CN118048420A - Preparation method of capped mRNA - Google Patents
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Abstract
The invention relates to a preparation method of capping mRNA. The preparation method of the invention comprises the following steps: providing a reaction system; incubating and capping a reaction system at 36-38 ℃, wherein the reaction system comprises the following components: mRNA;0.5~0.9mM MgCl2;0.1~1.5U/mL RNase III;40~60mM Tris-HCl;2~8mM KCl;0.5~2mM DTT;0.1~0.5U/μL vaccinia virus capping enzyme synthesized in vitro at a concentration of 1-3 mg/mL; 0.5-2U/. Mu.L Cap 2' -O methyltransferase; 0.1-1 mM GTP;0.1 to 0.5mM SAM; the balance being RNase-free water. The invention improves the recovery rate of mRNA, ensures the capping rate and reduces the dsRNA content, improves the integrity of mRNA, and can provide powerful support for the development of mRNA drugs.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a preparation method of capped mRNA.
Background
Messenger ribonucleic acids (mRNAs) are an emerging class of biotherapeutic drugs. Although they have considerable promise, the field is still in the start-up phase. The process of achieving drug-grade mRNA production by in vitro transcription reactions is briefly: constructing plasmid DNA, cutting the plasmid DNA by restriction enzyme to obtain linearization DNA, using the linearization DNA as DNA template for RNA in vitro transcription, carrying out transcription by RNA polymerase, and releasing template DNA and newly synthesized mRNA after transcription is stopped.
In mRNA vaccines and gene function studies, in vitro synthesized mRNA needs to be able to accomplish translation in vivo, capping and tailing are critical, they are effective in preventing degradation and facilitating its translation in eukaryotic cells. There are currently two ways in which cap structures can be added to transcripts, including co-transcriptional capping and enzymatic capping.
Co-transcriptional capping is the incorporation of a cap analogue as a first nucleotide into the transcript during the transcription reaction, directly producing mRNA with cap structure, but first generation cap analogues produce a mixture of transcripts with cap structure in both directions, which cannot be translated efficiently in the wrong direction, resulting in low yield of target protein. The second generation ARCA can avoid wrong ligation orientation, but the yield is much reduced. Third generation GAG-like cap analogues, while solving the above problems, are costly, with capping rates between 90-95%, which are still further to be improved. The enzymatic capping is to add cap0 structure at the 5 'end of mRNA with cap-free structure by using vaccinia virus capping system after transcription is completed, or to add cap 1 structure in one step by using vaccinia virus capping system and mRNA cap structure 2' -O methyltransferase, the capping efficiency can reach more than 99%, and all cap structures are added in correct direction. In addition, enzymatic capping is lower in cost, which is one of the important reasons for the current production of enzymatic capping.
In vitro transcription reactions, while producing large amounts of mRNA, inevitably contain double-stranded RNA (double-STRANDED RNA, DSRNA), which causes immunogenicity, and it is necessary to reduce the content thereof to a certain extent to reduce the immunogenicity. However, the related process of removing dsRNA from mRNA is not mature and has various drawbacks. Enzymatic dsRNA is difficult to balance dsRNA removal efficiency and mRNA integrity, and the better the removal effect on dsRNA, the greater the influence on mRNA integrity. And the reaction system for removing the dsRNA is different from the existing enzymatic capping reaction system, the step of removing the dsRNA is usually carried out before or after the capping reaction, and the operation is complicated and mRNA loss is easy to cause when the reaction system is switched, so that the mRNA yield is reduced. Patent CN101563457a uses porous reverse phase as stationary phase for purification to remove dsRNA using HPLC, with low loading and requiring the use of flammable, toxic solvents. Patent CN113166790a uses a denaturing salt and a reducing agent to purify mRNA, which introduces new impurities whose effect of dsRNA removal is unknown and may affect the post-capping effect. The patent CN115287282A removes dsRNA through a hydrophobic chromatography column, the operation is complex, the mRNA loss is large, and the dsRNA removing effect is unknown. The patent CN2023115690511 removes dsRNA by DEAE ion exchange chromatography, which is cumbersome to operate and can cause mRNA loss. In addition, the dsRNA removal process needs to be carried out before or after the capping reaction, has a plurality of procedures and long operation time, and is not beneficial to promoting the industrialized production of capped mRNA.
