Phi29 DNA polymerase variants and their applications

Abstract

This application provides a Phi29 DNA polymerase variant and its application. Compared with the wild-type enzyme, the Phi29 DNA polymerase variant described in this application, which contains substitutions of A484N+S395T, A484Q+S395T, A484Q+T534C, Y281H+S395T, D186E+G401K, or S395T+K402R, exhibits higher DNA synthesis fidelity, effectively reduces errors during amplification, thereby reducing the risk of non-specific amplification and showing good prospects for industrial application.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a Phi29 DNA polymerase variant and its applications. Background Technology

[0002] DNA vaccines have proven to be one of the most promising applications in gene therapy. Their unique ability to readily induce humoral and cellular immune responses attracted widespread attention from vaccine developers when the concept was first demonstrated in the early 1990s (Tang, DC et., al. (1992). Genetic immunization is a simple method for eliciting an immune response. Nature, 356(6365), 152–154; Ulmer, JB et., al. (1993). Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 259(5102), 1745–1749; Fynan, EF, et., al. (1993). DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations). Traditional methods for preparing DNA vaccines are based on intracellular replication, which is costly and requires avoiding contamination of the culture. In particular, when using the common Escherichia coli expression system, complex downstream purification processes are required to remove bacterial endotoxins that are toxic to mammals, which significantly increases production costs.

[0003] Touchlight, a UK-based company, has launched the doggyboneDNA (dbDNA) vector, which replaces the traditional intracellular replication method for preparing plasmid DNA with an in vitro enzymatic synthesis method. dbDNA is a small, linear, double-stranded DNA structure, resembling a dog bone. Synthesis mainly involves two steps: 1) DNA amplification: The starting template is a circular plasmid DNA containing the target gene sequence, the Phi29 DNA polymerase recognition sequence (telRL) derived from Escherichia phage N15, and the bacterial backbone sequence. Through denaturation, it is converted into two single-stranded circular DNA molecules, and then amplified using a polymerase derived from Bacillus subtilis phage phi29, generating a long linear double-stranded multiply DNA with telomerase recognition sequence spacers; 2) TelN telomerase cleavage and covalent closure: TelN telomerase binds to the telomerase recognition site, recognizing the telRL sites flanking the target gene expression element, performing cleavage and ligation to generate a linear gene, doggyboneDNA, with covalently linked ends.

[0004] Under current conditions, phi29 DNA polymerase reduces the generation of mispaired products during DNA product preparation through its 3′-5′ exonuclease activity. However, this may introduce incorrect substrates during DNA product preparation, resulting in impurities (Del Prado, A. et., al. (2019). New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in phi29 dna polymerase. Scientific Reports, 9(1)). Therefore, there is an urgent need for a phi29 DNA polymerase that is cost-effective and has high fidelity. Summary of the Invention

[0005] In a first aspect, this application provides a Phi29 DNA polymerase variant whose amino acid sequence, compared with SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites: A484, Q497, S395, M8, T534, G401, Y281, D186, K402, wherein the mutation type is selected from substitution or deletion.

[0006] Secondly, this application provides a class of biological materials selected from one or more of the following:

[0007] a) A polynucleotide molecule encoding the above variants;

[0008] b) An expression vector containing the polynucleotide molecule described in a);

[0009] c) A host cell containing a polynucleotide molecule as described in a), or a host cell containing an expression vector as described in b).

[0010] Thirdly, this application provides a method for preparing the above-mentioned Phi29 DNA polymerase variant.

[0011] Fourthly, this application provides a composition comprising at least one Phi29 DNA polymerase variant as described in this application.

[0012] Fifthly, this application provides a kit comprising at least one Phi29 DNA polymerase variant as described in this application.

[0013] Sixthly, this application provides the use of the above-mentioned Phi29 DNA polymerase variant, composition or kit in the preparation of DNA. Invention Details

[0015] Phi29 DNA polymerase variant

[0016] This application provides a Phi29 DNA polymerase variant having an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and containing, relative to SEQ ID NO: 1, at least one mutation selected from the following amino acid sites: A484, Q497, S395, M8, T534, G401, Y281, D186, K402, wherein the mutation type is selected from substitution or deletion.

[0017] In some embodiments, the mutation type is substitution. In some embodiments, the substitution at A484 is Q or N. In some embodiments, the substitution at Q497 is P. In some embodiments, the substitution at S395 is selected from T. In some embodiments, the substitution at M8 is R. In some embodiments, the substitution at T534 is C. In some embodiments, the substitution at G401 is K. In some embodiments, the substitution at Y281 is H. In some embodiments, the substitution at D186 is E. In some embodiments, the substitution at K402 is R.

