Method for improving purity of nucleic acid long chain synthesized by solid-phase phosphoramidite triester method and application thereof

Abstract

The invention provides a method for improving the purity of a nucleic acid long chain synthesized by a solid-phase phosphoramidite triester method and application of the method, the nucleic acid long chain is synthesized by being divided into 1-3 segments, the length of the synthesized first segment of nucleic acid is 1-30 basic groups, and the length of the synthesized second segment of nucleic acid is 31-60 basic groups; the length of the synthesized third segment of nucleic acid is more than 61 basic groups; the first segment of nucleic acid is synthesized into deprotection once, and each time lasts for 120 seconds; the synthesis of the second segment of nucleic acid is deprotected once, and each time is 150s; the third segment of nucleic acid is synthesized into deprotection twice, and each time is 150s; in the synthesis of each segment of nucleic acid, after deprotection, activation coupling needs to be carried out twice, 360 s each time, oxidation once, 180 s each time, cap covering once, and 60 s each time. By changing the time and frequency of the nucleic acid long chain deprotection reaction in stages, protection group residues are reduced, the quality and purity of a synthetic product are improved, and the synthetic efficiency of the nucleic acid long chain is further improved; the method is simple in process and can be used for synthesizing high-purity long-chain nucleic acid and nucleic acid drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid synthesis methods, and in particular to a method for improving the purity of nucleic acid long chains synthesized by a solid phase phosphoramidite triester method and an application thereof. Background Art

[0002] Nucleic acid synthesis by chemical synthesis can be applied to the preparation of nucleic acid drugs in the medical field, ensuring the consistency and repeatability of such drug molecules, promoting the development of personalized medicine, and improving the rapid transformation of nucleic acid drugs. The development of nucleic acid drugs not only represents technological progress in the medical field, but also heralds major changes in future treatment strategies. Therefore, how to efficiently and accurately synthesize oligonucleotides is particularly important.

[0003] Nucleic acid synthesis technology has evolved from manual synthesis to automatic synthesis. The most representative synthesis method is the solid-phase phosphoramidite triester method, which uses phosphoramidite triesters as active intermediates and connects nucleotide units through more stable phosphodiester bonds, further improving the synthesis efficiency and product purity. This technology uses the solid phase phosphoramidite triester method to synthesize DNA. The specific synthesis steps are mainly divided into the following four steps: the first step is deprotection. The solid phase carrier CPG reacts with dichloroacetic acid / dichloromethane solution to remove the dimethoxytrityl (DMT) protecting group of its 5'-hydroxyl group to obtain an active 5'-hydroxyl group; the second step is activation coupling. The DNA nucleic acid phosphoramidite monomer is mixed with the activator tetrazole 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 the first step of deprotection; the third step is oxidation reaction. Under the action of the oxidant iodine, the phosphoramidite form is converted into a more stable phosphotriester; the fourth step is capping reaction. There may be a small amount of 5'-hydroxyl group that does not participate in the condensation reaction. Acetic anhydride and 1-methylimidazole are used to perform acetylation reaction to block it and prevent it from continuing to react and produce by-products. Repeat the above steps until all the nucleotides required to be synthesized are connected in sequence.

[0004] The Chinese invention patent with publication number CN115010769 discloses a method for synthesizing long-chain RNA nucleic acid by solid-phase phosphoramidite triester method, wherein the RNA nucleic acid is divided into at least three segments for synthesis, and the length of each synthesized RNA nucleic acid segment is 30-60 bases. The synthesis of the first segment 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 RNA nucleic acid segment above the second segment is completed according to the five steps of deprotection, activation coupling, capping, oxidation, and capping of the solid-phase phosphoramidite triester method.

[0005] However, in this solid phase phosphoramidite triester method, the deprotection methods at different stages are consistent, which will cause the deprotection effect and the synthesis process to not match, and then cause the incomplete removal of the protecting group in the deprotection step. The protecting group that is not completely removed may cause the breakage or non-specific connection of the nucleic acid chain, thereby producing incorrect sequences or short fragments. The incomplete removal of the protecting group may cause the protecting group on the partial nucleic acid chain to remain, so that there may be n-1, n-2 nucleic acid chains in the product. Thereby reducing the efficiency and quality of the synthesized nucleic acid. And the protecting group that is not completely removed will hinder the connection of the next phosphoramidite triester monomer, reduce the efficiency of the reaction, reduce the product purity, and it is difficult to ensure the quality of the long-chain nucleic acid and the reliability of the application. Summary of the invention