Therefore, developing a method that can compromise the capping rate, integrity and dsRNA content of the capped mRNA synthesized in vitro remains a primary solution for the development and application of drugs for synthesizing mRNA in vitro.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of capping mRNA, which can remove dsRNA while capping to prepare high-quality mRNA with high capping rate, high integrity and low dsRNA content.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a preparation method of capped mRNA, which comprises the following steps:
Providing a reaction system;
placing the reaction system in a constant temperature culture shaker for incubation reaction at 36-38 ℃, then adopting Tris-EDTA buffer solution with pH value of 7.8-8.0 to terminate the reaction to obtain reaction solution containing capped mRNA,
Wherein the components of the reaction system are as follows: mRNA;0.5~0.9mM MgCl2;0.1~1.5U/mL RNase III;40~60mM Tris-HCl;2~8mM KCl;0.5~2mM DTT;0.1~0.5U/μL vaccinia virus capping enzyme synthesized in vitro at a concentration of 1-3 mg/mL; 0.5-2U/. Mu.L Cap 2' -O methyltransferase; 0.1-1 mM GTP;0.1 to 0.5mM SAM; the balance being RNase-free water.
Preferably, the content of MgCl 2 in the reaction system is 0.7-0.9 mM.
Further preferably, the content of MgCl 2 in the reaction system is 0.8-0.9 mM.
Preferably, tris-HCl, DTT, KCl, mgCl 2 in the reaction system is dosed in the form of a capping buffer comprising the following components: 400-600 mM Tris-HCl; 5-20 mM DTT; 20-80 mM KCl; 5-9 mM MgCl 2; the balance is RNase-free water, and the pH value of the capping buffer solution is 7.5-8.2.
Specifically, the capping buffer is 10×capping buffer, and may be prepared in advance and stored for later use.
Further preferably, the pH value of the capping buffer is 7.8-8.0.
Still more preferably, the capping buffer has a pH of 7.8.
According to some preferred embodiments, the components of the reaction system are: mRNA;0.8~0.9mM MgCl2;1~1.5U/mL RNase III;40~60mM Tris-HCl;4~6mM KCl;0.8~1.5mM DTT;0.1~0.5U/μL vaccinia virus capping enzyme synthesized in vitro at a concentration of 1-3 mg/mL; 0.5-1.5U/. Mu.L Cap 2' -O methyltransferase; 0.4-0.6 mM GTP;0.1 to 0.5mM SAM; the balance is RNase-free water, wherein Tris-HCl, DTT, KCl, mgCl 2 is dosed in the form of a capping buffer comprising the following components: 400-600 mM Tris-HCl; 8-15 mM DTT; 40-60 mM KCl; 8-9 mM MgCl 2; the balance was RNase-free water, and the pH of the capping buffer was 7.8.
In the invention, the in vitro synthesized mRNA is uncapped mRNA obtained by treatment of IVT reaction solution.
The treatment is conventional in the art, and comprises one or more of tangential flow ultrafiltration liquid exchange, oligo-dT monolithic column affinity chromatography or tangential flow ultrafiltration concentrated liquid exchange.
According to some embodiments, the treatment comprises sequentially performing tangential flow ultrafiltration exchange, oligo-dT monolithic column affinity chromatography, and tangential flow ultrafiltration concentrate exchange.
In the present invention, the length of the in vitro synthesized mRNA is preferably 1000 to 5000nt.
Preferably, the incubation time is 0.5-2 hours.
Further preferably, the incubation time is 0.5-1.5 h.
According to some embodiments, the capping rate of the capped mRNA obtained by the method is more than or equal to 99%, the mRNA integrity is more than 89%, the dsRNA content is less than 100ng/mg, and the quality is obviously improved.
