Preparation of single-stranded linear DNA and application of single-stranded linear DNA in production of RNA vaccine

Abstract

The invention provides a cell-free RNA (Ribonucleic Acid) synthesis method which is named as a single-stranded template in-vitro transcription method, wherein single-stranded linear DNA is prepared by amplifying double-stranded annular DNA and is used as an IVT template. Wherein the double-stranded circular DNA comprises a nicking enzyme binding and cleavage site and an RNA polymerase binding site, and the method comprises the following steps: a) contacting the double-stranded circular DNA with a nicking enzyme capable of recognizing the nicking enzyme binding and cleavage site to obtain a mixture; b) contacting the mixture in the step a) with constant-temperature DNA polymerase for rolling circle amplification to obtain single-chain linear multi-conjoined DNA; c) annealing the oligonucleotide fragment with the RNA polymerase binding site to form a local double strand; and d) contacting the local double strand with an RNA polymerase to initiate transcription. According to the method disclosed by the invention, large-scale RNA production can be carried out only by a small amount of plasmid templates, and a complete GMP three-level strain library does not need to be established. The DNA template is synthesized by adopting an in-vitro enzyme method, and then the RNA is synthesized, so that the raw materials are clear, and the system is simple.

Description

Technical Field

[0001] The present invention mainly relates to the field of biotechnology, and particularly relates to a method for cell-free synthesis of single-stranded linear DNA in vitro and its application in the production of RNA vaccines. Background Art

[0002] In the current mRNA vaccine production process, linearized plasmids are used as templates for in vitro transcription (IVT) to produce mRNA bulk. Since the plasmid production process includes multiple steps such as strain screening, fermentation process development, and purification process development, its cycle is long, and the requirements for equipment and plant facilities are high, which is not conducive to the rapid production of vaccines. And due to the strict requirements of the plasmid production process, the following problems are caused: 1) It is necessary to establish a GMP three-level library and conduct inspections, and at the same time conduct research on the genetic stability of the strain during passage, which takes a long time; 2) Using the Escherichia coli system for plasmid amplification, there are many process-related impurities, and the fermentation and purification steps are complex.

[0003] With the development of in vitro DNA synthesis technology, the in vitro synthesis of long fragments of DNA has become possible. For example, "Production of Closed Linear DNA Using Palindromic Sequences" uses rolling circle amplification (RCA) and TelN telomerase for in vitro DNA synthesis. This technology has been applied in the production of lentivirus, adeno-associated virus, and mRNA vaccines, greatly shortening the production cycle. However, the TelN telomerase and specific sequences used in this technology are restricted by patents. Summary of the Invention

[0004] To solve the disadvantages in the prior art that the plasmid process is used for DNA template synthesis, the production cycle is long, and the ability to respond to sudden epidemics is poor; using the bacterial cell system for DNA synthesis, there are many process-related impurities, and multiple purification steps are required to meet the quality requirements; and the plasmid is used as the production template for mRNA vaccines, which is managed according to raw materials, has high quality requirements, and requires strict inspection and release, and the inspection and release time is long, etc., the present invention has developed a method for cell-free synthesis of single-stranded linear DNA in vitro and provided its application in the production of RNA vaccines, named single-stranded template in vitro transcription method (ssTIVT).

[0005] On the one hand, the present invention provides a cell-free in vitro method for obtaining single-stranded linear DNA as an in vitro transcription template for synthesizing RNA by rolling circle amplification from double-stranded circular DNA, wherein the double-stranded circular DNA contains a nicking enzyme binding and cleavage site, and wherein the method comprises:

[0006] a) contacting the double-stranded circular DNA with a nicking enzyme that can recognize the binding and cleavage site of the nicking enzyme to obtain a mixture; and

[0007] b) contacting the mixture with a thermophilic DNA polymerase to perform rolling circle amplification to obtain single-stranded linear concatemeric DNA;

[0008] wherein the rolling circle amplification is extended from the 3'-hydroxyl group of the nick.

[0009] In some embodiments, the above method does not require the additional addition of any primers.

[0010] In the present invention, the nicking enzyme may be a restriction endonuclease having corresponding double-strand cleavage activity.

[0011] In some embodiments, the double-stranded circular DNA is followed by the 5'-terminal sequence of the in vitro transcription template strand (antisense strand) immediately after the binding and cleavage site of the nicking enzyme in the 5'-3' direction.

[0012] In some embodiments, the double-stranded circular DNA further contains a polyT sequence (antisense strand) after the binding and cleavage site of the nicking enzyme.

[0013] In some embodiments, step b) further includes cleaving the single-stranded linear concatemeric DNA into monomeric DNA.

