Thermal cycling transcription boosts RNA production and use thereof
Thermal cycling in vitro transcription addresses the challenges of secondary structure stabilization in mRNA production by enhancing T7 RNA polymerase tolerance, significantly increasing yields and reducing costs for mRNA and circRNA production, thus advancing mRNA therapeutics.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- MO DINGDING
- Filing Date
- 2024-12-31
- Publication Date
- 2026-07-09
AI Technical Summary
Current in vitro transcription methods, particularly using T7 RNA polymerase, face challenges in efficiently producing sequence-optimized mRNAs due to secondary structure stabilization, leading to transcription termination and DNA-RNA hybridization, which impedes the production of long and highly structured mRNAs, and limits the development of mRNA therapeutics.
Applying thermal cycling to in vitro transcription by incorporating a denature step up to 75 ℃, which allows T7 RNA polymerase to tolerate higher temperatures, effectively reducing secondary structures and enhancing transcription efficiency for both linear and circular RNA production.
Thermal cycling significantly increases the yield and reduces costs of producing sequence-optimized mRNAs, including large transcripts, and improves circRNA production through permuted intron-exon splicing, facilitating the development of mRNA therapeutics.
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Abstract
Description
Thermal cycling transcription boosts RNA production and use thereofTechnical Field
[0001] The disclosure relates to method of production of RNA and the using of the RNA produced in RNA vaccine and therapeutics.Background Art
[0002] mRNA therapeutics is rapidly developing. Several key obstacles for the development of mRNA medicine beyond mRNA vaccines are encountered. It is required to produce about 1,000-fold more protein to reach a therapeutic threshold than mRNA vaccines. Another issue is mRNA instability problem, which impede the efficient production, storage and delivery of mRNA drug. Recently studies showed that increased stability and translation capacity can be achieved by the increased GC ratio and stabilized RNA secondary structures, such as Linear Design algorithm optimized mRNAs. Thus, the next main step of mRNA therapeutics is the efficient production of such stability increased mRNAs.
[0003] Currently in vitro transcription (IVT) is the key approach of mRNA production. Significant increasing IVT efficiency will correspondingly dramatically reduce RNA production cost. IVT efficiency, especially the efficiency of T7 RNA polymerase, is extensively studied previously.
[0004] T7 RNA polymerase, the extensive-used in vitro transcription enzyme, is not always efficient to transcribe every individual mRNA. Sequence composition and size of the mRNA will affect transcription efficiency significantly. Particularly, large size and challenging template will hamper or even stop mRNA production. Thus, establishing a universal technology to produce the sequence optimized mRNA encoding any target protein is a must step of improving the modality and versatility of mRNA medicine.
[0005] For the transcription of these highly structured mRNAs, T7 RNA polymerase can be hindered down or even blocked by G-C rich sequences and highly stabilized hairpin-shaped secondary structures, which may resemble T7 terminator and cause intrinsic transcription termination, leading to low yields of run-off mRNA production or to the accumulation of prematurely truncated transcripts.
[0006] Finally, the accumulation of DNA-RNA complexes at constant temperature may prevent efficient RNA transcription. At constant temperature, the synthesized RNA product can hybridize or re-anneal with the DNA template, forming stable DNA-RNA complexes. The accumulation of these DNA-RNA hybrids can prevent the reinitiation of transcription by blocking access of the polymerase to the promoter region on the DNA template. This issue is particularly problematic when transcribing long mRNA sequences with high degrees of secondary structure. As the polymerase transcribes along the template, the nascent RNA transcript can start folding and annealing back to the DNA, leading to the formation of DNA-RNA hybrids that hinder further rounds of transcription.
[0007] Therefore, establishing a general technology to produce any sequence optimized mRNA encoding target protein is a necessary step to improve the pharmaceutical modality and versatility of mRNA.Summary of the Invention
[0008] To overcome at least one problem above, the inventor of the disclosure developed a new approach to robustly overcome the transcription obstacles of secondary structure stabilized mRNAs. Thermal cycling, well-used in PCR procedures, was applied to mRNA transcription. In the disclosure a denature step was added to mRNA transcription. T7 RNA polymerase, surprisingly tolerated the heating of IVT reaction up to 75 ℃. Fascinatingly, denature step effectively boost the transcription of these secondary structure stabilized mRNA, including big size transcripts (such as the 1.1 k nts mCherry mRNA and the 4.6 k nts Cas9 mRNA) . Specifically, the disclosure comprises the following contents.
