Plasmid and application thereof

Abstract

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a plasmid and its applications. Background Technology

[0002] Over the past 20 years, isothermal DNA / RNA amplification methods have developed rapidly. Among them, rolling circle amplification (RCA) is one of the earliest and most widely used techniques. RCA is a nucleic acid detection method established by mimicking the rolling circle replication process of circular DNA in natural microorganisms. Using circular DNA as a template, a short DNA primer (complementary to part of the circular template) extends along the circle under the action of phi29 DNA polymerase, continuously replacing the previously generated extended strands, eventually producing repeating long single-stranded DNA products. Based on RCA, introducing one or more primers that anneal and hybridize with the extended products can form superbranched RCA, achieving super-exponential amplification. RCA technology has the characteristics of high sensitivity, good specificity, and ease of operation, and therefore can be applied to SNP detection, in vitro enzymatic production of plasmids, and DNA sequencing template preparation. RCA is a simple and efficient isothermal enzymatic amplification strategy with mild reaction conditions, exhibiting stability and high efficiency in complex biological environments. In addition, RCA can amplify DNA templates at an isothermal and efficient rate, and can be applied to plasmid amplification, thereby avoiding the E. coli fermentation system, eliminating the drawbacks of bacterial plasmid production process, and eliminating the risk of plasmid DNA insertion into the host genome.

[0003] In vitro transcription synthesis of mRNA is the process of synthesizing mRNA using linearized plasmid DNA as a template in a cell-free system via RNA polymerase. Traditional DNA manufacturing techniques utilize E. coli fermentation. This method of producing plasmid DNA has inherent limitations in terms of speed, safety, and scalability. The most significant advantage of mRNA vaccines is their rapid research and development and production, which is based on the fact that the mRNA preparation process is entirely a chemical synthesis reaction, bypassing cellular fermentation. If the entire process of a biopharmaceutical relies entirely on chemical synthesis, batch uniformity can be guaranteed, and the entire production process becomes simpler and more controllable. However, reviewing the entire process of mRNA vaccine production reveals that the upstream template preparation still relies on biological fermentation. Therefore, is it possible to find a way to bypass biological fermentation and solely rely on a high-efficiency, high-yield in vitro synthesis system for large-scale plasmid production?

[0004] RCA can be applied to the in vitro transcription synthesis of mRNA. However, rolling circle amplification (RCA) can randomly stop, resulting in an incomplete DNA template, so the transcribed mRNA may not be full-length. Furthermore, DNA purity affects RNA purity; impure RNA can affect its expression level. In previous studies, we found that directly using the product from rolling circle amplification for linearization resulted in relatively impure DNA. Using this DNA as a template for in vitro transcription, the obtained mRNA showed comparable quality control levels to mRNA obtained from in vitro transcription of plasmids obtained from E. coli fermentation, but the protein translation levels differed significantly after transfection into cells. Touchlight has now successfully applied RCA technology to plasmid preparation. Doggybone DNA is a novel in vitro enzymatic method for synthesizing DNA vectors, developed by Touchlight in 2010. This enzymatic synthesis system uses a very special prokaryotic telomerase, TelN (derived from bacteriophage N15), which has cleavage-ligation activity and can be used to synthesize linear closed mini DNA vectors. This method allows for the purification of impure RCA products, yielding complete closed circular plasmids. However, the solution has been patented (see CN102301010B), and the TelN used in the solution is expensive, which greatly increases the cost. Summary of the Invention

[0005] As mentioned above, rolling circle amplification (RoBAM) randomly stops, resulting in an incomplete DNA template. In vitro transcription templates require a promoter, 5'UTR, CDS, 3'UTR, and polyA structure; an incomplete DNA template leads to incomplete mRNA transcription. Furthermore, DNA purity affects RNA purity; impure RNA impacts expression levels. In previous studies, we found that directly linearizing the product of RoBAM resulted in relatively impure DNA. Using this DNA as a template for in vitro transcription yielded mRNA with comparable quality control levels to mRNA obtained from in vitro transcription of plasmids from *E. coli* fermentation; however, after transfection into cells, the protein translation levels differed significantly.

[0006] To address the aforementioned issues, this invention employs a unique redesign and construction of the DNA template. Following rolling circle amplification, the DNA template is purified primarily through a purification process involving endonucleases, exonucleases, and oligonucleotides, significantly enhancing DNA purity. As one application, the DNA template prepared using this method can be used for in vitro transcription. The resulting mRNA, after transfection into cells, exhibits translation levels comparable to those of plasmids prepared from *E. coli*, and is no less than (and even slightly superior to) Touchlight patent (see CN102301010B).

