RNA (Ribonucleic Acid) as well as synthetic method and application thereof
By combining DCA and TCA reagents in the deprotection step of RNA synthesis, the problems of unstable quality and low purity in the micromole to mmol-molecular RNA production in the prior art are solved, and efficient and stable mass production of high-purity RNA is achieved.
Patent Information
- Application Number
- CN202411959609.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-05-06
AI Technical Summary
It is difficult to quickly and in batches for high-quality micromolar to millimolar grade RNA, and existing methods are prone to problems of unstable product quality and low purity during large-scale production.
The solid-phase phosphoramidide triester method is used to perform RNA synthesis. By first treating the solid-phase carrier with DCA reagent in the deprotection step, and then treating it with TCA reagent, combined with the two reagents to reduce the depurine phenomenon and the problem of incomplete deprotection, achieving more thorough deprotection and higher product purity.
The rapid and mass production of high-purity micromole to mmol-grade RNA is achieved, ensuring the quality stability of each batch of products and avoiding the instability of product quality caused by batch synthesis.
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Figure CN119930713A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to RNA and a synthesis method and application thereof. Background Art
[0002] Ribonucleic acid (abbreviated as RNA, i.e. Ribonucleic Acid) is a genetic information carrier present in biological cells and some viruses and viroids. RNA is a long chain molecule formed by condensation of ribonucleotides through phosphodiester bonds. A ribonucleotide molecule is composed of phosphate, ribose and bases. There are four main bases of RNA, namely A adenine, G guanine, C cytosine, and U uracil. RNA can be divided into messenger RNA (rRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), MicroRNAs (miRNAs), small molecule RNA (including snRNA and scRNA), telomerase RNA (telomeraseRNA), antisense RNA (antisenseRNA), non-coding RNA, etc.
[0003] The principle of solid phase synthesis is to connect the reactants to an insoluble solid phase carrier and form the target molecule in a directed manner through a chemical reaction. One of the most common methods for solid phase synthesis of nucleic acids is the phosphoramidite triester method, which mainly includes a deprotection step, a condensation step, a capping step, and an oxidation step. The phosphoramidite triester method is a method for fixing DNA or RNA on a solid phase carrier to complete the synthesis of a DNA chain or an RNA chain. The direction of synthesis is from the 3' end to the 5' end of the primer to be synthesized, and adjacent nucleotides are connected by a 3'→5' phosphodiester bond. The use of the phosphoramidite triester method to synthesize DNA or RNA has the characteristics of high efficiency, rapid coupling, and relatively stable starting reactants. Many methods for synthesizing RNA by the phosphoramidite triester method are disclosed in the prior art, such as:
[0004] Patent CN 115010769 B discloses a method for synthesizing long-chain RNA nucleic acid by solid-phase phosphoramidite triester method, wherein the RNA nucleic acid is synthesized in at least three sections, and each section of the synthesized RNA nucleic acid is 30-60 bases long. The synthesis of the first section of RNA nucleic acid is completed by the four steps of deprotection, activation coupling, capping, and oxidation of the solid-phase phosphoramidite triester method, and the synthesis of each section of RNA nucleic acid above the second section is completed by the five steps of deprotection, activation coupling, capping, oxidation, and capping of the solid-phase phosphoramidite triester method. The method of the present invention can stably synthesize long-chain RNA nucleic acids of up to about 100 bases, avoiding the problems of protein host residues and the like in RNA synthesis by biological transcription methods.
[0005] Patent CN 115010770 B discloses a method for synthesizing RNA nucleic acid using a mixed deprotecting agent, the method is a solid phase phosphoramidite triester method, the RNA nucleic acid is synthesized in at least three sections, each section of the synthesized RNA nucleic acid is 30-60 bases long, the first section of RNA nucleic acid is synthesized according to the four steps of deprotection, activation coupling, capping, and oxidation of the solid phase phosphoramidite triester method, and the synthesis of each section of RNA nucleic acid above the second section is completed according to the five steps of deprotection, activation coupling, capping, oxidation, and capping of the solid phase phosphoramidite triester method; the deprotecting agent used in the deprotection step is a dichloromethane solution of dichloroacetic acid and trichloroacetic acid. The mixed deprotecting agent of the present invention is used to achieve the stable synthesis of longer chain RNA nucleic acids.
[0006] However, the RNA production methods disclosed in the prior art are all applicable to the production of RNA at nanomolar level, and when more RNA needs to be produced, it is necessary to obtain multiple batches of products by multiple rounds of production and merge after screening, and not only the production time is long but also the batch product quality is easily different, and it is difficult to obtain the RNA product with stable quality. It is not reported that there is a method applicable to the production of RNA in micromole to millimole batches at present, and directly using a large-volume synthesis column and producing large quantities of RNA by the existing method for producing RNA is prone to problems such as poor product quality, low purity. Therefore, in order to meet the production demand for RNA in micromole to millimole batches on the market, it is urgently needed to provide a method applicable to the rapid production of each batch of RNA with output reaching micromole to millimole and stable quality. Summary of the invention
[0007] The purpose of the present invention is to provide an RNA and a synthesis method and application thereof. The synthesis method of the present invention can be used to synthesize RNA at the micromole level, and solves the problem of incomplete deprotection or the presence of depurination in the deprotection step of the existing synthesis method (especially when the existing synthesis method is applied to synthesize RNA at the micromole level), thereby providing a feasible method for synthesizing RNA at the micromole level and capable of mass-producing products with high quality stability.
[0008] The above objectives of the present invention are achieved through the following technical solutions.
