Method for synthesizing RNA nucleic acid using mixed deprotecting agents

Abstract

The present invention provides 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 synthesis is completed according to the deprotection, activation coupling, capping, oxidation of the solid phase phosphoramidite triester method, and the synthesis of the first section of RNA nucleic acid is completed according to the 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. Using the mixed deprotecting agent of the present invention, the stable synthesis of longer chain RNA nucleic acid is achieved.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid synthesis, and in particular to a method for synthesizing RNA nucleic acid by using a mixed deprotecting agent. Background Art

[0002] Single-stranded nucleic acids are composed of a certain number of deoxynucleotides or nucleotides. Long-stranded RNA nucleic acids are generally composed of more than 50 nucleotides (rA, rG, rC, rU). Long-stranded RNA nucleic acids are widely used in the development of new therapeutic strategies, Crispr gene editing, FISH (fluorescence in situ hybridization technology), and the effects of RNA modification on RNA-protein complexes.

[0003] Long-chain RNA nucleic acids play an increasingly important role in genome function research, drug discovery, and biosynthesis. However, the synthesis of long-chain RNA nucleic acids has always faced challenges such as complex sequence composition, secondary structure, and difficulty in modification and labeling. Although some clever strategies have been developed to optimize these problems and improve the synthesis efficiency of long-chain RNA, long-chain RNAs greater than 50 bases in length cannot be synthesized stably and efficiently due to the steric hindrance of the sequence structure. Therefore, there is an urgent need to find a new method for synthesizing long-chain RNA nucleic acids.

[0004] In the current solid-phase phosphoramidite triester method for nucleic acid synthesis, when the synthetic chain is too long, segmented synthesis can be used. When synthesizing 50 or more bases, the number of monomer reactions can be appropriately increased, and the reagent reaction time can be appropriately extended to allow for sufficient reaction. This method can achieve the synthesis of long-chain DNA nucleic acids, but it cannot be achieved when synthesizing long-chain RNA nucleic acids.

[0005] The main structures of DNA and RNA nucleic acids are similar. The biggest difference is that RNA has a hydroxyl group at the 2' of the sugar group compared to DNA, and this hydroxyl group is an active group. Their structural formulas are shown in Formula 1 below. Pre-protection must be performed during the synthesis process to avoid side reactions at the 2' end of the synthesis process. The protecting groups used to protect the 2' hydroxyl group (such as tert-butyldimethylsilyl (TBS), triisopropylsilyloxymethyl (TOM), etc.) will produce a large steric hindrance, thereby reducing the coupling efficiency of long-chain RNA nucleic acids. In comparison, the existing solid-phase phosphoramidite triester process can synthesize DNA nucleic acids with a length of up to 160 bases, while when used for RNA nucleic acid synthesis, it can generally only synthesize a maximum of 50-55 bases in length. When synthesizing RNA nucleic acids with a length of more than 50 bases, severe depurination and base deficiency will gradually occur. In addition, if the purity of the crude product of the synthesized RNA nucleic acid is less than 30%, it is difficult to obtain a qualified RNA nucleic acid product.

[0006]

[0007] When synthesizing nucleic acids using the solid phase phosphoramidite triester method, it is necessary to first remove the protecting group (dimethoxytrityl) at the 5th position of the phosphoramidite monomer before proceeding to the next step of the activated coupling reaction. The reagent for removing the protecting group at the 5th position is usually called a deprotecting agent, and its main component is a mixed solution of alkylbenzene or halogenated hydrocarbons of protonic acid.

[0008] The reaction mechanism of the deprotection step is shown in Formula 2. Proton acid (TCA, DCA) can easily react with the 5' oxygen of the phosphoramidite monomer to remove the DMT protecting group. It can be found that the base (Base1) is completely exposed to the acidic reagent during this reaction, which is the main reason for depurination in nucleic acid synthesis. The depurination reaction of nucleic acid refers to the process in which the chemical bond between ribose and purine on the nucleic acid chain is broken, thereby generating free purine and purine-free sites.

