A method for enzymatic amplification of DNA and its use

Abstract

The application provides a DNA enzymatic amplification method and application thereof, and the method comprises the following steps: performing rolling circle amplification on circular DNA containing a target DNA sequence in the presence of a DNA polymerase with strand displacement activity, the target DNA sequence containing a polyA-polyT structure, a forward primer in the at least one pair of specific primers containing a polyA sequence or a reverse primer containing a polyT sequence or the forward primer containing a polyA sequence and the reverse primer containing a polyT sequence, and the reaction system of the rolling circle amplification containing dNTP and a reaction buffer; and cutting the rolling circle amplification product into uniform linear double-stranded DNA fragments by using a restriction endonuclease. Through the cooperation of the polyA or polyT sequence in the specific primer and the polyA-polyT structure in the template, the DNA purity is improved, and the generation of dsRNA by-products in the subsequent in vitro transcription process is reduced.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to enzymatic synthesis technology of DNA, and in particular to a method for enzymatic amplification of DNA and its application. Background Technology

[0002] Following the success of preventative vaccines, mRNA therapy is rapidly expanding into fields such as tumor immunotherapy and protein replacement therapy. Personalized tumor immunotherapy is one of the most promising directions, demonstrating groundbreaking clinical effects in the treatment and recurrence prevention of malignant tumors such as melanoma, pancreatic cancer, and kidney cancer.

[0003] Personalized tumor immunotherapy requires customized mRNA drugs targeting the patient's tumor neoantigens. Due to the short survival time of cancer patients and the easy mutation of tumor antigens, the development cycle of personalized tumor immunotherapy should be at least 4 weeks, and the shorter the cycle, the better the efficacy.

[0004] The classic mRNA production process involves four main steps: molecular cloning, plasmid fermentation amplification, IVT synthesis, and LNP encapsulation. The first two steps take approximately three weeks in total, and the use of *E. coli* cells necessitates library construction for GMP production, adding an additional four weeks. Therefore, developing a cell-independent DNA synthesis technology is essential for personalized tumor immunotherapy. Recently, Pfizer developed a DNA synthesis technology based on oligonucleotide fragment assembly and rolling circle amplification (RCA) (Ghosh S). et al., (NPJ Vaccines 2026). However, the DNA prepared using this technology, when used as an IVT template, exhibits a significantly higher dsRNA byproduct compared to mRNA prepared using classical linearized plasmid DNA as a template. dsRNA is widely recognized as the most significant immunogenic impurity in mRNA drugs. The 2023 Nobel Prize in Physiology or Medicine was awarded for techniques that reduce mRNA immunogenicity. Therefore, developing a cell-independent DNA synthesis technology that can effectively control dsRNA production during IVT is urgently needed. Summary of the Invention

[0005] The purpose of this application is to provide a DNA enzymatic amplification method and its application, in order to solve the technical problem that the content of double-stranded RNA byproducts in the mRNA product is significantly increased when DNA templates prepared by cell-free DNA synthesis technology based on rolling circle amplification are used for in vitro transcription.

[0006] To achieve the above objectives, this application adopts the following technical solution: A method for DNA enzymatic amplification includes the following steps: (1) In the presence of a DNA polymerase with strand displacement activity, a rolling circle amplification is performed on a circular DNA containing a target DNA sequence using at least one pair of specific primers, wherein the circular DNA contains a polyA-polyT structure, and wherein the at least one pair of specific primers contains either a polyA sequence in the forward primer or a polyT sequence in the reverse primer, or both the forward primer and the reverse primer contain a polyT sequence; the reaction system for the rolling circle amplification includes dNTPs and a reaction buffer. (2) Use restriction endonucleases to cut the rolling circle amplification product into uniform linear double-stranded DNA fragments.

[0007] This method employs specific primers containing polyA or polyT sequences in rolling circle amplification, ensuring stable complementary pairing between the polyA or polyT sequence in the primers and the polyA-polyT structure in the circular DNA. This guarantees the specificity of the amplification and the uniformity of the product, thereby reducing non-specific transcription and the formation of dsRNA byproducts during subsequent in vitro transcription.

[0008] Furthermore, the length of the polyA sequence in the forward primer is selected from 10-25 nucleotides, preferably 13-22 nucleotides, more preferably 13, 19, or 22 nucleotides, and / or the length of the polyT sequence in the reverse primer is selected from 10-25 nucleotides, preferably 13-22 nucleotides, more preferably 13, 19, or 22 nucleotides. Suitable polyA or polyT sequence lengths ensure effective binding of the primer to the template and amplification efficiency, while maintaining a low level of dsRNA byproducts.