[0014] In the present invention, the single-stranded linear concatemeric DNA can be cleaved into monomeric DNA by enzymatic digestion. By annealing a synthetic oligonucleotide fragment with the binding and cleavage site of the nicking enzyme (such as the BspQI cleavage site), a local double strand is formed. The oligonucleotide fragment is reverse complementary to the BspQI cleavage site. Then, by enzymatic digestion with BspQI, the concatemeric DNA is further fragmented into monomeric DNA.

[0015] In some embodiments, step a) is carried out in a nicking buffer containing Tris-HCl, NaCl, MgCl2 and recombinant albumin.

[0016] In some embodiments, step b) is carried out in an amplification buffer containing Tris-HCl, MgCl2, (NH4)2SO4 and DTT.

[0017] In some embodiments, the nicking enzyme includes, but is not limited to, the following range: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI and Nt.CviPII.

[0018] In some preferred embodiments, the nickase is Nt.BspQI.

[0019] In some embodiments, the isothermal DNA polymerase includes but is not limited to the following range: phi29 DNA polymerase and EquiPhi29 DNA polymerase.

[0020] EquiPhi29 DNA polymerase is a mutant of wild-type phi29 DNA polymerase, which has improved thermal stability compared to wild-type phi29 DNA polymerase.

[0021] In some preferred embodiments, the isothermal DNA polymerase is phi29 DNA polymerase.

[0022] In some preferred embodiments, the isothermal DNA polymerase is EquiPhi29 DNA polymerase.

[0023] In some preferred embodiments, a) and b) can be carried out step by step or simultaneously in the same reaction system.

[0024] In some preferred embodiments, the double-stranded circular DNA further contains at least two restriction endonuclease cleavage sites, and the method further includes: c) digesting the reaction solution of the method with a restriction endonuclease that can recognize the restriction endonuclease cleavage site.

[0025] On the other hand, the present invention provides a buffer composition, which contains the above-mentioned nickase buffer and the above-mentioned amplification buffer. The buffer composition is used for the cell-free in vitro method of obtaining single-stranded linear DNA as an in vitro transcription template by rolling circle amplification of double-stranded circular DNA for synthesizing RNA.

[0026] On the other hand, the present invention provides an amplification reaction system, which contains the above-mentioned nickase, isothermal DNA polymerase, nickase buffer and amplification buffer. The amplification reaction system is used for the cell-free in vitro method of obtaining single-stranded linear DNA as an in vitro transcription template by rolling circle amplification of double-stranded circular DNA for synthesizing RNA.

[0027] On the one hand, the present invention provides a cell-free in vitro method for obtaining single-stranded linear DNA as an in vitro transcription template by rolling circle amplification of double-stranded circular DNA for synthesizing RNA, wherein the double-stranded circular DNA contains a nickase binding and cleavage site and an RNA polymerase binding site, and the method includes:

[0028] a) contacting the double-stranded circular DNA with a nickase that can recognize the nickase binding and cleavage site to obtain a mixture;

[0029] b) contacting the mixture described in a) with a thermophilic DNA polymerase to perform rolling circle amplification to obtain single-stranded linear concatemeric DNA;

[0030] c) annealing an oligonucleotide fragment to the RNA polymerase binding site to form a partial double strand; and

[0031] d) contacting the partial double strand with RNA polymerase to initiate transcription;

[0032] wherein the oligonucleotide fragment is reverse complementary to the RNA polymerase binding site.

[0033] In some embodiments, the double-stranded circular DNA is followed by the 5'-end sequence of the in vitro transcription template strand (antisense strand) immediately after the nicking enzyme binding and cleavage site in the 5'-3' direction.

[0034] In some embodiments, the double-stranded circular DNA further contains a polyT sequence (antisense strand) after the nicking enzyme binding and cleavage site.

[0035] In some embodiments, b) further includes cleaving the single-stranded linear concatemeric DNA into monomeric DNA.

[0036] In some embodiments, a) is carried out in a nicking buffer containing Tris-HCl, NaCl, MgCl2 and recombinant albumin.

[0037] In some embodiments, b) is carried out in an amplification buffer containing Tris-HCl, MgCl2, (NH4)2SO4 and DTT.

[0038] In some embodiments, d) is carried out in a transcription buffer containing Tris-HCl, MgCl2, DTT and spermidine.

[0039] In some embodiments, the nicking enzyme includes but is not limited to the following range: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI and Nt.CviPII.

[0040] In some preferred embodiments, the nicking enzyme is Nt.BspQI.

[0041] In some embodiments, the thermophilic DNA polymerase includes but is not limited to the following range: phi29 DNA polymerase and EquiPhi29 DNA polymerase.