[0007] Therefore, the first aspect of the present invention provides a plasmid comprising a recognition site for a first endonuclease, a target gene, and a recognition site for a second endonuclease in the 5'-3' direction;

[0008] The first endonuclease can generate a 3' protruding sticky end of 4 or more bases after recognizing and cutting the plasmid, and the second endonuclease can generate a blunt end or a 5' protruding sticky end after recognizing and cutting the plasmid.

[0009] In some implementations, the second endonuclease is a type IIs endonuclease.

[0010] In some preferred embodiments, the first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

[0011] In some preferred embodiments, the second endonuclease comprises BspQI, BsaI, DraI, Esp3I, and EcoRI.

[0012] In some embodiments, the plasmid also includes one or more elements selected from a promoter, a 5'UTR, a signal peptide, a 3'UTR, and a polyA tail.

[0013] If the plasmid contains a promoter, the promoter is located between the recognition site of the first endonuclease and the target gene.

[0014] If the plasmid contains a polyA tail, the polyA tail is located between the target gene and the recognition site of the second endonuclease.

[0015] In some implementations, the promoter comprises a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter comprises a CMV promoter and / or the prokaryotic promoter comprises a T7 promoter.

[0016] In one specific embodiment, the present invention first introduces an endonuclease, such as PstI, which generates a 3' protruding sticky end of 4 or more bases before the promoter (e.g., the T7 promoter) in the plasmid template design. This ensures that the T7 promoter is not digested by exonucleases (e.g., ExoIII), and that the T7 promoter portion remains in double-stranded DNA, facilitating the binding of T7 RNA polymerase. Simultaneously, an endonuclease that generates blunt ends or 5' protruding sticky ends (including ends generated by type IIs endonucleases) is introduced after the polyA tail. The cleavage site of this endonuclease is located outside the recognition site, leaving no "scar" on the linearized DNA template. This ensures the accuracy of the polyA tail length and the absence of additional nucleotide addition, thus preventing the addition of unwanted nucleotides during in vitro transcription. Furthermore, after generating the 5' protruding sticky end, ExoIII can release a 5'-mononucleotide at the 3' end of the 5'-3' strand gap in the DNA, generating a single-stranded DNA fragment. Using this template design, we can prepare double-stranded DNA with an unaffected promoter but with sticky 3' ends. This DNA can then be purified using corresponding oligonucleotide (e.g., Oligo dA) magnetic beads / packing material to obtain a high-purity DNA template.

[0017] In this invention, one example of an exonuclease is ExoIII, whose 3'→5' deoxyribonuclease activity is specific for double-stranded DNA. ExoIII degrades dsDNA from blunt ends, 5'-protrusions, or nicks, releasing a 5'-mononucleotide from the 3'-end of the DNA strand to produce a single-stranded DNA fragment. It is inactive at the 3'-protrusion of DNA (at least four bases long and without a 3'-terminal C-residue), single-stranded DNA, or phosphate-thioester-linked nucleotides.

[0018] A second aspect of the present invention provides a method for preparing a DNA template for rolling circle amplification, comprising:

[0019] a) Obtain a plasmid, wherein the plasmid contains a recognition site for a first endonuclease, a target gene, and a recognition site for a second endonuclease in the 5'-3' direction;

[0020] b) Amplify the plasmid using rolling circle amplification;

[0021] c) Contact the first and second endonucleases with the product of step b), wherein the first endonuclease recognizes and cleaves the plasmid to produce a 3' protruding sticky end of 4 or more bases, and the second endonuclease recognizes and cleaves the plasmid to produce a blunt end or a 5' protruding sticky end; and

[0022] d) Contact the product of step c) with an exonuclease (e.g., ExoIII) that can degrade dsDNA at blunt ends, 5'-protrusions, or notches, but not at 3'-protrusions and single-stranded DNA.

[0023] In some embodiments, the method further includes e) purifying the product of step d) using oligonucleotide magnetic beads or packing material, wherein the oligonucleotides are complementary to the sticky ends of the product of step d).

[0024] In this invention, the plasmid product after rolling circle amplification is digested by the first and second endonucleases, resulting in a DNA product with a 3' protruding sticky end of 4 or more bases at one end and a blunt end or a 5' protruding sticky end at the other end. After digestion with the exonucleases, the resulting DNA product has a longer sticky end at the 5' end. Based on this sticky end, an oligonucleotide that specifically binds to it can be designed. This oligonucleotide can be coupled to magnetic beads or packing material for further purification of the DNA product.

