WO2024067644A1 - Method for cell-free preparation of mrna transcription template - Google Patents

Method for cell-free preparation of mrna transcription template Download PDF

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WO2024067644A1
WO2024067644A1 PCT/CN2023/121770 CN2023121770W WO2024067644A1 WO 2024067644 A1 WO2024067644 A1 WO 2024067644A1 CN 2023121770 W CN2023121770 W CN 2023121770W WO 2024067644 A1 WO2024067644 A1 WO 2024067644A1
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template
dna
restriction endonuclease
mrna
primer
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PCT/CN2023/121770
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French (fr)
Chinese (zh)
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胡勇
张行
艾亮霞
肖雪婷
徐盼
吕彬
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深圳瑞吉生物科技有限公司
武汉瑞佶生物科技有限公司
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Publication of WO2024067644A1 publication Critical patent/WO2024067644A1/en

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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a method for preparing mRNA transcription template without cells.
  • PCR technology polymerase chain reaction for short, is a method of in vitro enzymatic amplification of specific nucleic acid fragments based on the semi-conservative replication mechanism of DNA.
  • PCR includes three steps of denaturation, annealing, and extension between template DNA and primers as one cycle, which is repeated in cycles to amplify DNA fragments.
  • the three repeated steps of denaturation, annealing, and extension of PCR are performed at three different temperatures, and each step lasts for a short time.
  • the PCR process is very sensitive to temperature, which makes it difficult to amplify the PCR process and difficult to apply in large-scale preparation of DNA.
  • Bacterial fermentation technology by introducing the target plasmid into bacteria, the plasmid DNA molecules are continuously and stably in a free state outside the chromosome, replicated as the chromosome replicates, and passed to the offspring through cell division. After a large amount of bacterial enrichment through bacterial fermentation, the cells are alkaline lysed, and a large amount of plasmid DNA can be obtained after separation and purification of the plasmid.
  • the plasmid fermentation process is an operation on living organisms, and the production environment and equipment need to be cleaned and verified, and the discharge of wastes has strict requirements.
  • bacterial fermentation requires strain library construction, which is more complicated and time-consuming in the entire process.
  • plasmid replication is a high-load process for bacteria, which can easily lead to instability of the plasmid sequence.
  • the polyA of the template plasmid is very easy to be lost during the plasmid fermentation process, but polyA is extremely important for the stability and translation effect of mRNA, which is a defect for template preparation in the mRNA field.
  • the existing method for preparing closed linear DNA is to contact a DNA template containing at least one protelomerase sequence with at least one DNA polymerase in the presence of at least one primer to perform an amplification reaction, and then after adding protelomerase, the protelomerase sequence that recognizes the amplified product forms a large amount of closed linear DNA.
  • the linear closed DNA produced by this technology is called dbDNA, and it has been proven that the effect of DNA vaccines in the form of dbDNA is equivalent to the expression effect of DNA vaccines produced by traditional fermentation.
  • This technology can continue to amplify and produce dbDNA using dbDNA as a template, completely avoiding the fermentation process.
  • the disadvantage of this technology is that it is limited to using protelomerase to cut into closed linear DNA, which is limited in sequence.
  • Holtkamp S et al. (Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. 2006 Dec 15; 108(13): 4009-17.) disclosed that enzymatic digestion of mRNA with type IIS restriction endonucleases and non-type IIS restriction endonucleases would cause a non-polyA sequence to protrude from the polyA sequence; and disclosed that compared with Sap I digestion, the polyA obtained had an additional sequence "ACUAG", and indicated that mRNA with redundant polyA sequences had poor expression effects.
  • CN103080337B discloses that when protelomerase is used to cut mRNA, an additional protelomeric residue will appear after polyA.
  • the extra sequence of telomerase recognition sequence is longer than that of general restriction endonucleases; and protelomerase cleavage will result in a circular structure at the end, which cannot guarantee the position of transcription termination when DNA is transcribed to RNA, which is very likely to fail to ensure the stable quality of mRNA drugs.
  • the present invention provides a method for preparing mRNA transcription templates in a cell-free manner.
  • the first aspect of the present invention provides a method for preparing an mRNA transcription template in a cell-free manner, wherein the method comprises the following steps:
  • the amplification includes a strand displacement replication method; more preferably, the amplification is a rolling circle amplification; further preferably, the amplification is an exponential amplification of a multi-branch amplification reaction under the action of a DNA polymerase; this step produces a DNA product;
  • the method further includes introducing corresponding restriction sites when designing the DNA template.
  • the method further comprises melting the circular DNA template.
  • the method further comprises annealing the primer to the DNA template and allowing the primer to be recognized by a DNA polymerase under conditions that promote amplification of the DNA template.
  • the circular DNA template comprises a single-stranded circular DNA obtained after denaturation and melting, which allows hybridization and binding of the primer.
  • the above conditions also include temperature and buffer, which allow the primer to anneal to the template. Appropriate annealing/hybridization conditions can be selected, which depends on the characteristics of the primer.
  • the above-mentioned amplification refers to the replication by replacement of the replication strand and by strand displacement of another strand.
  • the above-mentioned conditions include the use of any temperature that allows the amplification of DNA, usually in the range of 20 to 95° C.
  • the preferred temperature range can be about 20 to about 40 or about 25 to about 35° C., more preferably 30° C.
  • a suitable temperature range should be about 25 to about 35°C, preferably 30°C.
  • a technician can routinely determine a suitable temperature for effective amplification according to the method of the present invention. For example, the above method can be performed within a temperature range and the yield of amplified DNA can be monitored to determine the optimal temperature range for a given DNA polymerase.
  • conditions that promote amplification of the DNA template include the presence of a DNA polymerase and one or more primers.
  • the above conditions also include the presence of all four dNTPs, ATP, TTP, CTP and GTP, suitable buffer/pH, and other factors required for enzyme performance and stability.
  • Suitable conditions include any conditions that provide activity for DNA polymerases known in the art. Any conditions. Those skilled in the art can improve and optimize the amplification and incubation conditions for the method of the present invention. Likewise, the specific concentration of a particular preparation can be selected based on previous examples in the art and further optimized based on general knowledge.
  • the method further comprises performing rolling circle amplification on the circular DNA template to form a multi-branch amplification reaction.
  • the primers continue to amplify using the amplified product as a template to form a complementary double strand.
  • the system of rolling circle amplification includes: 10 ⁇ buffer 1 ⁇ ; dNTPs 0.5-8mM; primer 2.5 ⁇ M-2.5mM; DTT 1mM; pyrophosphatase 0.01-1U; plasmid DNA 0.05-7.5ng/ ⁇ L; phi29DNA polymerase 2.5-15U.
  • 10 ⁇ buffer 1 ⁇ dNTPs 0.5-8mM
  • primer 2.5 ⁇ M-2.5mM primer 2.5 ⁇ M-2.5mM
  • DTT 1mM pyrophosphatase 0.01-1U
  • plasmid DNA 0.05-7.5ng/ ⁇ L
  • phi29DNA polymerase 2.5-15U When preparing the reaction system, first configure the corresponding concentrations of 10 ⁇ buffer, dNTPs, primers, DTT, pyrophosphatase, and plasmid DNA, add enzyme-free water to the rest, denature at 95°C for 5min, and then add phi29DNA polymerase to carry out rolling circle amplification reaction.
  • the method further comprises a plasmid design and construction process.
  • the method further comprises a concentration and liquid replacement process after plasmid amplification.
  • the method further comprises a purification and/or recovery process of the DNA template after contacting with the restriction endonuclease.
  • the method further comprises a liquid replacement and concentration process of the linear DNA template obtained after contacting with the restriction endonuclease.
  • the palindromic sequence can be designed at any position of the template sequence.
  • the palindromic sequence is used for primer annealing.
  • the palindromic sequence of the present invention can also be written as a palindromic repeat sequence.
  • the DNA template comprises at least one restriction endonuclease action site
  • the restriction endonuclease action site includes a restriction endonuclease recognition site and/or a cleavage site;
  • the restriction endonuclease action site comprises a type IIS restriction endonuclease action site
  • restriction endonuclease action site includes the BspQ I endonuclease action site.
  • the primers of the present invention bind or specifically bind to a given palindromic sequence, which can minimize the occurrence of intra-primer and inter-primer binding.
  • the primer length/sequence can be selected based on temperature considerations, such as the ability to bind to the template at the temperature used in the amplification step.
  • the primers of the present invention bind to a given palindromic sequence in only half of the template sequence; it is understood that the primer length of the present invention can be less than half of the given palindromic sequence in the corresponding template sequence; preferably, the palindromic sequence length is twice as long as the corresponding primer.
  • the primers of the present invention are capable of specific binding to the template palindromic sequence.
  • the length of the primer can be extended to introduce additional palindromic sequences outside the existing palindromic sequence in a given template.
  • the primer can be unlabeled, or can include one or more labels, such as radionuclides or fluorescent dyes.
  • the primer can also include chemically modified nucleotides.
  • the primer can preferably include one or more thiophosphate bonds.
  • the annealing temperature of the primer is equivalent to the active temperature of the DNA polymerase.
  • the primer of the present invention can specifically bind to the template palindromic sequence at the temperature of rolling circle amplification.
  • the strand displacement DNA polymerase has a sustained amplification capacity equivalent to, or greater than, that of the phi29 DNA polymerase.
  • Two, three, four, five or more different DNA polymerases may be used, for example, a DNA polymerase that provides a proofreading function and one or more other DNA polymerases that do not provide a proofreading function.
  • the DNA polymerase is highly stable so that prolonged incubation under process conditions does not significantly reduce its activity. Therefore, under a range of processing conditions (including but not limited to temperature and pH), the DNA polymerase preferably has a long half-life.
  • the DNA polymerase has one or more characteristics suitable for the manufacturing process.
  • the DNA polymerase preferably has high fidelity, for example by having proofreading activity.
  • it is preferred that the DNA polymerase exhibits high sustained amplification capacity, high strand displacement activity, and a low Km for dNTPs and DNA.
  • the DNA polymerase described in the present invention includes phi29 DNA polymerase.
  • Restriction endonuclease sites may be added to the DNA template. Any suitable restriction endonuclease known to the skilled person may be used.
  • the restriction endonuclease of the present invention comprises a type IIS restriction endonuclease.
  • the restriction endonuclease of the present invention includes BspQ I endonuclease.
  • the DNA template of the present invention contains a polyA sequence.
  • the DNA template can be used for transcription by RNA polymerase; preferably, the RNA polymerase includes T7, T3 and SP6 RNA polymerase, etc.
  • the second aspect of the present invention proposes the application of the above-mentioned method for preparing mRNA transcription templates in the field of mRNA template preparation.
  • the mRNA domain includes a conventional mRNA domain and/or a specific mRNA domain
  • the specific mRNA domain comprises a circular RNA and/or a self-replicating RNA domain.
  • the present invention also provides a primer for use in the above method, wherein the primer is composed of a sequence selected from SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, and SEQ ID NO.8.