[0009] In a first aspect, the present invention provides a method for synthesizing RNA, wherein the synthesis method adopts a solid phase phosphoramidite triester method for synthesis, and the synthesis method specifically comprises a deprotection step, an activation condensation step, an oxidation / thiolation step, and a capping step performed in sequence, wherein the deprotection step comprises first treating a solid phase carrier with a DCA reagent for 1 to 5 times, and then treating the solid phase carrier with a TCA reagent for 1 to 2 times.
[0010] The inventors of the present invention have found that in order to synthesize RNA at a micromolar level or above in each batch, it is necessary to use a larger synthesis column. However, due to the large volume of the synthesis column, when the deprotection reagent is added from the reagent inlet at the upper end of the synthesis column, the contact time between the carrier in the upper part of the synthesis column and the deprotection reagent must be longer than the contact time between the carrier in the lower part and the deprotection reagent. In addition, when a larger synthesis column is used to synthesize RNA with a longer sequence length, the deprotection time also needs to be extended to ensure the deprotection effect. When the TCA reagent is used alone for deprotection, due to the strong acidity of the TCA reagent, the carrier in the upper part of the synthesis column is prone to depurination. The acidity of the DCA reagent is weaker than that of the TCA reagent. After using the DCA reagent alone for deprotection, although the degree of depurination can be greatly reduced, it is found in the actual production process that the amount of DCA reagent required for the deprotection process needs to be double the amount of the TCA reagent, and as the synthesis length increases, incomplete deprotection is prone to occur. Combining the advantages and disadvantages of TCA and DCA, the inventors found that the combined use of DCA reagent and TCA reagent can effectively reduce the phenomenon of depurination and incomplete deprotection, especially after using DCA reagent for deprotection 1 to 5 times and then using TCA reagent for deprotection 1 to 2 times, it can achieve basically no depurination phenomenon and thorough deprotection.
[0011] In some embodiments of the present invention, the volume percentage of dichloroacetic acid in the DCA reagent is 3% to 10%.
[0012] In some embodiments of the present invention, the DCA reagent is a solution of dichloroacetic acid in dichloromethane.
[0013] In some embodiments of the present invention, the volume percentage of trichloroacetic acid in the TCA reagent is 3% to 5%.
[0014] In some embodiments of the present invention, the TCA reagent is a dichloromethane solution of trichloroacetic acid.
[0015] In some embodiments of the present invention, the deprotection step further comprises washing the solid phase support from which the DMT group has been removed after treatment with a DCA reagent and a TCA reagent, preferably using anhydrous acetonitrile for washing.
[0016] In some embodiments of the present invention, the activation condensation step comprises mixing an activator with a phosphoramidite monomer, and then mixing the mixture with a solid support treated in a deprotection step and performing a condensation reaction; preferably, the activator is a benzylthiotetrazole reagent, preferably an acetonitrile solution of benzylthiotetrazole, more preferably a 0.3 M benzylthiotetrazole acetonitrile solution.
[0017] In the present invention, the benzylthiotetrazolyl reagent is selected as the activating agent, which can effectively shorten the activation time and improve the activation efficiency.
[0018] In some embodiments of the present invention, each reaction time of the activation condensation step is 6 to 9 minutes.
[0019] In some embodiments of the present invention, the oxidation / thiolation step comprises reacting the nucleotide product obtained by the capping step with an oxidizing agent or a thiolation agent.
[0020] In some embodiments of the present invention, the oxidation / thiolation step refers to selecting whether to use an oxidant or a thiolation agent to react with the nucleotide product according to whether the site of the nucleotide product is an oxidation site or a thiolation site; that is, an oxidant is selected to react with the oxidation site of the nucleotide product, and a thiolation agent is selected to react with the nucleotide product at the thiolation site.
[0021] In some embodiments of the present invention, the reaction time of each oxidation / thiolation step is 3 to 9 minutes.
[0022] In some embodiments of the present invention, the oxidant is an iodine-containing reagent, preferably an iodine-containing reagent with a mixed solution of tetrahydrofuran, pyridine and ultrapure water in a volume ratio of 7:2:1 as a solvent, and / or an iodine-containing reagent with an iodine content of 0.025M.
[0023] In some embodiments of the present invention, the sulfurizing agent is a DDTT reagent, preferably a DDTT reagent with a mixed solution of Sulfurizing Reagent II and pyridine in a ratio of 1 g: (25-50) mL as solvent, and / or a DDTT reagent containing 0.1 M to 0.2 M DDTT.
[0024] In some embodiments of the present invention, the capping step includes allowing a capping agent to undergo an acetylation reaction with the nucleotide product obtained by the oxidation / thiolation step; preferably, the capping agent is an acetic anhydride reagent and a 1-methylimidazole reagent, preferably a tetrahydrofuran solution containing 10% by volume of acetic anhydride and a tetrahydrofuran solution containing 16% by volume of 1-methylimidazole.
[0025] In some embodiments of the present invention, the reaction time of each capping step is 2 to 4 minutes.
[0026] The inventors of the present invention have found that a capping step is performed after the oxidation / thiolation step, and the reagent used in the capping step contains tetrahydrofuran, which can dissolve the iodine introduced by the use of an oxidant in the oxidation step, thereby preventing the final product from having a yellow appearance.
[0027] In some embodiments of the present invention, the capping step further comprises washing the nucleotide product treated with the capping agent, preferably using anhydrous acetonitrile for washing. Using anhydrous acetonitrile for washing can also wash away the water introduced by the oxidant used in the oxidation step.
[0028] In some embodiments of the present invention, the synthesis method further comprises a capping step performed after the activation condensation step and before the oxidation / thiolation step; preferably, the capping step here comprises allowing a capping agent to undergo an acetylation reaction with the nucleotide product obtained by the activation condensation step; preferably, the capping agent is an acetic anhydride reagent and a 1-methylimidazole reagent, preferably a tetrahydrofuran solution containing 10% by volume of acetic anhydride and a tetrahydrofuran solution containing 16% by volume of 1-methylimidazole.