[0025] In some implementations, the second endonuclease is a type IIs endonuclease.

[0026] In some preferred embodiments, the first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

[0027] In some preferred embodiments, the second endonuclease comprises BspQI, BsaI, DraI, Esp3I, and EcoRI.

[0028] In some implementations, the amount of ExoIII is saturated relative to the amount of plasmid.

[0029] In some preferred embodiments, the ratio of the plasmid to the ExoIII is 150 units of ExoIII per pmole of the plasmid.

[0030] In some embodiments, the plasmid in step a) further comprises one or more elements selected from promoter, 5'UTR, signal peptide, 3'UTR, and polyA tail.

[0031] In some preferred embodiments, the plasmid in step a) further comprises a polyA tail located between the target gene and the recognition site of the second endonuclease, and the method further comprises step e) purifying the product of step d) using oligonucleotide magnetic beads or packing material, the oligonucleotides being complementary to the polyA tail.

[0032] In some preferred embodiments, the plasmid in step a) further comprises a promoter located between the recognition site of the first endonuclease and the target gene, and the promoter comprises a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter comprises a CMV promoter and / or the prokaryotic promoter comprises a T7 promoter.

[0033] A third aspect of the present invention provides a method for preparing mRNA by in vitro transcription, comprising:

[0034] a) Obtain a plasmid, wherein the plasmid contains, from the 5'-3' direction, a recognition site for a first endonuclease, a promoter, a target gene, a polyA tail, and a recognition site for a second endonuclease;

[0035] b) Amplify the plasmid using rolling circle amplification;

[0036] c) Contact the first and second endonucleases with the product of step b), wherein the first endonuclease recognizes and cleaves the plasmid to produce a 3' protruding sticky end of 4 or more bases, and the second endonuclease recognizes and cleaves the plasmid to produce a blunt end or a 5' protruding sticky end.

[0037] d) Contact the product of step c) with an exonuclease (e.g., ExoIII) that can degrade dsDNA at blunt ends, 5'-protrusions, or notches, but not at 3'-protrusions and single-stranded DNA.

[0038] e) Purify the product of step d) using oligonucleotide magnetic beads or packing material; and

[0039] f) Using the product of step e) as a template, mRNA is obtained through in vitro transcription.

[0040] In some implementations, the second endonuclease is a type IIs endonuclease.

[0041] In some preferred embodiments, the first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

[0042] In some preferred embodiments, the second endonuclease comprises BspQI, BsaI, DraI, Esp3I, and EcoRI.

[0043] In some implementations, the amount of ExoIII is saturated relative to the amount of plasmid.

[0044] In some preferred embodiments, the ratio of the plasmid to the ExoIII is 150 units of ExoIII per pmole of the plasmid.

[0045] In some implementations, the promoter comprises a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter comprises a CMV promoter and / or the prokaryotic promoter comprises a T7 promoter.

[0046] This invention achieves significantly improved DNA purity by uniquely redesigning and constructing the DNA template, followed by purification after rolling circle amplification, primarily through a purification method involving endonucleases, exonucleases, and oligonucleotides. DNA templates prepared using this approach are then used for in vitro transcription, and the resulting mRNA, after transfection into cells, exhibits translation levels comparable to those achieved with plasmids prepared from *E. coli*.

[0047] The fourth aspect of this invention provides the application of the above-mentioned plasmid in the preparation of in vitro transcription templates or eukaryotic expression DNA.

[0048] The fifth aspect of the present invention provides the use of the above-described plasmid in the preparation of mRNA or eukaryotic expression DNA by in vitro transcription.

[0049] Using the above approach, we developed a rolling circle amplification (RBA) DNA template based on exonuclease and oligonucleotide purification for in vitro transcription, without the use of TelN. This established a simple, high-yield, economical, rapid, and scalable DNA template preparation process. Based on this approach, the initial DNA template preparation time can be shortened to days (unlike the month-long process of E. coli strain library construction, process development, and preparation); the reaction system can be scaled down from large fermenters to EP tubes or centrifuge tubes; and the isothermal reaction eliminates the need for precise temperature control, significantly reducing R&D and production costs, lowering the cost of bringing biopharmaceuticals to market, alleviating the economic burden on research institutions, companies, and users, and facilitating the advancement of more projects and research into more potential therapies. Attached Figure Description

[0050] Figure 1 Linearized electrophoresis diagrams of RCA products and E. coli fermentation plasmids.