  • the present invention further provides a kit comprising the components required for carrying out the method of the present invention.
  • the kit of the present invention comprises at least one primer according to the present invention.
  • the kit can be used for cell-free preparation of mRNA transcription templates.
  • the kit of the present invention further comprises at least one DNA polymerase.
  • the DNA polymerase is a strand-displacing DNA polymerase.
  • the kit may comprise two, three, four, five or more different DNA polymerases.
  • the kit comprises at least one strand-displacing DNA polymerase, and more preferably RCA DNA polymerase.
  • the kit comprises phi29 DNA polymerase.
  • the kit of the present invention further comprises at least one restriction endonuclease.
  • the kit of the present invention may also include at least one single-stranded binding protein (SSBP), preferably a T4 gene 32 protein.
  • the kit may further include a pyrophosphatase, preferably a pyrophosphatase is a saccharomyces cerevisiae pyrophosphatase.
  • the kit may include any DNA polymerase, restriction endonuclease, SSBP or pyrophosphatase described herein.
  • the kit may also include dNTPs and/or a suitable buffer.
  • the present invention preferably uses a plasmid template containing a palindromic sequence, and preferably uses a primer containing a palindromic sequence.
  • the primer and the template are complementary and paired, but the extended product is a sequence consistent with the template strand.
  • the palindromic sequence designed by the present invention enables the primer to be complementary and paired with the amplified product, extending to form a double-stranded DNA.
  • exponential amplification can be achieved.
  • the present invention can also achieve the purpose of exponential amplification by designing two primers without using a palindromic sequence.
  • the present invention relates to a method for preparing linear DNA molecules without cells, which utilizes the principle of rolling circle amplification, performs rolling circle amplification in the presence of one or more primers and in contact with at least one DNA polymerase, and the primers can continue to amplify using the amplified product as a template to form a complementary double strand, and form a multi-branch amplification reaction by the strand displacement function of the DNA polymerase, thereby achieving exponential amplification.
  • the polyA sequence can be stable without gain or loss during the amplification process.
  • the method can be used without considering the loss of polyA during bacterial fermentation, thereby reducing the workload of sequence screening.
  • the restriction endonuclease used in the method can maintain the original length of polyA.
  • a DNA template containing the original polyA without any increase or loss can be obtained, that is, the obtained DNA template polyA does not contain any redundant sequence, and the transcribed mRNA has a good translation effect; a DNA template with more thorough digestion can also be obtained, thereby reducing molecular loss, reducing the miscellaneous bands generated during subsequent transcription, increasing yield, and reducing the workload of the purification step and improving efficiency.
  • the cell-free method for preparing mRNA transcription templates of the present invention can achieve large-scale preparation, and compared with the bacterial fermentation method, the stability of polyA is increased and the process does not involve the cultivation of living organisms, thereby simplifying the preparation process. Therefore, it is a more advantageous method for preparing DNA templates in the mRNA field.
  • FIG1 is a flow chart of the technical solution of the present invention.
  • FIG. 2 is a graph showing the results of agarose gel electrophoresis of the enzyme-digested product in Example 1 of the present invention.
  • FIG. 3 is a graph showing the results of agarose gel electrophoresis of the products obtained by post-transcription enzyme cleavage in Example 1.
  • FIG. 4 is the result of fluorescence microscopy observation of 293T cells transfected in Example 1 after culturing for 24 hours.
  • FIG. 5 is the results of the flow cytometer detection of fluorescence intensity and positive rate in Example 1.
  • Figure 6 is a schematic diagram of the recognition sites and cleavage sites of restriction endonucleases BspQ I and Hind III.
  • Figure 7 shows the linearization after restriction endonuclease Hind III digestion.
  • Figure 8 shows the electrophoresis results of the linearized DNA template obtained by restriction endonuclease Hind III after transcription.
  • FIG. 9 shows the effect of the mRNA obtained after transcription and then transfected into 293T cells for translation.
  • FIG10 is a graph showing the rolling circle amplification results of Example 2.
  • FIG. 11 is a diagram showing the result of TelN restriction enzyme digestion after rolling circle amplification in Example 2.
  • Figure 12 is a diagram showing the result of BspQ I enzyme digestion after rolling circle amplification in Example 2.
  • FIG. 13 is a diagram showing the transcription results of the product after enzyme digestion in Example 2.
  • FIG. 14 is a diagram showing the results of observing the expression of transcription products using a fluorescence microscope in Example 2.
  • FIG. 15 is a comparison of the results of Example 3 and Example 1.
  • the present invention provides a method for preparing mRNA transcription templates in a cell-free manner.
  • the preparation process of the transcription templates in the traditional mRNA field is as follows: plasmid construction - strain library construction - bacterial fermentation - alkaline lysis - plasmid DNA purification - concentration and liquid replacement - linearization - linear DNA purification - liquid replacement and concentration; while the preparation process of the transcription templates in the present method is as follows: plasmid construction - rolling circle amplification - concentration and liquid replacement - linearization - linear DNA purification - liquid replacement and concentration; the preparation process of the mRNA transcription templates is greatly simplified.
  • FIG1 The operation flow of the method for preparing mRNA transcription template without cells of the present invention in practical application is shown in FIG1 .
  • the meaning of each step is as follows:
  • Primer anneals to circular template and is recognized by DNA polymerase
  • This example provides a method for preparing mRNA transcription templates without cells according to the present invention, and compares restriction endonucleases Effects of BspQ I and protelomerase TelN digestion.
  • a typical DNA plasmid contains a T7 promoter sequence, a 5'UTR sequence, a CDS sequence, a 3'UTR sequence and a polyA sequence from 5' to 3'.
  • This example modifies the existing plasmid SEQ ID NO.7 to obtain a circular DNA template containing at least one palindromic sequence. The modification steps are as follows:
  • Enzyme digestion The PCR product was digested with Dpn I from NEB, and then the digested product was treated with T5 nuclease from NEB.
  • plasmid containing at least two palindromic sequences was constructed through the same operation as above, and the plasmid was named pRCA2-GFP, and its sequence is SEQ ID NO.6.
  • the total volume is 95 ⁇ L, and the primer for rolling circle amplification is SEQ ID NO.8.
  • the product amplified in (3) was ultrafiltrated and the solution was replaced by adding 400 ⁇ l of enzyme-free water each time.
  • the solution was centrifuged at 3000 g for 7 min. The operation was repeated 5 times and the final volume was adjusted to 45 ⁇ l.
  • restriction endonuclease BspQ I and protelomerase TelN were used for enzyme digestion to facilitate subsequent comparison of the differences between the two.
  • the restriction endonuclease BspQ I was used for digestion.
  • the digestion system is shown in Table 2.
  • bands 5-6 contain fewer impurities than bands 1-4, and bands 11-12 and 7-10 also contain fewer impurities, which is most likely due to incomplete digestion of the concatemer. It can be seen that the restriction endonuclease BspQ I has a better digestion effect than the protelomerase TelN.
  • the target bands were recovered by gel recovery kit from Omega Company, and nucleic acid quantification was performed using nanodrop from ThermoFisher Company.
  • the main band recovered by protelomerase digestion in lanes 1 and 2 (the 7th band from the top of the marker) is lighter than the main band recovered by restriction endonuclease digestion in lanes 3 and 4, and there are many very light bands above the main bands in lanes 1 and 2. It can be seen that protelomerase is not thorough enough in digesting the DNA template, so it is easy to generate more miscellaneous bands in subsequent transcription (protelomerase digestion is not thorough, and multiple lengths of the minimum unit will appear, as shown in the schematic diagram of steps 6-7 in Figure 1).
  • RNA 500 ng was transfected into 293T cells using jetMESSENGER transfection reagent and tested after 24 hours of culture in 12-well plates.
  • This example provides the method for preparing mRNA transcription templates in a cell-free manner according to the present invention, and compares the effects of restriction endonuclease BspQ I and protelomerase TelN.
  • a circular DNA template pRCA1-GFP containing at least one palindromic sequence and a circular DNA template pRCA2-GFP containing at least two palindromic sequences were obtained.
  • the total volume is 95 ⁇ l, and the primer for rolling circle amplification is SEQ ID NO.8.
  • the product amplified in (1) was ultrafiltrated and the solution was replaced by adding 400 ⁇ l of enzyme-free water each time.
  • the solution was centrifuged at 3000 g for 4 min. The operation was repeated 5 times and the final volume was adjusted to 60 ⁇ l.
  • restriction endonuclease BspQ I and protelomerase TelN were used for enzyme digestion to facilitate subsequent comparison of the differences between the two.
  • the restriction endonuclease BspQ I was used for digestion.
  • the digestion system is shown in Table 6.
  • lanes 1 and 2 show the effect of TelN digestion after rolling circle amplification of pRCA1-GFP; lanes 3 and 4 show the effect of TelN digestion after rolling circle amplification of pRCA2-GFP. Since pRCA1-GFP contains only one cleavage point when designed, and pRCA2-GFP contains two cleavage points, lanes 1 and 2 should theoretically have only one band each, and the remaining lighter bands are miscellaneous bands produced by incomplete enzyme digestion; lanes 3 and 4 should theoretically have two bands each, and the remaining bands are miscellaneous bands produced by incomplete enzyme digestion.
  • lanes 1 and 2 are the results of BspQ I digestion after pRCA2-GFP rolling circle amplification, and theoretically there are two bands; lanes 3 and 4 are the results of BspQ I digestion after pRCA1-GFP rolling circle amplification, and theoretically there is one band.
  • the digestion results are basically consistent with the theory, and there are significantly fewer miscellaneous bands.
  • the product in 2 was subjected to gel recovery of the target band using the gel recovery kit of Omega Company, and the nucleic acid was quantified using the nanodrop of ThermoFisher Company.
  • lane 1 is the transcription result of the product of protelomerase TelN digestion after pRCA1-GFP rolling circle amplification
  • lane 2 is the transcription result of the product of restriction endonuclease BspQ I digestion after pRCA1-GFP rolling circle amplification
  • lane 3 is the transcription result of the product of restriction endonuclease BspQ I digestion after bacterial fermentation of GFP plasmid. It can be seen from the comparison that the RNA transcribed from the template digested by protelomerase has a lighter main band and obvious miscellaneous bands, and the effect is poor.
  • RNA 500 ng was transfected into 293T cells using jetMESSENGER transfection reagent and tested after 24 hours of culture in 12-well plates.
  • Example 1 Compared with Example 1, the rolling circle amplification system of this example is shown in Table 9, and the other conditions are the same as Example 1. After the amplification reaction, the products were quantitatively compared, and the results were as follows:
  • the total system was 94 ⁇ L, denatured at 95°C for 5 min, and then 6 ⁇ L of phi29 DNA polymerase was added for subsequent reaction.
  • This comparative example provides the expression status of the DNA template prepared by the fermentation method after being digested by restriction endonucleases BspQ I and HindIII.