[0009]

[0010] Due to the structural differences between RNA and DNA, RNA faces more challenges during the solid-phase phosphoramidite synthesis process, such as complex sequence composition, more secondary structures, lower reaction efficiency, and serious nucleic acid depurination. The most concerning issue is the occurrence of nucleic acid depurination during the synthesis process, which will directly lead to the failure of RNA nucleic acid synthesis. The reason is that the deprotection reagent is a proton acid solution, and frequent exposure of nucleic acids to strong acidic solutions will destroy their intact structure.

[0011] In order to minimize the problems of depurination, low deprotection efficiency and low purity of crude products during RNA nucleic acid synthesis and to achieve efficient reaction of phosphoramidite monomers during the synthesis process, it is crucial to deprotect in the first step to obtain effective 5'-OH. Summary of the invention

[0012] In order to solve the above technical problems, the present invention provides 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, the length of each synthesized RNA nucleic acid section is 30-60 bases, the synthesis of the first section of RNA nucleic acid is completed 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, wherein the mass ratio of dichloroacetic acid to trichloroacetic acid is 13:2-2:3.

[0013] In one embodiment, the total mass concentration of the dichloroacetic acid and trichloroacetic acid dichloromethane solution is 3 g / 100 mL.

[0014] In one embodiment, the mass ratio of dichloroacetic acid to trichloroacetic acid is 4:1.

[0015] In one embodiment, the RNA nucleic acid is synthesized in three sections: a first section of RNA nucleic acid of 1-40 bases; a second section of RNA nucleic acid of 41-80 bases; and a third section of RNA nucleic acid of more than 81 bases.

[0016] In one embodiment, the step of synthesizing the first segment of RNA nucleic acid is to deprotect twice, each time for 12-15 seconds, the step of synthesizing the second segment of RNA nucleic acid is to deprotect three times, each time for 12-13 seconds, and the step of synthesizing the third segment of RNA nucleic acid is to deprotect three times, each time for 15-18 seconds.

[0017] In one embodiment, the steps of synthesizing the first RNA nucleic acid are coupling twice, each time for 120 seconds, capping once, each time for 60 seconds, and oxidation once, each time for 60 seconds.

[0018] In one embodiment, the steps of synthesizing the second RNA nucleic acid are coupling twice, each time for 150 seconds, capping once, each time for 60 seconds, oxidation once, each time for 60 seconds, and capping once, each time for 60 seconds.

[0019] In one embodiment, the steps of synthesizing the third RNA nucleic acid are coupling 3 times, each time for 180 seconds, capping once, each time for 60 seconds, oxidation once, each time for 60 seconds, and capping once, each time for 60 seconds.

[0020] In one embodiment, the activated coupling agent in the coupling step is a 0.50M-0.75M ethylthiotetrazolium solution in acetonitrile.

[0021] The invention provides a chemical preparation method for RNA nucleic acid which can synthesize RNA with a maximum length of 120 bases based on a solid phase phosphoramidite triester method, the purity of the synthesized long-chain RNA crude product is improved, and a pure long-chain RNA product can be obtained after purification.

[0022] By adopting the segmented synthesis method of the present invention, using a higher concentration of an activated coupling agent and reducing the total coupling time can effectively improve the coupling efficiency, significantly improve the base deficiency in RNA nucleic acid synthesis, and greatly improve the situation of a small number of depurines; by adding a capping reaction, the side reaction during the deprotection after the first segment synthesis is reduced, and the purity of the crude RNA in RNA nucleic acid synthesis is improved.

[0023] By using the mixed deprotecting agent of the present invention, the stable synthesis of longer-chain RNA nucleic acids is achieved, which is increased from 100 bases to 120 bases. The purity of the synthesized long-chain RNA crude product is further improved, and the deprotection time used in the present invention is shortened, the time of the entire synthesis reaction is shortened, and the efficiency of the synthesis is improved.