Preferably, the preparation method further comprises a step of extracting capped mRNA in the reaction liquid by a magnetic bead method.
The reaction system in the preparation method of the capping mRNA is also within the protection scope of the invention as a novel capping reaction system of the mRNA synthesized in vitro.
Capping buffers in the above-described methods of preparing capped mRNA are also within the scope of the present invention as a novel capping buffer.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
Compared with two-step reaction, the preparation method of capping mRNA can carry out dsRNA removal reaction at the same time of capping reaction, not only simplifies the process and reduces the loss of mRNA, but also improves the integrity of mRNA while ensuring the capping rate and reducing the dsRNA content, thereby obtaining the capping mRNA with truly high quality and having great significance for the development of mRNA medicine.
Detailed Description
In the in vitro synthesis production of capping mRNA, the capping reaction and dsRNA removal need to be carried out in two steps, which can lead to low recovery rate of mRNA, and the removal rate of dsRNA and the integrity of mRNA are difficult to be simultaneously considered, so that the quality of the capping mRNA produced by the existing in vitro synthesis of capping mRNA still needs to be improved.
In order to obtain a higher quality capped mRNA product, the inventors of the present application have conducted intensive studies and extensive experimental verification to propose a reaction system suitable for simultaneously carrying out capping reactions and dsRNA removal reactions, thereby achieving a "one-pot" reaction. According to the application, through the integral design of a reaction system and the organic combination of the components, under one-step reaction, the dsRNA content is reduced, the high capping rate is obtained, meanwhile, the influence on the mRNA integrity is reduced, and particularly, the concentration of MgCl 2 in the system is controlled to be 0.5-0.9 mM, and the other components are matched, so that unexpected positive effects of ensuring the capping rate, reducing the dsRNA content and improving the mRNA integrity are achieved. The capping rate of the capped mRNA obtained by the one-step reaction is more than or equal to 99%, the mRNA integrity is more than 89%, and the dsRNA content is less than 100ng/mg. The preparation method of the capping mRNA is beneficial to realizing the industrialized production of the capping mRNA and provides powerful support for the development and application of mRNA medicaments.
The technical scheme of the present invention will be further described with reference to specific embodiments, but the present invention is not limited to the following examples. The implementation conditions employed in the examples may be further adjusted according to specific requirements, and the implementation conditions not specified are generally those in routine experiments.
The mRNA used in the following examples and comparative examples was in vitro synthesized mRNA (an mRNA encoding luciferase with a sequence length of about 2000 nt), and was subjected to in vitro transcription (In vitro transcription, IVT) using a linearized plasmid template according to conventional techniques in the art, followed by degradation of the linearized plasmid template by DNase I, followed by tangential flow ultrafiltration and Oligo-dT column affinity chromatography, followed by tangential flow ultrafiltration concentration of the stock to give uncapped mRNA, which was stored in ultrapure water at an mRNA concentration of 2.5mg/mL and used as a substrate for the subsequent capping reaction.
In the following examples and comparative examples, RNase III was purchased from Shanghai megadimension technology development Co., ltd. (cat. No.: ON-024); commercially available 10 Xcapping buffer was purchased from offshore protein Biotechnology Co., ltd (Novoprotein) (cat# GMP-EB 62-M010); the 10 x capping buffer in the remaining examples and comparative examples was self-made. Vaccinia virus capping enzyme was purchased from offshore protein biotechnology Co., ltd (Novoprotein) (cat# GMP-M062-M010); cap 2' -O methyltransferase was purchased from offshore protein Biotechnology Co., ltd (Novoprotein) (cat# GMP-M072-M010); t7 RNA polymerase was purchased from offshore protein Biotechnology Co., ltd (cat# GMP-E121-M010); GTP is purchased from Qingdao sugar Intelligence medicine technology Co., ltd. (product number: MR-1002); SAM is available from offshore protein biotechnology Co., ltd (Novoprotein) (cat# GMP-S062-M005); magnetic beads for RNA extraction were purchased from Baimei Biotechnology Co., ltd (cat# BMSX-60).