  • the DNA template was prepared by the same fermentation method and linearized by restriction endonucleases BspQ I and HindIII, respectively.
  • the recognition sites and restriction sites of restriction endonucleases BspQ I and HindIII are shown in Figure 6.
  • lane 1 corresponds to the DNA template linearized by restriction endonuclease BspQ I after transcription
  • lane 2 corresponds to the DNA template linearized by restriction endonuclease HindIII after transcription.
  • the d2EGFP-120A-PCR method indicates that the transcription template used is the PCR product
  • the restriction digestion method indicates that the transcription template used is the restriction digestion product of the plasmid.
  • mRNA In eukaryotic cells, mRNA generally needs a 3' end polyA structure to function.
  • the BspQI recognition site and the cleavage site are separate, so the sticky end after enzyme cleavage can be artificially designed so that the template chain ends with polyA after enzyme cleavage.
  • HindIII recognizes specific sites and cuts, which can also meet the requirements of being a transcription template, that is, the sticky end 5' protrudes and the end after cleavage is A, but it is impossible to design the terminal sequence of the transcription template chain to be a polyA sequence, that is, the mRNA product transcribed by the HindIII enzyme cleavage product will definitely have the sequence AGCU at the 3' end. This sequence has been proven to affect the translation ability of mRNA.

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Abstract

A method for cell-free preparation of an mRNA transcription template, the method comprising the following steps: 1) in the presence of at least one type of primer and while promoting the amplification of a DNA template, causing contact of a circular DNA template comprising at least one palindromic sequence with at least one type of DNA polymerase having strand displacement activity; and 2) causing contact of the DNA product prepared in step 1 with at least one type of restriction enzyme. Compared to PCR amplification methods, the present method for cell-free preparation of an mRNA transcription template can achieve large-scale preparation; compared to microbial fermentation methods,the present method had added polyA stability and does not involve culturing of live organisms, which simplifies the preparation process, and is a superior method for preparing a DNA template in the field of mRNA.

Description

一种无细胞制备mRNA转录模板的方法A method for preparing mRNA transcription template without cells 技术领域Technical Field
本发明属生物工程领域，具体涉及一种无细胞制备mRNA转录模板的方法。The invention belongs to the field of bioengineering, and in particular relates to a method for preparing mRNA transcription template without cells.
背景技术Background technique
PCR技术，聚合酶链式反应简称PCR，是以DNA半保留复制机制为基础，发展的体外酶促，扩增特定核酸片段的一种方法。通常PCR包括模板DNA与引物之间的变性、退火、延伸三步反应为一个周期，循环进行，使得DNA片段得以扩增。PCR的变性、退火、延伸的这三个循环重复的步骤在三个不同的温度下进行，并且每个步骤持续的时间较短，并且PCR过程对温度非常敏感，这使得PCR过程难以放大体系，在大规模制备DNA时难以应用。PCR technology, polymerase chain reaction for short, is a method of in vitro enzymatic amplification of specific nucleic acid fragments based on the semi-conservative replication mechanism of DNA. Usually, PCR includes three steps of denaturation, annealing, and extension between template DNA and primers as one cycle, which is repeated in cycles to amplify DNA fragments. The three repeated steps of denaturation, annealing, and extension of PCR are performed at three different temperatures, and each step lasts for a short time. In addition, the PCR process is very sensitive to temperature, which makes it difficult to amplify the PCR process and difficult to apply in large-scale preparation of DNA.
细菌发酵技术，通过将目标质粒导入到细菌中，质粒DNA分子持续稳定地处于染色体外的游离状态，随着染色体的复制而复制，并通过细胞分裂传递到后代，通过细菌的发酵，大量富集菌体后，碱性裂解细胞，再通过质粒的分离与纯化后，可得到大量的质粒DNA。质粒发酵过程是对活生物体的操作，对于生产环境及设备需要清洁及验证，废弃物的排放等要求较为严格，并且细菌发酵需要菌种建库，在整个工艺流程上更为复杂和耗时。在质粒发酵时，质粒复制对于细菌来说是高负荷过程，容易导致质粒序列的不稳定，对于mRNA领域，模板质粒的polyA极易在质粒发酵的过程中丢失，但polyA对于mRNA的稳定及翻译效果极为重要，这对于mRNA领域的模板制备是种缺陷。Bacterial fermentation technology, by introducing the target plasmid into bacteria, the plasmid DNA molecules are continuously and stably in a free state outside the chromosome, replicated as the chromosome replicates, and passed to the offspring through cell division. After a large amount of bacterial enrichment through bacterial fermentation, the cells are alkaline lysed, and a large amount of plasmid DNA can be obtained after separation and purification of the plasmid. The plasmid fermentation process is an operation on living organisms, and the production environment and equipment need to be cleaned and verified, and the discharge of wastes has strict requirements. In addition, bacterial fermentation requires strain library construction, which is more complicated and time-consuming in the entire process. During plasmid fermentation, plasmid replication is a high-load process for bacteria, which can easily lead to instability of the plasmid sequence. For the mRNA field, the polyA of the template plasmid is very easy to be lost during the plasmid fermentation process, but polyA is extremely important for the stability and translation effect of mRNA, which is a defect for template preparation in the mRNA field.
现有的闭合线性DNA制备方法，是在至少一种引物存在下，使包含至少一个原核端粒酶序列的DNA模板与至少一种DNA聚合酶接触后进行扩增反应，然后加入原核端粒酶后，识别扩增产物的原核端粒酶的序列形成大量的闭合线性DNA。该技术生产的线性闭合DNA被称为dbDNA，并被证明dbDNA形式的DNA疫苗效果与传统发酵生产的DNA疫苗的表达量效果相当。该技术可以dbDNA为模板继续扩增生产dbDNA，完全避免发酵过程。该技术的劣势在于限定了使用原核端粒酶酶切成闭合线性DNA，在序列上有限制。The existing method for preparing closed linear DNA is to contact a DNA template containing at least one protelomerase sequence with at least one DNA polymerase in the presence of at least one primer to perform an amplification reaction, and then after adding protelomerase, the protelomerase sequence that recognizes the amplified product forms a large amount of closed linear DNA. The linear closed DNA produced by this technology is called dbDNA, and it has been proven that the effect of DNA vaccines in the form of dbDNA is equivalent to the expression effect of DNA vaccines produced by traditional fermentation. This technology can continue to amplify and produce dbDNA using dbDNA as a template, completely avoiding the fermentation process. The disadvantage of this technology is that it is limited to using protelomerase to cut into closed linear DNA, which is limited in sequence.
Holtkamp S等(Modification of antigen-encoding RNA increases stability,translational efficacy,and T-cell stimulatory capacity of dendritic cells.2006 Dec 15；108(13):4009-17.)公开了在mRNA领域用ⅡS型限制性内切酶和非ⅡS型限制性内切酶酶切，会导致polyA序列突出一段非polyA序列；并公开了Spe I酶切和Sap I酶切相比，得到的polyA多出序列“ACUAG”，并且表明polyA存在多余序列的mRNA在表达时效果较差。Holtkamp S et al. (Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. 2006 Dec 15; 108(13): 4009-17.) disclosed that enzymatic digestion of mRNA with type IIS restriction endonucleases and non-type IIS restriction endonucleases would cause a non-polyA sequence to protrude from the polyA sequence; and disclosed that compared with Sap I digestion, the polyA obtained had an additional sequence "ACUAG", and indicated that mRNA with redundant polyA sequences had poor expression effects.
CN103080337B中公开了在mRNA领域采用原核端粒酶酶切时会在polyA后面多出原 核端粒酶识别序列，多出来的序列比一般限制性内切酶多余的序列还要长；并且原核端粒酶酶切会导致末端是环状的结构，无法保证在DNA转录到RNA时转录终止的位置，这极可能无法保证mRNA药物的质量稳定。CN103080337B discloses that when protelomerase is used to cut mRNA, an additional protelomeric residue will appear after polyA. The extra sequence of telomerase recognition sequence is longer than that of general restriction endonucleases; and protelomerase cleavage will result in a circular structure at the end, which cannot guarantee the position of transcription termination when DNA is transcribed to RNA, which is very likely to fail to ensure the stable quality of mRNA drugs.
发明内容Summary of the invention
为解决上述问题，本发明提出一种无细胞制备mRNA转录模板的方法。In order to solve the above problems, the present invention provides a method for preparing mRNA transcription templates in a cell-free manner.
本发明第一方面提出一种无细胞制备mRNA转录模板的方法，其中，所述方法包括以下步骤：The first aspect of the present invention provides a method for preparing an mRNA transcription template in a cell-free manner, wherein the method comprises the following steps:
1)在至少一种引物的存在下、在促进DNA模板扩增的条件下，使至少包含一个回文序列的环状DNA模板，与至少一种具链置换活性的DNA聚合酶接触；优选地，所述扩增包括链置换复制方法；更优选地，所述扩增为滚环扩增；进一步优选地，所述扩增为在DNA聚合酶作用下进行多分支扩增反应的指数扩增；该步骤制备得到DNA产物；1) contacting a circular DNA template containing at least one palindromic sequence with at least one DNA polymerase having strand displacement activity in the presence of at least one primer and under conditions that promote DNA template amplification; preferably, the amplification includes a strand displacement replication method; more preferably, the amplification is a rolling circle amplification; further preferably, the amplification is an exponential amplification of a multi-branch amplification reaction under the action of a DNA polymerase; this step produces a DNA product;
2)使1)中制备的DNA产物，与至少一种限制性内切酶接触。2) contacting the DNA product prepared in 1) with at least one restriction endonuclease.
根据本发明的具体实施方案，其中，所述方法还包在设计DNA模板时，引入相应的酶切位点。According to a specific embodiment of the present invention, the method further includes introducing corresponding restriction sites when designing the DNA template.
根据本发明的具体实施方案，其中，所述方法还包括环状DNA模板解链。According to a specific embodiment of the present invention, the method further comprises melting the circular DNA template.
根据本发明的具体实施方案，其中，所述方法还包括在促进DNA模板扩增的条件下，使引物与DNA模板退火并被DNA聚合酶识别。上述环状DNA模板包括变性解链后得到的单链环状DNA，其允许引物的杂交结合。According to a specific embodiment of the present invention, the method further comprises annealing the primer to the DNA template and allowing the primer to be recognized by a DNA polymerase under conditions that promote amplification of the DNA template. The circular DNA template comprises a single-stranded circular DNA obtained after denaturation and melting, which allows hybridization and binding of the primer.
上述条件还包括温度和缓冲液，其允许引物退火至模板。可以选择适当的退火/杂交条件，其取决于引物的特性。The above conditions also include temperature and buffer, which allow the primer to anneal to the template. Appropriate annealing/hybridization conditions can be selected, which depends on the characteristics of the primer.
