A method for in vitro mRNA transcription based on structural optimization elements

Abstract

This invention provides a method for in vitro mRNA transcription based on structurally optimized elements. The method includes: designing a DNA transcription template containing, sequentially along the transcription direction, an RNA polymerase promoter, a front-end regulatory sequence, a translation initiation enhancement sequence, a target protein coding sequence, a back-end stabilizing sequence, a polyA tail sequence, and a linearized restriction enzyme site; ligating the template into a plasmid vector and identifying it; cutting the template at the linearized restriction enzyme site to obtain the linearized DNA template; performing in vitro transcription using the template, introducing modified nucleotides and end-cap modifications; and obtaining the mRNA product after DNase I treatment, purification, and detection. This invention solves the problems of scattered template structure design, difficulty in controlling the length of the polyA tail, unclear transcription endpoint, and difficulty in simultaneously achieving product stability and expression efficiency in existing in vitro mRNA transcription processes.

Description

Technical Field

[0001] This invention relates to the field of gene editing, and more particularly to an in vitro transcription method for mRNA based on structural optimization elements. Background Technology

[0002] mRNA technology has received widespread attention in recent years in fields such as vaccines, cancer treatment, protein replacement therapy, and gene editing. In vitro transcription of mRNA typically uses linearized DNA as a template, synthesizing the target mRNA under the action of RNA polymerase. After end-cap modification, nucleotide modification, purification, and quality testing, it is used for cell expression or further formulation development. Compared with traditional protein expression or viral vector preparation methods, in vitro mRNA transcription has advantages such as shorter design cycles, convenient target sequence replacement, and relatively clear preparation procedures.

[0003] However, existing in vitro mRNA transcription methods still have certain shortcomings. On the one hand, transcription templates for different target proteins are often designed separately, and the layout of the front regulatory sequences, translation initiation enhancement sequences, back stabilizing sequences, polyA tail sequences, and linearized restriction enzyme sites lacks uniformity, resulting in high reproducibility in template construction and subsequent validation processes. On the other hand, some methods introduce polyA tail sequences through post-transcriptional tailing, which can easily lead to uneven tail lengths and affect batch-to-batch consistency of products. In addition, if the end-cap modification, modified nucleotide incorporation, and purification steps are not designed in conjunction with the template structure, they can also affect the integrity, stability, and expression efficiency of the target protein.

[0004] Therefore, it is necessary to provide a new in vitro mRNA transcription method based on structurally optimized elements to solve the above-mentioned technical problems. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an in vitro mRNA transcription method based on structural optimization elements, which solves the problems of scattered template structure design, difficulty in controlling the length of the polyA tail, unclear transcription endpoint, and difficulty in balancing product stability and expression efficiency in existing in vitro mRNA transcription processes.

[0006] The in vitro mRNA transcription method based on structure-optimized elements provided by this invention includes the following steps: S1. Design a DNA transcription template, wherein the DNA transcription template includes, in sequence along the transcription direction, an RNA polymerase promoter, a front regulatory sequence, a translation initiation enhancement sequence, a target protein coding sequence, a back stable sequence, a polyA tail sequence, and a linearized restriction enzyme site, wherein the front regulatory sequence, the translation initiation enhancement sequence, the back stable sequence, the polyA tail sequence, and the linearized restriction enzyme site constitute structural optimization elements; S2. The DNA transcription template is ligated into a plasmid vector to obtain a recombinant transcription vector, and the recombinant transcription vector is identified by PCR, enzyme digestion and sequencing. S3. The recombinant transcription vector is digested with a restriction endonuclease corresponding to the linearized restriction site, and the digestion product is purified to obtain a linearized DNA template. The cutting end of the linearized DNA template is located downstream of the polyA tail sequence. S4. Using the linearized DNA template as a template, add RNA polymerase, nucleoside triphosphate, modified nucleotides, reaction buffer and nuclease inhibitor for in vitro transcription, and add a cap analog during in vitro transcription or add a capped enzyme system after in vitro transcription for end-cap modification to obtain mRNA reaction product. S5. Add DNase I to the mRNA reaction product to remove residual DNA template, and then purify the mRNA reaction product to obtain the mRNA product. S6. The concentration, purity, and integrity of the mRNA product are detected, and the expression of the protein corresponding to the target protein coding sequence is verified by cell transfection.

[0007] Preferably, the RNA polymerase promoter is a T7 promoter, and the RNA polymerase is a T7 RNA polymerase.

[0008] Preferably, the front-end regulatory sequence is derived from the human β-globulin gene, the human α-globulin gene, or the Xenopus β-globulin gene, and the length of the front-end regulatory sequence is 40 to 150 nt.