[0042] In some preferred embodiments, the isothermal DNA polymerase is phi29 DNA polymerase.

[0043] In some preferred embodiments, the isothermal DNA polymerase is EquiPhi29 DNA polymerase.

[0044] In some preferred embodiments, the RNA polymerase binding site is the T7 promoter and the RNA polymerase is T7 RNA polymerase; or the RNA polymerase binding site is the SP6 promoter and the RNA polymerase is SP6 RNA polymerase.

[0045] In some preferred embodiments, a), b), c) and d) are carried out simultaneously.

[0046] In some preferred embodiments, the double-stranded circular DNA further comprises at least two restriction endonuclease cleavage sites, and the method further comprises: e) digesting the reaction solution of the method with a restriction endonuclease that can recognize the restriction endonuclease cleavage sites.

[0047] On the other hand, the present invention provides a buffer composition comprising the above-mentioned nicking buffer, amplification buffer and transcription buffer. The buffer composition is used for the cell-free in vitro method described above for obtaining single-stranded linear DNA by rolling circle amplification of double-stranded circular DNA as an in vitro transcription template for synthesizing RNA.

[0048] On the one hand, the present invention provides a cell-free in vitro method for producing an RNA vaccine, which amplifies single-stranded linear DNA as an in vitro transcription template from double-stranded circular DNA,

[0049] wherein the double-stranded circular DNA comprises a nicking enzyme binding and cleavage site, an RNA polymerase binding site and a target gene, and the method comprises:

[0050] a) contacting the double-stranded circular DNA with a nicking enzyme that can recognize the nicking enzyme binding and cleavage site to obtain a mixture;

[0051] b) contacting the mixture in a) with an isothermal DNA polymerase to carry out rolling circle amplification to obtain single-stranded linear concatemeric DNA;

[0052] c) annealing an oligonucleotide fragment to the RNA polymerase binding site to form a local double strand;

[0053] d) contacting the local double strand with an RNA polymerase to initiate transcription to obtain RNA containing the target gene.

[0054] In some embodiments, the double-stranded circular DNA is followed, in the 5'-3' direction, by the 5'-end sequence of the in vitro transcription template strand (antisense strand) immediately after the nickase binding and cleavage site.

[0055] In some embodiments, the double-stranded circular DNA further comprises a polyT sequence (antisense strand) after the nickase binding and cleavage site.

[0056] In some embodiments, step b) further comprises cleaving the single-stranded linear concatemer DNA into monomer DNA.

[0057] In some embodiments, step a) is carried out in a nickase buffer comprising Tris-HCl, NaCl, MgCl2 and recombinant albumin.

[0058] In some embodiments, step b) is carried out in an amplification buffer comprising Tris-HCl, MgCl2, (NH4)2SO4 and DTT.

[0059] In some embodiments, step d) is carried out in a transcription buffer comprising Tris-HCl, MgCl2, DTT and spermidine.

[0060] In some embodiments, the nickase includes, but is not limited to, the following range: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI and Nt.CviPII.

[0061] In some preferred embodiments, the nickase is Nt.BspQI.

[0062] In some embodiments, the isothermal DNA polymerase includes, but is not limited to, the following range: phi29 DNA polymerase and EquiPhi29 DNA polymerase.

[0063] In some preferred embodiments, the isothermal DNA polymerase is phi29 DNA polymerase.

[0064] In some preferred embodiments, the isothermal DNA polymerase is EquiPhi29 DNA polymerase.

[0065] In some preferred embodiments, the RNA polymerase binding site is the T7 promoter and the RNA polymerase is T7 RNA polymerase; or the RNA polymerase binding site is the SP6 promoter and the RNA polymerase is SP6 RNA polymerase.

[0066] In some preferred embodiments, a), b), c), and d) can be carried out step by step or simultaneously in the same reaction system.

[0067] In some preferred embodiments, the double-stranded circular DNA further comprises at least two restriction endonuclease cleavage sites, and the method further comprises: e) digesting the reaction solution of the method with a restriction endonuclease that can recognize the restriction endonuclease cleavage sites.

[0068] On the other hand, the present invention provides a buffer composition comprising the above-mentioned nicking buffer, amplification buffer, and transcription buffer. The buffer composition is used in a cell-free in vitro method for producing mRNA vaccines.

[0069] On the other hand, the present invention provides a transcription reaction system comprising the above-mentioned nicking enzyme, isothermal DNA polymerase, RNA polymerase, nicking buffer, amplification buffer, and transcription buffer. The transcription reaction system is used in the above-mentioned cell-free in vitro method for obtaining single-stranded linear DNA by rolling circle amplification of double-stranded circular DNA as an in vitro transcription template for synthesizing RNA or the above-mentioned cell-free in vitro method for producing RNA vaccines.