[0024] The method of the present invention avoids the problems of protein host residues and the like existing in the synthesis of RNA nucleic acids by biological transcription methods, while also minimizing the complexity of nucleotide combination sequences, and can be extended to label and modify RNA nucleic acids, while meeting the requirements of large-scale production; and solves the problem of low purity of long-chain RNA nucleic acids. DETAILED DESCRIPTION

[0025] In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the embodiments. Obviously, the described embodiments are only a 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 making creative work should belong to the scope of protection of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0026] Example 1 Method for synthesizing RNA nucleic acid using the solid phase phosphoramidite triester method of the present invention

[0027] 1. Principle of RNA nucleic acid synthesis by solid phase phosphoramidite triester method

[0028] The specific synthesis steps of RNA synthesis by solid phase phosphoramidite triester method in the prior art are divided into the following four steps:

[0029] The first step is deprotection, in which the solid phase carrier CPG reacts with a dichloroacetic acid / dichloromethane solution to remove the dimethoxytrityl (DMT) protecting group of its 5'-hydroxyl group to obtain an active 5'-hydroxyl group;

[0030] The second step is activation coupling, where the RNA nucleic acid phosphoramidite monomer is mixed with the activator tetrazolium to obtain a highly reactive nucleoside phosphorous acid activated intermediate (its 3' end is activated, and the 5'-hydroxyl group is still protected by DMT), which undergoes a condensation reaction with the active 5'-hydroxyl group obtained by deprotection in the first step;

[0031] The third step is the capping reaction. A small amount of 5'-hydroxyl groups may not participate in the condensation reaction. Acetic anhydride and 1-methylimidazole are used to perform an acetylation reaction to block the reaction and prevent the subsequent reaction from producing byproducts.

[0032] The fourth step is oxidation reaction. Under the action of iodine, the phosphite form is converted into a more stable phosphate triester.

[0033] After the above four steps, a nucleotide is connected to the nucleotide on the solid phase carrier. Then, the DMT protecting group on the 5'-hydroxyl group of the new nucleotide is removed with dichloroacetic acid / dichloromethane solution, and the above steps are repeated until all the nucleotides required to be synthesized are connected in sequence.

[0034] The RNA nucleic acid connected to the solid phase carrier CPG is cut off and the protecting group is removed by ammonia treatment. The crude product is purified by high performance liquid chromatography (HPLC), and the RNA nucleic acid is quantified by measuring the absorption value of OD260. The product is packaged according to requirements.

[0035] The present invention synthesizes and purifies the long-chain RNA nucleic acid of the present invention according to the following steps:

[0036] Step 1: Synthesis of long-chain RNA nucleic acid;

[0037] 1. According to the method for synthesizing RNA nucleic acid by solid phase phosphoramidite triester method, adjust the synthesis condition parameters 1 to 10 of the RNA nucleic acid synthesizer, and use the RNA nucleic acid synthesizer to synthesize long-chain RNA nucleic acid;

[0038] 2. Prepare the deprotecting agent, activating coupling agent, capping agent and oxidizing agent used in the synthesis, wherein the deprotecting agent is a 3% (w / v) dichloroacetic acid solution in dichloromethane, and the amount used each time is 200ul; the activating coupling agent is a 0.25M ethylmercaptotetrazole solution in acetonitrile, and the amount used each time is 75ul; the capping agent CAPA is a 10% (v / v) acetic anhydride solution in tetrahydrofuran, and the amount used each time is 80ul; the capping agent CAPB is a 16% (v / v) 1-methylimidazole solution in tetrahydrofuran, and the amount used each time is 80ul. The capping agents CAPA and CAPB are automatically added to the instrument at the same time by the machine; the oxidizing agent is a mixed solution of 0.05M iodine in tetrahydrofuran / pyridine / ultrapure water (v / v / v=7 / 2 / 1), and the amount used each time is 150ul. Dissolve 20g RNA monomer in 450ml anhydrous acetonitrile and protect with argon gas, using 63ul each time; load all the above synthesis reagents onto the RNA nucleic acid synthesizer.