[0009] Preferably, the translation initiation enhancement sequence is a Kozak sequence, the Kozak sequence is GCCACC, and it is located upstream of the start codon of the target protein coding sequence.

[0010] Preferably, the back-end stable sequence is derived from the human β-globulin gene, the human α-globulin gene, or the Xenopus β-globulin gene, and the back-end stable sequence is a single-copy sequence or a tandem double-copy sequence.

[0011] Preferably, the polyA tail sequence consists of 100 to 130 consecutive adenines, and the polyA tail sequence is positioned between the posterior stable sequence and the linearized restriction site.

[0012] Preferably, the modified nucleotide is N1-methylpseuuridine, pseudouridine, or 5-methylcytidine, the capping analog is ARCA or CleanCapAG, and the capping enzyme system includes vaccinia virus capping enzyme and 2-O-methyltransferase. Preferably, the purification in step S5 is carried out by lithium chloride precipitation, RNA purification column purification, cellulose purification, ion exchange chromatography or high performance liquid chromatography purification; the integrity detection in step S6 is carried out by agarose gel electrophoresis, denaturing gel electrophoresis, microarray electrophoresis or capillary electrophoresis; and the expression verification is carried out by Western blot, immunofluorescence or flow cytometry.

[0013] The beneficial effects of this invention are: 1. This invention integrates a front-end regulatory sequence, a translation initiation enhancement sequence, a back-end stabilizing sequence, a polyA tail sequence, and a linearized restriction enzyme site into the same DNA transcription template, making the transcription template structure clear and facilitating the construction, identification, and subsequent replacement of the target protein coding sequence.

[0014] 2. In this invention, the polyA tail sequence is pre-set in the DNA transcription template, and the linearization restriction site is located downstream of the polyA tail sequence. The transcription endpoint can be controlled by the linearization template, so that the resulting mRNA product has a relatively defined end structure.

[0015] 3. This invention introduces modified nucleotides during in vitro transcription, and can employ co-transcriptional end-cap modification or post-transcriptional enzymatic end-cap modification, which is beneficial for improving the stability of mRNA products and intracellular expression effects.

[0016] 4. The present invention incorporates DNase I treatment and purification steps after transcription, which can reduce the impact of residual DNA template, free nucleotides, enzyme proteins, short RNA fragments and other impurities on subsequent applications. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the in vitro mRNA transcription method of the present invention; Figure 2 A schematic diagram of the structure for preparing a linearized DNA template for recombinant transcription vector linearization; Figure 3 This is a schematic diagram of the in vitro transcription, purification, and expression validation process for mRNA. Detailed Implementation

[0018] The present invention will be further illustrated below with reference to the embodiments. It should be understood that the following embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make appropriate adjustments based on the target protein coding sequence, vector backbone, linearized restriction enzyme sites, end-cap modification methods, and purification methods without departing from the concept of the present invention.

[0019] Unless otherwise specified, the restriction endonucleases, T7 RNA polymerase, nucleoside triphosphates, modified nucleotides, RNA purification reagents, cell culture reagents, and transfection reagents used in the following examples are all commercially available products. All instruments, solutions, and consumables involving RNA manipulation are nuclease-free.

[0020] Example 1 like Figure 1-3 As shown, this embodiment provides an in vitro transcription method for mRNA using human β-globulin-derived structurally optimized elements and co-transcriptional end-cap modification, specifically including the following steps.

[0021] The nucleotide sequence encoding target protein A is selected as the target protein coding sequence. Target protein A can be a viral antigen protein, a tumor-associated antigen protein, a reporter protein, or a therapeutic protein. In this embodiment, the sequence encoding an antigen protein is used as the target protein coding sequence for illustration.

[0022] The DNA transcription template was designed to include, sequentially along the transcription direction, a T7 promoter, a front regulatory sequence, a Kozak sequence, a target protein coding sequence, a back stabilizing sequence, a polyA tail sequence, and a linearization restriction site. The front regulatory sequence was derived from human β-globulin; the Kozak sequence was set to GCCACC and positioned upstream of the start codon of the target protein coding sequence; the back stabilizing sequence was also derived from human β-globulin and configured as a tandem double copy; the polyA tail sequence consisted of 120 consecutive adenine residues; and the linearization restriction site was located downstream of the polyA tail sequence, using the XbaI restriction site in this embodiment.

[0023] To facilitate subsequent cloning, linker sequences or homologous arms matching the vector are designed at both ends of the target protein coding sequence. The target protein coding sequence is synthesized after codon optimization, without altering its encoded amino acid sequence, and avoiding the introduction of restriction enzyme sites identical to linearized restriction sites within the coding region.