[0070] On the other hand, the present invention provides a double-stranded circular DNA, the antisense strand of which sequentially comprises the following elements: BspQI binding and cleavage site, polyT sequence, target gene, and T7 promoter. The double-stranded circular DNA is used in the various methods described in the present invention.

[0071] In the method described in the present invention, double-stranded DNA intermediates will be generated during the reaction process, and they can be digested and purified using restriction endonucleases.

[0072] In some embodiments, the double-stranded circular DNA further comprises at least two restriction endonuclease cleavage sites.

[0073] In some preferred embodiments, the nucleotide spacing between two adjacent restriction endonuclease cleavage sites is 10 bp - 1000 bp.

[0074] The above is a detailed description of various aspects of this application. It should be noted that the above description is not limiting. Those skilled in the art can make any modifications and substitutions according to the common technical knowledge in the art without significantly hindering the technical effects of this application based on the following description.

[0075] The present invention has achieved the following excellent technical effects: The present invention has developed an enzymatic amplification process for single-stranded linear DNA and uses this single-stranded DNA as a template for in vitro RNA synthesis in the production process of RNA vaccines. Nickase is used to cut a nick on the target strand of the double-stranded circular DNA to be amplified. A DNA polymerase with strand displacement activity (such as phi29 DNA polymerase) can bind to the nick and, using the other strand of the double-stranded circular DNA as a template, start from the 3'-hydroxyl group at the nick and perform isothermal amplification to obtain a single-stranded target DNA sequence. Subsequently, after purifying the single-stranded DNA, it is used as an IVT template for RNA synthesis. Compared with the currently common plasmid process, this technology only requires a small amount of plasmid template for large-scale production, does not require the establishment of a complete GMP three-level bacterial strain bank, and saves the time for the establishment and screening of the bacterial strain bank and the study of passage stability. DNA synthesis is carried out by an in vitro enzymatic method, the enzyme proteins and raw materials related to the process in the system are clear, the system composition is simple, and large-scale purification work is omitted. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is a schematic diagram of the technology of the present invention. Among them, IVT is in vitro transcription.

[0077] Figure 2 It shows that the nicking enzyme Nt.BspQI introduces a nick on the circular closed double-stranded DNA. Among them, Iso is the restriction endonuclease BspQI that has corresponding double-strand cutting activity with the nicking enzyme Nt.BspQI.

[0078] Figure 3 It shows that the isothermal DNA polymerase can use the nicked plasmid as a template for rolling circle amplification. Among them, the nicking enzyme is Nt.BspQI; Iso is the restriction endonuclease BspQI that has corresponding double-strand cutting activity with the nicking enzyme Nt.BspQI; DNAP is the isothermal DNA polymerase phi29 DNA polymerase; DpnI is a restriction endonuclease that recognizes the GATC sequence and only has cutting activity when N6-methyladenine exists in its recognition sequence.

[0079] Figure 4 It shows in vitro transcription using the isothermal amplification product as a template. Among them, Iso is the restriction endonuclease BspQI that has corresponding double-strand cutting activity with the nicking enzyme Nt.BspQI; RCA is the purified isothermal amplification product; P1 and P2 are oligonucleotides that can anneal with the RNA polymerase binding site to form a local double strand.

[0080] Figure 5It is a "one-pot" nicking and rolling circle replication reaction. Iso is the restriction endonuclease BspQI that has corresponding double-strand cleavage activity with the nicking enzyme Nt.BspQI; DNAP is the thermostable isothermal DNA polymerase EquiPhi29; DpnI is a restriction endonuclease that recognizes the GATC sequence and only has cleavage activity when N6-methyladenine exists in its recognition sequence. Detailed implementation mode

[0081] The technological process of the present invention is as Figure 1 shown. This method includes a binary enzymatic reaction system composed of a nicking enzyme (Nickase) and an isothermal amplification DNA polymerase. For example, Nt.BspQI is a nicking enzyme that recognizes the BspQI enzyme binding site and cleaves the topstrand. Using Nt.BspQI to cleave double-stranded circular DNA introduces a Nick on the single strand containing the IVT template strand. The isothermal amplification DNA polymerase with strand displacement ability (such as phi29 DNA polymerase) can bind to the Nick site, use the other strand of the plasmid as a template to roll circle replicate the Nick strand, and regenerate the BspQI cleavage site. Therefore, multiple Nt.BspQI and phi29 cleavage and replication reactions can be carried out on the same plasmid to produce the target monomer single-stranded DNA (optional step: adding BspQI to fully cleave into monomer DNA). Further, purification processes such as chromatography and ultrafiltration are used to remove replication intermediates, enzymes, inorganic salts, dNTPs and other product- and process-related impurities to obtain a single-stranded IVT template that can be used for downstream in vitro transcription. The single-stranded template cannot effectively initiate the IVT reaction. After annealing with a reverse complementary oligonucleotide (oligo) to form a double strand in the promoter region, it can efficiently bind to T7 RNA polymerase and direct mRNA synthesis.