[0051] Figure 2 Electrophoresis diagram of the post-transcriptional products of a linearized plasmid template derived from RCA / E. coli plasmid.

[0052] Figure 3 A diagram showing the expression of mRNA after in vitro transcription using a linearized plasmid template derived from RCA / E. coli plasmids.

[0053] Figure 4OligodT affinity chromatography electrophoresis image of the product after in vitro transcription of a linearized plasmid template derived from an RCA / E. coli plasmid.

[0054] Figure 5 The graph shows the mRNA transfection and expression of products from linearized plasmid templates derived from RCA / E. coli plasmids after in vitro transcription followed by oligo dT affinity chromatography.

[0055] Figure 6 This diagram shows the mRNA transfection and expression after in vitro transcription following gel recovery from RCA-derived linearized plasmid template digestion.

[0056] Figure 7 This is an electrophoresis image of the DNA product after rolling circle amplification, enzyme digestion, and purification with Oligo dA magnetic beads.

[0057] Figure 8 The diagram shows the expression of RCA-derived linearized plasmid templates after enzyme digestion, purification with Oligo dA magnetic beads, and in vitro transcription followed by mRNA transfection.

[0058] Figure 9 We used rolled circle amplified DNA purified based on ExoIII and Oligo dA as a template for in vitro transcription. Detailed Implementation

[0059] The following provides a detailed description of each aspect of this application. It should be noted that the following description is not intended to be limiting, and those skilled in the art can make any modifications or substitutions based on the following description and their common technical knowledge, without significantly hindering the technical effect of this application.

[0060] Comparative Example 1.

[0061] 1. Configure the rolling circle amplification (RCA) reaction system according to Table 1.

[0062] Table 1 RCA reaction system

[0063]

[0064] 2. Rolling circle amplification was performed using plasmids of different lengths. The plasmids consisted of a pUC57 vector backbone, a T7 promoter, a 5'UTR, a CDS, a 3'UTR, a polyA tail, and restriction endonuclease recognition sites (EcoRI or BspQI). The CDS region contained the reporter gene GFP (SEQ ID NO:1), luciferase (SEQ ID NO:2), and S protein (SEQ ID NO:3), respectively.

[0065] The rolling circle amplification product and the normal *E. coli* fermentation plasmid were simultaneously digested with polyA restriction endonuclease to obtain a linearized plasmid template for in vitro transcription. The results showed that the DNA template obtained by direct linearization of the RCA-amplified plasmid had lower purity than the DNA template recovered after linearization of the *E. coli* fermentation plasmid, containing impurities and byproducts. Figure 1 ).

[0066] 3. In vitro transcription was performed on the linearized plasmid template derived from the RCA / E. coli plasmid obtained in step 2. The results showed that the GFP / luciferase in vitro transcription bands were normal, the electrophoretic bands were single, and there was no significant difference in in vitro transcription yield. However, byproducts were generated after in vitro transcription of the S protein using the linearized plasmid template derived from RCA. This may be because rolling circle amplification randomly stops, resulting in an incomplete DNA template, and therefore the transcribed mRNA may not be full-length. Figure 2 ).

[0067] 4. GFP and luciferase mRNA products obtained from the in vitro transcription of the linearized plasmid template derived from the RCA / E. coli plasmid in step 3 were used for HEK293 cell transfection experiments. The results showed that the protein expression levels of GFP and luciferase mRNA obtained from the in vitro transcription of the linearized plasmid template derived from the RCA were significantly lower than those obtained from the in vitro transcription of the linearized plasmid template derived from the E. coli plasmid, and the transfection caused greater damage to HEK293 cells. Figure 3 ).

[0068] 5. In eukaryotes, the poly(A) tail is present on almost every mRNA. The 3' poly(A) tail plays a crucial role in mRNA translation, protecting the mRNA from degradation, increasing its stability, and improving translation efficiency. Therefore, all plasmid templates contain a poly(A) tail. Products prematurely terminated after RCA may not contain a poly(A) tail. POROS TM Oligo(dT)25 affinity packing material specifically binds to the poly(A) tail of mRNA, facilitating the purification of mRNA from crude transcription mixtures to effectively remove plasmid DNA and other components.

