 endonuclease which can effectively improve the mismatch recognition and cleavage activity and better preserve the correct sequence. Based on this, the following technical solutions are proposed.

[0006] First, the present invention provides an improved T7 endonuclease, which, based on the wild-type T7 endonuclease, has the following site mutations: the 9th amino acid residue is mutated from isoleucine (I) to lysine (K), the 96th amino acid residue is mutated from serine (S) to asparagine (N), and the 146th amino acid residue is mutated from glycine (G) to lysine (K).

[0007] Preferably, the amino acid sequence of the wild-type T7 endonuclease is shown in SEQ ID No. 1.

[0008] SEQ ID No. 1: MAGYGAKGIRKVGAFRSGLEDKVSKQLESKGIKFEYEEWKVPYVIPASNHTYTPDFLLPNGIFVETKGLWESDDRKKHLLIREQHPELDIRIVFSSRTKLYKGSPTSYGEFCEKHGIKFADKLIPAEWIKEPKKEVPFDRLKRKGGKK Furthermore, the present invention provides a nucleic acid sequence encoding the improved T7 endonuclease.

[0009] Furthermore, the present invention provides biological materials containing the improved T7 endonuclease or the nucleic acid sequence.

[0010] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or cell.

[0011] Furthermore, the present invention provides reagents or kits containing the improved T7 endonuclease or the nucleic acid sequence.

[0012] Preferably, the reagent or kit is used for DNA sequence assembly error correction, gene mutation detection, gene editing efficiency determination, or genotyping.

[0013] Preferably, the reagent or kit includes: the modified T7 endonuclease and reaction buffer.

[0014] In practice, the reagents or kits may also include purified water to adjust the volume of the reaction system.

[0015] In the specific implementation process, the reagents or kits may also include at least one of metal ions, stabilizers, and inert dyes to optimize reaction conditions.

[0016] Furthermore, the present invention provides the application of the improved T7 endonuclease, the nucleic acid sequence, or the biological material in the preparation of reagents or kits.

[0017] Furthermore, the present invention provides the application of the improved T7 endonuclease in at least one of the following aspects: (1) DNA sequence assembly error correction; (2) Gene mutation detection for purposes other than disease diagnosis and treatment; (3) Determination of gene editing efficiency for non-disease diagnosis and treatment purposes; (4) Genotyping.

[0018] Preferably, the improved T7 endonuclease recognizes and cuts 1, 2, 3, 4 or 5 consecutive base mismatches.

[0019] Preferably, the improved T7 endonuclease recognizes and cleaves AC, AA, AG, GG, and CT.

[0020] Preferably, the improved T7 endonuclease has the highest efficiency in recognizing and cleaving AC and AA mismatches.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, through site-directed mutagenesis of the wild-type T7 endonuclease, yields an improved T7 endonuclease. This improved endonuclease exhibits superior ability to recognize and cleave mismatched DNA while better preserving the correct sequence, demonstrating better recognition and cleavage capabilities than both the wild-type and commercially available T7 endonucleases. Therefore, this improved T7 endonuclease possesses significant application value in the field of genome assembly technology and holds great potential for application in molecular biology and synthetic biology. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the mutant design structure of the improved T7 endonuclease; a is a schematic diagram of the mutation sites of 10 single-site improved T7 endonucleases; b is a schematic diagram of the mutation sites of the selected multi-site improved T7 endonuclease.

[0023] Figure 2 This is a schematic diagram of correct and mismatched double-stranded DNA design; PM: correct sequence; MM1: 1 base pair mismatch sequence (AC single base mismatch); MM2: 2 consecutive base pair mismatch sequences; MM3: 3 consecutive base pair mismatch sequences; MM4: 4 consecutive base pair mismatch sequences; MM5: 5 consecutive base pair mismatch sequences; red marks indicate mismatched base pairs.

[0024] Figure 3This is a TBE-PAGE electrophoresis image of DNA products digested by the single-site modified T7 endonuclease; L is the DNA molecular standard, Control is the negative control, WT is the wild-type T7 endonuclease, and NEB is the commercial T7 endonuclease.

[0025] Figure 4 This is a TBE-PAGE electrophoresis image of the DNA products digested by the multi-site modified T7 endonuclease.

