Method for synthesizing gRNA (guide Ribonucleic Acid)
By using phosphoramidite monomers with different protecting groups and optimizing solid-phase synthesis steps during gRNA synthesis, the problem of low gRNA purity was solved, achieving efficient and low-cost gRNA synthesis suitable for large-scale production.
Patent Information
- Application Number
- CN202511069369.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-18
AI Technical Summary
Existing technologies suffer from low purity when synthesizing gRNA, especially in solid-phase synthesis, where the purity of 100nt oligonucleotides is only 13.3%, which is insufficient to meet the needs of large-scale production.
A solid-phase synthesis cycle was employed, using phosphoramide monomers with different protecting groups at the 2' position of the sugar ring. With appropriate activators and optimized coupling reaction conditions, gRNA chains were formed through deprotection, coupling, oxidation, and capping steps. High-purity gRNA products were obtained through ammonolysis and purification.
It improves the purity and yield of gRNA, reduces synthesis costs, is suitable for large-scale production, and the crude product has an HPLC purity of over 30%.
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Figure CN120965780A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of chemical medicine synthesis, and particularly relates to a synthesis method of gRNA. BACKGROUND
[0002] In recent years of biological technology research, gRNA (guide RNA) as a key component of CRISPR / Cas9 gene editing technology is increasingly becoming an important tool in the fields of gene therapy, agricultural improvement and basic biological research. The function of gRNA is to guide the Cas9 enzyme to accurately cut the target DNA sequence, so as to realize specific editing of the genome. With the progress of science and technology, the application range of gRNA is continuously expanding, prompting the increasing demand for its large-scale, low-cost and high-efficiency synthesis.
[0003] At present, the main synthesis methods include chemical synthesis method and chemical synthesis + enzyme ligation method. The chemical method is to directly prepare the final product by chemical synthesis method, which does not need the participation of biological enzymes, and the operation process is relatively direct. Therefore, it is widely used in the preparation of short fragment tools in molecular biology experiments. The chemical synthesis + enzyme ligation method is to first synthesize small nucleic acid fragments by chemical synthesis, and then connect the small nucleic acid fragments into the final product by enzyme catalysis. This method combines the high efficiency of chemical synthesis of short fragments and the high fidelity of enzyme ligation, and is especially suitable for the preparation of long chain nucleic acids (such as gene fragments, vector skeletons, etc.), which to some extent breaks through the limitation of pure chemical synthesis method in chain length.
[0004] However, whether it is pure chemical synthesis method or the chemical synthesis step for preparing small fragments in the chemical synthesis + enzyme ligation method, the mainstream technology at present depends on solid-phase synthesis method. The reaction characteristics of this method make it inevitable to have purity problems in the synthesis process. In the solid-phase synthesis method, four steps of deprotection, coupling, oxidation and capping are needed for the introduction of each base. In theory, even if the reaction efficiency of each monomer reaches 98%, the purity of 100nt oligonucleotide can only reach about 13% (0.98 100 = 13.3%) after synthesis. The crude product will be mixed with a large amount of short chain impurities produced due to incomplete sub-reaction, and the purity is low. Therefore, it is urgent to develop a low-cost, high-efficiency synthesis method of high-purity guide RNA (gRNA).
[0005] Therefore, there is a need in the art to develop a low-cost, high-efficiency synthesis method of gRNA, which is easy to operate and has high product purity, so as to realize the large-scale production of gRNA. SUMMARY
[0006] To solve the above technical problems, the application provides a synthesis method of gRNA, comprising the following steps:
[0007] Step 1, solid-phase synthesis cycle: loading a solid-phase carrier into a synthesis column, then performing four cycles of deprotection, coupling, oxidation and capping reaction, and sequentially connecting nucleotide monomers to form a gRNA chain;
[0008] Step 2, post-treatment: after the solid-phase synthesis is completed, the gRNA chain is cut from the solid-phase carrier and the protecting group is removed, then purification and freeze-drying are performed to obtain a gRNA product;
[0009] In the application, the nucleotide monomer is a phosphoramidite monomer with a protecting group at the 2' position of a sugar ring, and in the coupling reaction, the phosphoramidite monomer is mixed with an activating agent in an organic solvent and then reacts with the free hydroxyl group on the solid-phase carrier.
[0010] Specifically, in step 1, the solid-phase carrier is selected from at least one of controllable pore size glass beads CPG or polystyrene microspheres PS.
[0011] Specifically, in step 1, the deprotection solution used for deprotection is a dichloroacetic acid toluene solution with a volume fraction of 3% to 5%.