[0029] In some embodiments of the present invention, the capping step performed after the activation condensation step and before the oxidation / thiolation step has a reaction time of 0 to 2 minutes per reaction.
[0030] In some embodiments of the present invention, the solid phase carrier is selected from controlled pore glass beads or polystyrene beads, preferably controlled pore glass beads.
[0031] In some embodiments of the present invention, in the synthesis method, each base connected to the target nucleotide chain needs to sequentially perform a deprotection step, an activation condensation step, an optional capping step, an oxidation / thiolation step, and a capping step, and by repeating the above steps, all bases required to be synthesized are connected to obtain the target nucleotide chain; preferably, the reaction of each step is performed in batches, preferably 2 to 5 times; more preferably, the entire target nucleotide chain is synthesized in segments, preferably 2 to 10 segments, more preferably 2 to 6 segments, the synthesis procedure of the nucleotides in each segment is the same, and the synthesis procedure of the nucleotides between segments is the same or different, wherein the synthesis procedure includes the steps for each base to be connected to the target nucleotide chain and the number of reactions in each step.
[0032] In one embodiment of the present invention, the target nucleotide chain is synthesized according to the procedure in Table 1 below, that is, the synthesis process of the entire target nucleotide chain is divided into three stages. When the bases corresponding to the first nucleotide chain are connected to form a nucleotide chain, the procedure for connecting each base is carried out according to the first stage procedure, that is, the deprotection step, the activation condensation step, the oxidation / thiolation step, and the capping step are carried out in sequence, and the reaction of each step is carried out according to the corresponding number of times in Table 1; when the bases corresponding to the second nucleotide chain are connected to the first nucleotide chain, the procedure for connecting each base is carried out according to the second stage procedure, that is, the deprotection step, the activation condensation step, the capping step, the oxidation / thiolation step, and the capping step are carried out in sequence, and the reaction of each step is carried out according to the corresponding number of times in Table 1; when the bases corresponding to the third nucleotide chain are connected to the nucleotide chain formed by the first nucleotide chain + the second nucleotide chain, the procedure for connecting each base is carried out according to the third stage procedure, that is, the deprotection step, the activation condensation step, the oxidation / thiolation step, and the capping step are carried out in sequence, and the reaction of each step is carried out according to the corresponding number of times in Table 1.
[0033] Table 1 Example of stepwise synthesis reaction procedure
[0034]
[0035] In Table 1, the number of reactions in the deprotection step in the first procedure is 3+1, which means that the solid phase carrier is treated with a DCA reagent for 3 times and then with a TCA reagent for 1 time; the number of reactions in the deprotection step in the second procedure is 3+1, which means that the solid phase carrier is treated with a DCA reagent for 3 times and then with a TCA reagent for 1 time; the number of reactions in the deprotection step in the third procedure is 4+1, which means that the solid phase carrier is treated with a DCA reagent for 4 times and then with a TCA reagent for 1 time.
[0036] In Table 1, the number of reactions of the activation condensation step in the first or third procedure is 3, which means that the activator is mixed with the phosphoramidite monomer, and then the mixture is mixed with the solid phase support treated in the deprotection step and subjected to a condensation reaction; after the reaction is completed, the product obtained by the condensation reaction is subjected to a condensation reaction with the mixture of the activator and the phosphoramidite monomer again; after the reaction is completed, the product obtained by the condensation reaction is continued to be subjected to a condensation reaction with the mixture of the activator and the phosphoramidite monomer. The number of reactions of the activation condensation step in the second procedure is 2, which means that the activator is mixed with the phosphoramidite monomer, and then the mixture is mixed with the solid phase support treated in the deprotection step and subjected to a condensation reaction; after the reaction is completed, the product obtained by the condensation reaction is subjected to a condensation reaction with the mixture of the activator and the phosphoramidite monomer again.
[0037] In Table 1, the number of reactions of the oxidation / thiolation step in the first or third procedure is 3, which means that the nucleotide product obtained by the activation condensation step is reacted with an oxidant or a thiolation agent; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant or a thiolation agent again; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant or a thiolation agent again. The number of reactions of the oxidation / thiolation step in the second procedure is 2, which means that the nucleotide product obtained by the capping step is reacted with an oxidant or a thiolation agent; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant or a thiolation agent again.
[0038] In Table 1, the number of reactions of the capping step in the second procedure is 2, which means that the capping agent is subjected to an acetylation reaction with the nucleotide product obtained by the previous step (activation condensation, oxidation / thiolation) of the capping step; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again. The number of reactions of the capping step in the first procedure or the third procedure is 4, which means that the capping agent is subjected to an acetylation reaction with the nucleotide product obtained by the oxidation / thiolation step; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again.
[0039] In some embodiments of the present invention, the synthesis method utilizes a nucleic acid synthesizer to perform the step of synthesizing nucleic acid on a solid phase carrier; preferably, the nucleic acid synthesizer uses a 12-channel negative pressure nucleic acid synthesizer produced by Beijing Qingke Biotechnology Co., Ltd., and / or uses a single synthesis column with a capacity of 50umol or more, preferably 50umol, to synthesize nucleic acid on the nucleic acid synthesizer.
[0040] In some embodiments of the present invention, the synthesis method further comprises cutting the synthesized RNA from the solid phase carrier; preferably, the synthesis method further comprises the steps of aminolysis and desilylation of the cut RNA.
[0041] In some embodiments of the present invention, the aminolysis step includes mixing the cut RNA with concentrated ammonia water and reacting at 75-85°C for 2.5-3.5 hours, then cooling at -20°C and filtering to recover the filtrate; preferably, the ratio of concentrated ammonia water to RNA is (2.0-3.0) g:50 mL.