[0009] In one aspect of the disclosure, it is provided a method for producing RNA through in vitro transcription comprising thermal cycling RNA transcription steps, wherein at least one cycle comprises a sub-step of enzyme-driven RNA transcription at a first temperature and a sub-step of melting at a second temperature; the first temperature is suitable for activating RNA polymerase, while the second temperature is suitable for melting nucleic acids, and the second temperature is higher than the first temperature.
[0010] In some embodiments of the method for producing RNA through in vitro transcription, wherein the first temperature is below 55℃.
[0011] In some embodiments of the method for producing RNA through in vitro transcription, wherein the second temperature is 56-75℃.
[0012] In some embodiments of the method for producing RNA through in vitro transcription, wherein the nucleic acid melting comprises the melting of at least one of DNA-DNA, DNA-RNA, and RNA-RNA.
[0013] In some embodiments of the method for producing RNA through in vitro transcription, wherein the time for melting the nucleic acid is 30 seconds to 1 minute.
[0014] In some embodiments of the method for producing RNA through in vitro transcription, wherein the RNA is selected from at least one of linear mRNA, circRNA, protein-coding RNA, and noncoding RNA.
[0015] In some embodiments of the method for producing RNA through in vitro transcription, wherein the cycle number is 5 or more, for example, 6 or more, 8 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more.
[0016] In some embodiments of the method for producing RNA through in vitro transcription, wherein the RNA polymerase comprises T7 RNA polymerase.
[0017] In some embodiments of the method for producing RNA through in vitro transcription, wherein the RNA sequence has undergone stability optimization.
[0018] In some embodiments of the method for producing RNA through in vitro transcription, wherein the RNA sequence is optimized using the Linear Design algorithm.
[0019] In some embodiments of the method for producing RNA through in vitro transcription, which comprises:
[0020] firstly, performing enzyme-driven RNA transcription at a first temperature, then performing nucleic acid melting at a second temperature, and repeating the above two steps multiple times.
[0021] In some embodiments of the method for producing RNA through in vitro transcription, which comprises:
[0022] firstly, performing the nucleic acid melting at a second temperature, then carrying out enzyme-driven RNA transcription at a first temperature, and repeating the above two steps multiple times.
[0023] In second aspect of the disclosure, it is provided the use of the method according to first aspect of the disclosure in the preparation of nucleic acid drugs.
[0024] In some embodiments of the use according to the second aspect of the disclosure, wherein the nucleic acid drug comprises a vaccine and / or a therapeutic agent.
[0025] Increased stability of sequence optimized mRNAs, such as the Linear Design algorithm, is very useful to overcome the mRNA instability problems in mRNA therapeutics. This new thermal cycling RNA transcription approach applies especially well with these high GC content and stability increased RNAs (Linear Design algorithm optimized mRNAs) .
[0026] For these long size (such as bigger than 2000 bp) and transcription challenging templates, the combination of sequence optimization by Linear Design algorithm and transcription by thermal cycling jointly improve their mRNA productions significantly, thus serving as universal approach of mRNA production.
[0027] Moreover, thermal cycling transcription also significantly increased the production of circRNA by permuted intron-exon (PIE) splicing strategy. It not only increased the production amount of the circRNA but also reduced the un-spliced precursor. Moreover, it reduced the processing step, thus saving the time and efforts.