[0039] 3. Weigh 10-16 mg of solid phase carrier CPG to make a synthesis column; load it onto the column base of the synthesizer.

[0040] 4. Check the equipment pressure, reagent bottle pressure, reagent dosage and other parameters. After confirming that they are correct, click the "Start Synthesis" button to start synthesis.

[0041] 5. Check the reagent dosage and equipment operating status during the synthesis process until the synthesis is completed.

[0042] Step 2: Aminolysis and desilylation protection

[0043] 1. After the synthesis is completed, the CPG powder connected with RNA nucleic acid is placed in 1 ml of concentrated ammonia water (28%) and heated to 45°C for 8 hours. After the reaction is completed, it is cooled to room temperature.

[0044] 2. Filter the turbid liquid after the above aminolysis to obtain a concentrated ammonia solution containing RNA nucleic acid, and concentrate it to a dry powder state, then add 1 ml of dimethyl sulfoxide (DMSO) to completely dissolve the RNA nucleic acid, and then continue to add 1 ml of triethylamine trihydrofluoride and heat to 80°C after dissolution, and react for 10 minutes. The crude RNA nucleic acid with the silicon-based protecting group (TBS / TOM) removed at the 2' end is obtained.

[0045] 3. Alcohol precipitation. Add 10 ml of anhydrous ethanol to the crude nucleic acid obtained in step 2, place in a -20°C refrigerator, wait for 2 hours and take out. Centrifuge at 12000 r / min to obtain a white RNA nucleic acid solid.

[0046] Step 3: Purification

[0047] The white solid was dissolved in nuclease-free water, loaded onto a high performance liquid chromatograph, and purified using acetonitrile and 0.2M triethylamine acetate (TEAA) aqueous solution. The proportion of acetonitrile was increased from 2% to 35% within 30 minutes by adjusting the proportional valve, while the proportion of 0.2M triethylamine acetate (TEAA) aqueous solution was decreased from 98% to 65%, and a high-purity long-chain RNA nucleic acid was purified.

[0048] Example 2: Solid-phase phosphoramidite triester method for synthesizing long-chain RNA nucleic acid Experiment 1

[0049] Solid phase phosphoramidite triester synthesis mainly depends on the synthesis parameters in the RNA nucleic acid synthesis instrument, in which the number of reactions and waiting time in each step will affect the reaction efficiency of the synthesis as the base length increases. Therefore, the optimal synthesis reaction parameters are obtained through testing and verification.

[0050] Parameter 1 shown in Table 1 below is a set of conventional RNA nucleic acid synthesis parameter combinations. The synthesis of one base requires a cycle of four steps: deprotection, activation coupling, capping and oxidation. The number of reactions in Table Parameter 1 refers to the number of times the corresponding reagent is injected into the solid phase carrier, and the single reaction time refers to the reaction waiting time after each reagent injection.

[0051] Table 1

[0052]

[0053] Parameter one was used to perform synthesis tests on RNA nucleic acids of different base lengths. The results showed that parameter one could only synthesize up to about 55 bases; the crude purity of RNA nucleic acids exceeding 55 bases was very poor and could not be purified to obtain products. The specific results are shown in Table 2 below.

[0054] Table 2

[0055]

[0056] According to the data in Table 2, the purity of RNA nucleic acid synthesized with 65 bases using parameter 1 is only 20.1%, and qualified products can no longer be obtained. The possible reason is that when synthesizing RNA nucleic acid with more than 55 bases, the total time and reagent dosage (i.e., the number of reactions) of the activated coupling reaction are insufficient. Therefore, the synthesis reaction is divided into two-stage synthesis, and the number and time of the activated coupling reactions after 50 bases are changed, the number of activated coupling reactions is increased and the time of a single reaction is reduced, which increases the reaction time overall. This embodiment screened three parameters, wherein the activation coupling number of parameter 2 was 2 times and the single reaction time was 360s when synthesizing 1-50 bases; the activation coupling number of parameter 3 was 3 times and the single reaction time was 245s; the activation coupling number of parameter 4 was 3 times and the single reaction time was 255s; after 51 bases, the activation coupling number of parameter 2 was 3 times and the single reaction time was 245s; the activation coupling number of parameter 3 was 3 times and the single reaction time was 255s; the activation coupling number of parameter 4 was 3 times and the single reaction time was 265s; the specific reaction parameters are shown in Table 3 below.