[0042] In some embodiments of the present invention, the concentrated ammonia water is concentrated ammonia water with a mass fraction of 28%.
[0043] In some embodiments of the present invention, the cooling time is 30 minutes.
[0044] In some embodiments of the present invention, the desilylation step comprises reacting the aminolyzed RNA with DMSO at 60-70°C for 15-25 min; then adding TEA and TEA.3HF and continuing the reaction at 60-70°C for 2.5-3.5 h; preferably, the dosage ratio of the DMSO to the aminolyzed RNA is (0.1-0.6) g:7 mL, and / or the volume ratio of TEA, TEA.3HF and DMSO is 7:3.75:5.
[0045] In the desilylation step of the present invention, a mixture of TEA and TEA.3HF is used to treat the RNA treated with DMSO. The reaction solution has a moderate pH value, a mild reaction, and a good desilylation effect, which can effectively avoid adverse reactions caused by the strong acidity of TEA.3HF.
[0046] In some embodiments of the present invention, the synthesis method further comprises purifying and / or lyophilizing the crude RNA product obtained by synthesis; preferably, the crude RNA product obtained by aminolysis and desilylation is purified and / or lyophilized.
[0047] In some embodiments of the present invention, the purification comprises an alcohol precipitation step and / or an elution step using a high performance liquid chromatography.
[0048] In some embodiments of the present invention, the alcohol precipitation comprises sequentially using isopropanol and 75% by volume ethanol to precipitate the RNA, performing solid-liquid separation and recovering the solid; preferably, the solid-liquid separation is performed by pouring out the liquid after centrifugation.
[0049] In some embodiments of the present invention, the ethanol with a volume fraction of 75% is icy ethanol at a temperature of -20°C.
[0050] In some embodiments of the present invention, the alcohol precipitation comprises the following steps:
[0051] 1) adding isopropanol and 5M NaCl to the synthesized RNA crude product, shaking, then transferring to a centrifuge tube, and placing in a -20°C refrigerator for 3 hours; preferably, the ratio of the RNA crude product to isopropanol and 5M NaCl is (0.1-0.6) g: (100-150) mL: (0.5-1) mL;
[0052] 2) placing the centrifuge tube obtained in step 1) in a high-speed refrigerated centrifuge at 8000 rpm at 4°C for 30 min, and then pouring out the upper layer of the centrifuge tube;
[0053] 3) adding 40 mL of 75% ethanol aqueous solution pre-frozen to -20°C to the centrifuge tube after pouring out the upper layer liquid in step 2), and then centrifuging the centrifuge tube again in a 4°C 8000 rpm high-speed refrigerated centrifuge for 30 min, and then pouring out the upper layer liquid of the centrifuge tube; preferably, the ratio of 75% ethanol aqueous solution to the crude RNA product in step 1) is (10-40) mL: (0.1-0.6) g.
[0054] In some embodiments of the present invention, the reagent used for elution is acetonitrile and a 0.05 M aqueous solution of triethylamine acetate.
[0055] In some embodiments of the present invention, the elution procedure includes increasing the proportion of acetonitrile from 2% to 55% and decreasing the proportion of 0.05 M triethylammonium acetate aqueous solution from 98% to 45% within 30 minutes.
[0056] In some embodiments of the present invention, the elution procedure includes increasing the proportion of acetonitrile from 2% to 55% at a constant rate, while decreasing the proportion of 0.05 M triethylammonium acetate aqueous solution from 98% to 45% at a constant rate within 30 minutes.
[0057] In a second aspect, the present invention provides an application of the synthesis method described in the first aspect in synthesizing RNA at the micromolar level.
[0058] In some embodiments of the present invention, the RNA is an RNA with a length of 20 to 103mer; more preferably, the RNA is an RNA with a length of 97 to 103mer.
[0059] In some embodiments of the present invention, the RNA includes sgRNA (small guide RNA), which is consistent with the small nucleic acid raw materials used in medical research and development, such as siRNA, miRNA, saRNA and Aptamer.
[0060] In a third aspect, the present invention provides RNA synthesized by the synthesis method described in the first aspect.
[0061] In some embodiments of the invention, the purity of the RNA is ≥ 95%.
[0062] The beneficial effects of the present invention are as follows:
[0063] The present invention proposes a method for synthesizing RNA. In the deprotection step, the solid phase carrier is first treated with a DCA reagent, and then with a TCA reagent, that is, the DCA reagent with lower acidity is first used for long-term multiple deprotection. In the deprotection process, most of the 5'-hydroxyl protecting groups, dimethoxytrityl (DMT), can be removed, and then the TCA reagent with higher acidity is used for deprotection 1 to 2 times. The TCA reagent can perform deep deprotection, making the deprotection more thorough; and because the two deprotection reagents are superimposed for deprotection, the entire deprotection time is shorter than the deprotection time of using the DCA reagent alone, and the deprotection intensity is weaker than the deprotection intensity of using the TCA reagent alone, which can effectively reduce the depurination phenomenon. At the same time, when RNA is synthesized by the method of the present invention, less impurities are generated during the synthesis process, the product recovery rate is high after purification, and the final product yield is high.
[0064] The present invention proposes the use of a single synthesis column with a capacity of more than 50 umol to synthesize RNA on a nucleic acid synthesizer and proposes a corresponding synthesis method. RNA synthesis is performed by using a large-volume synthesis column, and RNA of micromole to millimole level can be synthesized at one time. Moreover, the quality stability of products obtained from different batches is high, thus avoiding the problem of unstable product quality between different batches existing in batch synthesis. BRIEF DESCRIPTION OF THE DRAWINGS
[0065] Figure 1 When RNA is synthesized using the method of Example 1, 100 nmol of the sample is eluted and purified using a high performance liquid chromatograph to obtain a peak spectrum.