[0018] In some embodiments, the mutation type is substitution, and the variant comprises a substitution or substitution group selected from the following: A484N, A484Q, Q497P, S395T, M8R, A484N+S395T, A484Q+S395T, A484Q+T534C, Y281H+S395T, D186E+G401K, or S395T+K402R. In some embodiments, the mutation type is substitution, and the variant occurs within the following substitution or substitution group: A484N, A484Q, Q497P, S395T, M8R, A484N+S395T, A484Q+S395T, A484Q+T534C, Y281H+S395T, D186E+G401K, or S395T+K402R.

[0019] In some implementations, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1.

[0020] In some embodiments, the amino acid sequence of the variant has at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher sequence identity compared to any sequence selected from SEQ ID NO: 2-12. In some embodiments, the amino acid sequence of the variant is as shown in any of SEQ ID NO: 2-12.

[0021] The Phi29 DNA polymerase variant provided in this application has higher fidelity compared to the wild-type Phi29 DNA polymerase.

[0022] biomaterials

[0023] This application provides a polynucleotide encoding a variant of the Phi29 DNA polymerase. Due to codon degeneracy or codon bias in the host cell expressing the polypeptide, the polynucleotide sequence can be any polynucleotide sequence encoding the variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the Phi29 DNA polymerase variant of this application may be selected from SEQ ID NO: 13-24.

[0024] The expression vector provided in this application comprises a multinucleotide molecule encoding a variant of the Phi29 DNA polymerase of this application. In some embodiments, the expression vector further comprises one or more regulatory sequences, including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequences are operatively linked to the multinucleotide molecule encoding the variant.

[0025] In some embodiments, the expression vector may be a linear or circular DNA molecule, typically containing elements such as a multiple cloning site, an antibiotic resistance gene, and a replication origin site. In some embodiments, the expression vector described in this application is pET-30a(+).

[0026] The host cell provided in this application refers to any cell that is favorable for the expression of the Phi29 DNA polymerase variant of this application, that is, any cell that is susceptible after being transformed, transfected or transduced with the expression vector described in this application, including any daughter cells that are different from the parent cells due to mutations that occur during replication.

[0027] In some embodiments, the host cell is a prokaryotic cell, selectable from Gram-positive or Gram-negative bacteria. In some embodiments, the host cell is a Gram-positive bacterium, including but not limited to: *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Bacillus cereus*, *Staphylococcus*, *Streptococcus*, and *Streptomyces*. In some embodiments, the host cell is a Gram-negative bacterium, including but not limited to: *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, and *Ureaplasma*. In some embodiments, the host cell is *Escherichia coli* BL21(DE3).

[0028] Preparation method of Phi29 DNA polymerase variant

[0029] The method for preparing a Phi29 DNA polymerase variant provided in this application includes 1) culturing the host cells described in this application under suitable variant expression conditions; and 2) recovering the variant.

[0030] In some embodiments, the method for recovering variants can be a method known in the art, such as centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof.

[0031] In some embodiments, the preparation method further includes a purification step of the variant, which can be a method known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), ammonium sulfate precipitation, etc.

[0032] Composition

[0033] The composition provided in this application comprises at least one variant of the Phi29 DNA polymerase described in this application.

[0034] The composition described in this application can be a composition for storing a Phi29 DNA polymerase variant. In some embodiments, in addition to the Phi29 DNA polymerase variant described above, the composition described in this application may optionally contain: buffer components (such as Tris base, Tris-HCl, HEPES, MOPS), salts (such as NaCl, MgCl2), enzyme inhibitors (such as EDTA, EDTA-2Na), reducing agents (such as DTT), stabilizers (such as glycerol), etc. In some embodiments, the composition for storing Phi29 DNA polymerase described in this application contains: Phi29 DNA polymerase variant, Tris-HCl, NaCl, EDTA-2Na, DTT, and glycerol. In some embodiments, the composition for storing Phi29 DNA polymerase described in this application contains: Phi29 DNA polymerase variant, Tris-HCl, MgCl2, (NH4)2SO4, and DTT.

[0035] Reagent test kit

[0036] This application provides a kit comprising: at least one Phi29 DNA polymerase variant as described in this application; or at least one nucleic acid encoding a Phi29 DNA polymerase variant as described in this application; or at least one expression vector as described in this application; or at least one host cell as described in this application; or a composition as described in this application.

[0037] application

[0038] This application provides the use of the above-described Phi29 DNA polymerase variants, compositions, or kits in the preparation of DNA products.