[0024] The DNA transcription template obtained in step S1 is inserted into the pcDNA3.1 vector or the pGEM vector to obtain a recombinant transcription vector. Specifically, the plasmid vector is linearized, and the target protein coding sequence and its upstream and downstream structural optimization elements are used as insert fragments and ligated to the linearized vector via homologous recombination or restriction enzyme digestion.

[0025] The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar containing the appropriate antibiotic, and incubated overnight at 37°C. Single clones were picked for initial screening by bacterial PCR, and positive clones were expanded and plasmids were extracted. The extracted plasmids were identified by PCR, double enzyme digestion, and sequencing, confirming that the order and orientation of the T7 promoter, front regulatory sequence, Kozak sequence, target protein coding sequence, posterior stable sequence, polyA tail sequence, and linearized restriction enzyme sites were correct.

[0026] The correctly sequenced recombinant transcription vector was digested with XbaI restriction endonuclease to break the plasmid downstream of the polyA tail sequence. After the digestion reaction, a small amount of the reaction solution was subjected to agarose gel electrophoresis to confirm that the plasmid had changed from a circular or supercoiled state to a single linear band.

[0027] The linearized restriction enzyme digestion products were purified by column chromatography or gel extraction to remove restriction endonucleases, salt ions, short DNA fragments, and incompletely digested plasmids. The purified linearized DNA template was eluted with nuclease-free water, and its concentration was determined. The resulting linearized DNA template was used for subsequent in vitro transcription reactions.

[0028] The linearized DNA template obtained in step S3 was used as a template to prepare the in vitro transcription reaction system. 1 μg of linearized DNA template, 2 μL of 10×T7 reaction buffer, ATP, CTP, GTP, N1-methylpseudouridine triphosphate, nuclease inhibitor, T7 RNA polymerase, and CleanCap AG were added to every 20 μL of the reaction system.

[0029] N1-methylpseudouridine triphosphate was used to replace UTP in the in vitro transcription reaction; CleanCapAG was used to modify the end cap of mRNA during transcription. The reaction mixture was gently mixed and briefly centrifuged, then incubated at 37°C for 3 to 4 hours. After the reaction, DNase I was added to the mixture, and the mixture was further treated at 37°C for 15 to 30 minutes to remove residual DNA template.

[0030] In another alternative, CleanCapAG can be replaced with ARCA, while other reaction conditions remain unchanged.

[0031] The reaction system treated with DNase I was purified. Preliminary purification can be performed using lithium chloride precipitation. Specifically, lithium chloride precipitate is added to the reaction system, mixed, and then placed at low temperature to allow mRNA precipitation. The precipitate is collected by centrifugation, washed 2 to 3 times with pre-cooled 70% ethanol, dried, and then dissolved in nuclease-free water.

[0032] For mRNA products requiring high precision in subsequent expression validation or in vivo application, further purification using RNA purification columns, cellulose purification, or high-performance liquid chromatography (HPLC) can be employed to reduce the content of short RNA fragments, residual proteins, free nucleotides, and double-stranded RNA impurities. Purified mRNA products should be aliquoted and stored at -80°C to avoid repeated freeze-thaw cycles.

[0033] The concentration and purity of the mRNA product were determined by ultraviolet spectrophotometry, and the A260 / A280 and A260 / A230 values ​​were recorded. An appropriate amount of mRNA product was mixed with RNA loading buffer, heated to 65°C, and then quickly placed on ice. The mixture was subsequently analyzed by denaturing agarose gel electrophoresis or microarray electrophoresis. The electrophoresis results showed that the main mRNA band was consistent with the theoretical length, and there were no obvious tails or degradation bands, indicating good mRNA integrity.

[0034] The purified mRNA product was transfected into HEK293T cells or HeLa cells. Cells were seeded in cell culture plates one day prior to transfection to achieve 70% to 80% confluency at transfection. The mRNA was mixed with the transfection reagent according to the instructions, incubated at room temperature, and then added to the cell culture system. Cells were collected 24 to 48 hours after transfection, and the expression of target protein A was detected by Western blot or immunofluorescence. If a specific band matching the theoretical molecular weight of target protein A was detected, or a specific fluorescent signal was observed in immunofluorescence, it indicates that the mRNA product prepared in this embodiment can express the corresponding target protein in cells.

[0035] This embodiment integrates the front regulatory sequence and back stable sequence, Kozak sequence, fixed-length polyA tail sequence, and downstream linearized restriction enzyme site of polyA tail from human β-globulin into the same DNA transcription template, making the structure of the mRNA product more defined. At the same time, the use of co-transcriptional end-cap modification and modified nucleotides to participate in transcription is beneficial to improve the stability of the mRNA product and the cell expression effect.