上述扩增是指借助于复制链的替换并通过另一条链的链置换复制。上述条件包括使用允许DNA的扩增的任何温度，通常在20至95℃的范围内。优选的温度范围可以是约20至约40或约25至约35℃，更优选为30℃。The above-mentioned amplification refers to the replication by replacement of the replication strand and by strand displacement of another strand. The above-mentioned conditions include the use of any temperature that allows the amplification of DNA, usually in the range of 20 to 95° C. The preferred temperature range can be about 20 to about 40 or about 25 to about 35° C., more preferably 30° C.
本领域技术人员可以根据一般性知识对温度选择适用。例如，在使用phi29 DNA聚合酶的情况下，合适的温度范围应是约25至约35℃，优选为30℃。技术人员能够常规地确定用于按照本发明的方法的有效扩增的合适温度。例如，可以在温度范围内进行上述方法，并监测扩增DNA的产量以确定用于给定DNA聚合酶的最佳温度范围。A person skilled in the art can select suitable temperatures based on general knowledge. For example, in the case of using phi29 DNA polymerase, a suitable temperature range should be about 25 to about 35°C, preferably 30°C. A technician can routinely determine a suitable temperature for effective amplification according to the method of the present invention. For example, the above method can be performed within a temperature range and the yield of amplified DNA can be monitored to determine the optimal temperature range for a given DNA polymerase.
促进DNA模板的扩增的其它条件包括DNA聚合酶和一种或多种引物的存在。上述条件还包括存在所有四种dNTP，ATP、TTP、CTP和GTP、合适的缓冲剂/pH以及酶的性能和稳定性所需要的其它因素。合适的条件包括用来为本领域已知的DNA聚合酶提供活性的任 何条件。本领域技术人员能够改进和优化用于本发明的方法的扩增和温育条件。同样，可以基于本领域中的先前的实例来选择特定制剂的具体浓度并基于一般知识加以进一步优化。Other conditions that promote amplification of the DNA template include the presence of a DNA polymerase and one or more primers. The above conditions also include the presence of all four dNTPs, ATP, TTP, CTP and GTP, suitable buffer/pH, and other factors required for enzyme performance and stability. Suitable conditions include any conditions that provide activity for DNA polymerases known in the art. Any conditions. Those skilled in the art can improve and optimize the amplification and incubation conditions for the method of the present invention. Likewise, the specific concentration of a particular preparation can be selected based on previous examples in the art and further optimized based on general knowledge.
根据本发明的具体实施方案，其中，所述方法还包括环状DNA模板进行滚环扩增，并形成多分支扩增反应。所述引物以扩增的产物为模板继续扩增而形成互补双链。According to a specific embodiment of the present invention, the method further comprises performing rolling circle amplification on the circular DNA template to form a multi-branch amplification reaction. The primers continue to amplify using the amplified product as a template to form a complementary double strand.
根据本发明的具体实施方案，滚环扩增的体系包括：10×buffer 1×；dNTPs 0.5～8mM；引物2.5μM～2.5mM；DTT 1mM；焦磷酸酶0.01～1U；质粒DNA 0.05～7.5ng/μL；phi29DNA聚合酶2.5～15U。配制反应体系时，先将10×buffer、dNTPs、引物、DTT、焦磷酸酶、质粒DNA配置对应的浓度，其余补加无酶水，于95℃变性5min，然后加入phi29DNA聚合酶进行滚环扩增反应。According to a specific embodiment of the present invention, the system of rolling circle amplification includes: 10×buffer 1×; dNTPs 0.5-8mM; primer 2.5μM-2.5mM; DTT 1mM; pyrophosphatase 0.01-1U; plasmid DNA 0.05-7.5ng/μL; phi29DNA polymerase 2.5-15U. When preparing the reaction system, first configure the corresponding concentrations of 10×buffer, dNTPs, primers, DTT, pyrophosphatase, and plasmid DNA, add enzyme-free water to the rest, denature at 95°C for 5min, and then add phi29DNA polymerase to carry out rolling circle amplification reaction.
根据本发明的具体实施方案，其中，所述方法还包括质粒的设计构建过程。According to a specific embodiment of the present invention, the method further comprises a plasmid design and construction process.
根据本发明的具体实施方案，其中，所述方法还包括质粒扩增后的浓缩换液过程。According to a specific embodiment of the present invention, the method further comprises a concentration and liquid replacement process after plasmid amplification.
根据本发明的具体实施方案，其中，所述方法还包括与限制性内切酶接触后，DNA模板的纯化和/或回收过程。According to a specific embodiment of the present invention, the method further comprises a purification and/or recovery process of the DNA template after contacting with the restriction endonuclease.
根据本发明的具体实施方案，其中，所述方法还包括与限制性内切酶接触后，得到的线性DNA模板换液浓缩过程。According to a specific embodiment of the present invention, the method further comprises a liquid replacement and concentration process of the linear DNA template obtained after contacting with the restriction endonuclease.
根据本发明的具体实施方案，其中，所述回文序列可以设计在模板序列的任意位置。优选地，所述回文序列是用以供引物退火的。本发明所述回文序列亦可写作回文重复序列。According to a specific embodiment of the present invention, the palindromic sequence can be designed at any position of the template sequence. Preferably, the palindromic sequence is used for primer annealing. The palindromic sequence of the present invention can also be written as a palindromic repeat sequence.
根据本发明的具体实施方案，其中，所述DNA模板包含至少一个限制性内切酶作用位点；According to a specific embodiment of the present invention, wherein the DNA template comprises at least one restriction endonuclease action site;
优选地，所述限制性内切酶作用位点包括限制性内切酶识别位点和/或切割位点；Preferably, the restriction endonuclease action site includes a restriction endonuclease recognition site and/or a cleavage site;
优选地，所述限制性内切酶作用位点包括ⅡS型限制性内切酶作用位点；Preferably, the restriction endonuclease action site comprises a type IIS restriction endonuclease action site;
进一步优选地，所述限制性内切酶作用位点包括BspQ I内切酶作用位点。Further preferably, the restriction endonuclease action site includes the BspQ I endonuclease action site.
本发明的引物结合或特异性地结合于给定回文序列，可以最大限度地减少引物内和引物间结合的发生。可以基于温度考虑，如在用于扩增步骤的温度下能够结合于模板，来选择引物长度/序列。优选地，本发明的引物结合于仅一半的模板序列中给定的回文序列；可以理解，本发明的引物长度可以小于对应模板序列中给定的回文序列的一半；优选地，所述回文序列长度为对应引物的两倍长。The primers of the present invention bind or specifically bind to a given palindromic sequence, which can minimize the occurrence of intra-primer and inter-primer binding. The primer length/sequence can be selected based on temperature considerations, such as the ability to bind to the template at the temperature used in the amplification step. Preferably, the primers of the present invention bind to a given palindromic sequence in only half of the template sequence; it is understood that the primer length of the present invention can be less than half of the given palindromic sequence in the corresponding template sequence; preferably, the palindromic sequence length is twice as long as the corresponding primer.
优选地，本发明所述引物能够特异性结合到模板回文序列处。可以延长引物的长度以在给定模板中现有回文序列之外引入的另外的回文序列。引物可以是未标记的，或可以包含一个或多个标记，例如放射性核素或荧光染料。引物还可以包含化学修饰核苷酸。例如，引物可以优选包含一个或多个硫代磷酸酯键。 Preferably, the primers of the present invention are capable of specific binding to the template palindromic sequence. The length of the primer can be extended to introduce additional palindromic sequences outside the existing palindromic sequence in a given template. The primer can be unlabeled, or can include one or more labels, such as radionuclides or fluorescent dyes. The primer can also include chemically modified nucleotides. For example, the primer can preferably include one or more thiophosphate bonds.
根据本发明的具体实施方案，其中，所述引物的退火温度与DNA聚合酶的活性温度相当。优选地，本发明所述引物能够在滚环扩增的温度特异性结合到模板回文序列处。According to a specific embodiment of the present invention, the annealing temperature of the primer is equivalent to the active temperature of the DNA polymerase. Preferably, the primer of the present invention can specifically bind to the template palindromic sequence at the temperature of rolling circle amplification.
任何市售具有链置换活性的DNA聚合酶适用于本发明的这种方法。优选地，链置换DNA聚合酶具有相当于、或大于phi29 DNA聚合酶的持续扩增能力。可以使用两种、三种、四种、五种或更多种不同DNA聚合酶，例如，一种提供校对功能的DNA聚合酶以及一种或多种并不提供校对功能的其它DNA聚合酶。优选的是，DNA聚合酶是高度稳定的，以致在过程条件下的长时间温育并不显著降低它的活性。因此，在一系列的处理条件(包括但不限于温度和pH)下，DNA聚合酶优选具有长半衰期。还优选的是，DNA聚合酶具有适合于制造过程的一种或多种特性。DNA聚合酶优选具有高保真度，例如通过具有校对活性。另外，优选的是，DNA聚合酶显示高持续扩增能力、高链替换活性以及对于dNTP和DNA的低Km。Any commercially available DNA polymerase with strand displacement activity is suitable for use in this method of the present invention. Preferably, the strand displacement DNA polymerase has a sustained amplification capacity equivalent to, or greater than, that of the phi29 DNA polymerase. Two, three, four, five or more different DNA polymerases may be used, for example, a DNA polymerase that provides a proofreading function and one or more other DNA polymerases that do not provide a proofreading function. Preferably, the DNA polymerase is highly stable so that prolonged incubation under process conditions does not significantly reduce its activity. Therefore, under a range of processing conditions (including but not limited to temperature and pH), the DNA polymerase preferably has a long half-life. It is also preferred that the DNA polymerase has one or more characteristics suitable for the manufacturing process. The DNA polymerase preferably has high fidelity, for example by having proofreading activity. In addition, it is preferred that the DNA polymerase exhibits high sustained amplification capacity, high strand displacement activity, and a low Km for dNTPs and DNA.
优选地，本发明所述DNA聚合酶包括phi29 DNA聚合酶。Preferably, the DNA polymerase described in the present invention includes phi29 DNA polymerase.
可以将限制性内切酶位点加入DNA模板。可以使用技术人员已知的任何适宜的限制性内切酶。Restriction endonuclease sites may be added to the DNA template. Any suitable restriction endonuclease known to the skilled person may be used.
优选地，本发明所述限制性内切酶包括ⅡS型限制性内切酶。Preferably, the restriction endonuclease of the present invention comprises a type IIS restriction endonuclease.
优选地，本发明所述限制性内切酶包括BspQ I内切酶。Preferably, the restriction endonuclease of the present invention includes BspQ I endonuclease.
优选地，本发明所述DNA模板含有polyA序列。Preferably, the DNA template of the present invention contains a polyA sequence.
根据本发明的具体实施方案，其中，所述DNA模板可用于RNA聚合酶来转录；优选地，所述RNA聚合酶包括T7、T3和SP6 RNA聚合酶等。According to a specific embodiment of the present invention, the DNA template can be used for transcription by RNA polymerase; preferably, the RNA polymerase includes T7, T3 and SP6 RNA polymerase, etc.