[0082] Example 1 Nt.BspQI digestion introduces a nick on the circular closed plasmid

[0083] Using Nt.BspQI can cleave one strand of DNA at a specific cleavage sequence to form a nick on one of the two strands. Table 1 shows the Nt.BspQⅠ digestion conditions. Prepare a 100 μL digestion system according to the order in Table 1 and perform 10 μg double-stranded circular plasmid digestion. After mixing, react at 50 °C for 1.5 h. The sequence of the double-stranded circular plasmid pssTIVT1.0 is shown in SEQ ID NO:3.

[0084] Table 1

[0085]

[0086]

[0087] After the reaction, 0.8% agarose gel electrophoresis was used to detect the digestion effect. From Figure 2 the electrophoresis results, it was observed that only a single band could be obtained after digestion with BspQⅠ. The band position after digestion with Nt.BspQⅠ was much higher than the linearized position (since a nick was made resulting in plasmid nicking and much slower electrophoresis speed). The top tailing might be due to plasmids hybridizing together, leading to too large molecular weight or volume, thus slowing down the electrophoresis rate or even preventing entry into the gel lane. After digestion with Nt.BspQⅠ and then with BspQⅠ, the band became a single band and its position was the same as that of the band obtained only with BspQⅠ, which also proved that after digestion with Nt.BspQⅠ, it was in an open circular state that was not supercoiled.

[0088] Example 2 Rolling circle replication of single-stranded DNA

[0089] Phi29 DNA polymerase was used for isothermal amplification starting from the 3'-hydroxyl group of the nicked site in Example 1. Phi29 DNA polymerase not only has the function of DNA synthase but also has strand displacement activity, and can perform DNA synthesis using the uncut circular closed single-stranded DNA as a template while displacing the non-template strand. A 100 μL isothermal amplification system was prepared according to the order in Table 2, and the reaction was carried out at 30 °C for 6 h for single-stranded DNA template synthesis.

[0090] Table 2

[0091]

[0092] After the reaction, DpnI was added to the above reaction solution at a ratio of 16 U DpnI per μg plasmid input amount and incubated at 37 °C for 1.5 h to remove the plasmid template in the reaction solution.

[0093] After incubation, the amplification products were visualized by 1.5% agarose gel electrophoresis. DpnI is a restriction endonuclease that recognizes the GATC sequence and only has cleavage activity when N6-methyladenine exists in its recognition sequence. The GATC sequence in the plasmid DNA used in Example 1 comes from a dam+ strain and contains N6-methyladenine, while the isothermal amplification products in Example 2 lack this modification. Therefore, the isothermal amplification products can be distinguished from the plasmids by DpnI digestion. As Figure 3 the results showed, compared with the sample lacking DpnI (lane DpnI-), DpnI successfully and completely digested the plasmids (lane DpnI+), demonstrating the cleavage activity of DpnI under these conditions.

[0094] In addition, by introducing DpnI into the reaction product of DNAP (lane DNAP+DpnI), it was compared with the DNAP lane without DpnI. This comparison confirmed that the upper band in the DNAP lane represented the isothermal amplification product, while the lower band corresponded to the plasmid template. The above results indicate that phi29 DNA polymerase can perform rolling circle replication using the nicked plasmid as a template without adding primers.

[0095] Example 3 Purification of the isothermal amplification product by phenol-chloroform extraction

[0096] Prepare a phenol-chloroform DNA extraction solution according to the ratio of Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1. Add an equal volume of the phenol-chloroform DNA extraction solution to the reaction solution of Example 2. After thoroughly inverting and mixing, centrifuge at 10000g for 15 min at 4°C. After centrifugation, carefully transfer the supernatant to a new centrifuge tube, add 2 volumes of isopropanol, gently invert and mix, let stand in a -20°C refrigerator for more than 30 min, and centrifuge at 10000g for 10 min at 4°C. After centrifugation, discard the supernatant, leave the white precipitate, add pre-cooled 70% ethanol, gently invert several times, centrifuge at 10000g for 5 min at 4°C, and repeat the above ethanol washing step once more. Discard the supernatant, retain the white precipitate, air-dry at room temperature for 10 min, add TE buffer for dissolution, mix thoroughly, store in a -20°C low-temperature storage box for the next experiment.