[0012] Specifically, in step 1, the protecting group at the 2' position of the sugar ring of the phosphoramidite monomer is selected from a combination of one or more of fluorine (-F), t-butyldimethylsilyl (-TBDMS), methoxy (-OMe), methoxyethoxy (-OMOE), cyanoethoxymethyl (-cyanoethoxymethyl, CEM), acetoxyethyloxymethyl (-acetoxyethyloxymethyl, ACE), triisopropylsilyloxymethyl (-triisopropylsilyloxymethyl, TOM), t-butyldithiomethyl (-t-butyldithiomethyl, DTM), 1,1-dioxo-λ6-thiomorpholine-4-carbothioate (-1,1-dioxo-λ6-thiomorpholine-4-carbothioate, TC), trimethylsilyl ethoxymethyl (-SEM), acetal acetylpropyl ester (-O-ALE) and 1-(2-cyanoethoxy) ethyl (-O-CEE).
[0013] Specifically, in step 1, when the solution of the phosphoramidite monomer is prepared, acetonitrile or N,N-dimethylformamide is used as the solvent, and the mass ratio of the solvent to the phosphoramidite monomer is 3:1 to 6:1.
[0014] Specifically, the activator is selected from one or a combination of 1H-tetrazole and 5-ethylthiotetrazole, 5-benzylthiotetrazole, 4,5-dicyanoimidazole or 5-[3,5-bis(trifluoromethyl)phenyl]tetrazole, preferably one or a combination of 5-ethylthiotetrazole, 5-benzylthiotetrazole or 4,5-dicyanoimidazole.
[0015] Specifically, the molar ratio of the phosphoramidite monomer and the activator is 1:2.0-1:6.0.
[0016] Specifically, in step 1, the reaction temperature for coupling is 15-25℃, and the reaction time is 8-20 minutes, preferably the reaction temperature is 18-22℃, and the preferred reaction time is 8-16 minutes.
[0017] Specifically, in step 1, the oxidant used for oxidation is 0.05 mol / L iodopyridine aqueous solution, and the reagent used for capping is a mixed solution composed of 20% acetic anhydride acetonitrile solution and 20% azidomethyl imidazole acetonitrile solution.
[0018] Specifically, in step 2, the cleavage refers to the use of ammonia solution to ammonolyze the solid phase carrier to cut the gRNA from the solid phase carrier, the ammonolysis time is 8-24 hours, preferably 12-18 hours, and the ammonolysis temperature is 15-25℃, preferably 18-22℃, the removal of the protecting group refers to adding triethylamine trifluoride in the ammonolysis solution to remove the protecting group on the product, and then adding an alkaline phosphate buffer solution to quench the reaction for subsequent purification and freeze-drying.
[0019] Specifically, the length of the gRNA is 80-120 nt, and the gRNA product is purified by ultrafiltration or high performance liquid chromatography after filtration.
[0020] The beneficial effects of the present application include:
[0021] 1) The gRNA synthesis method provided in the present application uses different structures of solid phase carriers as starting materials and phosphoramidite monomers with different protecting groups at the 2' position of the sugar ring as raw materials in the synthesis process, which reduces the steric hindrance of the molecules in the coupling process. In addition, by selecting appropriate activators and optimizing the addition ratio of the activators, the coupling efficiency is greatly improved.
[0022] 2) The gRNA synthesis method provided in the present application has simple process operation and is easy to control, which improves the purity and yield of the crude gRNA product, reduces the synthesis cost, improves the synthesis efficiency, and is beneficial to the large-scale production development of gRNA. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1A solid phase synthesis cycle schematic diagram of the gRNA synthesis method provided in the present application;
[0024] Figure 2 A HPLC detection result graph in Example 1 of the present application;
[0025] Figure 3 A HPLC detection result graph in Example 2 of the present application;
[0026] Figure 4 A HPLC detection result graph in Example 3 of the present application. DETAILED DESCRIPTION
[0027] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.
[0028] In the present application, percentages and parts are calculated by weight unless otherwise specified. The experimental materials involved in the present application are commercial reagents unless otherwise specified, which can be obtained from commercial channels.
[0029] Example 1
[0030] This embodiment shows the specific operation of a certain embodiment of the gRNA synthesis method provided in the present application. The solid phase synthesis cycle schematic diagram is referred to Figure 1 , and the steps are as follows:
[0031] (1) The phosphoramidite monomer with tert-butyldimethylsilyl (-TBDMS) as the protecting group at the 2' position of the sugar ring and acetonitrile are used to prepare a phosphoramidite monomer solution, the amount of acetonitrile used is 5.5 times the mass of the phosphoramidite monomer, molecular sieves are added for drying for more than 12 h, and the solution is sealed and stored for use.