[0066] Figure 2 This is the mass spectrometry analysis of the RNA product synthesized by the method of Example 1, with a main peak of 32322.1.
[0067] Figure 3 This is a purity analysis chart of the RNA product synthesized using the method of Example 1.
[0068] Figure 4 When RNA is synthesized using the method of Comparative Example 1, 100 nmol of the sample is eluted and purified using a high performance liquid chromatograph to obtain a peak spectrum.
[0069] Figure 5 This is the mass spectrometry analysis of the RNA product synthesized by the method of Comparative Example 1, with a main peak of 32337.7.
[0070] Figure 6 This is a purity analysis chart of the RNA product synthesized using the method of Comparative Example 1.
[0071] Figure 7When RNA is synthesized using the method of Comparative Example 2, 100 nmol of the sample is eluted and purified using a high performance liquid chromatograph to obtain a peak spectrum.
[0072] Figure 8 This is the mass spectrometry analysis of the RNA product synthesized by the method of Comparative Example 2, with a main peak of 32320.0.
[0073] Fig. 9 This is a purity analysis chart of the RNA product synthesized using the method of Comparative Example 2. DETAILED DESCRIPTION
[0074] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.
[0075] Example 1
[0076] Step 1: The sequence is mC*mC*mA*
[0077] rUrUrCrArCrCrCrGrArCrGrCrUrCrArGrGrGrUrUrUrUrArGrArGrCrUrArGrArArArUr
[0078] ArGrCrArArGrUrUrArArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCr UrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrUrGrCrUrGrCrU*mU*mU*mU RNA synthesis, the synthesis process specifically includes the following steps:
[0079] 1. Use the 12-channel negative pressure nucleic acid synthesizer produced by Beijing Qingke Biotechnology Co., Ltd., set the working pressure parameters according to Table 3, set the drain parameters according to Table 4, and set the parameters of the segmented synthesis program according to Table 5.
[0080] Table 3 Working pressure parameters
[0081] project Standard parameter (psi) Total pressure 25±5.0 Large reagent pressure 6.0±0.1 Monomer pressure 6.0±0.1 Negative pressure 1 -0.8±1.0 Negative pressure 2 -0.8±1.0
[0082] Table 4 Drainage parameters
[0083] Drying procedure Total reaction time after draining(s) Deblock-50umol_DCA 80 Deblock-50umol_TCA 50 Couple-50umol 180 Wash-50umol 30 Cap-50umol 60 Ox-50umol 60 Sul-50umol 180
[0084] Table 5 Parameters of the segmented synthesis procedure
[0085]
[0086]
[0087] In Table 5, the number of reactions in the deprotection step in the first procedure is 3 times (80s) + 1 time (50s), which means that the solid phase support is treated with a DCA reagent 3 times, each treatment time is 80s, and then the solid phase support is treated with a TCA reagent once, and the treatment time is 50s; the number of reactions in the deprotection step in the second procedure is 3 times (80s) + 1 time (50s), which means that the solid phase support is treated with a DCA reagent 3 times, each treatment time is 80s, and then the solid phase support is treated with a TCA reagent once, and the treatment time is 50s; the number of reactions in the deprotection step in the third procedure is 4 times (80s) + 1 time (50s), which means that the solid phase support is treated with a DCA reagent 4 times, each treatment time is 80s, and then the solid phase support is treated with a TCA reagent once, and the treatment time is 50s.
[0088] In Table 5, the number of reactions of the activation condensation step in the first or third procedure is 3 times (180s), which means that the activator is mixed with the phosphoramidite monomer, and then the mixture is mixed with the solid phase support treated by the deprotection step and subjected to condensation reaction; after the reaction is completed, the product obtained by the condensation reaction is subjected to condensation reaction with the mixture of the activator and the phosphoramidite monomer again; after the reaction is completed, the product obtained by the condensation reaction is continued to be subjected to condensation reaction with the mixture of the activator and the phosphoramidite monomer; the duration of each treatment (raw material mixing + condensation reaction) is 180s, and the total duration is 540s. The number of reactions of the activation condensation step in the second procedure is 2 (180s), which means that the activator is mixed with the phosphoramidite monomer, and then the mixture is mixed with the solid phase support treated by the deprotection step and subjected to condensation reaction; after the reaction is completed, the product obtained by the condensation reaction is subjected to condensation reaction with the mixture of the activator and the phosphoramidite monomer again; the duration of each treatment (raw material mixing + condensation reaction) is 180s, and the total duration is 360s.
[0089] In Table 5, the number of reactions of the oxidation / thiolation step in the first or third procedure is 3 times (60s / 180s), which means that the nucleotide product obtained by the activation condensation step is reacted with an oxidant for 60s or with a thiolation agent for 180s; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant for 60s or with a thiolation agent for 180s; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant for 60s or with a thiolation agent for 180s; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant for 60s or with a thiolation agent for 180s. The number of reactions of the oxidation / thiolation step in the second procedure is 2 times (60s / 180s), which means that the nucleotide product obtained by the capping step is reacted with an oxidant for 60s or with a thiolation agent for 180s; after the reaction is completed, the nucleotide product obtained by the reaction is reacted with an oxidant for 60s or with a thiolation agent for 180s.
[0090] In Table 5, the number of reactions of the capping step in the second procedure is 2 times (60s), which means that the capping agent is subjected to an acetylation reaction with the nucleotide product obtained by the previous step (activation condensation, oxidation / thiolation) of the capping step for 60s; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again for 60s. The number of reactions of the capping step in the first procedure or the third procedure is 4 times (60s), which means that the capping agent is subjected to an acetylation reaction with the nucleotide product obtained by the oxidation / thiolation step for 60s; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again for 60s; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again for 60s; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again for 60s; after the reaction is completed, the nucleotide product treated with the capping agent is subjected to an acetylation reaction with the capping agent again for 60s.