[0069] Therefore, the mRNA products obtained from the in vitro transcription of the linearized plasmid template derived from the RCA / E. coli plasmid in step 3 were purified by oligo dT affinity chromatography. The results show that after affinity chromatography, various byproducts shorter than the target band were removed, demonstrating that the products from the in vitro transcription reaction of the RCA-derived linearized plasmid template contained many incomplete fragments that terminated prematurely. CE purity, capping rate, poly(A) distribution, and endotoxin residue were detected in each sample after affinity chromatography. The results indicate that there were no significant differences in the various tests after affinity chromatography between the mRNA products obtained from the in vitro transcription of the RCA / E. coli plasmid template (Table 2 and 3). Figure 4 ).

[0070] Table 2. Quality assay results of the products from the linearized plasmid template derived from RCA / E. coli plasmid after in vitro transcription, followed by oligo dT affinity chromatography.

[0071] Linearized pDNA template purity(%) Capping rate (%) Endotoxin (EU / mL) E. coli 81.8 98.5 <0.1 RCA 81.6 97.2 <0.1 E. coli 83.0 97.8 <0.1 RCA 89.0 97.4 <0.1 E. coli 89.9 94.0 <0.1 RCA 87.0 94.1 <0.1

[0072] 6. HEK293T cell transfection experiments were performed on the GFP and luciferase mRNA products obtained from the linearized plasmid template derived from the RCA / E. coli plasmid in step 5 after in vitro transcription. The results showed that the protein expression levels of GFP and luciferase mRNA obtained from the linearized plasmid template derived from the RCA were still significantly lower than those obtained from the linearized plasmid template derived from the E. coli plasmid after in vitro transcription, but slightly higher than before affinity chromatography. Figure 5 ).

[0073] 7. Efficient in vitro transcription requires a high-quality template. Template quality not only affects the efficiency of in vitro transcription but also determines the integrity of the synthesized RNA. The synthesis yield also largely depends on template purity. Furthermore, studies have shown that the purity of the DNA template significantly affects the protein expression of mRNA after in vitro transcription.

[0074] Therefore, we performed gel extraction and recovery of the target band from the linearized plasmid template derived from RCA. The results showed that ( Figure 6The plasmid amplified by E. coli, after digestion with phenol-chloroform and subsequent in vitro transcription, showed almost no tailing. However, the product after RCA digestion, regardless of whether it was recovered with phenol-chloroform or gel, showed significant tailing after IVT. Subsequently, we performed HEK293T cell transfection experiments with these three mRNAs. The results showed that using the gel-recovered RCA template significantly improved the protein expression level of the mRNA, demonstrating that the purity of the in vitro transcription template is crucial for mRNA translation. However, the gel-recovered RCA template still could not achieve the same level of expression as the plasmid amplified by E. coli, possibly because gel recovery cannot obtain the target fragment precisely, only a general region.

[0075] In summary, linearizing the product directly after rolling circle amplification resulted in relatively impure DNA. Using this DNA as a template for in vitro transcription yielded mRNA with quality control levels comparable to mRNA obtained from in vitro transcription of plasmids obtained from E. coli fermentation. However, after transfection into cells, the protein translation levels differed significantly.

[0076] Example 1. Template Sequence Design

[0077] The template for in vitro transcription includes a promoter, 5'UTR (optional), signal peptide (optional), CDS, 3'UTR (optional), and polyA structure. Therefore, the template sequence design also includes these structures accordingly.

[0078] ExoIII is an exonuclease whose 3'→5' deoxyribonuclease activity is specific for double-stranded DNA. ExoIII degrades dsDNA at blunt ends, 5'-protrusions, or nicks, releasing a 5'-mononucleotide from the 3' end of the DNA strand to produce a single-stranded DNA fragment. It is inactive at DNA 3'-protrusions (at least four bases long and without a 3'-terminal C-residue), single-stranded DNA, or phosphate-thioester-linked nucleotides.

[0079] Therefore, in the plasmid template design of this invention, an endonuclease such as PstI, which can generate a 3' protruding sticky end of 4 or more bases, is introduced before the T7 promoter. This ensures that the T7 promoter is not digested by ExoIII, and that the T7 promoter portion remains in double-stranded DNA, facilitating T7 RNA polymerase binding. Simultaneously, a type IIs endonuclease such as BspQI is introduced after the polyA tail. Its cleavage site is located outside the recognition site, leaving no "scar" on the linearized DNA template, ensuring the accuracy of the polyA tail length and preventing the addition of extra nucleotides. Therefore, no unwanted nucleotides are added during in vitro transcription. Furthermore, a 5' protruding sticky end can be generated, allowing ExoIII to release a 5'-mononucleotide at the 3' end of the 5'-3' strand gap in the DNA, producing a single-stranded DNA fragment. Using this template design, we can prepare double-stranded DNA with an unaffected T7 promoter but with a polyT sticky end at the 3' end. Purification of this DNA using Oligo dA magnetic beads / packing material yields a high-purity DNA template.