[0028] Thermal cycling RNA transcription dramatically increase the efficiency of most, if not all, linear RNA and circRNA productions and reduce their costs, thus significantly facilitating RNA therapeutics development.Brief Description of the Drawings
[0029] Fig. 1. Thermal cycling transcription boost mCherry mRNA production. A. Schematic of the DNA template of mCherry mRNA (the mCherry ORF was optimized by Linear Design program) . 5’ UTR, 5' UTR of human hemoglobin subunit alpha 2 (HBA2) ; 3’ UTR, 3’ UTR of human hemoglobin subunit alpha (HBA1) ; poly A (97 nt) , the 97 nts poly A tail. B. Agarose gel (1.5%) electrophoresis of mCherry mRNA transcription products (phenol-chloroform purified) ; “37 ℃” , RNA transcription at 37 ℃ for 1 hour; “37 ℃, 55 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 55 ℃ for 1 minute; “37 ℃, 60 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 60 ℃ for 1 minute; “37 ℃, 65 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 65 ℃ for 1 minute; “37 ℃, 70 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 70 ℃ for 1 minute. DNA ladder was used a size marker. C. 8 M urea-polyacrylamide gel (3.75%) electrophoresis of phenol-chloroform purified mCherry mRNA transcription products; -, mCherry mRNA (1.1 k nt) ; *, transcription by-products. D.quantification of the mCherry mRNA amount in B. E. Agarose gel (1.5%) electrophoresis of purified mCherry mRNA transcribed in A and then capped with Vaccinia Virus Capping Enzyme. F. Western blot analysis of the HEK293T and A549 cells transfected with the purified and 5’ capped mCherry mRNA of two transcription methods ( “37 ℃” , “37 ℃, 70 ℃” ) ; -, mCherry protein; *, mCherry protein with the N-terminal 2.4 kDa additional protein sequence starting from the upstream AUG in the 5’ UTR region of mCherry mRNA. NC, negative control cells without transfection. G. Fluorescence of mCherry in the HEK293T and A549 cells transfected with the purified mCherry mRNA of two transcription methods ( “37 ℃” , “37 ℃, 70 ℃” ) ; BW, black and white view of cells; Red, red fluorescence of mCherry.
[0030] Fig. 2. Thermal cycling transcription boost other mRNA productions. A, B, C. Agarose gel (1.0%) electrophoresis of ISAam1 / SpuFz1-V2 / Cas9 mRNA transcription products (phenol-chloroform purified) ; “37 ℃” , RNA transcription at 37 ℃ for 1 hour; “37 ℃, 70 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 70 ℃ for 1 minute; Original, the original ISAam1 / SpuFz1-V2 / Cas9 sequence; Linear Design, Optimized ISAam1 / SpuFz1-V2 / Cas9 sequence by Linear Design algorithm; DNA ladder was used a size marker.
[0031] Fig. 3. Thermal cycling transcription boost circRNA productions. A, B, C, D. Agarose gel (1.0%) electrophoresis of circRNA production (phenol-chloroform purified) from various transcription conditions; “37 ℃” , RNA transcription at 37 ℃ for 1 hour; “37 ℃, 70 ℃” , 20 times thermal cycling of transcription at 37 ℃ for 3 minutes and denature at 70 ℃ for 1 minute; the other conditions were similarly performed. *and **, unspliced precursors.Detailed Description of Embodiments
[0032] A number of exemplary embodiments of the disclosure will now be described in detail. The detailed description should not be considered as a limitation on the disclosure, but should be construed as a more detailed description of certain aspects, features and embodiments of the disclosure.
[0033] It should be understood that the terminology described in the disclosure is only for describing specific embodiments and is not used to limit the disclosure. In addition, with regard to a numerical range in the disclosure, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Intermediate values between any stated values or within any stated range as well as any other stated values and every smaller range between intermediate values within the stated range are also included in the disclosure. The upper and lower limits of these smaller ranges can be independently included in or excluded from the range.
[0034] Unless otherwise specified, all the technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the field to which the disclosure belongs. Although the disclosure only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosure. All documents mentioned in the description are incorporated by reference to disclose and describe methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of the description shall prevail.
[0035] Efficient RNA production is the prerequisite of RNA therapeutics. Currently mRNA is produced by in vitro transcription through RNA polymerase, such as T7 or SP6 RNA polymerase. The classic in vitro RNA transcription is the incubation of DNA template with rNTP and T7 RNA polymerase in reaction buffer at a constant temperature (normally 37 ℃) . But the success of such in vitro transcription is always depended on the sequence composition and size of the template. In some cases, especially for the long size RNA, efficient transcription is always challenging or even impossible.