[0006] The object of the present invention is to solve the above technical problems and provide a method for improving the purity of long-chain nucleic acid synthesized by the solid phase phosphoramidite triester method, which can significantly improve the efficiency and purity of nucleic acid synthesis; another object of the present invention is to provide an application of the method.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The invention discloses a method for improving the purity of a long nucleic acid chain synthesized by a solid phase phosphoramidite triester method. The long nucleic acid chain is synthesized in 1-3 sections, wherein the length of the first section of the nucleic acid is 1-30 bases, the length of the second section of the nucleic acid is 31-60 bases, and the length of the third section of the nucleic acid is more than 61 bases. The synthesis steps of the first section of the nucleic acid are deprotection once, each time for 120 s, activation coupling twice, each time for 360 s, oxidation once, each time for 180 s, and capping once, each time for 60 s. The synthesis steps of the second section of the nucleic acid are deprotection once, each time for 150 s, activation coupling twice, each time for 360 s, oxidation once, each time for 180 s, and capping once, each time for 60 s. The synthesis steps of the third section of the nucleic acid are deprotection twice, each time for 150 s, activation coupling twice, each time for 360 s, oxidation once, each time for 180 s, and capping once, each time for 60 s.

[0009] The present invention studies the deprotection process of nucleic acid long chains in stages based on the traditional solid phase phosphoramidite triester method for nucleic acid synthesis, and reduces the residual protecting groups by changing the deprotection reaction time and number of times in stages, thereby improving the quality and purity of the synthetic product, and further improving the synthesis efficiency of the nucleic acid long chain.

[0010] Furthermore, the nucleic acid is DNA.

[0011] Furthermore, the nucleic acid has a length of 16-100 nt.

[0012] Preferably, the nucleic acid is 60-90 nt in length.

[0013] The method for improving the purity of long-chain nucleic acid synthesized by solid-phase phosphoramidite triester method is applicable to DNA, but has relatively poor effect on RNA synthesis.

[0014] Furthermore, the deprotection is carried out using a 3% mixed solvent of dichloroacetic acid (DCA) and dichloromethane (DCM).

[0015] Furthermore, the reaction flow rate of deprotection in the synthesis step of the first nucleic acid segment is 12.6 mL / min; the reaction flow rate of deprotection in the synthesis step of the second nucleic acid segment is 15.7 mL / min; and the reaction flow rate of deprotection in the synthesis step of the third nucleic acid segment is 18.9 mL / min.

[0016] The present invention has found through experiments that the flow rate of the deprotection reagent has a promoting effect on the sufficiency of the reaction. Changing the flow rate and coordinating the deprotection reaction time and number can ensure the sufficiency of the reaction and help the protecting groups to be completely removed, thereby significantly improving the efficiency and purity of DNA synthesis.

[0017] Furthermore, the activation coupling uses a mixed solution of 0.3M benzotriazole, 0.5% N-methylimidazole, and 99.5% anhydrous acetonitrile.

[0018] Furthermore, the oxidation uses 0.05M iodine-pyridine / water (volume ratio 90:10).

[0019] Furthermore, the capping material used in the invention is CAP A which is 5% phenoxyacetic anhydride, and CAP B which is methyl acetimidate / pyridine / anhydrous acetonitrile (volume ratio 20:30:50).

[0020] Furthermore, the reaction flow rate of the activation coupling is 15.7 mL / min; the reaction flow rate of the oxidation is 4 mL / min; and the reaction flow rate of the capping is 6.3 mL / min.

[0021] The method of the present invention can significantly improve the quality and purity of the synthetic product and reduce the residual impurities such as protecting groups. Therefore, the present invention also provides the use of the above method for improving the purity of long-chain nucleic acid synthesized by solid-phase phosphoramidite triester method in the preparation of nucleic acid drugs.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The present invention studies the deprotection process of nucleic acid long chains in stages based on the traditional solid phase phosphoramidite triester method for nucleic acid synthesis, and reduces the residual protecting groups by changing the deprotection reaction time and number in stages, thereby improving the quality and purity of the synthesized product, thereby improving the synthesis efficiency of nucleic acid long chains. The present invention solves the problem that in the process of nucleic acid synthesis by the solid phase phosphoramidite triester method, the incomplete removal of the protecting groups may lead to the breakage of nucleic acid chains, non-specific connection, the generation of wrong sequences or short fragments, and the reduction of synthesis efficiency and product quality.

[0024] (2) The present invention studies the flow rate of the deprotection reagent and finds that changing the flow rate and coordinating the deprotection reaction time and number can ensure the sufficiency of the reaction and help the protecting groups to be completely removed, thereby significantly improving the efficiency and purity of nucleic acid synthesis.

[0025] (3) The process of the present invention is simple, and the efficiency and purity of nucleic acid synthesis are high. It can be used to synthesize high-purity long-chain nucleic acids and prepare nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic structural diagram of the phosphoramidite triester method of the present invention.