[0057] Table 3

[0058]

[0059] Test verification is performed based on the three set parameters, and the verification data is entered into Tables 4, 5, and 6 below.

[0060] Table 4

[0061]

[0062] Table 5

[0063]

[0064] Table 6

[0065]

[0066]

[0067] By testing and verifying the three sets of synthesis parameters, the results showed that the two-stage synthesis and the increase in the total activation coupling time and number of parameters showed that the data of parameter 2 only increased the purity of the crude RNA nucleic acid of 55 bases by about 3%, while the crude purity of parameters 3 and 4 was worse than that of parameter 1. At the same time, it can be seen that the purity of the crude products of 65 and 75 bases is less than 30%, which does not meet the purification requirements. Through mass spectrometry analysis, the main impurity components are base missing and depurinated short-chain impurities. More by-product impurities will seriously affect the coupling efficiency, resulting in low purity of the crude product.

[0068] Example 3 Experiment 2 of Synthesis of Long-chain RNA by Solid-phase Phosphoramidite Trisester Method

[0069] According to the test data of Example 2, it can be concluded that simply increasing the reaction time cannot improve the synthesis efficiency of RNA nucleic acid. The results show that serious depurination and base deficiency occur, which also causes the crude product purity to be very poor. It shows that simply increasing the reaction time cannot solve the steric hindrance problem of RNA nucleic acid. In order to minimize the steric hindrance problem of RNA nucleic acid synthesis, the concentration of the activated coupling reagent in the activated coupling step is increased to improve the coupling efficiency as much as possible through the concentration effect. The screening concentration is shown in Table 7.

[0070] Table 7

[0071]

[0072] The activator concentration was screened using the synthesis parameter 2 in Example 2, and the screening results are shown in Table 8 below.

[0073] Table 8

[0074]

[0075] The screening results of the activator concentration in Table 8 show that when the concentration is increased, the synthesis efficiency of RNA nucleic acid with the same number of bases will be improved. The mass spectrometry analysis shows that the base deficiency accounts for about 20% to 40%, and the depurination accounts for 1% to 4%. When the concentration is increased to 0.5M, the base deficiency phenomenon of RNA nucleic acid synthesis is improved. The mass spectrometry analysis shows that the base deficiency accounts for about 14% to 25%, and the depurination accounts for 4.3% to 5.1%. When the concentration is increased to 0.75M, the base deficiency is significantly improved, but there will still be a small number of depurinations. The mass spectrometry analysis shows that the base deficiency accounts for about 9% to 14%, and the depurination accounts for 7% to 9%. The possible reason is that the activation coupling reagent is acidic. After increasing the concentration of the activation coupling agent, its acidity also increases. As the activation coupling time increases, the depurination becomes more serious.

[0076] Therefore, considering the need to reduce the time that nucleic acid bases are exposed to the acidic activation coupling reagent, and considering that the longer the base, the greater the coupling difficulty caused by steric hindrance, the synthesis parameters are set to three-stage synthesis. As the number of bases increases, the time of the second and third stage coupling procedures is appropriately increased. See Table 9 for setting parameters five, six, and seven

[0077] Table 9

[0078]

[0079] The best reaction time was screened out by reducing the coupling time. The test results are shown in Tables 10, 11, and 12 below.