[0026] Figure 5 This is an analysis of the preference of the improved T7 endonuclease for single-base mismatched double-stranded DNA; A is a schematic diagram of the design of single-base mismatched double-stranded DNA; B is the TBE-PAGE electrophoresis identification and analysis results of the enzyme digestion products of wild-type (C), KNK improved T7 endonuclease and commercial T7 endonuclease (NEB) for single-base mismatched DNA. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0028] This invention relates to molecular biology experiments. Unless otherwise specified, references can be made to *Molecular Cloning: A Laboratory Manual (4th Edition)* (Mr. Green and J. Sambrook, Science Press) and *Protein Purification: A Laboratory Manual (2nd Edition)* (R.R. Burgess and MP. Deutsch, Science Press). These references are the most commonly used and instructive books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as oligonucleotide synthesis, DNA sequencing, and molecular weight determination.

[0029] In the following examples, I9K indicates that the 9th amino acid residue of the wild-type T7 endonuclease is mutated from isoleucine to lysine, and the mutations at other sites are consistent with this naming rule.

[0030] Example 1: Design and preparation of an improved T7 endonuclease 1. The amino acid sequence of the wild-type T7 endonuclease is shown in SEQ ID No. 1 (WT), and the single-point amino acid mutation design is as follows: Figure 1 As shown in 'a', the multi-site amino acid mutation design is as follows: (1) I9K: F63I: S96N (denoted as "KIN"); (2) S24A: F63I: S96N (denoted as "AIN"); (3) F63I: S96N: F112W (denoted as "INW"); (4) I9K: S24A: S96N (denoted as "KAN"); (5) I9K: S24A: F63I (referred to as "KAI"); (6) I9K: S96N: F112W (referred to as "KNW112"); (7) I9K: F63V: S96N (denoted as "KVN"); (8) I9K: S96N: G146K (denoted as "KNK"); (9) I9K: I92F: S96N (denoted as "KFN"); (10) I9K: S96N: F120W (referred to as "KNW120").

[0031] 2. Plasmid Vector Construction: Candidate improved T7 endonuclease gene sequences were synthesized (e.g., the nucleic acid sequence of KNK is shown in SEQ ID No. 2, the nucleic acid sequence of KNW112 is shown in SEQ ID No. 3, and the nucleic acid sequence of KNW120 is shown in SEQ ID No. 4), constructed in the pET-28a plasmid vector, and sequenced to verify that the DNA sequences were completely correct. DNA synthesis, plasmid construction, and sequencing verification were all completed by Beijing Qingke Biotechnology Co., Ltd.

[0032] 3. Prepare the solution: (1) Isopropyl-β-D-thiogalactoside (IPTG) solution (1M): Weigh 2.38g IPTG and add it to 10mL of enzyme-free water. After dissolving, filter it through a 0.22µm filter membrane and store it at -20℃ for later use.

[0033] (2) Lysis buffer: 50mM Tris HCl (pH 8.0), 500mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0034] (3) Imidazole stock solution (2M): Weigh 1.36g of imidazole and add it to 10mL of enzyme-free water. After dissolving, adjust the pH to 8 with hydrochloric acid and store at 4℃ for later use.

[0035] (4) Gradient elution buffer: 50mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 50mM imidazole; 100mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 100mM imidazole; 500mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 500mM imidazole.

[0036] (5) Equilibrium solution: 20mM Tris, 500mM NaCl.

[0037] (6) Dialysis solution / protein storage solution: 50mM Tris HCl (pH 8.0), 100mM NaCl.

[0038] 4. Protein Induction Expression: 1 μL of plasmids encoding wild-type and modified T7 endonucleases were added to BL21(DE3) competent cells (Qingke Biotechnology, catalog number: TSC-E01), placed on ice for 20 minutes, heat-shocked at 42℃ for 90 seconds, and immediately placed on ice for 5 minutes. 600 μL of LB medium was added. The cells were cultured at 37℃ with shaking at 220 rpm for 1 hour. After centrifugation, all cells were plated onto solid LB plates containing 50 μg / mL kanamycin and incubated overnight at 37℃. Single colonies were picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin. The cells were cultured at 37℃ with shaking at 220 rpm for 2 hours. Afterward, the cells were scaled up 1:100 and cultured until OD was reached. 600 When the value reaches 0.5-0.8, add IPTG solution to the culture medium to a final concentration of 0.2 mM, and culture at 16℃ with shaking at 220 rpm for 18 hours to induce protein expression.