[0091] 2. Take out 30mL synthetic column and fill the lower filter membrane; weigh 2.000-2.1000g The solid phase carrier CPG (Loading value = 24.7 umol / g) was loaded into a single synthesis column with a capacity of 50 umol, and a filter membrane was added; and loaded onto the column holder of the synthesizer.
[0092] 3. Prepare the deprotecting agent, activating agent, monomer reagent, capping agent, oxidizing agent and thiolation agent used in the synthesis. The deprotecting agent is 3% (w / v) trichloroacetic acid in dichloromethane solution and 3% (v / v) dichloroacetic acid in dichloromethane solution; the activating agent is 0.3M benzylthiotetrazole in acetonitrile solution; the monomer reagent is 0.1M phosphoramidite in acetonitrile solution; the capping agent is 10% (v / v) acetic anhydride in tetrahydrofuran solution and 16% (v / v) 1-methylimidazole in tetrahydrofuran solution; the oxidizing agent is a mixed solution of 0.025M iodine in tetrahydrofuran / pyridine / ultrapure water (v / v / v=7 / 2 / 1); the thiolation agent is a mixed solution of 0.1M di-p-chlorophenyltrichloroethane in Sulfurizing Reagent II / pyridine (w / v=1 / 50).
[0093] 4. Add the deprotecting agent, activating agent, monomer reagent, oxidizing agent, thiolation agent, and capping agent prepared in step 3 into the synthesizer.
[0094] 5. Import the sequence to be synthesized into the instrument, check the pressure of the synthesizer, the pressure of the reagent bottle and other parameters, confirm that the reagent volume is twice the theoretical reagent volume, and click the "Start Synthesis" button to start synthesis after confirmation until the sequence synthesis is completed.
[0095] Step 2: RNA aminolysis and desilylation deprotection, specifically including the following steps:
[0096] 6. After the synthesis in step 5 above is completed, the CPG powder in the synthesis column is poked into a 150 mL explosion-proof bottle, 50 mL of concentrated ammonia water (mass fraction is 28%) is added, the bottle cap is tightened, and the bottle is placed in a constant temperature magnetic stirrer heated to 80°C in advance, and the reaction is stirred at a constant temperature for 3 hours.
[0097] 7. After the reaction in step 6 is completed, place the explosion-proof bottle and the reaction solution in the bottle in a -20°C refrigerator for cooling for 30 minutes. Then take the explosion-proof bottle out of the refrigerator and filter the turbid solution in the bottle using a 0.45 μm filter membrane to obtain a concentrated ammonia solution containing RNA, which is then concentrated to a dry powder state.
[0098] 8. Add 7 mL of DMSO (dimethyl sulfoxide) to the dry powder sample obtained in step 7, and heat it in a 65°C oven for 20 min. Then take it out and add 3.75 mL of TEA (triethylamine) and shake it well. Continue to add 5 mL of TEA.3HF (triethylamine trihydrofluoride), and heat it in a 65°C oven again for 3 h to obtain the RNA crude product with the 2'-end silicon-based protecting group (TBDMS) removed.
[0099] Step 3: Precipitation of micromolar RNA, specifically including the following steps:
[0100] 9. Add 1 mL of 5M NaCl to the crude RNA obtained in step 8, shake well, then add 140 mL of isopropanol, shake well, and then transfer to 50 mL centrifuge tubes, each with a volume of 40 mL, and place the centrifuge tubes in a -20°C refrigerator for 3 hours. Place the frozen centrifuge tubes in a 4°C 8000 rpm high-speed refrigerated centrifuge for 30 minutes, and then pour out the upper liquid of the centrifuge tubes.
[0101] 10. After pouring out the upper layer of liquid in step 9, add 40 mL of 75% ethanol aqueous solution pre-frozen to -20°C to each centrifuge tube, then place the centrifuge tube in a high-speed refrigerated centrifuge at 8000 rpm at 4°C and centrifuge for 30 minutes, then pour out the upper layer of liquid in the centrifuge tube.
[0102] Step 4: Purification and analysis of micromole-level RNA and freeze-drying and storage, specifically including the following steps:
[0103] 11. After pouring out the upper layer of the centrifuge tube for the second time in step 10, add 10 mL of RNase Free sterile water to the centrifuge tube to dissolve the white solid at the bottom of the centrifuge tube, and then filter it using a 0.22 μm filter membrane. Load the filtrate onto a high performance liquid chromatograph (take 100 nmol of the filtrate each time), and use acetonitrile and 0.05 M TEAA (triethylammonium acetate) aqueous solution as eluents for elution and purification. During the elution and purification process, the proportion of acetonitrile is uniformly increased from 2% to 55% within 30 minutes by adjusting the proportional valve, while the proportion of 0.05 M TEAA aqueous solution is uniformly decreased from 98% to 45%, and high-purity RNA is purified. The peaks from 10 minutes to 22 minutes during the elution and purification process are as follows: Figure 1 shown.
[0104] 12. The high-purity RNA sample obtained in step 11 is subjected to mass spectrometry analysis and purity analysis, wherein the spectrum obtained by mass spectrometry analysis is as follows: Figure 2 The purity analysis results are shown in Figure 3 When the error between the molecular weight of the sample and the target molecular weight (32303.55) is within ≤0.10%, it indicates that the product corresponding to the sample meets the mass spectrometry analysis standard. When the analytical purity of the sample is ≥95%, it indicates that the product corresponding to the sample meets the purity analysis standard. Figure 2 and Figure 3 It can be seen that the product prepared in this example meets the mass spectrometry analysis standards and the purity analysis standards.