The other reagents, instruments, etc., as used in the following examples and comparative examples, are commercially available products conventionally used in the art.
The detection methods used in the following examples and comparative examples are as follows:
1. Integrity of mRNA: the integrity of the mRNA was checked using an agilent fragmentation Analyzer 5300 (AGILENT FRAGMENT Analyzer 5300). The operation follows the user manual of the agilent fragment analyzer 5300. The sample to be tested was diluted to 100 ng/. Mu.L, incubated at 70℃for 2 minutes, and then frozen for 5 minutes. mu.L of the diluted sample was mixed with 22. Mu.L of the diluent label (15-nt) in a 96-well plate. The isolation was performed using DNF-471-33-SS Total RNA (15-nt) method. mRNA integrity was assessed using Agilent data analysis software (Agilent ProSize), and with reference to the specification, a vertical line was drawn at the peak inflection point before and after the main peak, the area between the two vertical lines divided by the total area from which the 15-nt marker from the diluent was subtracted, defined as mRNA integrity.
The capping rate detection method comprises the following steps:
The DNA primer sequence was designed based on the mRNA 5' sequence, mRNA and DNA primers were annealed at 95℃for 5 minutes, frozen on ice for 5 minutes, and then incubated with RNase H for 30 minutes, and the capping efficiency, defined as the area of cap1 mRNA/(the area of cap1 mRNA+the area of cap0 mRNA+the area of G cap mRNA+the area of uncapped mRNA). Times.100%, was determined by theoretical molecular weight for each fragment using LC-MS. The G cap mRNA indicates that methyl was not added to the cap at position 7.
The dsRNA content detection method comprises the following steps:
dsRNA was quantified by enzyme-linked immunosorbent assay (ELISA) according to the user instructions. ELISA kit for dsRNA quantification was purchased from Nanjinofuzan medical science and technology Co., ltd (Vazyme Biotech), and the product number was DD3508-01, and the dsRNA quantification was performed according to the instruction.
Example 1
The present example provides a method for preparing capped mRNA, comprising the steps of:
The reaction system is shown in Table 1.
The 10 x capping buffer in table 1 is pre-formulated and comprises the following components: 500mM Tris-HCl,10mM DTT,50mM KCl,9mM MgCl 2; pH 7.8 (25 ℃ C.).
The experimental steps are as follows:
1. taking 80 mu L of mRNA solution (2.5 mg/mL) to a 1.5mL centrifuge tube, adding 4 mu L of RNase-free water, uniformly mixing, incubating for 15min at 37 ℃ in a constant temperature culture oscillator, taking out the centrifuge tube, placing the centrifuge tube at room temperature, adding the rest components according to the table 1, and uniformly mixing to obtain a reaction system.
2. The reaction system of step 1 was incubated at 37℃for 1 hour in a constant temperature incubator shaker, and then 11. Mu.L of EDTA (0.5M pH 8.0) was added to terminate the reaction.
3. RNA was extracted by the magnetic bead method. Taking out the magnetic beads from the refrigerator at 4 ℃, uniformly mixing by vortex, recovering to room temperature, and mixing the reaction liquid and the magnetic beads in the step 2 according to the volume ratio of 1:2 mixing, standing at room temperature for 10min, then placing on a magnetic rack for adsorption for 10min, carefully sucking liquid with a gun head (taking care not to touch magnetic beads), adding 800 mu L of 80% ethanol (for use at present), gently reversing and washing for 2 times, carefully removing ethanol, volatilizing ethanol in a fume hood for 10min, adding 150 mu L of sterile water for injection, placing on the magnetic rack for adsorption for 10min, transferring the liquid to a new centrifuge tube, and simultaneously detecting mRNA capping rate, mRNA integrity and dsRNA content.