[0039] A method for preparing DNA, characterized in that the method comprises contacting a DNA template, primers, modified or unmodified nucleoside triphosphates with the Phi29 DNA polymerase variant described in this application, incubating in a reaction system to obtain target DNA.

[0040] The application of the Phi29 DNA polymerase variant in this application in a DNA preparation method that improves the fidelity of the product DNA.

[0041] Other implementation plans

[0042] 1. A Phi29 DNA polymerase variant, characterized in that the amino acid sequence of the variant, relative to SEQ ID NO: 1, contains at least one mutation selected from the following amino acid sites:

[0043] A484, Q497, S395, M8, T534, G401, Y281, D186, K402.

[0044] 2. The variant as described in item 1, characterized in that:

[0045] (1) The substitution at position A484 is Q or N;

[0046] (2) The substitution at position Q497 is P;

[0047] (3) The substitution at position S395 is T;

[0048] (4) The substitution at position M8 is R;

[0049] (5) The substitution at position T534 is C;

[0050] (6) The substitution at position G401 is K;

[0051] (7) The substitution at position Y281 is H;

[0052] (8) The substitution at position D186 is E;

[0053] (9) The substitution at position K402 is R.

[0054] 3. The variant as described in item 1, characterized in that the amino acid sequence of the variant is shown in any of SEQ ID NO: 2-12.

[0055] 4. A biological material, characterized in that it is selected from one or more of the following:

[0056] a) A multinucleotide molecule encoding a Phi29 DNA polymerase variant as described in any of items 1-3;

[0057] b) An expression vector containing the polynucleotide molecule described in a);

[0058] c) A host cell containing a polynucleotide molecule as described in a), or a host cell containing an expression vector as described in b).

[0059] 5. A method for preparing a variant as described in any of items 1-3, characterized in that it comprises: (1) culturing host cells in the biological material as described in item 4, and (2) recovering the Phi29 DNA polymerase variant.

[0060] 6. A composition, characterized in that it comprises a Phi29 DNA polymerase variant as described in any one of items 1-3.

[0061] 7. A kit, characterized in that it comprises a Phi29 DNA polymerase variant as described in any one of items 1-3.

[0062] 8. Use of any of the Phi29 DNA polymerase variants described in items 1-3 in the synthesis of DNA.

[0063] 9. A method for preparing DNA, characterized in that the method comprises contacting a DNA template, primers, modified or unmodified nucleoside triphosphates with the Phi29 DNA polymerase variant described in item 1, incubating in a reaction system to obtain target DNA.

[0064] Beneficial Effects: The Phi29 DNA polymerase variant provided in this application exhibits higher DNA synthesis fidelity compared to the wild-type Phi29 DNA polymerase with the amino acid sequence shown in SEQ ID NO:1, effectively reducing errors during amplification and thus lowering the risk of nonspecific amplification. This improvement significantly enhances the safety and efficacy of DNA drugs in therapeutic applications. Attached Figure Description

[0065] Figure 1 This is a schematic diagram illustrating the construction of recombinant plasmids;

[0066] Figure 2 Results of detection of Phi29 DNA polymerase wild-type and mutant. Detailed Implementation

[0067] The technical solution of this application will be further described below with reference to specific embodiments. However, the following embodiments are merely examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application is determined by the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the art, and the experimental methods and techniques used are conventional methods and techniques in the art.

[0068] The definition of DNA polymerase activity (phi29): 1U unit refers to the amount of enzyme required to catalyze the incorporation of 0.5 pmoldNTPs into acid-insoluble matter in 10 minutes at 30°C.

[0069] Example 1: Preparation and expression determination of Phi29 DNA polymerase variant

[0070] A His tag was added to the front of the amino acid sequence SEQ ID NO: 1-12 of wild-type Phi29 DNA polymerase and its variants. After DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NO: 13-24). The DNA was then introduced into the NdeI and XhoI restriction sites of the expression vector pET-30a(+) to obtain a recombinant expression vector, which was transformed into E. coli BL21(DE3). After screening by antibiotic resistance plate plating, cloned strains were obtained. After successful recombination, the obtained strain was activated overnight at 37°C in LB medium, then added to 200 mL of fermentation broth (LB medium) and cultured until the OD600 value reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the strain was cultured for another 18 h (16°C) or 6 h (37°C). The strain was then collected by centrifugation at 12000 rpm at 5°C. The collected strain was washed with 0.2 M PBS buffer (pH 7.0), and 15 mL of buffer was added for ultrasonic disruption to obtain crude enzyme solution. The crude enzyme solution was pretreated with 0.3% PEI to remove nucleic acid impurities, precipitated with 60% saturated ammonium sulfate to remove PEI, and resuspended in buffer to the original volume. Purification was performed by nickel column affinity chromatography (His trap HP) to obtain Phi29 DNA polymerase solution, which was then dialyzed into a storage buffer (10×, 500 mM Tris-HCl, 50% v / v glycerol, 1M NaCl, 4 mM...). DTT (1 mM EDTA-2Na, pH 8.0) was prepared and sent to HPLC for concentration determination.