本发明第二方面提出上述无细胞制备mRNA转录模板的方法在mRNA领域的模板制备中的应用。The second aspect of the present invention proposes the application of the above-mentioned method for preparing mRNA transcription templates in the field of mRNA template preparation.
根据本发明的具体实施方案，其中，所述mRNA领域包括常规mRNA领域和/或特定的mRNA领域；According to a specific embodiment of the present invention, wherein the mRNA domain includes a conventional mRNA domain and/or a specific mRNA domain;
优选地，所述特定的mRNA领域包括环状RNA和/或自复制RNA领域。Preferably, the specific mRNA domain comprises a circular RNA and/or a self-replicating RNA domain.
本发明还提供一种用于上述方法的引物，所述引物由选自SEQ ID NO.1-SEQ ID NO.2、SEQ ID NO.4-SEQ ID NO.5、SEQ ID NO.8的序列组成。The present invention also provides a primer for use in the above method, wherein the primer is composed of a sequence selected from SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, and SEQ ID NO.8.
本发明进一步提供了试剂盒，其包括为进行本发明的方法所需要的成分。本发明所述试剂盒包括至少一种按照本发明的引物。所述试剂盒可用于无细胞制备mRNA转录模板。The present invention further provides a kit comprising the components required for carrying out the method of the present invention. The kit of the present invention comprises at least one primer according to the present invention. The kit can be used for cell-free preparation of mRNA transcription templates.
根据本发明的具体实施方案，本发明所述的试剂盒还包括至少一种DNA聚合酶。优选地，DNA聚合酶是链替换型DNA聚合酶。试剂盒可以包含两种、三种、四种、五种或更多种不同DNA聚合酶。优选地，试剂盒包括至少1种链替换型DNA聚合酶，还更优选 RCA DNA聚合酶。特别优选的是，试剂盒包括phi29 DNA聚合酶。According to a specific embodiment of the present invention, the kit of the present invention further comprises at least one DNA polymerase. Preferably, the DNA polymerase is a strand-displacing DNA polymerase. The kit may comprise two, three, four, five or more different DNA polymerases. Preferably, the kit comprises at least one strand-displacing DNA polymerase, and more preferably RCA DNA polymerase. Particularly preferably, the kit comprises phi29 DNA polymerase.
根据本发明的具体实施方案，本发明所述的试剂盒还包括至少一种限制性内切酶。According to a specific embodiment of the present invention, the kit of the present invention further comprises at least one restriction endonuclease.
根据本发明的具体实施方案，本发明所述的试剂盒还可以包含至少一种单链结合蛋白(SSBP)，优选的SSBP是T4基因32蛋白。试剂盒可以进一步包括焦磷酸酶，优选的焦磷酸酶是酿酒酵母焦磷酸酶。试剂盒可以包含本文描述的任何DNA聚合酶、限制性内切酶、SSBP或焦磷酸酶。试剂盒还可以包括dNTP和/或合适的缓冲液。According to a specific embodiment of the present invention, the kit of the present invention may also include at least one single-stranded binding protein (SSBP), preferably a T4 gene 32 protein. The kit may further include a pyrophosphatase, preferably a pyrophosphatase is a saccharomyces cerevisiae pyrophosphatase. The kit may include any DNA polymerase, restriction endonuclease, SSBP or pyrophosphatase described herein. The kit may also include dNTPs and/or a suitable buffer.
本发明优选采用包含回文序列的质粒模板，同时优选采用包含回文序列的引物，在滚环扩增时，引物与模板是互补配对结合的，但是延伸出来的产物，是与模板链一致的序列。本发明设计的回文序列，使得引物能够与扩增出来的产物进行互补配对，延伸形成双链DNA，同时，由于扩增的产物可以作为模板，可以达到指数扩增。本发明亦可不采用回文序列，通过设计两条引物来达到指数扩增的目的。The present invention preferably uses a plasmid template containing a palindromic sequence, and preferably uses a primer containing a palindromic sequence. During rolling circle amplification, the primer and the template are complementary and paired, but the extended product is a sequence consistent with the template strand. The palindromic sequence designed by the present invention enables the primer to be complementary and paired with the amplified product, extending to form a double-stranded DNA. At the same time, since the amplified product can be used as a template, exponential amplification can be achieved. The present invention can also achieve the purpose of exponential amplification by designing two primers without using a palindromic sequence.
本发明技术方案带来的有益效果Beneficial effects brought by the technical solution of the present invention
本发明涉及无细胞制备线性DNA分子的方法，利用滚环扩增的原理，在一种或者多种引物的存在下，在至少一种DNA聚合酶的接触下进行滚环扩增，并且引物能以扩增的产物为模板继续扩增而形成互补双链，并由DNA聚合酶的链置换功能形成多分支扩增反应，从而达到指数扩增。并且由于DNA聚合酶的高保真性及持续合成能力可使polyA序列在扩增过程中稳定无增损。The present invention relates to a method for preparing linear DNA molecules without cells, which utilizes the principle of rolling circle amplification, performs rolling circle amplification in the presence of one or more primers and in contact with at least one DNA polymerase, and the primers can continue to amplify using the amplified product as a template to form a complementary double strand, and form a multi-branch amplification reaction by the strand displacement function of the DNA polymerase, thereby achieving exponential amplification. In addition, due to the high fidelity and continuous synthesis ability of the DNA polymerase, the polyA sequence can be stable without gain or loss during the amplification process.
采用本方法一方面可以不用考虑polyA在细菌发酵过程中的丢失，从而减少了序列筛选的工作量。另一方面，本方法筛选利用的限制性内切酶可以保持polyA的原始长度，与原核端粒酶酶切相比，既可以得到含有无增无损的原始polyA的DNA模板，即得到的DNA模板polyA不含有多余序列，经转录后的mRNA具有良好的翻译效果；也可以得到酶切更为彻底的DNA模板，从而减少分子损失、减少后续转录时生成的杂带，提高产量、并减轻纯化步骤的工作量、提高效率。On the one hand, the method can be used without considering the loss of polyA during bacterial fermentation, thereby reducing the workload of sequence screening. On the other hand, the restriction endonuclease used in the method can maintain the original length of polyA. Compared with protelomerase digestion, a DNA template containing the original polyA without any increase or loss can be obtained, that is, the obtained DNA template polyA does not contain any redundant sequence, and the transcribed mRNA has a good translation effect; a DNA template with more thorough digestion can also be obtained, thereby reducing molecular loss, reducing the miscellaneous bands generated during subsequent transcription, increasing yield, and reducing the workload of the purification step and improving efficiency.
本发明的无细胞制备mRNA转录模板的方法，与其他制备方法如PCR扩增法相比可实现大规模制备，与细菌发酵法相比增加了polyA的稳定性并且在工艺上不涉及活体生物的培养，简化了制备工艺，所以在mRNA领域是更具有优势的制备DNA模板的方法。Compared with other preparation methods such as PCR amplification method, the cell-free method for preparing mRNA transcription templates of the present invention can achieve large-scale preparation, and compared with the bacterial fermentation method, the stability of polyA is increased and the process does not involve the cultivation of living organisms, thereby simplifying the preparation process. Therefore, it is a more advantageous method for preparing DNA templates in the mRNA field.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明技术方案流程图。FIG1 is a flow chart of the technical solution of the present invention.
图2为本发明实施例1中对酶切后的产物进行琼脂糖凝胶电泳检测结果图。FIG. 2 is a graph showing the results of agarose gel electrophoresis of the enzyme-digested product in Example 1 of the present invention.
图3为实施例1转录后酶切得到的产物进行琼脂糖凝胶电泳检测结果图。 FIG. 3 is a graph showing the results of agarose gel electrophoresis of the products obtained by post-transcription enzyme cleavage in Example 1.
图4为实施例1转染的293T细胞培养24小时后荧光显微镜观察结果。FIG. 4 is the result of fluorescence microscopy observation of 293T cells transfected in Example 1 after culturing for 24 hours.
图5为实施例1流式细胞仪检测荧光强度与阳性率结果。FIG. 5 is the results of the flow cytometer detection of fluorescence intensity and positive rate in Example 1.
图6为限制性内切酶BspQ I和Hind Ⅲ的识别位点和酶切位点示意图。Figure 6 is a schematic diagram of the recognition sites and cleavage sites of restriction endonucleases BspQ I and Hind III.
图7为限制性内切酶Hind Ⅲ酶切后的线性化情况。Figure 7 shows the linearization after restriction endonuclease Hind Ⅲ digestion.
图8为限制性内切酶Hind Ⅲ得到的线性化DNA模板经转录后的电泳结果。Figure 8 shows the electrophoresis results of the linearized DNA template obtained by restriction endonuclease Hind Ⅲ after transcription.
图9为转录后得到的mRNA在转染至293T细胞进行翻译的效果。FIG. 9 shows the effect of the mRNA obtained after transcription and then transfected into 293T cells for translation.
图10为实施例2滚环扩增结果图。FIG10 is a graph showing the rolling circle amplification results of Example 2.
图11为实施例2滚环扩增后采用TelN酶切的结果图。FIG. 11 is a diagram showing the result of TelN restriction enzyme digestion after rolling circle amplification in Example 2.
图12为实施例2滚环扩增后采用BspQ I酶切的结果图。Figure 12 is a diagram showing the result of BspQ I enzyme digestion after rolling circle amplification in Example 2.
图13为实施例2酶切后产物的转录结果图。FIG. 13 is a diagram showing the transcription results of the product after enzyme digestion in Example 2.
图14为实施例2荧光显微镜观察转录产物的表达的结果图。FIG. 14 is a diagram showing the results of observing the expression of transcription products using a fluorescence microscope in Example 2.
图15为实施例3与实施例1的结果比较。FIG. 15 is a comparison of the results of Example 3 and Example 1.
具体实施方式Detailed ways
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be construed as limiting the applicable scope of the present invention.
本发明提供了一种无细胞制备mRNA转录模板的方法，传统mRNA领域转录模板的制备过程为：质粒构建——菌种建库——细菌发酵——碱裂解——质粒DNA纯化——浓缩换液——线性化——线性DNA纯化——换液浓缩；而本方法的转录模板制备过程为：质粒构建——滚环扩增——浓缩换液——线性化——线性DNA纯化——换液浓缩；大大简化了mRNA转录模板的制备流程。The present invention provides a method for preparing mRNA transcription templates in a cell-free manner. The preparation process of the transcription templates in the traditional mRNA field is as follows: plasmid construction - strain library construction - bacterial fermentation - alkaline lysis - plasmid DNA purification - concentration and liquid replacement - linearization - linear DNA purification - liquid replacement and concentration; while the preparation process of the transcription templates in the present method is as follows: plasmid construction - rolling circle amplification - concentration and liquid replacement - linearization - linear DNA purification - liquid replacement and concentration; the preparation process of the mRNA transcription templates is greatly simplified.