[0097] Example 4 Digestion of the amplified product with BspQI

[0098] Anneal with the synthetic oligonucleotide fragment (oligo B: AAAAAGGAAGAGCCCTCG (SEQ ID NO:1)) to the above product to form a double-stranded with a local BspQⅠ digestion site, and then digest with BspQI to further fragmentize the single strand of the isothermal amplification.

[0099] 1. Annealing

[0100] Mix 5 μL of the purified product with 0.3 μL of oligo B and incubate at 70°C for 5 min.

[0101] 2. Linearization by digestion with BspQI

[0102] Add BspQI enzyme and digestion buffer to the above reaction system in the order shown in Table 3 and incubate at 50°C for 50 min:

[0103] Table 3

[0104]

[0105] Example 5 In vitro transcription (IVT)

[0106] Mix the oligonucleotides (oligo P1: TAATACGACTCACT (SEQ ID NO: 2); oligo P2: TAATACGACTCACTATA (SEQ ID NO: 4)) that can target and bind to the T7 RNA polymerase binding site with the RCA product, incubate at 70 °C for 5 min for annealing to form a partial double strand, and then add each reaction component in the order shown in Table 4 to allow T7 RNA polymerase to bind to the template and initiate transcription. Incubate at 37 °C for 2.5 h.

[0107] Table 4

[0108]

[0109]

[0110] After the reaction is completed, add 0.5 μL of DNase I (1 U / μL) to the above system and react at 37 °C for 50 min. The RNA product generated by IVT can be purified, for example, by the LiCl precipitation method, the RNA magnetic bead purification method, or the chromatography method.

[0111] Visualize the product by 1.5% agarose gel electrophoresis. The results are as Figure 4 shown. The purified RCA product cannot effectively serve as a transcription template for RNA (lane RCA), but adding the promoter oligonucleotide (lane RCA+oligo P1, lane RCA+oligo P2) can effectively carry out the transcription reaction and has the same size as the transcription product of the traditional linearized plasmid template (lane Iso).

[0112] Example 6 "One-pot" nicking and rolling circle replication reaction.

[0113] Preferably, introducing a nick on the circular closed double-stranded DNA by Nt.BspQI and rolling circle amplification by EquiPhi29 DNA polymerase can be carried out in the same reaction system. Prepare a 40 μL reaction system in the order shown in Table 5 and react at 45 °C for 6 h to synthesize the DNA single-stranded template. The sequence of the double-stranded circular plasmid pssTIVT1.0 is shown in SEQ ID NO: 3.

[0114] Table 5

[0115]

[0116] After the reaction is completed, add DpnI to the above reaction solution at a ratio of 16 U of DpnI enzyme per μg of plasmid input amount and incubate at 37 °C for 1.5 h to remove the plasmid template in the reaction solution. The amplified product can be purified, for example, by the phenol-chloroform extraction method, the agarose gel recovery method, or the chromatography method. The results are asFigure 5 As shown, Nt.BspQI and EquiPhi29 DNA polymerase were mixed for a one-pot test. The same amount of Nt.BspQI was added to the four rightmost lanes. As the amount of EquiPhi29 DNA polymerase added increased (lane 0.5U DNAP, lane 1U DNAP), the amplification yield also increased, and the position of the band with the strongest signal was the same as that of the ISO sample, indicating that the single-copy DNA was the main amplification product. By adding DpnI to the amplification product and comparing it with the lane without DpnI addition, it can be seen that the trailing signal above 15 kb disappeared, indicating that these signals were large-molecular-weight amplification intermediates, while there was no obvious difference in the main band signal, further confirming that we successfully amplified the single-copy DNA and proving that the one-pot method could significantly improve the yield of single-copy DNA.

[0117] It should be understood that although the present invention has been described exemplarily according to its preferred embodiments, it should not be limited to the above embodiments. For those skilled in the art, the present invention can have various changes and modifications. Therefore, for those skilled in the art, within the scope of the concept and principle of the present invention, several simple substitutions can also be made, and these should all be included in the protection scope of the present invention.

[0118] The sequences used in the present invention are as follows:

[0119] pssTIVT1.0 (SEQ ID NO: 3):

[0120]

Claims

1. A cell-free in vitro method for synthesizing RNA, which uses single-stranded linear DNA obtained by rolling circle amplification of double-stranded circular DNA as an in vitro transcription template, characterized in that, The double-stranded circular DNA contains a nicking enzyme binding and cleavage site, and the method includes: a) contacting the double-stranded circular DNA with a nicking enzyme that can recognize the nicking enzyme binding and cleavage site to obtain a mixture; b) contacting the mixture with a thermophilic DNA polymerase to perform rolling circle amplification to obtain single-stranded linear concatemer DNA; wherein, the rolling circle amplification is extended with the 3'-hydroxyl group of the nick generated by the nicking enzyme.