[0009] In some embodiments, the specific primers further contain 6-11 nucleotides that specifically bind to the circular DNA. By introducing specific recognition sequences into the primers, the specificity of primer-template binding is further improved, optimizing amplification efficiency and product purity.

[0010] In some embodiments, the at least one pair of specific primers is selected from one pair of specific primers, two pairs of specific primers, or three pairs of specific primers.

[0011] In some embodiments, the concentration of the at least one pair of specific primers is selected from 5-50 μM, preferably 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM or 50 μM.

[0012] In some implementations, the specifically binding nucleotide sequence is located at the 5' end of the polyA or polyT sequence and is directly linked to it. This design allows the specifically recognized sequence to bind to the template first, followed by the extension of the polyA or polyT sequence, ensuring the accuracy and efficiency of amplification.

[0013] In some embodiments, the DNA polymerase with strand displacement activity is phi29 DNA polymerase. It is understood that the DNA polymerase with strand displacement activity is not limited to phi29 DNA polymerase, but may be other DNA polymerases with strand displacement activity, such as one or more of Bst DNA polymerase, Bsm DNA polymerase, and Bca DNA polymerase.

[0014] In some embodiments, the concentration of the phi29 DNA polymerase is 2 U / μl. In some embodiments, the recognition site of the restriction endonuclease is located at the 5' end of the target DNA or the specific primer. The rolling circle amplification product contains the recognition sites arranged at equal intervals, such that the restriction endonuclease cleavage in step (2) yields a uniformly long linear double-stranded DNA fragment.

[0015] Furthermore, the restriction endonuclease is BspQI. BspQI can recognize and cleave specific sequences in rolling circle amplification products, homogenizing long-chain DNA into linear double-stranded DNA fragments of specific lengths, meeting the requirements of in vitro transcription templates. It is understood that the restriction endonuclease is not limited to BspQI, but can also be other restriction endonucleases capable of recognizing and cleaving specific sequences in RCA products, such as BsaI, BsmBI, Esp3I, XbaI, PmeI, EcoRI, SapI, HindIII, BamHI, XhoI, EcoRV, SalI, PstI, SmaI, NotI, KpnI, SacI, SphI, EcoRII, BglII, AvaI, NdeI, SstI, BstEII, and HpaI.

[0016] In some embodiments, the rolling circle amplification is performed under isothermal conditions, with a reaction temperature of 20°C to 70°C and a reaction time of 30 minutes to 24 hours. In some embodiments, the rolling circle amplification reaction temperature is 30°C and the reaction time is 24 hours.

[0017] In some embodiments, the final concentration of dNTPs in the rolling circle amplification reaction system is 0.2–5.0 mM, preferably 1.0–3.0 mM, and more preferably 2.0 mM. In some embodiments, the Mg in the rolling circle amplification reaction system... 2+The concentration is 1.0–20.0 mM, preferably 10.0–20.0 mM, and more preferably 11.0 mM, 12.0 mM, 13.0 mM, 14.0 mM, 15.0 mM, 16.0 mM, 17.0 mM, 18.0 mM, or 19.0 mM. In some embodiments, the reaction buffer contains one or more of Tris-HCl, KCl, (NH4)2SO4, and DTT.

[0018] Specifically, the circular DNA is plasmid DNA. Plasmid DNA, as a circular DNA template, has a stable structure, is easy to prepare, and is suitable for rolling circle amplification reactions.

[0019] In some embodiments, the circular DNA is artificially synthesized circular DNA.

[0020] This application also provides a method for preparing mRNA, comprising the following steps: (1) Linear double-stranded DNA was prepared using the above-mentioned DNA enzymatic amplification method; (2) Using the obtained linear double-stranded DNA as a template, in vitro transcription was performed to obtain mRNA.

[0021] Using linear double-stranded DNA prepared by the DNA enzymatic amplification method of this application as a template can significantly reduce the generation of dsRNA byproducts during in vitro transcription and improve the quality of mRNA products.

[0022] Furthermore, the in vitro transcription employs T7 RNA polymerase. T7 RNA polymerase recognizes the T7 promoter sequence and efficiently catalyzes RNA synthesis. It is understood that the RNA polymerase used in the in vitro transcription is not limited to T7 RNA polymerase, but may also be other enzymes such as SP6 RNA polymerase or T3 RNA polymerase.