Example 2
Essentially the same as in example 1, except that the concentration of MgCl 2 in the pre-prepared 10 Xcapping buffer was 8mM.
Example 3
Essentially the same as in example 1, except that the pH of the pre-prepared 10 Xcapping buffer was 8.0 (25 ℃).
Example 4
Essentially the same as in example 1, except that the pH of the preformed 10 Xcapping buffer was 8.2 (25 ℃).
Example 5
The procedure was substantially as in example 1, except that RNase III was used in an amount of 3. Mu.L in the reaction system.
Comparative example 1
Essentially the same as in example 1, except that the 10 x capping buffer in the reaction system was a commercially available product comprising the following components: 500mM Tris-HCl,10mM DTT,50mM KCl,10mM MgCl 2; pH 8.0 (25 ℃ C.).
Comparative example 2
Essentially the same as in example 1, except that the concentration of MgCl 2 in the pre-prepared 10 Xcapping buffer was 1mM.
Comparative example 3
Essentially the same as in example 1, except that the concentration of MgCl 2 in the pre-prepared 10 Xcapping buffer was 4mM.
Comparative example 4
Essentially the same as in example 1, except that the concentration of MgCl 2 in the pre-prepared 10 Xcapping buffer was 20mM.
Comparative example 5
Essentially the same as in example 1, except that the concentration of MgCl 2 in the pre-prepared 10 Xcapping buffer was 30mM.
Comparative example 6
Essentially the same as in example 1, except that the pH of the pre-prepared 10 Xcapping buffer was 7.2.
Comparative example 7
The comparative example uses dsRNA removal followed by capping reaction, and is specifically as follows:
the dsRNA removal reaction system is shown in Table 2:
the 10 XRNase III reaction buffer composition in Table 2 was: 500mM Tris-HCl,50mM KCl,10mM DTT,20mM MgCl 2, pH7.8 (25 ℃).
The experimental steps are as follows:
1. mu.L of mRNA (2.5 mg/mL) was taken into a 1.5mL centrifuge tube, 9. Mu.L of RNase-free water was added, and after mixing, the relevant components were added according to Table 2, and the mixture was mixed to obtain a dsRNA-free reaction system.
2. The reaction system of step 1 was incubated at 37℃for 30min in a constant temperature incubator shaker, and then 11. Mu.L of EDTA (0.5M pH 8.0) was added to terminate the reaction.
3. RNA was extracted by the magnetic bead method. Taking out the magnetic beads from the refrigerator at 4 ℃, uniformly mixing by vortex, recovering to room temperature, and mixing the reaction liquid and the magnetic beads in the step 2 according to the volume ratio of 1:2, mixing, standing at room temperature for 10min min, then placing on a magnetic rack to adsorb 10min, carefully sucking the liquid off with a gun head (taking care not to touch the magnetic beads), adding 800 μl of 80% ethanol (for use now), gently washing upside down for 2 times, carefully removing the ethanol, volatilizing the ethanol in a fume hood for 10min, adding 80 μl of sterile water for injection, placing on the magnetic rack to adsorb for 10min, and transferring the liquid into a new 1.5mL centrifuge tube to serve as mRNA substrate for capping reaction.
The capping reaction system is shown in table 3:
The 10 x capping buffer in table 3 is a commercially available product with the following components: 500mM Tris-HCl,10mM DTT,50mM KCl,10mM MgCl 2; pH 8.0 (25 ℃ C.).
The experimental steps are as follows:
1. Adding 4.87 mu L of RNase-free water into a centrifuge tube containing mRNA substrates obtained by dsRNA removal experiments, uniformly mixing, incubating for 15min at 37 ℃ in a constant temperature culture shaker, taking out the centrifuge tube, placing the centrifuge tube at room temperature, adding relevant components according to Table 3, and uniformly mixing to obtain a capping reaction system.
2. The capping reaction system of step 1 was incubated at 37℃for 1h in a constant temperature culture shaker, and then the reaction was terminated by adding 11.1. Mu.L of EDTA (0.5M pH 8.0).