2. The method according to claim 1, characterized in that, The double-stranded circular DNA is followed by the 5'-terminal sequence of the in vitro transcription template strand (antisense strand) in the 5'-3' direction immediately after the nicking enzyme binding and cleavage site.

3. The method according to claim 1 or 2, characterized in that, In step b), it also includes cleaving the single-stranded linear concatemer DNA into monomer DNA.

4. The method according to any one of claims 1-3, characterized in that, Step a) is carried out in a nicking buffer containing Tris-HCl, NaCl, MgCl2 and recombinant albumin.

5. The method according to any one of claims 1-4, characterized in that, Step b) is carried out in an amplification buffer containing Tris-HCl, MgCl2, (NH4)2SO4 and DTT.

6. The method according to any one of claims 1-5, characterized in that, The nicking enzymes include but are not limited to the following range: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI and Nt.CviPII.

7. The method according to any one of claims 1-6, characterized in that, The thermophilic DNA polymerases include but are not limited to the following range: phi29 DNA polymerase and EquiPhi29 DNA polymerase.

8. The method according to any one of claims 1-7, characterized in that, Steps a) and b) can be carried out step by step or simultaneously in the same reaction system.

9. A cell-free in vitro method for synthesizing RNA, which uses single-stranded linear DNA obtained by rolling circle amplification of double-stranded circular DNA as an in vitro transcription template, characterized in that, The double-stranded circular DNA contains a nicking enzyme binding and cleavage site and an RNA polymerase binding site, and the method includes: a) contacting the double-stranded circular DNA with a nicking enzyme that can recognize the nicking enzyme binding and cleavage site to obtain a mixture; b) contacting the mixture in a) with a thermophilic DNA polymerase to perform rolling circle amplification to obtain single-stranded linear concatemer DNA; c) annealing an oligonucleotide fragment to the RNA polymerase binding site to form a partial double strand; and d) contacting the partial double strand with RNA polymerase to initiate transcription; wherein, the oligonucleotide fragment is reverse complementary to the RNA polymerase binding site.

10. The method according to claim 9, characterized in that, The double-stranded circular DNA is followed by the 5'-terminal sequence of the in vitro transcription template strand (antisense strand) in the 5'-3' direction immediately after the nicking enzyme binding and cleavage site.

11. The method according to claim 9 or 10, characterized in that, In step b), it also includes cleaving the single-stranded linear concatemer DNA into monomer DNA.

12. The method according to any one of claims 9-11, characterized in that, Step a) is carried out in a nicking buffer containing Tris-HCl, NaCl, MgCl2 and recombinant albumin.

13. The method according to any one of claims 9-12, characterized in that, Step b) is carried out in an amplification buffer containing Tris-HCl, MgCl2, (NH4)2SO4 and DTT.

14. The method according to any one of claims 9-13, characterized in that, Step d) is carried out in a transcription buffer containing Tris-HCl, MgCl2, DTT and spermidine.

15. The method according to any one of claims 9-14, characterized in that, The nickase includes, but is not limited to, the following: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI, and Nt.CviPII.

16. The method according to any one of claims 9-15, characterized in that, The isothermal DNA polymerase includes, but is not limited to, the following: phi29 DNA polymerase and EquiPhi29 DNA polymerase.

17. The method according to any one of claims 9-16, characterized in that, The RNA polymerase binding site is the T7 promoter and the RNA polymerase is T7 RNA polymerase; or the RNA polymerase binding site is the SP6 promoter and the RNA polymerase is SP6 RNA polymerase.

18. The method according to any one of claims 9-17, characterized in that, The steps a), b), c), and d) can be carried out step by step or simultaneously in the same reaction system.

19. A cell-free in vitro method for producing an RNA vaccine, characterized in that, Amplify single-stranded linear DNA as an in vitro transcription template from double-stranded circular DNA, wherein the double-stranded circular DNA contains a nickase binding and cleavage site, an RNA polymerase binding site, and a target gene, and the method includes: a) contacting the double-stranded circular DNA with a nickase that can recognize the nickase binding and cleavage site to obtain a mixture; b) contacting the mixture in a) with an isothermal DNA polymerase for rolling circle amplification to obtain single-stranded linear concatemeric DNA; c) annealing an oligonucleotide fragment to the RNA polymerase binding site to form a partial double strand; d) contacting the partial double strand with an RNA polymerase to initiate transcription to obtain RNA containing the target gene.