[0023] In some embodiments, the in vitro transcription reaction is carried out at a temperature of 37°C for 2 hours. These reaction conditions ensure transcription efficiency while maintaining enzyme activity and the stability of the reaction system.

[0024] This application also provides the use of the linear double-stranded DNA prepared by the above-described DNA enzymatic amplification method in RNA preparation. In some embodiments, the RNA is mRNA. Using the linear double-stranded DNA prepared by the method of this application as an in vitro transcription template can significantly reduce the content of dsRNA byproducts in the mRNA product, thereby improving the quality and safety of the mRNA.

[0025] Furthermore, the mRNA is used in vaccine preparation. Low dsRNA content in the mRNA can reduce immunogenic impurities in the vaccine, improving its safety and efficacy.

[0026] Specifically, the vaccine is a personalized tumor immunotherapy vaccine. Personalized tumor immunotherapy vaccines require rapidly customized mRNA drugs targeting the patient's tumor antigens. The method described in this application can prepare high-quality mRNA templates in a short time, meeting the needs of personalized tumor immunotherapy.

[0027] Other implementation plans: 1. A method for DNA enzymatic amplification, comprising the following steps: (1) In the presence of a DNA polymerase with strand displacement activity, a rolling circle amplification is performed on a circular DNA containing a target DNA sequence using at least one pair of specific primers, wherein the circular DNA contains a polyA-polyT structure, and wherein the at least one pair of specific primers contains either a polyA sequence in the forward primer or a polyT sequence in the reverse primer, or both the forward primer and the reverse primer contain a polyT sequence; the reaction system for the rolling circle amplification includes dNTPs and a reaction buffer. (2) Use restriction endonucleases to cut the rolling circle amplification product obtained in step (1) into uniform linear double-stranded DNA fragments.

[0028] 2. The DNA enzymatic amplification method according to item 1, characterized in that the length of the polyA sequence is selected from 10-25 nucleotides, and / or the length of the polyT sequence is selected from 10-25 nucleotides.

[0029] 3. The DNA enzymatic amplification method according to item 1, characterized in that the specific primer further comprises 6-11 nucleotides that specifically bind to the circular DNA.

[0030] 4. The DNA enzymatic amplification method according to item 3, characterized in that the specifically bound nucleotide sequence is located at the 5' end of the polyA sequence or the polyT sequence and is directly linked to it.

[0031] 5. The DNA enzymatic amplification method according to item 1, characterized in that the DNA polymerase with strand displacement activity is phi29 DNA polymerase.

[0032] 6. The DNA enzymatic amplification method according to item 5, characterized in that the concentration of the phi29 DNA polymerase is 2 U / μl.

[0033] 7. The DNA enzymatic amplification method according to item 1, characterized in that the restriction endonuclease is BspQI.

[0034] 8. The DNA enzymatic amplification method according to item 5, characterized in that the rolling circle amplification reaction temperature is 30°C and the reaction time is 24 hours.

[0035] 9. The DNA enzymatic amplification method according to item 1, wherein the circular DNA is plasmid DNA.

[0036] 10. The DNA enzymatic amplification method according to item 1, characterized in that the recognition site of the restriction endonuclease is pre-existing at the 5' end of the circular DNA or the specific primer.

[0037] 11. A method for preparing mRNA, characterized by comprising the following steps: (1) Linear double-stranded DNA was prepared using the DNA enzymatic amplification method described in items 1-10; (2) Using the linear double-stranded DNA obtained in step (1) as a template, in vitro transcription was performed to obtain mRNA.

[0038] 12. The method for preparing mRNA according to item 11, characterized in that the in vitro transcription is performed using T7 RNA polymerase.

[0039] 13. The method for preparing mRNA according to item 11, characterized in that the in vitro transcription reaction temperature is 37°C and the reaction time is 2 hours.

[0040] 14. Use of the linear double-stranded DNA prepared by the method described in any one of items 1-10 in the preparation of mRNA.

[0041] 15. The use according to item 14, characterized in that the mRNA is used to prepare a vaccine.

[0042] 16. The use according to item 15, characterized in that the vaccine is a personalized tumor immunotherapy vaccine.