[0039] 5. Cell Lysis: Collect the bacterial suspension, centrifuge to remove the supernatant, wash the cells twice with PBS, centrifuge again to remove the supernatant, add 4 mL of lysis buffer to the cell pellet, resuspend thoroughly, add 20 µL of 100 mg / mL lysozyme solution, add PMSF to a final concentration of 1 mM, and mix well. Place the mixture in a four-dimensional rotary mixer and rotate at room temperature for 15 minutes. Heat the lysed cell suspension in a 75°C water bath for 1 hour, centrifuge at 16000 × g for 10 minutes at 4°C, collect the supernatant, filter through a 0.22 µm filter membrane, add 400 µL of dialysis buffer to the filtrate, mix well, and store at -80°C for later use.

[0040] 6. Protein purification: A Ni-NTA affinity chromatography column was used. Equilibration buffer was added, and the column was equilibrated for 10 minutes. The equilibration buffer was removed, and the protein sample solution was added, allowing it to bind for 30 minutes. Unbound protein was washed away with equilibration buffer, and the target protein was eluted sequentially using a concentration gradient elution buffer.

[0041] 7. Protein Quantitative Analysis: The eluted fractions were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Based on the staining results, fractions with high purity of the target protein were collected and dialyzed overnight. The product was added to a 50 kDa ultrafiltration tube and then concentrated by centrifugation and ultrafiltration. The concentrate was collected, and the protein concentration was determined using the Qubit protein assay kit (Thermo Fisher Scientific, catalog number: Q33212). The protein solution was stored at -20°C to obtain solutions of different modified T7 endonucleases.

[0042] Example 2: Detection of the DNA mismatch recognition activity of wild-type and modified T7 endonucleases 1. Sequence Design and Synthesis: Design correct and consecutive double-stranded DNA sequences with 1 (MM1), 2 (MM2), 3 (MM3), 4 (MM4), or 5 (MM5) base mismatches. Figure 2 The positive strand sequence is shown in SEQ ID No. 5, the correct negative strand sequence is shown in SEQ ID No. 6, and the consecutive mismatched negative strand sequences are shown in SEQ ID No. 7 ~ SEQ ID No. 11. Design double-stranded DNA sequences with single-base mismatch types ( Figure 5 The positive strand sequence is shown in SEQ ID No. 5, and the negative strand sequences with single base mismatches are SEQ ID No. 7 (AC mismatch), SEQ ID No. 12 (AA mismatch), SEQ ID No. 13 (AG mismatch), SEQ ID No. 14 (GG mismatch), and SEQ ID No. 15 (CT mismatch). All oligonucleotide sequences were synthesized by Genscript Biotech Inc.

[0043] 2. Double-stranded DNA hybridization reaction: Dissolve the above oligonucleotide sequences in enzyme-free water to prepare a 50 µM solution. Prepare the hybridization reaction system as follows: 5 μL positive-strand oligonucleotide solution (50 µM), 5 μL negative-strand oligonucleotide solution (50 µM), 5 μL annealing buffer (Beyotime, catalog number: D0251), and 10 μL enzyme-free water. Reaction program: 95℃, 2 minutes; 90℃, 5 seconds; decrease by 0.1℃ for each cycle, for 650 consecutive cycles; 4℃, 5 minutes. After the reaction, the product concentration was determined using the Qubit 1×dsDNA detection kit (Thermo Fisher Scientific, catalog number: Q33266).

[0044] 3. Enzyme Digestion Reaction: The protein concentrations of wild-type, improved, and commercially available T7 endonuclease were uniformly balanced. The enzyme digestion reaction system was prepared as follows: 1.5 μL 10×NE Buffer 2 (NEB, catalog number: B7002S), 300 ng dsDNA annealing product, 400 ng T7 endonuclease or an equal volume of enzyme-free water (as a negative control), and enzyme-free water to a total volume of 15 μL. Reaction program: 37℃, 30 minutes; 60℃, 5 minutes; 4℃, 5 minutes.