[0027] Figure 1 The meanings of the labels are as follows: Step 1 detrityation: react the solid phase carrier CPG with dichloroacetic acid / dichloromethane solution to remove the dimethoxytrityl (DMT) protecting group of its 5'-hydroxyl group to obtain an active 5'-hydroxyl group; Step 2 activation coupling: mix the DNA nucleic acid phosphoramidite monomer 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 the first deprotection step; Step 3 oxidation: under the action of the oxidant iodine, the phosphoramidite form is converted into a more stable phosphotriester; Step 4 capping: a small amount of 5'-hydroxyl group may not participate in the condensation reaction, and is blocked by acetylation reaction with acetic anhydride and 1-methylimidazole to prevent it from continuing to react and produce by-products; Repeat the above steps until all the nucleotides required to be synthesized are connected in sequence.

[0028] Figure 2 Synthesis experiment spectrum when the DNA length is 16 nt in Example 1.

[0029] Figure 3 Synthesis experiment spectrum when the DNA length is 90 nt in Example 4.

[0030] Figure 4 Mass spectrometry results when the DNA length is 90 nt in Example 4.

[0031] Figure 5 The injection results when the DNA length is 90 nt in Example 4. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] The invention provides a method for improving the purity of a long nucleic acid chain synthesized by a solid phase phosphoramidite triester method. The long nucleic acid chain is synthesized in 1-3 sections, the length of the first section of the nucleic acid is 1-30 bases, the length of the second section of the nucleic acid is 31-60 bases; the length of the third section of the nucleic acid is more than 61 bases; the steps of synthesizing the first section of the nucleic acid are deprotection once, each time for 120 seconds, activation coupling twice, each time for 360 seconds, oxidation once, each time for 180 seconds, and capping once, each time for 60 seconds; the steps of synthesizing the second section of the nucleic acid are deprotection once, each time for 150 seconds, activation coupling twice, each time for 360 seconds, oxidation once, each time for 180 seconds, and capping once, each time for 60 seconds; the steps of synthesizing the third section of the nucleic acid are deprotection twice, each time for 150 seconds, activation coupling twice, each time for 360 seconds, oxidation once, each time for 180 seconds, and capping once, each time for 60 seconds.

[0034] Preferably, the synthesis of nucleic acid using the improved solid phase phosphoramidite triester method is divided into three stages, and the number of reactions and reaction time of each stage are shown in Table 1.

[0035] Table 1 Reaction times and reaction times for the three stages of nucleic acid synthesis

[0036]

[0037] Preferably, the synthesis of nucleic acid using the improved solid phase phosphoramidite triester method is divided into three stages, and the reaction flow rate and reagents of each stage are shown in Table 2 and Table 3.

[0038] Table 2 Reaction flow rates of nucleic acid synthesis in three stages

[0039]

[0040] Table 3 Reagents for nucleic acid synthesis stage 3

[0041]

[0042] Example 1

[0043] This embodiment provides a method for improving the purity of nucleic acid synthesized by solid phase phosphoramidite triester method, wherein the number of nucleic acid synthesis reactions, reaction time and reaction flow rate are as follows:

[0044] Table 4 Reaction flow rate, reaction number and reaction time of nucleic acid synthesis

[0045]

[0046]

[0047] 1. Target nucleic acid DNA synthesis

[0048] The DNA sequence is shown in SEQ ID NO. 1, specifically 3'-AGCTAGCTAGCTAGCT-5'.

[0049] 2. Synthesis Method

[0050] This embodiment provides a method for improving the purity of nucleic acid chains synthesized by a solid phase phosphoramidite triester method, and the reaction flow rate, reaction times and reaction time are shown in Table 1.

[0051] The specific steps are as follows:

[0052] The present invention synthesizes and purifies the DNA nucleic acid of the present invention according to the following steps:

[0053] 2.1 Step 1: Synthesis of DNA

[0054] (1) According to the method of synthesizing DNA nucleic acid by solid phase phosphoramidite triester method, the synthesis conditions of DNA nucleic acid synthesizer are adjusted, and long-chain DNA nucleic acid is synthesized using Bio-Oligo series nucleic acid synthesizer;

[0055] (2) Prepare the deprotecting agent, activating coupling agent, oxidizing agent and capping agent used in the synthesis. The reagents are all from Suzhou Kelema Biotechnology Co., Ltd., wherein the deprotecting agent is a 3% mixed solvent of dichloroacetic acid (DCA) and dichloromethane (DCM), and the amount used each time is 39.25 mL; the activating coupling agent is a mixed solution of 0.3 M benzotriazole, 0.5% N-methylimidazole, and 99.5% anhydrous acetonitrile, and the amount used each time is 4.5 mL; the oxidizing agent is a mixed solution of 0.05 M iodine-pyridine / water (v / v=9 / 1), and the amount used each time is 4.8 mL; the capping agent CAP A is a 5% phenoxyacetic anhydride solution, and the amount used each time is 6.3 mL; the capping agent CAP B is a solution of methyl acetimidate / pyridine / anhydrous acetonitrile (v / v / v=2 / 3 / 5), and the amount used each time is 6.3 mL. The capping agents CAPA and CAP B are automatically added to the instrument by the machine at the same time. Dissolve 5 g of DNA monomer in 25 mL of anhydrous acetonitrile and protect with nitrogen. Use 1.58 mL each time. Load all the above synthesis reagents onto a DNA nucleic acid synthesizer.