[0043] Beneficial effects 1. By using specific primers containing polyA or polyT sequences for rolling circle amplification, the polyA or polyT sequences in the primers form a stable complementary pair with the polyA-polyT structure in the circular DNA template, ensuring the specificity of amplification and the uniformity of the product. After homogenization with restriction endonucleases, the DNA purity is significantly improved. When used as a template for in vitro transcription, compared with rolling circle amplification using random primers, the content of dsRNA byproducts in the mRNA product is reduced by about 70%, approaching the level of plasmid DNA template. This effectively reduces the immunogenicity impurities of mRNA drugs and improves the safety and efficacy of the drugs. 2. The method of this application adopts cell-free DNA amplification technology, which does not require molecular cloning and plasmid fermentation amplification steps, avoids the cumbersome process of E. coli cell culture and strain library construction during GMP production, and greatly shortens the development cycle of mRNA drugs. It is particularly suitable for application scenarios with strict requirements for development cycle, such as personalized tumor immunotherapy. 3. When the linear double-stranded DNA prepared by the method of this application is used as an in vitro transcription template, the integrity of the IVT product is significantly improved, and the concentration of the mRNA product is comparable to that of the plasmid DNA template, ensuring efficient synthesis and high yield of mRNA, which is more in line with the requirements of mRNA drug manufacturing. Attached Figure Description

[0044] Figure 1 This is a bar chart comparing the concentrations of rolling circle amplification products from random primers and specific primers in Example 1. Figure 2 The image shows the agarose gel electrophoresis of the RCA product after BspQI digestion and homogenization in Example 2. Lane 1: RCA (N6), Lane 2: RCA (G1), Lane 3: RCA (G2), Lane 4: RCA (G3), Lane 5: RCA (G4), Lane 6: RCA (G5), Lane 7: RCA (G6), Lane 8: RCA (G7), Lane 9: Marker (5000bp), Lane 10: pDNA. Figure 3 This is a bar chart comparing the mRNA concentrations of IVT products from different primer sets in Example 3. Figure 4 This is a bar chart comparing the dsRNA content of IVT products from different primer sets in Example 3; Figure 5 This is a comparative bar chart showing the concentrations of rolling circle amplification products from various poly(A) and / or poly(T) specific primers in Example 4; Figure 6 This is a bar chart comparing the dsRNA residual rate and plasmid DNA ratio of different primer sets in Example 5. Figure 7 This is a schematic diagram of plasmid DNA from Example 1. Detailed Implementation

[0045] 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.

[0046] Example 1: Rolling circle amplification using random primers (NNNNNN, i.e., N6, 3' end 2nt modified with thiophosphate ester bond) or specific primers. Prepare the annealing reaction solution according to the reaction system (10 μl) in Table 1, place it in a PCR instrument at 95℃ for 3 minutes, and then immediately place it on ice for 10 minutes.

[0047] Table 1:

[0048] Prepare the reaction solution according to the reaction system (10 μl) in Table 2, add it to the annealed reaction solution in Table 1, place it in a PCR instrument at 30°C for 24 hours, and then terminate the reaction by treating at 65°C for 10 minutes.

[0049] Table 2:

[0050] The concentration of rolling circle amplification products was quantified using the Equalbit 1×dsDNA HS Assay Kit (Vazyme EQ121-01). After following the instructions, the samples were analyzed using a Qubit fluorometer (ThermoFisher Scientific). Results are as follows: Figure 1 As shown, both random primers and specific primers can be used for effective amplification, and the product concentrations are comparable.

[0051] Example 2: Homogenization of Rolling Circle Amplification Products Prepare the reaction solution according to the reaction system (20 μl) in Table 3, and place it in a PCR instrument at 50℃ for 4 hours.

[0052] Table 3:

[0053] The reaction products were detected by agarose gel electrophoresis. The results are as follows: Figure 2 As shown, BspQI effectively homogenized the RCA product, obtaining two target linear DNA fragments of 1,242 bp and 2,434 bp. The DNA product was purified using DNA purification magnetic beads (VAHTS DNA Clean Beads, Vazyme N411-01) according to the manufacturer's instructions, and the DNA concentration was then determined using a micro-volume UV spectrophotometer (One Drop 1000+, Wuyi Technology).

[0054] Example 3: In vitro transcription Prepare the reaction solution according to the reaction system (20 μl) in Table 4, and place it in a PCR instrument at 37℃ for 2 hours.