[0105] 13. The high-purity RNA obtained in step 11 was quantitatively analyzed, and the recovery value of the purified product obtained after purification by high-performance liquid chromatography per 100 nmol of filtrate was calculated to be 7 nmol, which met the recovery rate ≥ 5% standard; all the purified products obtained in step 11 were freeze-dried, and a total of 1.05 μmol of product was obtained by weighing (that is, a single 50 μmol synthesis column can synthesize a total of 1.05 μmol of RNA product at a time), and finally the freeze-dried product was stored in a -20°C refrigerator for standby use.
[0106] Comparative Example 1
[0107] The synthesis method is the same as that in Example 1, except that only a 3% (w / v) trichloroacetic acid solution in dichloromethane is used as a deprotecting agent, that is, the solid phase support is treated 3 times with a 3% (w / v) trichloroacetic acid solution in dichloromethane in the first and second procedures, and each treatment time is 50 s; in the third procedure, the solid phase support is treated 4 times with a 3% (w / v) trichloroacetic acid solution in dichloromethane, and each treatment time is 50 s; that is, each base is connected to the solid phase support according to the segmented synthesis reaction program parameters in Table 6 to obtain the target nucleotide chain.
[0108] Table 6 Parameters of the stepwise synthesis reaction program using TCA reagent deprotection throughout
[0109]
[0110] The peaks of the RNA synthesized in this comparative example from the 13th minute to the 22nd minute when eluted and purified by high performance liquid chromatography are as follows: Figure 4 As shown, the mass spectrometry analysis results of the high-purity RNA purified by high-performance liquid chromatography are as follows Figure 5 The purity analysis results are shown in Figure 6 As shown. Figure 4 It can be seen that the solid phase carrier was treated with TCA reagent for deprotection throughout the process, and the synthesized RNA product formed more impurity peaks during the elution and purification process, and the main peak was not obvious; Figure 5 It can be seen that the solid phase carrier was treated with TCA reagent for deprotection throughout the process, and the synthesized RNA product had obvious depurination phenomenon; Figure 6 It can be seen that the solid phase carrier was treated with TCA reagent for deprotection throughout the process. The synthesized RNA product had severe bifurcation during purity analysis, and the main peak accounted for a low proportion, that is, the product purity was low.
[0111] Comparative Example 2
[0112] The synthesis method is the same as that in Example 1, except that a dichloromethane solution containing 1.5% (w / v) dichloroacetic acid and 1.5% (w / v) trichloroacetic acid is used as a deprotecting agent, that is, in the first and second procedures, a dichloromethane solution containing 1.5% (w / v) dichloroacetic acid and 1.5% (w / v) trichloroacetic acid is used to treat the solid phase support 3 times, and each treatment time is 60 s; in the third procedure, a dichloromethane solution containing 1.5% (w / v) dichloroacetic acid and 1.5% (w / v) trichloroacetic acid is used to treat the solid phase support 4 times, and each treatment time is 60 s; that is, each base is connected to the solid phase support according to the segmented synthesis reaction program parameters in Table 7 to obtain the target nucleotide chain.
[0113] Table 7 Parameters of the stepwise synthesis reaction program using a mixed reagent of TCA and DCA for deprotection throughout the process
[0114]
[0115] The peaks of the RNA synthesized in this comparative example from the 13th minute to the 22nd minute when eluted and purified by high performance liquid chromatography are as follows: Figure 7 As shown, the mass spectrometry analysis results of the high-purity RNA purified by high-performance liquid chromatography are as follows Figure 8 The purity analysis results are shown in Fig. 9 As shown. Figure 7It can be seen that the solid phase carrier is treated with a mixed reagent of TCA and DCA for deprotection throughout the process. The main peak of the synthesized RNA product in the elution and purification process is obvious, but the main peak is low. It is estimated that this will lead to a low recovery rate of the purified product and a low yield of the final product. Figure 8 It can be seen that the solid phase carrier is treated with a mixed reagent of TCA and DCA for deprotection throughout the process, and the main peak of the mass spectrum of the RNA product obtained by synthesis is correct, and the error between the molecular weight and the target molecular weight is within the range of ≤0.10%, which meets the standard; Fig. 9 It can be seen that the solid phase carrier is treated with a mixed reagent of TCA and DCA for deprotection throughout the process, and the purity of the RNA product obtained by synthesis is 97.46%, which meets the standard sample analysis purity ≥95%, but the purity of the RNA product is lower than the purity of the RNA product obtained in Example 1 (98.63%). In order to verify the yield of the final product, the high-purity RNA obtained in step 11 was quantitatively analyzed, and the recovery value of the purified product obtained after purification by high-performance liquid chromatography per 100nmol of filtrate was calculated to be 5nmol, which meets the recovery rate ≥5% standard; all the purified products obtained in step 11 were lyophilized, and a total of 0.71μmol of product was obtained by weighing (that is, a single 50umol synthesis column can synthesize a total of 0.71μmol of RNA product at a time).
[0116] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.
Claims
1. A method for synthesizing RNA, characterized in that: The synthesis method adopts the solid phase phosphoramidite triester method for synthesis, and the synthesis method specifically includes a deprotection step, an activation condensation step, an oxidation / thiolation step, and a capping step performed in sequence, wherein the deprotection step includes first treating the solid phase carrier with a DCA reagent for 1 to 5 times, and then treating the solid phase carrier with a TCA reagent for 1 to 2 times.
2. The synthesis method according to claim 1, characterized in that The volume percentage of dichloroacetic acid in the DCA reagent is 3% to 10%; And / or, the DCA reagent is a dichloroacetic acid solution in dichloromethane; And / or, the volume percentage of trichloroacetic acid in the TCA reagent is 3% to 5%; And / or, the TCA reagent is a dichloromethane solution of trichloroacetic acid; And / or, the deprotection step further comprises washing the solid phase carrier treated with the DCA reagent and the TCA reagent, preferably using anhydrous acetonitrile for washing.