[0036] Example 2 This embodiment provides an in vitro transcription method for mRNA using structurally optimized elements derived from Xenopus laevis β-globulin and post-transcriptional enzymatic end-cap modification, specifically including the following steps.

[0037] The nucleotide sequence encoding the target protein B is selected as the target protein coding sequence. Target protein B may differ from target protein A in Example 1, illustrating the applicability of the method of the present invention to different coding sequences.

[0038] The DNA transcription template was designed to include, sequentially along the transcription direction, a T7 promoter, a front regulatory sequence, a Kozak sequence, a target protein coding sequence, a back stabilizing sequence, a polyA tail sequence, and a linearized restriction enzyme site. Specifically, the front regulatory sequence was derived from Xenopus β-globulin; the Kozak sequence was set to GCCACC; the back stabilizing sequence was also derived from Xenopus β-globulin; the polyA tail sequence consisted of 100 consecutive adenine residues; and the linearized restriction enzyme site was located downstream of the polyA tail sequence, using HindIII or XbaI restriction enzyme sites in this embodiment.

[0039] During the design process, it was ensured that the linearized restriction site was not located within the target protein coding sequence, the front regulatory sequence, the back stabilizing sequence, or the polyA tail sequence, in order to avoid the linearization process disrupting the integrity of the transcription template.

[0040] The DNA transcription template designed in step S1 is inserted into the pXT7 vector or pGEM vector to obtain a recombinant transcription vector. Specifically, the vector is linearized, the target protein coding sequence is amplified by PCR or synthesized, and the amplified product is purified and ligated into the linearized vector.

[0041] The ligation product was transformed into *E. coli* DH5α competent cells, and single clones were picked after culture. Positive clones were screened by bacterial PCR, expanded, and plasmids were extracted. The recombinant plasmid was identified by restriction enzyme digestion and sequencing, confirming that the target protein coding sequence was correctly inserted, and that the T7 promoter, front regulatory sequence, Kozak sequence, target protein coding sequence, posterior stable sequence, polyA tail sequence, and linearized restriction sites were correctly arranged, with no base deletions, insertions, or frameshift mutations.

[0042] The recombinant transcription vector was digested using restriction endonucleases corresponding to the linearized restriction sites in step S1. For example, when the linearized restriction site was HindIII, HindIII was used for single digestion; when the linearized restriction site was XbaI, XbaI was used for single digestion.

[0043] The enzyme digestion reaction was carried out at 37°C for 1 to 3 hours. After the reaction, agarose gel electrophoresis was performed. If a single linear band was observed and no obvious undigested plasmid bands were found, further purification was performed. The purified linearized DNA template was eluted with nuclease-free water, and the template concentration was determined.

[0044] Using the linearized DNA template obtained in step S3 as a template, an in vitro transcription reaction system was prepared. The linearized DNA template, T7 RNA polymerase, ATP, CTP, GTP, UTP or pseudouridine triphosphate, 5-methylcytidine triphosphate, T7 reaction buffer, and a nuclease inhibitor were added to the reaction system. After mixing, the reaction system was incubated at 37°C for 3 to 4 hours to obtain the initial mRNA product without end-cap modification.

[0045] After the reaction was complete, DNase I was added to the system and the mixture was incubated at 37°C for 15 to 30 minutes to remove residual DNA template. The initial mRNA product was then purified to remove DNase I, salt ions, and free nucleotides.

[0046] The purified mRNA precursor was used for enzymatic end-cap modification. Specifically, the mRNA precursor was mixed with GTP, SAM, vaccinia virus capping enzyme, capping reaction buffer, and nuclease inhibitor, and reacted at 37°C for 1 hour. Subsequently, 2-O-methyltransferase was added, and the reaction continued for 1 to 2 hours to further methylate the mRNA product. After the reaction was completed, the enzymatically end-cap modified mRNA product was obtained.

[0047] The mRNA reaction product obtained in step S4 is then purified. Rapid purification can be performed using an RNA purification column, or a combination of lithium chloride precipitation and column purification can be used. For samples with high impurity control requirements, cellulose purification or high-performance liquid chromatography (HPLC) can be used to remove double-stranded RNA impurities and short RNA fragments.

[0048] After purification, the mRNA product was dissolved in nuclease-free water, its concentration was determined, and it was aliquoted and stored. The storage temperature was -80℃. Repeated freeze-thaw cycles were avoided during storage to reduce mRNA degradation.