3. RNA was extracted by the magnetic bead method. Taking out the magnetic beads from the refrigerator at 4 ℃, uniformly mixing by vortex, recovering to room temperature, and mixing the reaction liquid and the magnetic beads in the step 2 according to the volume ratio of 1:2 mixing, standing at room temperature for 10min, then placing on a magnetic rack for adsorption for 10min, carefully sucking liquid with a gun head (taking care not to touch magnetic beads), adding 800 μL of 80% ethanol (for use at present), gently reversing and washing for 2 times, carefully removing ethanol, volatilizing ethanol in a fume hood for 10min, adding 150 μL of sterile water for injection, placing on the magnetic rack for adsorption for 10min, transferring the liquid to a new centrifuge tube, and detecting mRNA capping rate, mRNA integrity and dsRNA content.
The results of each example and comparative example are shown in table 4:
The limit of detection of dsRNA in Table 4 was 23.0ng/mg, and the dsRNA content of comparative example 4, comparative example 5 and comparative example 7 was lower than the limit of detection.
Table 4 shows that the preparation method adopted in examples 1 to 5 ensures that the capping rate reaches 99% and above, and the dsRNA content is reduced and the integrity of mRNA is maintained to reach 89% or above, wherein the capping mRNA quality is the best in example 1, and the next in examples 2 and 5. In comparative examples 1-7, capping reaction systems or methods with lower dsRNA content resulted in significantly reduced mRNA integrity (comparative example 4, comparative example 5 and comparative example 7), and also may affect capping rate (comparative example 4 and comparative example 5), capping reaction systems with good mRNA integrity resulted in significantly reduced capping rate (comparative example 2) or dsRNA content exceeding 100ng/mg (comparative example 2 and comparative example 3). In addition, the recovery rate of mRNA in each of examples 1 to 5 was more than 95%, which is significantly improved compared to the two-step reaction (comparative example 7).
The present invention has been described in detail in order to make those skilled in the art understand the present invention and implement it, but the present invention is not limited to the above embodiments, and all equivalent changes or modifications according to the spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for preparing a capped mRNA, characterized by: the preparation method comprises the following steps:
Providing a reaction system;
Placing the reaction system in a constant temperature culture oscillator for incubation reaction at 36-38 ℃, then adopting EDTA buffer solution with pH value of 7.8-8.0 to terminate the reaction to obtain reaction solution containing capped mRNA,
Wherein the components of the reaction system are as follows: mRNA;0.5~0.9mM MgCl2;0.1~1.5U/mL RNase III;40~60mM Tris-HCl;2~8mM KCl;0.5~2mM DTT;0.1~0.5U/μL vaccinia virus capping enzyme synthesized in vitro at a concentration of 1-3 mg/mL; 0.5-2U/. Mu.L Cap 2' -O methyltransferase; 0.1-1 mM GTP;0.1 to 0.5mM SAM; the balance being RNase-free water.
2. The method of preparing capped mRNA according to claim 1, wherein: the content of MgCl 2 in the reaction system is 0.7-0.9 mM.
3. The method of preparing capped mRNA according to claim 1, wherein: tris-HCl, DTT, KCl, mgCl 2 in the reaction system is fed in the form of capping buffer solution, and the capping buffer solution comprises the following components:
400~600mM Tris-HCl；
5~20mM DTT；
20~80mM KCl；
5~9mM MgCl2；
The rest is water without RNase,
The pH value of the capping buffer solution is 7.5-8.2.