3. The synthesis method according to claim 1 or 2, characterized in that The activation condensation step comprises mixing an activator with a phosphoramidite monomer, and then mixing the mixture with a solid support treated in the deprotection step and performing a condensation reaction; preferably, the activator is a benzylthiotetrazole reagent, preferably an acetonitrile solution of benzylthiotetrazole, more preferably a 0.3 M benzylthiotetrazole acetonitrile solution.
4. The synthesis method according to any one of claims 1 to 3, characterized in that The oxidation / thiolation step comprises reacting the nucleotide product obtained by the activation condensation step with an oxidant or a thiolation agent; preferably, the oxidant is an iodine-containing reagent, preferably an iodine-containing reagent with a mixed solution of tetrahydrofuran, pyridine and ultrapure water in a volume ratio of 7:2:1 as solvent and an iodine content of 0.025M, and / or, the thiolation agent is a DDTT reagent, preferably a mixed solution of Sulfurizing Reagent II and pyridine in a ratio of 1g:(25-50)mL as solvent and a DDTT content of 0.1M-0.2M.
5. The synthesis method according to any one of claims 1 to 4, characterized in that The capping step includes allowing the capping agent to undergo an acetylation reaction with the nucleotide product obtained by the oxidation / thiolation step; preferably, the capping agent is an acetic anhydride reagent and a 1-methylimidazole reagent, preferably a tetrahydrofuran solution containing 10% by volume of acetic anhydride and a tetrahydrofuran solution containing 16% by volume of 1-methylimidazole; more preferably, the capping step here also includes washing the nucleotide product treated with the capping agent, preferably using anhydrous acetonitrile for washing.
6. The synthesis method according to any one of claims 1 to 5, characterized in that The synthesis method further comprises a capping step after the activation condensation step and before the oxidation / thiolation step; preferably, the capping step comprises causing a capping agent to undergo an acetylation reaction with the nucleotide product obtained by the activation condensation step; preferably, the capping agent is an acetic anhydride reagent and a 1-methylimidazole reagent, preferably a tetrahydrofuran solution containing 10% by volume of acetic anhydride and a tetrahydrofuran solution containing 16% by volume of 1-methylimidazole; And / or, the solid phase carrier is selected from controlled micropore glass beads or polystyrene microbeads, preferably controlled micropore glass beads; And / or, in the synthesis method, each base connected to the target nucleotide chain needs to be sequentially subjected to a deprotection step, an activation condensation step, an optional capping step, an oxidation / thiolation step, and a capping step, and by repeating the above steps, all bases required to be synthesized are connected to obtain the target nucleotide chain; preferably, the reaction of each step is performed in batches, preferably 2 to 5 times; more preferably, the entire target nucleotide chain is synthesized in segments, preferably 2 to 10 segments, more preferably 2 to 6 segments, the synthesis procedure of the nucleotides in each segment is the same, and the synthesis procedure of the nucleotides between segments is the same or different, wherein the synthesis procedure includes the steps for each base to be connected to the target nucleotide chain and the number of reactions in each step; And / or, the synthesis method utilizes a nucleic acid synthesizer to perform the step of synthesizing nucleic acid on a solid phase carrier; preferably, the nucleic acid synthesizer uses a 12-channel negative pressure nucleic acid synthesizer produced by Beijing Qingke Biotechnology Co., Ltd., and / or, a single synthesis column with a capacity of 50umol or more is used to synthesize nucleic acid on the nucleic acid synthesizer.
7. The synthesis method according to any one of claims 1 to 6, characterized in that The synthesis method further comprises cutting the synthesized RNA from the solid phase carrier; preferably, the synthesis method further comprises the steps of aminolysis and desilylation of the cut RNA; more preferably, The aminolysis step comprises mixing the cut RNA with concentrated ammonia water and reacting at 75-85°C for 2.5-3.5h, then cooling at -20°C and filtering to recover the filtrate; preferably, the ratio of the concentrated ammonia water to the RNA is (2.0-3.0) g: 50 mL; and / or The desilylation step comprises reacting the aminolyzed RNA with DMSO at 60-70° C. for 15-25 min; then adding TEA and TEA.3HF and continuing the reaction at 60-70° C. for 2.5-3.5 h; preferably, the dosage ratio of the DMSO to the aminolyzed RNA is (0.1-0.6) g:7 mL, and / or the volume ratio of TEA, TEA.3HF and DMSO is 7:3.75:
5.
8. The synthesis method according to any one of claims 1 to 7, characterized in that The synthesis method further comprises purifying and / or freeze-drying the synthesized RNA crude product; preferably, the purification comprises an alcohol precipitation step and / or an elution step using a high performance liquid chromatography instrument; more preferably, The alcohol precipitation comprises sequentially using isopropanol and 75% by volume ethanol to precipitate the RNA, and then performing solid-liquid separation and recovering the solid; preferably, the solid-liquid separation is performed by pouring out the liquid after centrifugation; and / or The reagents used for elution are acetonitrile and 0.05M aqueous solution of triethylammonium acetate; preferably, the elution procedure includes increasing the proportion of acetonitrile from 2% to 55% and decreasing the proportion of 0.05M aqueous solution of triethylammonium acetate from 98% to 45% within 30 minutes.
9. Use of the synthesis method according to any one of claims 1 to 8 in synthesizing RNA at the micromole level; preferably, the RNA is an RNA with a length of 20 to 103mer; more preferably, the RNA is an RNA with a length of 97 to 103mer.
10. RNA synthesized by the synthesis method according to any one of claims 1 to 8; preferably, the purity of the RNA is ≥ 95%.