[0080] The sequence of the plasmid template used in this embodiment is shown in SEQ ID NO:4. Positions 1-6 are the recognition site for the first endonuclease PstI, positions 13-32 are the T7 promoter, positions 33-88 are the 5' UTR (containing the Kozak sequence), positions 89-808 are the GFPCDS region, positions 809-942 are the 3' UTR, positions 951-1064 are polyA, and positions 1066-1072 are the recognition site for the second endonuclease BspQI. The vector backbone used is pUC57.

[0081] Example 2. Rolling circle amplification (RCA)

[0082] Configure the rolling circle amplification (RCA) reaction system according to Table 2.

[0083] Table 2 RCA reaction system

[0084]

[0085]

[0086] The plasmid from Example 1 was subjected to rolling circle amplification using the above reaction system and conditions to obtain DNA amplification products.

[0087] Example 3. Endonuclease digestion

[0088] Add 50 μL of BspQI and 50 μL of PstI to every 100 μg of DNA amplification product obtained in Example 2, add 50 μL of the corresponding 10× buffer to each, and make up the rest with water. The total reaction volume is 1 mL, and digest at 37°C for 3 h.

[0089] Example 4. ExoIII digestion

[0090] Add 10 μL of ExoIII (200 U / μL) to 1 mL of the enzyme digestion product in Example 3, digest at 37 °C for 15 min, and inactivate ExoIII at 70 °C for 10 min after the reaction is complete.

[0091] Example 5. Purification of Oligo dA

[0092] Using Oligo dA magnetic beads (BeaverBeads) TM The product in Example 4 was purified using Oligo A25 (Suzhou Beaver Biomedical Engineering Co., Ltd.) or a packing material containing polyT sticky ends (200 μL of DNA).

[0093] The purification steps are as follows:

[0094] a. Place the magnetic bead bottle on a vortex mixer for 20 seconds to fully resuspend the magnetic beads. Transfer 400 μL of magnetic beads to a new centrifuge tube using a pipette. Add the same volume (400 μL) of binding buffer [10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM EDTA] to resuspend the magnetic beads.

[0095] b. Place the centrifuge tube on a magnetic separator and let it stand for 1 minute (this operation will be referred to as magnetic separation below). Use a pipette to remove the supernatant and remove the centrifuge tube from the magnetic separator. Add 200 μL of binding buffer and set aside.

[0096] c. Add 200 μL of binding buffer to 200 μL of DNA (the product in Example 4), heat at 65°C for 2 min, and then quickly transfer to ice.

[0097] d. Add 400 μL of total DNA solution to 200 μL of washed magnetic beads and mix thoroughly by pipetting. Incubate at room temperature for 10 min by rotation.

[0098] e. Perform magnetic separation for 1 minute, then carefully remove the supernatant. Remove the centrifuge tube from the magnetic separator.

[0099] Add 1 mL of binding buffer and gently mix by pipetting.

[0100] f. Perform magnetic separation for 1 min, then carefully remove the supernatant. Remove the centrifuge tube from the magnetic separator. Add 1 mL of washing buffer ① [10 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 1 mM EDTA], and gently pipette to mix.

[0101] g. Perform magnetic separation for 1 min, then carefully remove the supernatant. Remove the centrifuge tube from the magnetic separator. Add 1 mL of washing buffer ② [10 mM Tris-HCl (pH 7.5), 0.10 M NaCl, 1 mM EDTA], and gently pipette to mix.

[0102] h. Carefully remove the clean washing buffer ② (be careful not to pick up the magnetic beads), then add 30 μL of enzyme-free water, mix thoroughly by pipetting, and heat at 70°C for 2 minutes to elute.

[0103] i. Place the centrifuge tube on a magnetic rack and transfer the DNA-containing supernatant to a new RNase-free centrifuge tube.

[0104] The results showed that the DNA purity was greatly improved, and the main band encoding GFP was clearly visible in the purified DNA product. Although there were some impurities, there were no residues in the wells. Figure 7 ).