[0049] The purified mRNA product was subjected to UV absorbance detection, and the A260 / A280 and A260 / A230 values ​​were recorded. Integrity was also assessed using denaturing agarose gel electrophoresis, microarray electrophoresis, or capillary electrophoresis. The mRNA product should show a main band or peak consistent with the theoretical length in the test results.

[0050] The mRNA product was transfected into HEK293T cells, HeLa cells, or other cell lines suitable for target protein expression. Cells were collected 24 to 48 hours post-transfection, and total protein was extracted using cell lysis buffer. Target protein B expression was detected by Western blot. Alternatively, transfected cells were fixed, permeabilized, blocked, and incubated with antibodies, and target protein B expression was detected by immunofluorescence. If a specific band of the corresponding molecular weight was detected by Western blot, or a specific signal was observed by immunofluorescence, it indicates that the mRNA product obtained in this embodiment can express target protein B in cells.

[0051] The difference between this embodiment and Embodiment 1 is that this embodiment uses the Xenopus laevis β-globulin-derived sequence as the front-end regulatory sequence and the back-end stabilizing sequence, and uses post-transcriptional enzymatic end-cap modification to prepare the mRNA product. This embodiment illustrates that the method of the present invention is applicable not only to co-transcriptional end-cap modification, but also to post-transcriptional enzymatic end-cap modification; and not only to human β-globulin-derived structural optimization elements, but also to Xenopus laevis β-globulin-derived structural optimization elements.

[0052] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for in vitro transcription of mRNA based on structural optimization elements, characterized in that, Includes the following steps: S1. Design a DNA transcription template, wherein the DNA transcription template includes, in sequence along the transcription direction, an RNA polymerase promoter, a front regulatory sequence, a translation initiation enhancement sequence, a target protein coding sequence, a back stable sequence, a polyA tail sequence, and a linearized restriction enzyme site, wherein the front regulatory sequence, the translation initiation enhancement sequence, the back stable sequence, the polyA tail sequence, and the linearized restriction enzyme site constitute structural optimization elements; S2. The DNA transcription template is ligated into a plasmid vector to obtain a recombinant transcription vector, and the recombinant transcription vector is identified by PCR, enzyme digestion and sequencing. S3. The recombinant transcription vector is digested with a restriction endonuclease corresponding to the linearized restriction site, and the digestion product is purified to obtain a linearized DNA template. The cutting end of the linearized DNA template is located downstream of the polyA tail sequence. S4. Using the linearized DNA template as a template, add RNA polymerase, nucleoside triphosphate, modified nucleotides, reaction buffer and nuclease inhibitor for in vitro transcription, and add a cap analog during in vitro transcription or add a capped enzyme system after in vitro transcription for end-cap modification to obtain mRNA reaction product. S5. Add DNase I to the mRNA reaction product to remove residual DNA template, and then purify the mRNA reaction product to obtain the mRNA product. S6. The concentration, purity, and integrity of the mRNA product are detected, and the expression of the protein corresponding to the target protein coding sequence is verified by cell transfection.

2. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The RNA polymerase promoter is a T7 promoter, and the RNA polymerase is a T7 RNA polymerase.

3. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The front-end regulatory sequence is derived from the human β-globulin gene, the human α-globulin gene, or the Xenopus laevis β-globulin gene, and the length of the front-end regulatory sequence is 40 to 150 nt.

4. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The translation initiation enhancement sequence is a Kozak sequence, which is GCCACC and is located upstream of the start codon of the target protein coding sequence.

5. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The back-end stable sequence is derived from the human β-globulin gene, the human α-globulin gene, or the Xenopus laevis β-globulin gene, and the back-end stable sequence is a single-copy sequence or a tandem double-copy sequence.

6. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The polyA tail sequence consists of 100 to 130 consecutive adenines, and the polyA tail sequence is positioned between the posterior stable sequence and the linearized restriction site.

7. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The modified nucleotide is N1-methylpseuuridine, pseudouridine, or 5-methylcytidine, the capping analog is ARCA or CleanCapAG, and the capping enzyme system includes vaccinia virus capping enzyme and 2-O-methyltransferase.

8. The in vitro mRNA transcription method based on structure-optimized elements according to claim 1, characterized in that, The purification described in step S5 uses lithium chloride precipitation, RNA purification column purification, cellulose purification, ion exchange chromatography, or high performance liquid chromatography. The integrity detection described in step S6 uses agarose gel electrophoresis, denaturing gel electrophoresis, microarray electrophoresis, or capillary electrophoresis. The expression verification uses Western blot, immunofluorescence, or flow cytometry.

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