[0080] Table 10

[0081]

[0082]

[0083] Table 11

[0084]

[0085] Table 12

[0086]

[0087] The test results of parameter seven show that segmented synthesis and the use of 0.75M activation coupling agent can effectively improve the coupling efficiency by reducing the total activation coupling time, and greatly improve the depurination and base deficiency in RNA nucleic acid synthesis. The parameter seven program is set to activate coupling twice for 1-40 bases, the activation coupling time is 120s, the activation coupling times for 41-80 bases are 2 times, the activation coupling time is 150s, and the activation coupling is 3 times for 81 bases to the end, and the activation coupling time is 180s. 85 bases are synthesized using this parameter. The crude product purity is increased from 26.8% of parameter five to 33.2% of parameter seven, and qualified products can be obtained for RNA nucleic acid synthesis. However, when using this program to synthesize longer bases, some bases are still missing, and the crude product purity is low. The test results are shown in Table 13.

[0088] Table 13

[0089]

[0090] The test results in Table 13 show that when parameter seven is used and the concentration of the activated coupling agent is 0.75 M, only 95 bases can be synthesized at most, and the purity of the 100-base RNA nucleic acid crude product is less than 30%, and no qualified product can be obtained.

[0091] Example 4 Experiment 3 of Synthesis of Long-chain RNA by Solid-phase Phosphoramidite Trisester Method

[0092] Compared with the solid phase phosphoramidite triester synthesis of DNA nucleic acid, the RNA nucleic acid solid phase phosphoramidite triester method has higher requirements for the moisture content of the deprotection process. In the oxidation step, the oxidant is a water-containing reagent. Although it will be washed with acetonitrile after oxidation, there will still be trace amounts of water remaining. In the next synthesis cycle, these water contents affect the efficiency of deprotection, thereby affecting the efficiency of the entire RNA nucleic acid reaction step and the purity of the crude product. In order to further improve the purity of the crude RNA nucleic acid and the average reaction efficiency, the solid phase carrier is washed again with a capping reagent after the oxidation is completed to remove residual moisture. The principle is that water can be removed after reacting with the acetic anhydride / methylimidazole system in the capping reagent, thereby greatly reducing the basic water content, reducing the side reactions of the deprotection process in the next cycle, and improving the purity of the crude nucleic acid synthesis. Therefore, the instrument parameters are set as follows. Parameters 8, 9, and 10 are specifically shown in Table 14.

[0093] Table 14

[0094]

[0095] For the results of parameter eight, parameter nine and parameter ten, please refer to Tables 15, 16 and 17.

[0096] Table 15

[0097]

[0098] Table 16

[0099]

[0100] Table 17

[0101]

[0102]

[0103] The test results show that the test results of parameter eight are worse than those of parameter nine and parameter ten. The possible reason is that during the synthesis of the first 40 bases, the solid phase carrier has a high permeability, the water is easier to wash, and the basic water is very small. As the number of bases increases, the permeability of the solid phase carrier continues to decrease, and the water is gradually difficult to remove, which affects the synthesis efficiency. It is necessary to add a capping reagent dehydration step. The test results of parameters nine and ten are not much different, but parameter ten adds a capping step in the first 40 bases, which wastes time and reagents. Therefore, parameter nine is the optimal synthesis parameter setting and can ensure the highest synthesis efficiency.

[0104] Example 5 Experiments on different deprotection agents for synthesizing long RNA nucleic acids

[0105] 1. Use an RNA nucleic acid synthesizer to synthesize, and set the program method for RNA nucleic acid synthesis according to the synthesis parameters in the table below. The program method remains unchanged during the synthesis process. For details, see Table 18.

[0106] Table 18

[0107]

[0108] The amount of reagents required for each step of the reaction: 1) the deprotecting agent is a dichloroacetic acid / trichloroacetic acid dichloromethane mixed solution, and the amount used each time is 200ul; 2) the activating agent is a 0.75M ethylmercaptotetrazole acetonitrile solution, and the amount used each time is 75ul; 3) the capping agent CAPA is a 10% (v / v) acetic anhydride tetrahydrofuran solution, and the amount used each time is 80ul; 4) the capping agent CAPB is a 16% (v / v) 1-methylimidazole tetrahydrofuran solution, and the amount used each time is 80ul; 5) the oxidizing agent is a 0.05M iodine tetrahydrofuran / pyridine / ultrapure water (v / v / v=7 / 2 / 1) mixed solution, and the amount used each time is 150ul. 20g RNA nucleic acid monomers are dissolved in 450ml of anhydrous acetonitrile solution and protected by argon gas, and the amount used each time is 63ul.