本发明所述无细胞制备mRNA转录模板的方法在实际应用时的操作流程如图1所示。其中，各步骤的含义为：The operation flow of the method for preparing mRNA transcription template without cells of the present invention in practical application is shown in FIG1 . The meaning of each step is as follows:
1-2：质粒DNA变性；1-2: Plasmid DNA denaturation;
2-3：引物与环状模板退火并被DNA聚合酶识别；2-3: Primer anneals to circular template and is recognized by DNA polymerase;
3-6：滚环扩增并形成多分支扩增反应；3-6: Rolling circle amplification and formation of multi-branch amplification reaction;
6-7：限制性内切酶对不同长度的扩增产物进行酶切得到均一长度的线性DNA。6-7: Restriction endonucleases digest amplified products of different lengths to obtain linear DNA of uniform length.
下面结合具体实施例说明本发明所述无细胞制备mRNA转录模板的方法及达到的效果。The method for preparing mRNA transcription template without cells according to the present invention and the effects achieved are described below with reference to specific examples.
实施例1Example 1
本实施例提供本发明所述无细胞制备mRNA转录模板的方法，并比较限制性内切酶 BspQ I和原核端粒酶TelN酶切的效果。This example provides a method for preparing mRNA transcription templates without cells according to the present invention, and compares restriction endonucleases Effects of BspQ I and protelomerase TelN digestion.
(1)得到至少包含一个回文序列的环状DNA模板pRCA1-GFP：(1) Obtaining a circular DNA template pRCA1-GFP containing at least one palindromic sequence:
典型的DNA质粒从5’到3’包含T7启动子序列，5’UTR序列，CDS序列，3’UTR序列和polyA序列。本实例在已有质粒SEQ ID NO.7上进行改造，以得到至少包含一个回文序列的环状DNA模板，改造步骤如下：A typical DNA plasmid contains a T7 promoter sequence, a 5'UTR sequence, a CDS sequence, a 3'UTR sequence and a polyA sequence from 5' to 3'. This example modifies the existing plasmid SEQ ID NO.7 to obtain a circular DNA template containing at least one palindromic sequence. The modification steps are as follows:
①PCR：使用SEQ ID NO.1和SEQ ID NO.2为引物，已有质粒为模板，takara公司的PrimeSTAR Max DNA Polymerase为聚合酶进行PCR扩增，得到PCR产物。①PCR: Use SEQ ID NO.1 and SEQ ID NO.2 as primers, existing plasmid as template, and Takara's PrimeSTAR Max DNA Polymerase as polymerase to perform PCR amplification to obtain PCR product.
②酶切：对PCR产物采用NEB公司的Dpn I进行模板消化，再对消化后的产物采用NEB公司的T5核酸外切酶进行处理。② Enzyme digestion: The PCR product was digested with Dpn I from NEB, and then the digested product was treated with T5 nuclease from NEB.
③转化：将T5核酸外切酶的酶切产物经大肠杆菌化学转化转至Stbl2菌株中。③Transformation: The cleavage product of T5 exonuclease is transferred into Stbl2 strain via chemical transformation of Escherichia coli.
④测序：对转化子进行测序，得到包含一个回文序列的质粒，命名为pRCA1-GFP，其序列为SEQ ID NO.3。④ Sequencing: The transformants were sequenced to obtain a plasmid containing a palindromic sequence, named pRCA1-GFP, and its sequence is SEQ ID NO.3.
(2)得到至少包含两个回文序列的环状DNA模板pRCA2-GFP：(2) Obtaining a circular DNA template pRCA2-GFP containing at least two palindromic sequences:
再使用SEQ ID NO.4和SEQ ID NO.5为引物，pRCA1-GFP为模板，经过上述同样操作构建至少含有两个回文序列的质粒，命名为pRCA2-GFP，其序列为SEQ ID NO.6。






Then, using SEQ ID NO.4 and SEQ ID NO.5 as primers and pRCA1-GFP as a template, a plasmid containing at least two palindromic sequences was constructed through the same operation as above, and the plasmid was named pRCA2-GFP, and its sequence is SEQ ID NO.6.






(3)使pRCA1-GFP和pRCA2-GFP与DNA聚合酶接触，对其进行滚环扩增，：(3) Contacting pRCA1-GFP and pRCA2-GFP with DNA polymerase and performing rolling circle amplification:
①配置滚环扩增的体系，如表1所示。① Configure the rolling circle amplification system as shown in Table 1.
表1
Table 1
总体积为95μL，滚环扩增的引物为SEQ ID NO.8。The total volume is 95μL, and the primer for rolling circle amplification is SEQ ID NO.8.
②DNA模板与DNA聚合酶接触：② DNA template contacts DNA polymerase:
于95℃变性5min，再加入5μL的phi29DNA聚合酶，于30℃反应3h。Denature at 95°C for 5 min, add 5 μL of phi29 DNA polymerase, and react at 30°C for 3 h.
(4)扩增的DNA产物与限制性内切酶接触：(4) The amplified DNA product is contacted with a restriction endonuclease:
①首先将扩增产物进行超滤换液：①First, ultrafilter the amplified product and replace the solution:
对(3)中扩增的产物进行超滤换液，每次加400μl无酶水，3000g离心7min，重复操作5次，最终定容至45μl。The product amplified in (3) was ultrafiltrated and the solution was replaced by adding 400 μl of enzyme-free water each time. The solution was centrifuged at 3000 g for 7 min. The operation was repeated 5 times and the final volume was adjusted to 45 μl.
②然后进行酶切实验：②Then carry out enzyme digestion experiment:
本次反应中同时采用限制性内切酶BspQ I和原核端粒酶TelN进行酶切，方便后续对比二者的差异。In this reaction, restriction endonuclease BspQ I and protelomerase TelN were used for enzyme digestion to facilitate subsequent comparison of the differences between the two.
限制性内切酶BspQ I酶切，酶切体系如表2所示。 The restriction endonuclease BspQ I was used for digestion. The digestion system is shown in Table 2.
表2
Table 2
50℃反应60min，75℃灭活5min。React at 50℃ for 60min and inactivate at 75℃ for 5min.
原核端粒酶TelN酶切，酶切体系如表3所示。Protelomerase TelN digestion, the digestion system is shown in Table 3.
表3
table 3
30℃反应60min，75℃灭活5min。React at 30℃ for 60min and inactivate at 75℃ for 5min.
对酶切后的产物进行琼脂糖凝胶电泳检测，结果如图2所示。其中，条带1-4：pRCA1-GFP用原核端粒酶TelN酶切产物。条带5-6：pRCA1-GFP用限制性内切酶BspQ I酶切产物。条带7-10：pRCA2-GFP用原核端粒酶TelN酶切产物。条带11-12：pRCA2-GFP用限制性内切酶BspQ I酶切产物。The digested products were detected by agarose gel electrophoresis, and the results are shown in Figure 2. Bands 1-4: products of pRCA1-GFP digested with protelomerase TelN. Bands 5-6: products of pRCA1-GFP digested with restriction endonuclease BspQ I. Bands 7-10: products of pRCA2-GFP digested with protelomerase TelN. Bands 11-12: products of pRCA2-GFP digested with restriction endonuclease BspQ I.
由图2可知，条带5-6与1-4相比含有更少的杂带，11-12与7-10同样杂带更少，极有可能是多连体酶切不彻底。可以看出限制性内切酶BspQ I比原核端粒酶TelN具有更优的酶切效果。As shown in Figure 2, bands 5-6 contain fewer impurities than bands 1-4, and bands 11-12 and 7-10 also contain fewer impurities, which is most likely due to incomplete digestion of the concatemer. It can be seen that the restriction endonuclease BspQ I has a better digestion effect than the protelomerase TelN.
③回收酶切产物并定量：③Recover the enzyme digestion products and quantify them:
使用Omega公司胶回收试剂盒对目的条带进行胶回收，采用ThermoFisher公司nanodrop进行核酸定量。The target bands were recovered by gel recovery kit from Omega Company, and nucleic acid quantification was performed using nanodrop from ThermoFisher Company.
(5)使用(4)中所得到的DNA模板进行转录，配置体系如表4所示。(5) Using the DNA template obtained in (4) for transcription, the configuration system is shown in Table 4.
表4
Table 4
①转录：配置好后，分别加入pRCA1-GFP/pRCA2-GF分别加入原核端粒酶TelN和限制性内切酶BspQ I酶切后回收的DNA模板，以及原有质粒发酵后酶切并回收的DNA模板为对照。于37℃反应4h。① Transcription: After preparation, add pRCA1-GFP/pRCA2-GF, DNA templates recovered after protelomerase TelN and restriction endonuclease BspQ I digestion, and DNA templates recovered after digestion of the original plasmid after fermentation as controls. React at 37°C for 4 hours.
②消化：然后加入DNaseⅠ于37℃反应30min消化模板。试剂均来源于ThermoFisher。②Digestion: Then add DNase I and react at 37℃ for 30min to digest the template. All reagents are from ThermoFisher.
③纯化：然后采用ThermoFisher公司MEGAclearTM Transcription Clean-Up Kit进行RNA纯化，然后进行琼脂糖凝胶电泳检测，结果如图3所示。其中，泳道1：mRNA-pRCA1-GFP–TelN。泳道2：mRNA-pRCA2-GFP–TelN。泳道3：mRNA-pRCA1-GFP-BspQ I。③Purification: ThermoFisher MEGAclear TM Transcription Clean-Up Kit was then used to purify RNA, and then agarose gel electrophoresis was performed. The results are shown in Figure 3. Lane 1: mRNA-pRCA1-GFP–TelN. Lane 2: mRNA-pRCA2-GFP–TelN. Lane 3: mRNA-pRCA1-GFP-BspQ I.
4：mRNA-pRCA2-GFP-BspQ I。4: mRNA-pRCA2-GFP-BspQ I.
5：阳性对照mRNA-GFP。5: Positive control mRNA-GFP.
由图3可知，泳道1和2中原核端粒酶酶切回收的主条带(Marker自上往下数第7条带)较泳道3和4中限制性内切酶酶切回收的主条带浅，并且泳道1和2的主带上方还有多条极浅的条带。可知原核端粒酶在DNA模板酶切时不够彻底，从而容易在后续转录时生成更多的杂带(原核端粒酶酶切时不彻底，会出现最小单元的多倍长度，如图1中6-7步骤示意图)。As shown in Figure 3, the main band recovered by protelomerase digestion in lanes 1 and 2 (the 7th band from the top of the marker) is lighter than the main band recovered by restriction endonuclease digestion in lanes 3 and 4, and there are many very light bands above the main bands in lanes 1 and 2. It can be seen that protelomerase is not thorough enough in digesting the DNA template, so it is easy to generate more miscellaneous bands in subsequent transcription (protelomerase digestion is not thorough, and multiple lengths of the minimum unit will appear, as shown in the schematic diagram of steps 6-7 in Figure 1).
(6)转录产物转染至293T细胞并观测荧光强度：(6) Transfect the transcription product into 293T cells and observe the fluorescence intensity:
采用jetMESSENGER transfection reagent将500ng的RNA转染至293T细胞，于12孔板培养24小时后进行检测。500 ng of RNA was transfected into 293T cells using jetMESSENGER transfection reagent and tested after 24 hours of culture in 12-well plates.