[0036] The inventor of the disclosure developed a new approach to robustly overcome the transcription obstacles of secondary structure stabilized mRNAs. Thermal cycling, well-used in PCR procedures, was applied to mRNA transcription. In the disclosure a denature step was added to mRNA transcription. T7 RNA polymerase, surprisingly tolerated the heating of IVT reaction up to 75 ℃. Fascinatingly, denature step effectively boost the transcription of these secondary structure stabilized mRNA, including big size transcripts (such as the 1.1 k nts mCherry mRNA and the 4.6 k nts Cas9 mRNA) .
[0037] The denature step may reduce or even remove the intrinsic RNA secondary structure, thus minimizing the T7 terminator resembling structures mediated transcription terminations. The denature step may also promote the release of RNA transcript from DNA and facilitate downstream sequence transcription, or the diffusion of T7 RNA polymerase back to the promoter region of DNA template again to reinitiate transcription. Thus, thermal cycling transcription enabled the efficient production of highly stabilized mRNA, significantly promoting the development of mRNA medicine.
[0038] For the transcription of difficult templates (such as 2459 nts SpuFz1-V2 mRNA and 4630 nts Cas9 mRNA) , the combination of sequence optimization by Linear Design algorithm and transcription by thermal cycling jointly improve their mRNA productions significantly, thus representing a universal mRNA production approach of any individual protein, correspondingly boosting the therapeutic modality and versatility of mRNA therapeutics.
[0039] Furthermore, thermocycling transcription also significantly increased circRNA production via the permuted intron-exon (PIE) splicing strategy. It not only increases circRNA production but also reduces un-spliced precursors. Additionally, it reduces processing steps, saving time and effort.
[0040] Thermocycling RNA transcription significantly increases the efficiency and reduces the cost of most, if not all, linear RNA and circRNA production, thereby significantly facilitating the development of RNA therapeutics.
[0041] Example 1
[0042] Material and Methods
[0043] DNA template preparation
[0044] The mRNA expression plasmid is synthesized at Genscript. The expression cassette includes T7 promoter, 5’ UTR, mCherry coding sequence, 3’ UTR and 97 nt polyA. The expression cassette was PCR amplified, BspQI digested and phenol-chloroform purified as transcription template. mCherry mRNA sequence was optimized by Linear Design algorithm.
[0045] The DNA sequence of mCherry mRNA:
[0046] RNA transcription and 5’ capping
[0047] RNA transcription was performed with following conditions: 100 mM Hepes-K (pH 7.9) , 24 mM MgCl2, 30 mM DTT, 4 mM Spermidine, 4 mM rNTP each, 200 U / ml RNase inhibitor (10777019, Invitrogen) , 10 U / ml thermostable inorganic pyrophosphatase (M0296L, NEB) , 0.16 mg / ml T7 RNA polymerase (lab-made) , 50 μg / ml DNA template with T7 promoter. For mRNA synthesis, N1-Me-Pseudo UTP (HBP002501, Hzymes) was used instead of UTP. RNA transcription was performed either at constant temperature, such as 37 ℃ or 42 ℃, or at thermo cycling temperatures. Thermal cycling RNA transcription: 20 times thermal cycling of transcription at RNA polymerase active temperature (37 ℃, 42 ℃) for 3 minutes and denature at high temperature (55 ℃-75 ℃) for 1 minute. mRNA was capped with vaccinia virus capping enzyme (HBP000611, Hzymes) .
[0048] RNA purification
[0049] RNA transcripts were digested with 80 U / ml DNase I (04716728001, Roche) to remove DNA templates and then phenol-chloroform purification was performed to remove proteins. Finally, RNA was purified from agarose gel electrophoresis-electroelution.
[0050] Cell culture and RNA transfection
[0051] HEK293T were cultured in DMEM medium (C11995500CP, Gibco) , supplemented with 10%fetal bovine serum (10091148, Gibco) , 100 U / ml penicillin and 100 U / ml streptomycin (15140122, Gibco) at 37 ℃ in 5% (v / v) CO2.3 μg mRNA was transfected with 5 μl LipofectamineTM MessengerMAX (LMRNA003, Invitrogen) in 6 well plate.