[0109] The deprotecting agent, activating coupling agent, capping agent, and oxidizing agent used in the synthesis were prepared according to the following table. See Table 19 for details.

[0110] Table 19

[0111]

[0112] In the solid phase phosphoramidite triester synthesis method, the first step of the reaction is to remove the dimethoxytrityl (DMT) protecting group at the 5 ends. The most commonly used deprotection reaction reagent is a 3% (w / v) dichloroacetic acid in dichloromethane solution. Some researchers also use a 3% (w / v) dichloroacetic acid in dichloromethane solution as a deprotection agent, wherein the pKa of a 3% (w / v) dichloroacetic acid mixed solution in dichloromethane is 0.8, and the pKa of a 3% (w / v) dichloroacetic acid mixed solution in dichloromethane is 1.5. It can be seen from the pKa value that the acidity of trichloroacetic acid is stronger, while the acidity of dichloroacetic acid is relatively weak. Therefore, in order to obtain a deprotection agent with moderate acidity, the present invention uses two solutions mixed as a deprotection agent. By comparing the three deprotection agents, it can be seen that the present invention can well improve the efficiency of the reaction synthesis and improve the reaction purity of the crude product, see Table 20.

[0113] Table 20

[0114]

[0115]

[0116] The test data in Table 20 show that although 3% DCA can be used as a deprotection agent to synthesize RNA nucleic acids up to 100 bases, the crude purity is low, and when 3% TCA is used as a deprotection agent, the synthesis fails at 45 bases. Using a 3% TCA / 3% DCA mixture as a deprotection agent can significantly improve the crude purity of RNA nucleic acids.

[0117] Example 6 Experiment of synthesizing long RNA nucleic acid with different ratios of deprotection agents

[0118] From the above examples, it can be seen that the crude product purity can be significantly improved after the two are mixed, and its possible reason is that trichloroacetic acid can progressively destroy the structure of nucleic acid as strong acid in the RNA nucleic acid synthesis process, and dichloroacetic acid reacts as weak acid in the deprotection process and is not sufficient, and after the two are mixed, the acidity of the mixed solution is relatively neutralized, so as to achieve the more stable synthesis of long-chain RNA nucleic acid synthesis. By changing the relative mass concentration of dichloroacetic acid and trichloroacetic acid, the sequence (RNA-TD-5) of 100 base lengths of parallel synthesis, more appropriate mixing ratio (as shown in Table 21) is screened, to further improve the crude product purity of RNA nucleic acid synthesis. Specific results are shown in Table 21.

[0119] Table 21

[0120]

[0121] From the above results, it can be seen that 100 base RNA can be effectively synthesized when the mass ratio of dichloroacetic acid: trichloroacetic acid is in the range of 13:2-1:2, among which the crude product purity is high when the mass ratio of dichloroacetic acid: trichloroacetic acid is in the range of 13:2-2:3, and the crude product purity is highest when the mass ratio of the two is 4:1; after exceeding 1:1, the crude product purity decreases significantly.

[0122] Example 7 Experiments with different deprotection times

[0123] According to the above verification results, the crude purity can be improved by changing the concentration of dichloroacetic acid and trichloroacetic acid, because after changing the concentration of the mixed deprotecting agent, its acidity will be stronger than 3% dichloroacetic acid; in order to increase the production rate, it is possible to consider lowering the time required for deprotection in the synthesis process, thereby shortening the time of the entire synthesis reaction. This example selects the deprotection reaction time of different procedures, see Table 22 and Table 23 for details.