4. A method of preparing a capped mRNA according to claim 3, wherein: the pH of the capping buffer was 7.8.
5. The method of preparing capped mRNA according to claim 1, wherein: the components of the reaction system are as follows: mRNA;0.8~0.9mM MgCl2;1~1.5U/mL RNase III;40~60mM Tris-HCl;4~6mM KCl;0.8~1.5mM DTT;0.1~0.5U/μL vaccinia virus capping enzyme synthesized in vitro at a concentration of 1-3 mg/mL; 0.5-1.5U/. Mu.L Cap 2' -O methyltransferase; 0.4-0.6 mM GTP;0.1 to 0.5mM SAM; the rest is water without RNase,
Wherein Tris-HCl, DTT, KCl, mgCl 2 is dosed in the form of a capping buffer comprising the following components: 400-600 mM Tris-HCl; 8-15 mM DTT; 40-60 mM KCl; 8-9 mM MgCl 2; the balance was RNase-free water, and the pH of the capping buffer was 7.8.
6. The method of preparing capped mRNA according to claim 1, wherein: the in vitro synthesized mRNA is uncapped mRNA obtained by treatment of IVT reaction solution.
7. The method of preparing capped mRNA according to claim 6, wherein: the treatment comprises one or more of tangential flow ultrafiltration exchange, oligo-dT monolithic column affinity chromatography, or tangential flow ultrafiltration concentrate exchange.
8. The method of preparing capped mRNA according to claim 1, wherein: the incubation time is 0.5-2 h.
9. A capping reaction system for mRNA synthesized in vitro, characterized in that: the capping reaction system is the reaction system described in the production method according to any one of claims 1 to 8.
10. A capping buffer for mRNA synthesized in vitro, characterized in that: the capping buffer is the capping buffer in the production method described in claim 3 or 5.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200071689A1 (en) * | 2016-12-13 | 2020-03-05 | Modernatx, Inc. | Rna affinity purification |
| CN113234776A (en) * | 2020-08-20 | 2021-08-10 | 深圳市瑞吉生物科技有限公司 | Cap analog 5' with Cap2 structure and preparation method and application thereof |
| WO2022256597A1 (en) * | 2021-06-04 | 2022-12-08 | Translate Bio, Inc. | Assay for quantitative assessment of mrna capping efficiency |
| CN115948382A (en) * | 2022-12-22 | 2023-04-11 | 中国科学院过程工程研究所 | mRNA capping method |
| CN116536306A (en) * | 2023-05-11 | 2023-08-04 | 中国科学院过程工程研究所 | Kit for in vitro transcription synthesis of mRNA capable of reducing or inhibiting formation of dsRNA and using method thereof |
| CN116836974A (en) * | 2023-09-01 | 2023-10-03 | 苏州近岸蛋白质科技股份有限公司 | Method for synthesizing capped mRNA in vitro |
| TW202402306A (en) * | 2022-05-18 | 2024-01-16 | 比利時商ｅＴｈｅＲＮＡ免疫治療公司 | Method for reducing dsrna formation and/or increasing capping efficiency during ivt |
-
2024
- 2024-04-16 CN CN202410451298.1A patent/CN118048420A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200071689A1 (en) * | 2016-12-13 | 2020-03-05 | Modernatx, Inc. | Rna affinity purification |
| CN113234776A (en) * | 2020-08-20 | 2021-08-10 | 深圳市瑞吉生物科技有限公司 | Cap analog 5' with Cap2 structure and preparation method and application thereof |
| WO2022256597A1 (en) * | 2021-06-04 | 2022-12-08 | Translate Bio, Inc. | Assay for quantitative assessment of mrna capping efficiency |
| TW202402306A (en) * | 2022-05-18 | 2024-01-16 | 比利時商ｅＴｈｅＲＮＡ免疫治療公司 | Method for reducing dsrna formation and/or increasing capping efficiency during ivt |
| CN115948382A (en) * | 2022-12-22 | 2023-04-11 | 中国科学院过程工程研究所 | mRNA capping method |
| CN116536306A (en) * | 2023-05-11 | 2023-08-04 | 中国科学院过程工程研究所 | Kit for in vitro transcription synthesis of mRNA capable of reducing or inhibiting formation of dsRNA and using method thereof |
| CN116836974A (en) * | 2023-09-01 | 2023-10-03 | 苏州近岸蛋白质科技股份有限公司 | Method for synthesizing capped mRNA in vitro |
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