[0045] 4. TBE-PAGE electrophoresis identification: Take 8 μL of the enzyme digestion product, add 2 μL of 6×TriTrack loading buffer (Thermo Fisher Scientific, catalog number: R1161), and add 2 μL of enzyme-free water. Add the DNA ultra-low range molecular weight standard (Thermo Fisher Scientific, catalog number: SM1212) and the product sample to a 15% TBE-PAGE precast gel (Beyotime, catalog number: D0183S), and perform electrophoresis with 1×TBE buffer and a constant voltage of 100V. After the electrophoresis is completed, image the gel.

[0046] The experimental results are as follows: Figure 3 and Figure 4 The results of gel electrophoresis experiments on the digestion products of the single-site and multi-site modified T7 endonuclease are shown, respectively. The equal volume water treatment group is the negative control (Control), and is compared with wild-type (WT) and commercial (NEB) T7 endonuclease.

[0047] For single-point mutations, it can be seen that the six improved T7 endonucleases, I9K, F63I, I92F, S96N, F112W, and G146K, have superior mismatch cleavage activity compared to wild-type T7 endonucleases. However, I9K, I92F, and G146K also have varying degrees of cleavage activity on the correct sequence.

[0048] For multi-site mutations, KAN can degrade all sequences, including the correct sequence, and therefore cannot be used as a DNA error-correcting enzyme. The three improved T7 endonucleases, KNK, KNW112, and KNW120, showed the highest efficiency in recognizing and cleaving mismatched sequences, significantly outperforming wild-type and commercial (NEB) T7 endonucleases, while also exhibiting high retention of the correct sequence. Among them, the KNK improved T7 endonuclease showed the best performance. Figure 1 (as shown in b) is suitable for development into a DNA error-correcting enzyme.

[0049] Figure 5 The gel electrophoresis results of the KNK-modified T7 endonuclease, the best among three preferred improved T7 endonucleases (KNK, KNW112, and KNW120), show that the improved KNK endonuclease is superior to the wild-type enzyme and the commercial (NEB) T7 endonuclease in recognizing and cleaving various single-base mismatches. In particular, it has the highest recognition and cleavage efficiency for AC and AA mismatches, relatively high recognition and cleavage efficiency for AG mismatches, and some cleavage activity for GG and CT, indicating a preference for these mismatches.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An improved T7 endonuclease, characterized in that, Based on the wild-type T7 endonuclease, it has the following site mutations: the 9th amino acid residue is changed from isoleucine to lysine, the 96th amino acid residue is changed from serine to asparagine, and the 146th amino acid residue is changed from glycine to lysine.

2. The nucleic acid sequence encoding the improved T7 endonuclease of claim 1.

3. Biological material containing the improved T7 endonuclease of claim 1 or the nucleic acid sequence of claim 2.

4. The biomaterial according to claim 3, characterized in that, The biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or cells.

5. A reagent or kit containing the improved T7 endonuclease of claim 1 or the nucleic acid sequence of claim 2.

6. The reagent or kit according to claim 5, characterized in that, The reagents or kits are used for DNA sequence assembly error correction, gene mutation detection, gene editing efficiency determination, or genotyping.

7. The use of the improved T7 endonuclease of claim 1, the nucleic acid sequence of claim 2, or the biomaterial of claim 3 or 4 in the preparation of reagents or kits.

8. The use of the improved T7 endonuclease according to claim 1 in at least one of the following aspects: (1) DNA sequence assembly error correction; (2) Gene mutation detection for purposes other than disease diagnosis and treatment; (3) Determination of gene editing efficiency for non-disease diagnosis and treatment purposes; (4) Genotyping.

9. The application according to claim 8, characterized in that, The improved T7 endonuclease recognizes and cuts 1, 2, 3, 4, or 5 consecutive base mismatches.

10. The application according to claim 8 or 9, characterized in that, The improved T7 endonuclease recognizes and cleaves AC, AA, AG, GG, and CT.

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