[0124] Table 22

[0125]

[0126] Table 23

[0127]

[0128]

[0129] The test results in Table 23 show that when the deprotection time is Scheme 3, the purity of the synthesized crude product drops sharply; the purity of the crude products of Schemes 1 and 2 is not much different from before, so Scheme 1 with a shorter time is selected as the optimal deprotection reaction parameters.

[0130] Example 8 Ultra-long RNA Nucleic Acid Synthesis Experiment

[0131] The optimal deprotection reaction reagent formula and deprotection scheme obtained by verification were applied to the synthesis of ultra-long chain RNA nucleic acid, and the synthesis primer sequence information is shown in Table 24 below.

[0132] Table 24

[0133]

[0134]

[0135] The synthesis steps were synthesized according to the following method, and the program method for RNA nucleic acid synthesis was set according to the synthesis parameters in the table below. The program method remained unchanged during the synthesis process, see Table 25.

[0136] Table 25

[0137]

[0138] Table 26

[0139]

[0140]

[0141] Table 26 is the result of synthesizing long-chain RNA nucleic acid. It can be seen that the mixed deprotection reagent invented by this patent can realize the synthesis of long-chain RNA nucleic acid between 90-120 bases. The purity of the crude product is greater than 30%, which meets the purification requirements. After purification, the purity can reach more than 90%, which meets the application needs of customers.

[0142] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0143] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A deprotecting agent for synthesizing RNA nucleic acid by a solid phase phosphoramidite triester method, characterized in that The deprotection agent used in the deprotection step is a dichloromethane solution of dichloroacetic acid and trichloroacetic acid, wherein the mass ratio of dichloroacetic acid to trichloroacetic acid is 13:2-3:

2.

2. The deprotecting agent according to claim 1, characterized in that The total mass concentration of the dichloroacetic acid and trichloroacetic acid dichloromethane solutions is 3 g / 100 mL.

3. The deprotecting agent according to claim 1, characterized in that The mass ratio of dichloroacetic acid to trichloroacetic acid is 4:

1.

4. Use of the deprotecting agent according to any one of claims 1 to 3 in synthesizing RNA nucleic acid, wherein the RNA nucleic acid is synthesized in at least three sections, each section of the RNA nucleic acid synthesized is 30-60 bases in length, the first section of the RNA nucleic acid is synthesized in accordance with the four steps of deprotection, activation coupling, capping and oxidation by the solid phase phosphoramidite triester method to complete the synthesis of the first section of the RNA nucleic acid, and the second section of each section of the RNA nucleic acid is synthesized in accordance with the five steps of deprotection, activation coupling, capping, oxidation and capping by the solid phase phosphoramidite triester method to complete the synthesis of each section of the RNA nucleic acid; in the coupling step, the activated coupling agent is an acetonitrile solution of 0.50M-0.75M ethylthiotetrazolium; the RNA nucleic acid is synthesized in three sections, synthesizing a first section of 1-40 bases; synthesizing a second section of 41-80 bases; and synthesizing a third section of 81 bases or more.

5. The use according to claim 4, characterized in that: The steps of synthesizing the first RNA nucleic acid are deprotecting twice, each time for 12-15 seconds, the steps of synthesizing the second RNA nucleic acid are deprotecting three times, each time for 12-13 seconds, and the steps of synthesizing the third RNA nucleic acid are deprotecting three times, each time for 15-18 seconds.

6. The use according to claim 5, characterized in that: The steps of the first RNA nucleic acid synthesis are activation coupling twice, each time for 120 seconds, capping once, each time for 60 seconds, and oxidation once, each time for 60 seconds.

7. The use according to claim 5, characterized in that: The steps of the second RNA nucleic acid synthesis are activation coupling twice, each time for 150 seconds, capping once, each time for 60 seconds, oxidation once, each time for 60 seconds, and capping once, each time for 60 seconds.

8. The use according to claim 5, characterized in that: The steps of the third RNA nucleic acid synthesis are activation coupling 3 times, each time for 180 seconds, capping once, each time for 60 seconds, oxidation once, each time for 60 seconds, and capping once, each time for 60 seconds.

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