[0105] Example 6. In vitro transcription application

[0106] The purified template obtained in Example 5 was used for in vitro transcription (Table 3) to obtain mRNA, which was then transfected into cells (Table 4) to detect the translation level of the target gene.

[0107] Table 3 In vitro transcription reaction system

[0108]

[0109]

[0110] The purified DNA template obtained in Example 5 was transcribed in vitro using the system described in Table 3. After reacting at 37°C for 3 hours, 1 μL of DNase was added and digested at 37°C for 15 minutes. The mRNA was purified using VAHTS RNA Clean Beads, as follows:

[0111] a. Remove VAHTS RNA Clean Beads from the 4°C refrigerator about half an hour in advance to allow them to reach room temperature. Before use, vortex to mix.

[0112] b. Add 40 μL of magnetic bead solution to the RNA sample and gently pipette 10 times to mix thoroughly.

[0113] c. Incubate at room temperature for 5 minutes to allow the RNA to bind to the magnetic beads.

[0114] d. Place the sample on a magnetic rack and carefully remove the supernatant after the solution has clarified.

[0115] e. Keep the sample on the magnetic rack at all times, add 1 mL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant.

[0116] f. Repeat the above steps once, for a total of two rinses.

[0117] g. Keep the sample on the magnetic rack at all times, and dry the magnetic beads at room temperature for 5 minutes with the lid off.

[0118] h. Remove the sample from the magnetic rack, add 100 μL of nuclease-free water, vortex or pipette to mix thoroughly, and let stand at room temperature for 2 min.

[0119] i. After the solution has clarified, let it stand on a magnetic rack for 5 minutes. Then carefully aspirate the supernatant into a new nuclease-free centrifuge tube.

[0120] j. The concentration of the obtained mRNA and A were determined using a spectrophotometer. 260 / A 280 The value should be stored in a -80℃ refrigerator.

[0121] Cell transfection:

[0122] HEK293T cells were fed at a concentration of 0.38 × 10⁻⁶. 6 The mRNA was seeded at a density of cells / mL in 24-well plates. The next day, the mRNA was removed from the -80°C freezer and placed at room temperature. After the mRNA thawed, the transfection system was prepared according to Table 4.

[0123] Table 4. Preparation of Cell Transfection System

[0124]

[0125] Add the prepared RNA solution to the prepared transfection reagent, mix well, and incubate at room temperature for 10 minutes. Slowly add the RNA-transfection reagent solution along the well wall into the cells, and then place the cells in a cell culture incubator.

[0126] 24 hours after transfection, the cells in each group were photographed and recorded using an inverted fluorescence microscope (10× microscope).

[0127] Experimental results showed that, using the E. coli group as a control, the mRNA bands were clearly visible after BspQI linearization and IVT. Although the product transcribed in vitro in this invention exhibited slight tailing of the bands, its fluorescence level after transfection into cells was comparable to that of the control E. coli group. Figure 8 Therefore, this method enables the products of rolling circle amplification to achieve translational levels similar to those of traditional E. coli amplification.

[0128] DNA templates were prepared according to Touchlight's patented method and applied to in vitro transcription. The resulting mRNA was transfected into cells, and the translation level of the target gene was detected.

[0129] In summary, the DNA template prepared using the method of this invention, when applied to in vitro transcription, produces mRNA that, after transfection into cells, achieves translation levels comparable to those of plasmids prepared from *E. coli*, and is no less than (and even slightly superior to) Touchlight patented technology. Figure 9 ).

[0130] It should be understood that although the present invention has been described by way of example according to its preferred embodiments, it should not be limited to the above embodiments. Various modifications and variations can be made to the present invention by those skilled in the art. Therefore, several simple substitutions can be made by those skilled in the art without departing from the concept and principles of the present invention, and these should all be included within the scope of protection of the present invention.

[0131] The sequence used in this invention is as follows

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

Claims

1. A plasmid, characterized in that, The plasmid contains the recognition sites of the first endonuclease, the target gene, and the recognition sites of the second endonuclease from the 5'-3' direction. The first endonuclease can generate a 3' protruding sticky end of 4 or more bases after recognizing and cutting the plasmid, and the second endonuclease can generate a blunt end or a 5' protruding sticky end after recognizing and cutting the plasmid.

2. The plasmid as described in claim 1, characterized in that, The second endonuclease includes type IIs endonuclease.

3. The plasmid as described in claim 1, characterized in that, The first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

4. The plasmid as described in claim 1, characterized in that, The second endonuclease includes BspQI, BsaI, DraI, Esp3I, and EcoRI.