荧光显微镜观察结果如图4所示，由于原核端粒酶酶切回收的模板转录的RNA质量较差，转然后只能观察到极少的荧光。The results of fluorescence microscopy observation are shown in Figure 4. Since the quality of the RNA transcribed from the template recovered by protelomerase digestion is poor, only very little fluorescence can be observed after transcription.
流式细胞仪检测荧光强度与阳性率，三次重复实验数据绘图如图5所示。Fluorescence intensity and positive rate were detected by flow cytometry, and the data of three repeated experiments are shown in Figure 5.
图4中，原核端粒酶酶切后得到转录模板，再转录得到的mRNA表达效果较差，在GFP为报道基因时，荧光显微镜只能观察到极少数绿色荧光，而限制性内切酶BspQ I所得到的转录模板，再转录得到的mRNA能明细观察到绿色荧光。由图5可知，滚环扩增再由限制性内切酶酶切得到的转录模板，其转录的mRNA在表达效果上与传统质粒发酵得来的模板再转录mRNA效果相当。由图4和图5可知，原核端粒酶酶切不彻底，导致模板回收量降低，进而导致转录的mRNA杂带严重，回收量进一步降低；与之相比，限制性内切酶BspQ I酶切产物具有更优的完整性，具有更优的表达效果。In Figure 4, the expression effect of the mRNA transcribed from the transcription template obtained after protelomerase digestion is poor. When GFP is the reporter gene, only a few green fluorescence can be observed under the fluorescence microscope, while the mRNA transcribed from the transcription template obtained by restriction endonuclease BspQ I can clearly observe green fluorescence. As shown in Figure 5, the mRNA transcribed from the transcription template obtained by rolling circle amplification and then digested by restriction endonuclease has the same expression effect as the mRNA transcribed from the template obtained by traditional plasmid fermentation. As shown in Figures 4 and 5, the incomplete digestion by protelomerase leads to a decrease in the template recovery, which in turn leads to serious mixed bands in the transcribed mRNA and further reduces the recovery; in comparison, the restriction endonuclease BspQ I digestion product has better integrity and better expression effect.
实施例2Example 2
本实施例提供本发明所述无细胞制备mRNA转录模板的方法，并比较限制性内切酶BspQ I和原核端粒酶TelN酶切的效果。 This example provides the method for preparing mRNA transcription templates in a cell-free manner according to the present invention, and compares the effects of restriction endonuclease BspQ I and protelomerase TelN.
依实施例1中所述方法得到至少包含一个回文序列的环状DNA模板pRCA1-GFP和至少包含两个回文序列的环状DNA模板pRCA2-GFP。According to the method described in Example 1, a circular DNA template pRCA1-GFP containing at least one palindromic sequence and a circular DNA template pRCA2-GFP containing at least two palindromic sequences were obtained.
(1)使pRCA1-GFP和pRCA2-GFP与DNA聚合酶接触，对其进行滚环扩增：(1) Expose pRCA1-GFP and pRCA2-GFP to DNA polymerase and perform rolling circle amplification:
①配置滚环扩增的反应体系，如表5所示。① Configure the rolling circle amplification reaction system as shown in Table 5.
表5
table 5
总体积为95μl，滚环扩增的引物为SEQ ID NO.8。The total volume is 95μl, and the primer for rolling circle amplification is SEQ ID NO.8.
②使DNA模板与DNA聚合酶接触：②Contact the DNA template with DNA polymerase:
于95℃变性5min，再加入5μl的phi29DNA聚合酶，于30℃反应4h。Denature at 95°C for 5 min, add 5 μl of phi29 DNA polymerase, and react at 30°C for 4 h.
反应结束后，取2μl滚环扩增产物加入1μl DNA loading buffer，点样于1％琼脂糖凝胶中，160V 30min电泳后，结果如图10所示，其中，泳道1和2为pRCA1-GFP滚环扩增结果；泳道3和4为pRCA2-GFP滚环扩增结果。After the reaction, take 2 μl of rolling circle amplification product, add 1 μl DNA loading buffer, spot it on 1% agarose gel, and electrophoresed at 160V for 30 minutes. The results are shown in Figure 10, where lanes 1 and 2 are the results of pRCA1-GFP rolling circle amplification; lanes 3 and 4 are the results of pRCA2-GFP rolling circle amplification.
(2)扩增的DNA产物与限制性内切酶接触：(2) The amplified DNA product is contacted with a restriction endonuclease:
①首先将扩增产物进行超滤换液：①First, ultrafilter the amplified product and replace the solution:
对(1)中扩增的产物进行超滤换液，每次加400μl无酶水，3000g离心4min，重复操作5次，最终定容至60μl。The product amplified in (1) was ultrafiltrated and the solution was replaced by adding 400 μl of enzyme-free water each time. The solution was centrifuged at 3000 g for 4 min. The operation was repeated 5 times and the final volume was adjusted to 60 μl.
②然后进行酶切实验：②Then carry out enzyme digestion experiment:
本次反应中同时采用限制性内切酶BspQ I和原核端粒酶TelN进行酶切，方便后续对比二者的差异。In this reaction, restriction endonuclease BspQ I and protelomerase TelN were used for enzyme digestion to facilitate subsequent comparison of the differences between the two.
限制性内切酶BspQ I酶切，酶切体系如表6所示。The restriction endonuclease BspQ I was used for digestion. The digestion system is shown in Table 6.
表6
Table 6
50℃反应60min，75℃反应5min灭活。React at 50℃ for 60min and inactivate at 75℃ for 5min.
原核端粒酶TelN酶切，酶切体系如表7所示。 Protelomerase TelN digestion, the digestion system is shown in Table 7.
表7
Table 7
30℃反应60min，75℃反应5min灭活。React at 30℃ for 60min and inactivate at 75℃ for 5min.
原核端粒酶酶切和BspQ I酶切鉴定结果分别为图11和图12。The results of protelomerase digestion and BspQ I digestion identification are shown in Figures 11 and 12 , respectively.
图11中，泳道1和2为pRCA1-GFP滚环扩增后采用TelN酶切后效果；泳道3和4为pRCA2-GFP滚环扩增后采用TelN酶切后效果。由于pRCA1-GFP在设计时只包含一个切点，pRCA2-GFP包含两个切点，泳道1和2理论上应该各自只有一条带，其余较浅的条带为酶切不彻底产生的杂带；泳道3和4理论上各自均有两条带，其余为酶切不彻底产生的杂带。In Figure 11, lanes 1 and 2 show the effect of TelN digestion after rolling circle amplification of pRCA1-GFP; lanes 3 and 4 show the effect of TelN digestion after rolling circle amplification of pRCA2-GFP. Since pRCA1-GFP contains only one cleavage point when designed, and pRCA2-GFP contains two cleavage points, lanes 1 and 2 should theoretically have only one band each, and the remaining lighter bands are miscellaneous bands produced by incomplete enzyme digestion; lanes 3 and 4 should theoretically have two bands each, and the remaining bands are miscellaneous bands produced by incomplete enzyme digestion.
图12中，泳道1和2为pRCA2-GFP滚环扩增后BspQ I酶切后效果，理论上有两条带；泳道3和4为pRCA1-GFP滚环扩增后BspQ I酶切后效果，理论上有一条带。由图12可知，酶切结果与理论基本一致，杂带明显更少。In Figure 12, lanes 1 and 2 are the results of BspQ I digestion after pRCA2-GFP rolling circle amplification, and theoretically there are two bands; lanes 3 and 4 are the results of BspQ I digestion after pRCA1-GFP rolling circle amplification, and theoretically there is one band. As shown in Figure 12, the digestion results are basically consistent with the theory, and there are significantly fewer miscellaneous bands.
③回收酶切产物并定量：③Recover the enzyme digestion products and quantify them:
将②中的产物使用Omega公司胶回收试剂盒对目的条带进行胶回收，采用ThermoFisher公司nanodrop进行核酸定量。The product in ② was subjected to gel recovery of the target band using the gel recovery kit of Omega Company, and the nucleic acid was quantified using the nanodrop of ThermoFisher Company.
(3)使用回收到的DNA作为模板进行转录，转录体系如表8。(3) Using the recovered DNA as a template for transcription, the transcription system is shown in Table 8.
表8
Table 8
①转录：配置好后，分别加入pRCA1-GFP/pRCA2-GF分别加入原核端粒酶TelN和限制性内切酶BspQ I酶切后回收的DNA模板，以及原有质粒发酵后酶切并回收的DNA模板为对照。于37℃反应4h。① Transcription: After preparation, add pRCA1-GFP/pRCA2-GF, DNA templates recovered after protelomerase TelN and restriction endonuclease BspQ I digestion, and DNA templates recovered after digestion of the original plasmid after fermentation as controls. React at 37°C for 4 hours.
②消化：然后加入DNaseⅠ于37℃反应30min消化模板。试剂均来源于ThermoFisher。 ②Digestion: Then add DNase I and react at 37℃ for 30min to digest the template. All reagents are from ThermoFisher.
③纯化：然后采用ThermoFisher公司MEGAclearTM Transcription Clean-Up Kit进行RNA纯化，然后取等质量600ng的RNA进行琼脂糖凝胶电泳检测，结果如图13所示。图13中，泳道1为pRCA1-GFP滚环扩增后用原核端粒酶TelN酶切产物转录结果；泳道2为pRCA1-GFP滚环扩增后用限制性内切酶BspQ I酶切产物转录结果；泳道3为GFP质粒细菌发酵后用限制性内切酶BspQ I酶切产物转录结果。比较可知原核端粒酶酶切得到的模板进行转录得到的RNA，主条带较浅，并且有明显杂带，效果较差。③Purification: Then, the RNA was purified using the ThermoFisher MEGAclear TM Transcription Clean-Up Kit, and then 600 ng of RNA of equal mass was taken for agarose gel electrophoresis detection, and the results are shown in Figure 13. In Figure 13, lane 1 is the transcription result of the product of protelomerase TelN digestion after pRCA1-GFP rolling circle amplification; lane 2 is the transcription result of the product of restriction endonuclease BspQ I digestion after pRCA1-GFP rolling circle amplification; lane 3 is the transcription result of the product of restriction endonuclease BspQ I digestion after bacterial fermentation of GFP plasmid. It can be seen from the comparison that the RNA transcribed from the template digested by protelomerase has a lighter main band and obvious miscellaneous bands, and the effect is poor.
(4)转录产物转染至293T细胞并观测荧光强度：(4) Transfect the transcription product into 293T cells and observe the fluorescence intensity:
采用jetMESSENGER transfection reagent将500ng的RNA转染至293T细胞，于12孔板培养24小时后进行检测。500 ng of RNA was transfected into 293T cells using jetMESSENGER transfection reagent and tested after 24 hours of culture in 12-well plates.