20. The method according to claim 19, characterized in that, The double-stranded circular DNA has, in the 5'-3' direction, the 5' end sequence of the in vitro transcription template strand (antisense strand) immediately following the nickase binding and cleavage site.

21. The method according to claim 19 or 20, characterized in that, Step b) further includes cleaving the single-stranded linear concatemeric DNA into monomeric DNA.

22. The method according to any one of claims 19-21, characterized in that, Step a) is carried out in a nickase buffer containing Tris-HCl, NaCl, MgCl2, and recombinant albumin.

23. The method according to any one of claims 19-22, characterized in that, Step b) is carried out in an amplification buffer containing Tris-HCl, MgCl2, (NH4)2SO4, and DTT.

24. The method according to any one of claims 19-23, characterized in that, Step d) is carried out in a transcription buffer containing Tris-HCl, MgCl2, DTT, and spermidine.

25. The method according to any one of claims 19-24, characterized in that, The nickase includes, but is not limited to, the following: Nt.BspQI, Nb.BbvCI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.AlwI, Nt.BbvCI, and Nt.CviPII.

26. The method according to any one of claims 19-25, characterized in that, The isothermal DNA polymerase includes, but is not limited to, the following: phi29 DNA polymerase or EquiPhi29 DNA polymerase.

27. The method according to any one of claims 19-26, characterized in that, The RNA polymerase binding site is the T7 promoter and the RNA polymerase is T7 RNA polymerase; or the RNA polymerase binding site is the SP6 promoter and the RNA polymerase is SP6 RNA polymerase.

28. The method according to any one of claims 19-27, characterized in that, The steps a), b), c), and d) can be carried out step by step or simultaneously in the same reaction system.

29. The method according to claims 19-28, characterized in that, The double-stranded circular DNA also contains at least two restriction endonuclease cleavage sites, and the method further includes: e) digesting the reaction solution of the method with a restriction endonuclease that can recognize the restriction endonuclease cleavage sites.

30. A buffer composition for use in the method according to any one of claims 1-8, characterized in that, The buffer composition contains the nicking buffer as defined in the method of claim 4 and the amplification buffer as defined in the method of claim 5.

31. An amplification reaction system for use in the method according to any one of claims 1 - 8, characterized in that, The amplification reaction system contains the nicking enzyme and the isothermal DNA polymerase as defined in the method of any one of claims 1-8, and the nicking buffer as defined in the method of claim 4 and the amplification buffer as defined in the method of claim 5.

32. A buffer composition for use in the method according to any one of claims 9 - 18, characterized in that, The buffer composition contains the nicking buffer as defined in the method of claim 12, the amplification buffer as defined in the method of claim 13, and the transcription buffer as defined in the method of claim 14.

33. A transcription reaction system for use in the method according to any one of claims 9 - 18, characterized in that, The transcription reaction system contains the nicking enzyme, the isothermal DNA polymerase, and the RNA polymerase as defined in the method of any one of claims 9-18, and the nicking buffer as defined in the method of claim 12, the amplification buffer as defined in the method of claim 13, and the transcription buffer as defined in the method of claim 14.

34. A buffer composition for use in the method according to any one of claims 19 - 29, characterized in that, The buffer composition contains the nicking buffer as defined in the method of claim 22, the amplification buffer as defined in the method of claim 23, and the transcription buffer as defined in the method of claim 24.

35. An RNA production reaction system for use in the method according to any one of claims 19 - 29, characterized in that, The transcription reaction system contains the nicking enzyme, the isothermal DNA polymerase, and the RNA polymerase as defined in the method of any one of claims 19-29, and the nicking buffer as defined in the method of claim 22, the amplification buffer as defined in the method of claim 23, and the transcription buffer as defined in the method of claim 24.

36. The RNA production reaction system according to claim 35, characterized in that, The RNA production reaction system further contains a capping enzyme and / or an enzyme related to nucleotide modification.

37. A double-stranded circular DNA for use in the method according to any one of claims 1 - 29, characterized in that, The antisense strand of the double-stranded circular DNA sequentially contains the following elements from 5'-3': BspQI binding and cleavage site, polyT sequence, target gene, and T7 promoter.

38. The double-stranded circular DNA according to claim 37, characterized in that, The double-stranded circular DNA also contains at least two restriction endonuclease cleavage sites.

39. The double-stranded circular DNA according to claim 38, characterized in that, The nucleotide interval between two adjacent restriction endonuclease cleavage sites is 10bp-1000bp.

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