[0071] WT is the wild-type Phi29 DNA polymerase. The correspondence between Phi29 DNA polymerase variants and amino acid sequences is shown in Tables 1-1, 1-2, and 1-3.

[0072] Table 1-1:

[0073]

[0074] Table 1-2:

[0075]

[0076] Table 1-3:

[0077]

[0078] Example 2: Preparation of DNA by Rolling Circle Amplification

[0079] Add 1 μL of 10×phi29 polymerase buffer (1× formulation: 50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, (pH 7.5, 25°C)), 2 μL of modified random primers (100 μM), and 10 ng of DNA template I (SEQ ID NO: 25) to a PCR tube, and bring the volume up to 10 μL with water. The modified random primers are NNNN*N*N (N6), where * represents β-thiophosphate modification. After mixing, incubate at 95°C for 3 min; then incubate on ice for 20 min.

[0080] Table 2: Reaction System

[0081]

[0082] After the above reaction system was incubated on ice, the enzyme stock solution was diluted to 10 U / μL using a dilution buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, 0.05% Tween-20 (pH 7.5 25°C)) for the next reaction. The reaction system is shown in Table 3. The preparation conditions were as follows: 10 U phi29 DNA polymerase, 2 μL dNTPs, 1 μL inorganic pyrophosphatase, and 1 μL 10×phi29 DNA polymerase reaction buffer were added to the above reaction solution, and the volume was brought up to 20 μL with H2O. The reaction was carried out at 30°C for 18 h. After the reaction, the sample was denatured at 65°C for 10 min and the sample concentration was detected using an Equalbit 1× dsDNA HS Assay Kit. The mutation rate was detected by NGS sequencing at Sangon Biotech. The total point mutation rate was calculated as: total point mutations after subtracting the template from the AD / total number of sequencing bases (bp) * 100%. Relative fidelity (100%) = Total point mutation rate of DNA products prepared by wild-type phi29 DNA polymerase / Point mutation rate of DNA products prepared by mutant catalysis * 100%.

[0083] Table 3: Reaction System

[0084]

[0085] After testing, such as Figure 2 As shown in Table 4, compared with wild-type WT, all mutants significantly improved the fidelity of DNA products. Among them, the A484Q+S395T mutant significantly improved the product fidelity to 1074.63%, and the Y281H+S395T mutant significantly improved the product fidelity to 3242.60%.

[0086] Table 4: DNA Product Fidelity

[0087] 。

Claims

1. A variant of Phi29 DNA polymerase, characterized in that, The amino acid sequence of the variant is replaced by any of the following substitutions compared to SEQ ID NO: 1: A484N+S395T, A484Q+S395T, Y281H+S395T or S395T+K402R, and the amino acid sequence of the variant is shown in any of SEQ ID NO: 7, 8, 10 or 12.

2. A biomaterial, characterized in that, Selected from one or more of the following: a) Encoding a polynucleotide molecule of the Phi29 DNA polymerase variant as described in claim 1; b) An expression vector containing the polynucleotide molecule described in a); c) A host cell containing a polynucleotide molecule as described in a), or a host cell containing an expression vector as described in b).

3. The biomaterial as described in claim 2, characterized in that, The nucleotide sequence of the polynucleotide molecule is shown in any of SEQ ID NO: 19, 20, 22 or 24.

4. The method for preparing the variant as described in claim 1, characterized in that, include: (1) Culturing host cells in the biomaterial as described in claim 2, and (2) Recovering the Phi29 DNA polymerase variant.

5. A composition, characterized in that, It contains the Phi29 DNA polymerase variant as described in claim 1.

6. A reagent kit, characterized in that, It contains the Phi29 DNA polymerase variant as described in claim 1.

7. The use of the Phi29 DNA polymerase variant as described in claim 1 in the in vitro synthesis of DNA.

8. A method for preparing DNA, characterized in that, The method involves contacting a DNA template, primers, modified or unmodified nucleoside triphosphates with the Phi29 DNA polymerase variant described in claim 1, incubating them in a reaction system, and obtaining the target DNA.

9. The use of the Phi29 DNA polymerase variant as described in claim 1 in a DNA preparation method for improving the fidelity of the product DNA.

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