[0032] (2) The solid support of Universal CPG is used for column loading, and acetonitrile solution is used for rinsing.
[0033] (3) The loaded synthesis column is rinsed with 3% dichloroacetic acid (DCA) in toluene to remove the dimethoxytrityl (DMTr) protecting group on the solid support.
[0034] (4) The dried phosphoramidite monomer solution is activated with an activating agent, acetonitrile solution of 5-ethylthiotetrazole (mass ratio of acetonitrile and 5-ethylthiotetrazole activating agent is 6-10:1), to form a highly active phosphoramidite tetrazole, which is injected into the synthesis column at a certain flow rate to couple with the exposed hydroxyl groups on the solid support, and the reaction time is 8-15 min. In the cyclic reaction, the second monomer is coupled with the hydroxyl group on the first phosphoramidite monomer, and the cycle is repeated in turn, and finally the target product is formed.
[0035] (5) Oxidation: The phosphoramidite monomer is connected to the solid support through a phosphite bond, and the phosphite bond between monomers is not stable and is easily hydrolyzed, so an oxidizing agent is used to oxidize the phosphite to a phosphoric acid triester, making the oligonucleotide chain more stable and not easy to break. The oxidizing agent used is 0.05 mol / L iodopyridine aqueous solution, which includes 90% pyridine and 10% ultrapure water.
[0036] (6) Capping: In order to prevent unreacted hydroxyl groups from entering the next coupling cycle and generating impurities, acetic anhydride is used for acetylation to block them, which can greatly improve the separation effect of product purification. The capping reagent is Cap A and Cap B, wherein Cap A is acetonitrile solution containing 20% (v / v) acetic anhydride, and Cap B is acetonitrile solution containing 20% (v / v) azolemethyl imidazole.
[0037] (7) Anhydrous acetonitrile is used to wash the residual reagents after the reaction in steps (3)-(6);
[0038] (8) Steps (3)-(7) are repeated once.
[0039] (9) After the above steps, the phosphoramidite monomers are coupled together in a specific order on the solid support to form a gRNA oligonucleotide with a specific sequence. Finally, the gRNA is cut from the solid support with an ammonia solution, the ammonia hydrolysis temperature is 18-22°C, the ammonia hydrolysis time is 16 h, and the protecting groups on the base and phosphate groups are removed. Then, a solution of triethylamine trifluoride hydrochloride is added, which is equivalent to 1.5-4.0 times the volume of the ammonia hydrolysis solution, to remove the protecting group on the 2' position of the sugar ring of the phosphoramidite monomer, and finally a buffer solution of disodium hydrogen phosphate with a concentration of 0.1-0.2 mol / L is added, which is equivalent to 8.0-16.0 times the volume of the ammonia hydrolysis solution, to quench the ammonia hydrolysis solution. After filtration, a crude gRNA solution is obtained.
[0040] (10) The crude solution is analyzed for purity, and the HPLC detection result is shown in Figure 2 , and the HPLC purity of the crude product is about 30%.
[0041] Example 2
[0042] This embodiment illustrates the specific operation of another embodiment of the gRNA synthesis method provided by the present application, and the solid-phase synthesis cycle schematic diagram is referred to Figure 1 , the steps are the same as those in Embodiment 1, except that: in step (1), the phosphoramidite monomer used is replaced by a phosphoramidite monomer with a methoxy (-OMe) protecting group at the 2' position of the sugar ring, and the amount of acetonitrile or N,N-dimethylformamide used in the phosphoramidite monomer solution is 4.5 times the mass of the phosphoramidite monomer, the solid support in step (2) is replaced by mU CPG solid support, and the activating agent in step (4) is replaced by 5-benzylthio tetrazole, and the reaction time of the coupling reaction is 12 min.
[0043] Finally, the purity of the crude product solution was analyzed, and the HPLC detection results are shown in Table 1. Figure 3 The HPLC purity of the crude product is about 32%.
[0044] Embodiment 3
[0045] This embodiment illustrates the specific operation of another embodiment of the gRNA synthesis method provided by the present application, and the solid-phase synthesis cycle schematic diagram is referred to Figure 1 , the steps are the same as those in Embodiment 1, except that: in step (1), the phosphoramidite monomer used is replaced by a phosphoramidite monomer with a methoxy (-OMe) protecting group at the 2' position of the sugar ring, and the amount of acetonitrile or N,N-dimethylformamide used in the phosphoramidite monomer solution is 4.5 times the mass of the phosphoramidite monomer, the solid support in step (2) is replaced by mU CPG solid support, and the activating agent in step (4) is replaced by 5-benzylthio tetrazole, and the reaction time of the coupling reaction is 12 min.