[0052] Thermal cycling boosts mCherry mRNA production
[0053] The routine RNA transcription was performed at constant temperature, such as 37 ℃.
[0054] A mCherry mRNA in vitro transcription system was constructed. The DNA template includes T7 promoter, 5’ UTR (from human hemoglobin subunit alpha 2) , mCherry open reading frame, 3’ UTR (from human hemoglobin subunit alpha 1) and poly A tail (97 nt) (Fig. 1A) . As shown in Fig. 1B, mCherry mRNA transcription at 37 ℃ generated a moderate level of mRNA. The inventor performed thermal cycling RNA transcription which is described in details in the methods section. Agarose gel electrophoresis shown that the addition of denature step at 55 ℃, 60 ℃, 65 ℃, or 70 ℃ dramatically increased the mRNA yield (Fig. 1B) . Quantification of Fig. 1B shown that thermal cycling increased RNA production from 1.7-fold to 2.8-fold (Fig. 1D) .
[0055] To verify the mRNA length of thermal cycling transcription, we performed 8 M urea-polyacrylamide gel (3.75%) electrophoresis of these mCherry mRNAs. As shown in Fig. 1C, thermal cycling transcription produced the same length of mCherry mRNA run-off product as the constant 37 ℃ transcription, plus with significantly increased yields. The short by-products were only moderate increased (Fig. 1C) . After capped with vaccinia virus capping enzyme, mCherry mRNAs were purified through an agarose gel electrophoresis-electroelution procedure. The purified mCherry mRNAs produced at constant 37 ℃ or at thermal cycling transcription ( “37 ℃, 70 ℃” ) were analyzed by agarose gel electrophoresis. They demonstrated the same unique migration pattern in agarose gel (Fig. 1E) . Furthermore, I transfected these two purified mRNAs into HEK293T and A549 cells. After 18 hours transfection, both of them expressed almost the same level of mCherry protein in both HEK293T and A549 cells, demonstrated by both Western blot analysis of mCherry protein with anti-mCherry antibody (Fig. 1F) and the direct mCherry red fluorescence (Fig. 1G) . All of these evidences prove that thermal cycling generated significantly more intact mCherry mRNA transcription run-off molecules than the constant 37 ℃ transcription, and these mRNAs maintained the same translation capacity.
[0056] Thermal cycling boosts the transcription of LinearDesign algorithm optimized mRNAs
[0057] As thermal cycling transcription significantly improve mCherry mRNA production, we would like to explore whether such new approach could apply to other mRNA synthesis. The open reading frame of three genome editors (ISAam1, SpuFz1-V2 and Cas9) were inserted into the mRNA expression cassette (in replace of mCherry) , including two types of mRNA, one was the original mRNA sequence and the other type was optimized by LinearDesign algorithm. Six DNA templates were prepared as similarly as the mCherry DNA template by PCR.
[0058] Surprisingly, as shown in Fig. 2A, mRNA transcription of the original 1661 nts ISAam1 sequence at constant 37 ℃ produced little more intact mRNA than thermal cycling ( “37 ℃, 70 ℃” ) transcription (Fig. 2A) . Such result is possibly caused by the instability of the original ISAam1 mRNA sequence, as thermal cycling has a denature step at 70 ℃. But for the LinearDesign algorithm optimized ISAam1 mRNAs, its transcription at constant 37 ℃ had much better performance (Fig. 2A) . Thermal cycling further increased its transcription significantly (Fig. 2A) .
[0059] Interestingly, the transcription of original 2459 nts SpuFz1-V2 mRNA at both constant 37 ℃ and thermal cycling ( “37 ℃, 70 ℃” ) only produced very limited mRNA (Fig. 2B) , reflecting that it is a challenging template for transcription. LinearDesign algorithm optimized SpuFz1-V2 has improved mRNA transcription output at 37 ℃ (Fig. 2B) . Thermal cycling ( “37 ℃, 70 ℃” ) further dramatically boosts its transcription (Fig. 2B) .