荧光显微镜观察结果如图14所示，原核端粒酶酶切得到的RNA表达效果较差，一方面是因为原核端粒酶酶切不彻底，转录的RNA质量很差，表达的效果差；另一方面是因为原核端粒酶酶切后产物转录得到的RNA的3’端多出较长一段非polyA序列，从而翻译效果差。The results of fluorescence microscopy observation are shown in Figure 14. The expression effect of RNA obtained by protelomerase digestion is poor. On the one hand, this is because the protelomerase digestion is not thorough, the transcribed RNA is of poor quality, and the expression effect is poor; on the other hand, the 3' end of the RNA transcribed from the product of protelomerase digestion has a long non-polyA sequence, resulting in poor translation effect.
实施例3Example 3
本实施例与实施例1相比，滚环扩增体系如表9所示，其余条件同实施例1。进行扩增反应后，对产物进行定量对比，结果如下：Compared with Example 1, the rolling circle amplification system of this example is shown in Table 9, and the other conditions are the same as Example 1. After the amplification reaction, the products were quantitatively compared, and the results were as follows:
表9
Table 9
总体系94μL，于95℃变性5min，再加入6μL的phi29DNA聚合酶，进行后续反应。The total system was 94 μL, denatured at 95°C for 5 min, and then 6 μL of phi29 DNA polymerase was added for subsequent reaction.
本实施例与实施例1的结果比较见图15。The comparison of the results of this embodiment and embodiment 1 is shown in FIG15 .
对比例1Comparative Example 1
本对比例提供发酵方法制得的DNA模板经限制性内切酶BspQ I和HindⅢ酶切后的表达情况。This comparative example provides the expression status of the DNA template prepared by the fermentation method after being digested by restriction endonucleases BspQ I and HindⅢ.
采用同样的发酵方法制得DNA模板，分别采用限制性内切酶BspQ I和HindⅢ酶切线性化。限制性内切酶BspQ I和HindⅢ的识别位点和酶切位点如图6所示。The DNA template was prepared by the same fermentation method and linearized by restriction endonucleases BspQ I and HindⅢ, respectively. The recognition sites and restriction sites of restriction endonucleases BspQ I and HindⅢ are shown in Figure 6.
其中限制性内切酶Hind Ⅲ酶切后的线性化情况如图7所示，这说明Hind Ⅲ能彻底线 性化质粒。The linearization after restriction endonuclease Hind Ⅲ digestion is shown in Figure 7, which shows that Hind Ⅲ can completely linearize Personalized plasmid.
经限制性内切酶BspQ I和Hind Ⅲ线性化的DNA模板经转录后的电泳结果如图8所示。其中，泳道1对应限制性内切酶BspQ I线性化的DNA模板转录后，泳道2对应限制性内切酶HindⅢ线性化的DNA模板转录后。The electrophoresis results of DNA templates linearized by restriction endonucleases BspQ I and Hind Ⅲ after transcription are shown in Figure 8. Among them, lane 1 corresponds to the DNA template linearized by restriction endonuclease BspQ I after transcription, and lane 2 corresponds to the DNA template linearized by restriction endonuclease HindⅢ after transcription.
转录后得到的mRNA在转染至293T细胞进行翻译的情况如图9所示。其中，d2EGFP-120A-PCR法表示转录模板所用的是PCR产物，酶切法表示转录模板所用的是质粒的酶切产物。The situation of the mRNA obtained after transcription and transfected into 293T cells for translation is shown in Figure 9. Among them, the d2EGFP-120A-PCR method indicates that the transcription template used is the PCR product, and the restriction digestion method indicates that the transcription template used is the restriction digestion product of the plasmid.
结果分析Result analysis
由图7和图8可知，两种线性方法在转录水平没有太大差异。As shown in Figures 7 and 8 , there is not much difference between the two linear methods at the transcription level.
但是由图9来看，在以d2EGFP为报告基因进行检测时，两种线性方法得到的DNA模板的转录产物翻译水平差异较大：HindⅢ酶切的模板进行转录所得的mRNA进行转染后，GFP的翻译量低于BspQⅠ相应操作后得到的mRNA，极大程度上低于PCR法得到的转录模板。However, as shown in Figure 9, when d2EGFP was used as the reporter gene for detection, the translation levels of the transcription products of the DNA templates obtained by the two linear methods were quite different: after transfection of the mRNA transcribed from the HindⅢ-digested template, the translation amount of GFP was lower than that of the mRNA obtained after the corresponding operation of BspQⅠ, and was much lower than that of the transcription template obtained by PCR.
分析该结果可能的原因有两个方面：There are two possible reasons for this result:
1)质粒法所得的模板进行转录后的mRNA质量低于PCR法，导致翻译受到影响。1) The quality of mRNA transcribed from the template obtained by the plasmid method is lower than that of the PCR method, which affects the translation.
2)HindⅢ酶切后的产物进行转录后得到mRNA的3’末端引入了额外的序列，可能使mRNA翻译收到影响。2) After transcription of the product of HindⅢ digestion, additional sequences are introduced to the 3’ end of the mRNA, which may affect the translation of the mRNA.
在真核细胞中mRNA发挥功能一般需要3’末端polyA结构，BspQⅠ识别位点和切割位点是分开的，所以可以通过人为设计酶切后的粘性末端，使酶切之后的模板链以polyA结束。而HindⅢ识别特异性位点并切割，虽然也能满足作为转录模板的要求，即粘性末端5’突出且切割后的末端为A，但是无法设计转录模板链末端序列为polyA序列，即HindⅢ酶切后的产物进行转录所得的mRNA产物3’末端必定会带上序列AGCU。这段序列经验证会影响mRNA的翻译能力。 In eukaryotic cells, mRNA generally needs a 3' end polyA structure to function. The BspQⅠ recognition site and the cleavage site are separate, so the sticky end after enzyme cleavage can be artificially designed so that the template chain ends with polyA after enzyme cleavage. HindⅢ recognizes specific sites and cuts, which can also meet the requirements of being a transcription template, that is, the sticky end 5' protrudes and the end after cleavage is A, but it is impossible to design the terminal sequence of the transcription template chain to be a polyA sequence, that is, the mRNA product transcribed by the HindⅢ enzyme cleavage product will definitely have the sequence AGCU at the 3' end. This sequence has been proven to affect the translation ability of mRNA.

Claims (15)

  1. 一种无细胞制备mRNA转录模板的方法，其中，所述方法包括以下步骤：A method for preparing an mRNA transcription template in a cell-free manner, wherein the method comprises the following steps:
    1)在至少一种引物的存在下、在促进DNA模板扩增的条件下，使至少包含一个回文序列的环状DNA模板，与至少一种具链置换活性的DNA聚合酶接触；1) contacting a circular DNA template comprising at least one palindromic sequence with at least one DNA polymerase having strand displacement activity in the presence of at least one primer and under conditions that promote amplification of the DNA template;
    2)使1)中制备的DNA产物，与至少一种限制性内切酶接触。2) contacting the DNA product prepared in 1) with at least one restriction endonuclease.
  2. 根据权利要求1所述的方法，其中，所述方法还包括环状DNA模板解链。The method according to claim 1, wherein the method further comprises melting the circular DNA template.
  3. 根据权利要求1或2所述的方法，其中，所述方法还包括引物与DNA模板退火并被DNA聚合酶识别。The method according to claim 1 or 2, wherein the method further comprises the step of annealing the primer to the DNA template and being recognized by a DNA polymerase.
  4. 根据权利要求1-3任一项所述的方法，其中，所述方法还包括环状DNA模板进行滚环扩增，并形成多分支扩增反应。The method according to any one of claims 1 to 3, wherein the method further comprises performing rolling circle amplification on the circular DNA template and forming a multi-branch amplification reaction.
  5. 根据权利要求1-4任一项所述的方法，其中，所述回文序列可以设计在模板序列的任意位置。The method according to any one of claims 1 to 4, wherein the palindromic sequence can be designed at any position of the template sequence.
  6. 根据权利要求1-5任一项所述的方法，其中，所述DNA模板包含至少一个限制性内切酶作用位点；The method according to any one of claims 1 to 5, wherein the DNA template comprises at least one restriction endonuclease action site;
    优选地，所述限制性内切酶作用位点包括限制性内切酶识别位点和/或切割位点；Preferably, the restriction endonuclease action site includes a restriction endonuclease recognition site and/or a cleavage site;
    优选地，所述限制性内切酶作用位点包括ⅡS型限制性内切酶作用位点；Preferably, the restriction endonuclease action site comprises a type IIS restriction endonuclease action site;
    进一步优选地，所述限制性内切酶作用位点包括BspQ I内切酶作用位点。Further preferably, the restriction endonuclease action site includes the BspQ I endonuclease action site.
  7. 根据权利要求1-6任一项所述的方法，其中，所述引物的退火温度与DNA聚合酶的活性温度相当。The method according to any one of claims 1 to 6, wherein the annealing temperature of the primer is equivalent to the active temperature of the DNA polymerase.
  8. 根据权利要求1-7任一项所述的方法，其中，DNA聚合酶包括phi29 DNA聚合酶。A method according to any one of claims 1-7, wherein the DNA polymerase comprises phi29 DNA polymerase.
  9. 根据权利要求1-8任一项所述的方法，其中，所述限制性内切酶包括ⅡS型限制性内切酶。The method according to any one of claims 1 to 8, wherein the restriction endonuclease comprises a type IIS restriction endonuclease.
  10. 根据权利要求1-9任一项所述的方法，其中，所述限制性内切酶包括BspQ I内切酶。The method according to any one of claims 1 to 9, wherein the restriction endonuclease comprises BspQ I endonuclease.
  11. 根据权利要求1-10任一项所述的方法，其中，所述DNA模板含有polyA序列。The method according to any one of claims 1 to 10, wherein the DNA template contains a polyA sequence.
  12. 权利要求1-11任一项所述的方法在mRNA领域的模板制备中的应用。Use of the method according to any one of claims 1 to 11 in template preparation in the mRNA field.
  13. 根据权利要求12所述的应用，其中，所述mRNA领域包括常规mRNA领域和/或特定的mRNA领域；The use according to claim 12, wherein the mRNA domain includes a conventional mRNA domain and/or a specific mRNA domain;
    优选地，所述特定的mRNA领域包括环状RNA和/或自复制RNA领域。Preferably, the specific mRNA domain comprises a circular RNA and/or a self-replicating RNA domain.
  14. 一种用于权利要求1-11任一项所述的方法的引物，所述引物由选自SEQ ID NO.1-SEQ ID NO.2、SEQ ID NO.4-SEQ ID NO.5、SEQ ID NO.8的序列组成。A primer for use in the method described in any one of claims 1 to 11, wherein the primer consists of a sequence selected from SEQ ID NO.1-SEQ ID NO.2, SEQ ID NO.4-SEQ ID NO.5, and SEQ ID NO.8.
  15. 一种试剂盒，其包括至少一种如权利要求14所述的引物。 A kit comprising at least one primer according to claim 14.
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