[0046] Finally, the purity of the crude product solution was analyzed, and the HPLC detection results are shown in Table 1. Figure 4 The HPLC purity of the crude product is about 32%.
[0047] In summary, by using the gRNA synthesis method of the present application, using the phosphoramidite monomers and solid supports described in the present application, in the coupling step, by using an appropriate proportion of phosphoramidite monomer solution and a suitable activating agent solution, and under appropriate coupling reaction conditions, the crude gRNA synthesized has high purity, and its HPLC purity is > 30%, which is beneficial to factory-scale production.
[0048] The above embodiments are only preferred embodiments of the present application and do not limit the protection scope of the present application, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.
Claims
1. A method for synthesizing gRNA, characterized in that, Includes the following steps: Step 1, Solid-phase synthesis cycle: The solid-phase support is loaded into the synthesis column, and then a four-step cycle of deprotection, coupling, oxidation and capping reactions is performed to sequentially link nucleotide monomers to form gRNA chains; Step 2, Post-processing: After solid-phase synthesis is completed, the gRNA chain is cut from the solid-phase support and the protecting group is removed. Then, it is purified and lyophilized to obtain the gRNA product. The nucleotide monomer is a phosphorus amide monomer with a protecting group at the 2' position of the sugar ring. In the coupling reaction, the phosphorus amide monomer is mixed with an activator in an organic solvent for activation, and then reacts with the free hydroxyl group on the solid support.
2. The method according to claim 1, characterized in that, In step 1, the solid support is selected from at least one of controllable pore size glass beads (CPG) or polystyrene microspheres (PS).
3. The method according to claim 1, characterized in that, In step 1, the deprotection solution used for deprotection is a dichloroacetic acid toluene solution with a volume fraction of 3% to 5%.
4. The method according to claim 1, characterized in that, In step 1, the protecting group at the 2' position of the sugar ring of the phosphoramidamide monomer is selected from one or more combinations of fluorine (-F), tert-butyldimethylsilyl (-TBDMS), methoxy (-OMe), methoxyethoxy (-OMOE), cyanoethoxymethyl (-cyanoethoxymethyl, CEM), acetoxyethoxymethyl (-acetoxyethyloxymethyl, ACE), triisopropylsilyloxymethyl (-triisopropylsilyloxymethyl, TOM), tert-butyldithiomethyl (-t-butyldithiomethyl, DTM), 1,1-dioxo-λ6-thiomorpholine-4-carbothioate (-1,1-dioxo-λ6-thiomorpholine-4-carbothioate, TC), trimethylsilylethoxymethyl (-SEM), acetal acetylacetyl ester (-O-ALE), and 1-(2-cyanoethoxy)ethyl (-O-CEE).
5. The method according to claim 1, characterized in that, The activator is selected from one or a combination of several of 1H-tetrazole and 5-ethylthiotetrazole, 5-benzylthiotetrazole, 4,5-dicyanimidazolium or 5-[3,5-bis(trifluoromethyl)benzyl]tetrazole.
6. The method according to claim 1, characterized in that, The molar ratio of the phosphoramide monomer to the activator is 1:2.0 to 1:6.
0.
7. The method according to claim 1, characterized in that, In step 1, the coupling reaction temperature is 15–25°C and the time is 8–20 minutes.
8. The method according to claim 1, characterized in that, In step 1, the oxidant used for oxidation is a 0.05 mol / L aqueous solution of iodopyridine, and the reagent used for capping is a mixed solution composed of a 20% (v / v) acetic anhydride acetonitrile solution and a 20% (v / v) N-methylimidazolium acetonitrile solution.
9. The method according to claim 1, characterized in that, In step 2, cleavage refers to ammonolyzing the solid-phase support with an ammonia solution to cleave the gRNA from the solid-phase support. The ammonolysis time is 8-24 hours and the ammonolysis temperature is 15-25℃. Deprotection involves adding triethylamine trihydrofluoride to the ammonolysis solution to remove the protecting group on the product. Then, an alkaline phosphate buffer solution is added to quench the reaction for subsequent purification and lyophilization.
10. The method according to claim 1, characterized in that, The gRNA is 80-120 nt in length, and the gRNA product is purified by ultrafiltration or high performance liquid chromatography after filtration.