[0060] Furthermore, the original 4630 nts Cas9 mRNA expression cassette produced very limited mRNA output at constant 37 ℃ (Fig. 2C) . Thermal cycling transcription ( “37 ℃, 70 ℃” ) dramatically improved the mRNA output (Fig. 2C) . Surprisingly, LinearDesign algorithm optimized Cas9 mRNA also had low level of transcription at constant 37 ℃. But thermal cycling transcription ( “37 ℃, 70 ℃” ) once again significantly improved the mRNA output (Fig. 2C) .
[0061] In conclusion, thermal cycling boosts the transcription of LinearDesign algorithm optimized mRNAs.
[0062] Thermal cycling boosts circRNA production
[0063] Circular RNA (circRNA) has recently been shown the high efficiency of translation capacity and low immunogenicity both in cell lines and tested animals, thus representing the next generation of mRNA therapeutics. Currently, circRNA is produced by permuted intron-exon (PIE) splicing strategy of Group I intron or Group II intron. The transcription of circRNA precursor is followed by a splicing step. Un-spliced by-products are inevitably accumulated in the process. In some cases, only about half of the transcripts were circRNA and a lot by-products were accumulated, which make the purification complicated.
[0064] The inventor applied the invented thermal cycling strategy to circRNA synthesis. Such approach not only improved RNA transcription, but also boosted the splicing of permuted intron-exon, thus significantly improving the production of circRNA. As shown in Figure 3A, when the transcription was at 37 ℃, at the denature temperature from 56 ℃ to 75 ℃ could dramatically improve circRNA production. Figure 3B, C shown that the transcription temperature could be up to 55 ℃. when the first transcription temperature was beyond 55 ℃, the denature step would not improve RNA transcription and circRNA production (Figure 3D) .
[0065] Although the disclosure has been described with reference to exemplary embodiments, it should be understood that the disclosure is not limited to the disclosed exemplary embodiments. Without departing from the scope or spirit of the disclosure, various adjustments or changes can be made to the exemplary embodiments in the description of the disclosure. The scope of the claims should be based on the broadest interpretation in order to cover all modifications and equivalent structures and functions.
Claims
1.A method for producing RNA through in vitro transcription, characterized in that it comprises thermal cycling RNA transcription steps, wherein at least one cycle comprises a sub-step of enzyme-driven RNA transcription at a first temperature and a sub-step of melting at a second temperature; the first temperature is suitable for activating RNA polymerase, while the second temperature is suitable for melting nucleic acids, and the second temperature is higher than the first temperature.2.The method for producing RNA through in vitro transcription according to claim 1, wherein the first temperature is below 55℃.3.The method for producing RNA through in vitro transcription according to claim 1, wherein the second temperature is 56-75℃.4.The method for producing RNA through in vitro transcription according to claim 1, wherein the nucleic acid melting comprises the melting of at least one of DNA-DNA, DNA-RNA, and RNA-RNA.5.The method for producing RNA through in vitro transcription according to claim 1, wherein the time for melting the nucleic acid is 30 seconds to 1 minute.6.The method for producing RNA through in vitro transcription according to claim 1, wherein the RNA is selected from at least one of linear mRNA, circRNA, protein-coding RNA, and noncoding RNA.7.The method for producing RNA by in vitro transcription according to claim 1, wherein the cycle number is 5 or more, for example, 6 or more, 8 or more, 10 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more.8.The method for producing RNA through in vitro transcription according to claim 1, wherein the RNA polymerase comprises T7 RNA polymerase.9.The method for producing RNA through in vitro transcription according to claim 1, wherein the RNA sequence has undergone stability optimization.10.The method for producing RNA through in vitro transcription according to claim 1, wherein the RNA sequence is optimized using the Linear Design algorithm.11.The method for producing RNA through in vitro transcription according to claim 1, which comprises:firstly, performing enzyme-driven RNA transcription at a first temperature,then performing nucleic acid melting at a second temperature, andrepeating the above two steps multiple times.12.The method for producing RNA through in vitro transcription as claimed in claim 1 which comprises:firstly, performing the nucleic acid melting at a second temperature,then carrying out enzyme-driven RNA transcription at a first temperature, andrepeating the above two steps multiple times.13.The use of the method according to any one of claims 1-12 in the preparation of nucleic acid drugs.14.The use according to claim 13, wherein the nucleic acid drug comprises a vaccine and / or a therapeutic agent.