[0055] Table 4:

[0056] The obtained mRNA product was purified, and the concentration of the purified product was determined using a micro-volume UV spectrophotometer (One Drop 1000+, Wuyi Technology). Impurity dsRNA was quantified using a dsRNA (modification) quantification kit (Vazyme DD3509). The results are as follows: Figure 3 , 4 As shown.

[0057] Example 4: Rolling circle amplification using various specific primers containing poly(A) and / or poly(T) Using random primers as a control, rolling circle amplification was performed using a variety of specific primers (SEQ ID NO: 2-16) containing poly(A) and / or poly(T), as described in Example 1. The amplification results are as follows: Figure 5 As shown.

[0058] Example 5: Homogenization of rolling circle amplification products and in vitro transcription using them as templates Using plasmid DNA (SEQ ID NO: 1) as a control, the RCA product obtained in Example 4 was homogenized and transcribed in vitro according to the methods of Examples 2 and 3. The ratio of dsRNA residual rate to plasmid DNA in the RCA group is shown in the figure. Figure 6 As shown.

[0059] The comparative data from Example 5 show that using specific primers containing polyA and / or polyT sequences for rolling circle amplification, followed by homogenization, as a template for in vitro transcription significantly reduced the content of dsRNA byproducts in the mRNA product compared to the method using random primers. The dsRNA content of the random primer N6 group was approximately 10 times that of the plasmid DNA group, while the optimal polyA / polyT specific primer group (such as 11NF / 19A, 22T / 22A, 6N13T / 10NR) reduced the dsRNA content to approximately 3 times that of the plasmid DNA group, a reduction of about 70%, approaching the plasmid DNA template level. This indicates that the introduction of specific primers containing polyA / polyT sequences significantly improved the quality of the RCA product as an IVT template and effectively reduced dsRNA immunogenic impurities, an effect that was unexpected.

[0060] Table 6: List of Sequences .

Claims

1. A method for DNA enzymatic amplification, comprising the following steps: (1) In the presence of a DNA polymerase with strand displacement activity, a rolling circle amplification is performed on a circular DNA containing a target DNA sequence using at least one pair of specific primers, wherein the circular DNA contains a polyA-polyT structure, and wherein the at least one pair of specific primers contains either a polyA sequence in the forward primer or a polyT sequence in the reverse primer, or both the forward primer and the reverse primer contain a polyT sequence; the reaction system for the rolling circle amplification includes dNTPs and a reaction buffer. (2) Use restriction endonucleases to cut the rolling circle amplification product obtained in step (1) into uniform linear double-stranded DNA fragments.

2. The DNA enzymatic amplification method according to claim 1, characterized in that, The length of the polyA sequence is selected from 10-25 nucleotides, and / or the length of the polyT sequence is selected from 10-25 nucleotides.

3. The DNA enzymatic amplification method according to claim 1, characterized in that, The specific primer also contains 6-11 nucleotides that specifically bind to the circular DNA.

4. The DNA enzymatic amplification method according to claim 3, characterized in that, The specifically bound nucleotide sequence is located at the 5' end of the polyA or polyT sequence and is directly linked to it.

5. The DNA enzymatic amplification method according to claim 1, characterized in that, The DNA polymerase with strand displacement activity is selected from phi29 DNA polymerase, Bst DNA polymerase, Bsm DNA polymerase or Bca DNA polymerase.

6. The DNA enzymatic amplification method according to claim 1, characterized in that, The restriction endonuclease is selected from BspQI, BsaI, BsmBI, Esp3I, XbaI, PmeI, EcoRI, SapI, HindIII, BamHI, XhoI, EcoRV, SalI, PstI, SmaI, NotI, KpnI, SacI, SphI, EcoRII, BglII, AvaI, NdeI, SstI, BstEII, or HpaI.

7. The DNA enzymatic amplification method according to claim 1, characterized in that, The restriction endonuclease recognition site is located at the 5' end of the target DNA or the specific primer.

8. A method for preparing mRNA, characterized in that, Includes the following steps: (1) Linear double-stranded DNA is prepared using the DNA enzymatic amplification method described in claim 1; (2) Using the linear double-stranded DNA obtained in step (1) as a template, in vitro transcription was performed to obtain mRNA.

9. The method for preparing mRNA according to claim 8, characterized in that, The in vitro transcription was performed using T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase.

10. Use of the linear double-stranded DNA prepared by the method of any one of claims 1-7 in the preparation of RNA.

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