5. The plasmid as described in claim 1, characterized in that, The plasmid also contains one or more of the following elements: promoter, 5'UTR, signal peptide, 3'UTR, and polyA tail.

6. The plasmid as described in claim 5, characterized in that, The promoter is located between the recognition site of the first endonuclease and the target gene.

7. The plasmid as described in claim 6, characterized in that, The promoter includes a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter includes a CMV promoter and / or the prokaryotic promoter includes a T7 promoter.

8. The plasmid as described in claim 5, characterized in that, The polyA tail is located between the target gene and the recognition site of the second endonuclease.

9. A method for preparing a DNA template for rolling circle amplification, characterized in that, The method includes: a) Obtain a plasmid, wherein the plasmid contains a recognition site for a first endonuclease, a target gene, and a recognition site for a second endonuclease in the 5'-3' direction; b) Amplify the plasmid using rolling circle amplification; c) Contact the first and second endonucleases with the product of step b), wherein the first endonuclease recognizes and cleaves the plasmid to produce a 3' protruding sticky end of 4 or more bases, and the second endonuclease recognizes and cleaves the plasmid to produce a blunt end or a 5' protruding sticky end; and d) Contact the product of step c) with an exonuclease (e.g., ExoIII) that can degrade dsDNA at blunt ends, 5'-protrusions, or notches, but not at 3'-protrusions and single-stranded DNA.

10. The method as described in claim 9, characterized in that, The method further includes e) purifying the product of step d) using oligonucleotide magnetic beads or packing material, wherein the oligonucleotides are complementary to the sticky ends of the product of step d).

11. The method as described in claim 9, characterized in that, The second endonuclease includes type IIs endonuclease.

12. The method as described in claim 9, characterized in that, The first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

13. The method as described in claim 9, characterized in that, The second endonuclease includes BspQI, BsaI, DraI, Esp3I, and EcoRI.

14. The method as described in claim 9, characterized in that, The plasmid in step a) further comprises one or more of the following elements: promoter, 5'UTR, signal peptide, 3'UTR, and polyA tail.

15. The method as described in claim 9, characterized in that, The plasmid in step a) further comprises a polyA tail located between the target gene and the recognition site of the second endonuclease, and the method further comprises step e) purifying the product of step d) using oligonucleotide magnetic beads or packing material, wherein the oligonucleotide is complementary to the polyA tail.

16. The method as described in claim 9, characterized in that, The plasmid in step a) further includes a promoter located between the recognition site of the first endonuclease and the target gene, and the promoter includes a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter includes a CMV promoter and / or the prokaryotic promoter includes a T7 promoter.

17. A method for preparing mRNA by in vitro transcription, characterized in that, The method includes: a) Obtain a plasmid, wherein the plasmid contains, from the 5'-3' direction, a recognition site for a first endonuclease, a promoter, a target gene, a polyA tail, and a recognition site for a second endonuclease; b) Amplify the plasmid using rolling circle amplification; c) Contact the first and second endonucleases with the product of step b), wherein the first endonuclease recognizes and cleaves the plasmid to produce a 3' protruding sticky end of 4 or more bases, and the second endonuclease recognizes and cleaves the plasmid to produce a blunt end or a 5' protruding sticky end. d) Contact the product of step c) with an exonuclease (e.g., ExoIII) that can degrade dsDNA at blunt ends, 5'-protrusions, or notches, but not at 3'-protrusions and single-stranded DNA. e) Purify the product of step d) using oligonucleotide magnetic beads or packing material; and f) Using the product of step e) as a template, mRNA is obtained through in vitro transcription.

18. The method as described in claim 17, characterized in that, The second endonuclease includes type IIs endonuclease.

19. The method as described in claim 17, characterized in that, The first endonuclease comprises PstI, KpnI, PaeI, SacI, and PacI.

20. The method as described in claim 18, characterized in that, The second endonuclease includes BspQI, BsaI, DraI, Esp3I, and EcoRI.

21. The method as described in claim 17, characterized in that, The promoter includes a prokaryotic promoter and a eukaryotic promoter; preferably, the eukaryotic promoter includes a CMV promoter and / or the prokaryotic promoter includes a T7 promoter.

22. The use of the plasmid according to any one of claims 1-8 in the preparation of in vitro transcription templates or eukaryotic expression DNA.

23. Use of the plasmid according to any one of claims 1-8 in the preparation of mRNA or eukaryotic expression DNA by in vitro transcription.

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