[0056] (3) Weigh 1.5 g of solid phase carrier CPG and use a 6.3 mL synthesis column; load it onto the column base of the synthesizer.

[0057] (4) Check the equipment pressure, reagent bottle pressure, reagent dosage and other parameters, and start synthesis after confirming that they are correct.

[0058] (5) During the synthesis process, check the reagent dosage and equipment operating status until the synthesis is completed.

[0059] 2.2 Step 2: Ammonolysis

[0060] (1) After the synthesis, the CPG powder connected with nucleic acid was placed in 20 mL of concentrated ammonia water and heated to 65° C. for 12 h. After the reaction, it was cooled to room temperature.

[0061] (2) The turbid liquid after the above aminolysis is filtered to obtain a concentrated ammonia solution containing nucleic acid, which is then concentrated to a dry powder state, and 1 mL of dimethyl sulfoxide (DMSO) is added to completely dissolve the nucleic acid. After dissolution, 1.5 mL of triethylamine trihydrofluoride is added and heated to 65° C. for 2.5 hours to obtain a crude nucleic acid with the 2'-end silicon-based protecting group (TBS / TOM) removed.

[0062] (3) Alcohol precipitation. Add 35 mL of isopropanol to the crude nucleic acid, place in a -20°C refrigerator, wait for 2 hours, and then take out. Centrifuge at 12,000 r / min to obtain a white nucleic acid solid.

[0063] 2.3 Step 3: Detection

[0064] 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 high-purity long-chain nucleic acids were purified.

[0065] 3. Synthesis Results and Purification

[0066] 16nt DNA chain synthesis was performed using these synthesis parameters, as shown in Table 5 below.

[0067] Table 5 16nt DNA chain synthesis sequence and purity

[0068]

[0069] Nucleic acid test spectrum Figure 2 As shown, the test results show that according to this synthesis parameter, the 16nt DNA long chain is synthesized, and the purity of the crude product is 92.2%.

[0070] Example 2

[0071] The method was the same as in Example 1, except that the deprotection flow rate was changed to 15.7 mL / min. The purification results were as follows.

[0072] 1. Target nucleic acid DNA synthesis

[0073] The DNA sequence is shown in SEQ ID NO.1.

[0074] 2. Synthesis method is the same as in Example 1

[0075] 3. Synthesis Results and Purification

[0076] 16nt DNA chain synthesis was performed using these synthesis parameters, as shown in Table 6 below.

[0077] Table 6 16nt DNA chain synthesis sequence and purity

[0078]

[0079] The test results show that the 16nt DNA long chain synthesized according to this synthesis parameter has a crude product purity of 91.6%.

[0080] Example 3

[0081] The method was the same as in Example 1, except that the deprotection flow rate was changed to 18.9 mL / min. The purification results were as follows.

[0082] 1. Target nucleic acid DNA synthesis

[0083] The DNA sequence is 3'-AGCTAGCTAGCTAGCT-5', SEQ ID NO.1

[0084] 2. Synthesis method is the same as in Example 1

[0085] 3. Synthesis Results and Purification

[0086] 16nt DNA chain synthesis was performed using these synthesis parameters, as shown in Table 7 below.

[0087] Table 7 16nt DNA chain synthesis sequence and purity

[0088]

[0089] The test results show that the 16ntDNA long chain was synthesized according to this synthesis parameter, and the crude product purity was 91.3%. It was found that with the increase of flow rate, the product purity changed less. Preliminary analysis showed that the increase of flow rate in the short chain made the protective group be removed faster. However, when the flow rate increased to 18.9mL / min, the possible reason was that the deprotection reagent feed time remained unchanged, the flow rate increased, and the feed increased, which may cause the purine bases (adenine and guanine) on the DNA chain to fall off, resulting in damage to the DNA chain and incompleteness of the sequence, so the purity was slightly reduced. In order to further verify the influence of the number of reactions and reaction time, experiments were conducted on different variables.

[0090] Table 8 Reaction flow rate of nucleic acid synthesis of Examples 1-3:

[0091]

[0092] Comparative Example 1

[0093] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 1, and the reaction time was 150 s; the purity result was as follows: 90.3%.

[0094] Comparative Example 2

[0095] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 1, and the reaction time was 180 s; the purity result was as follows: 90%.

[0096] Comparative Example 3

[0097] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 2, and the reaction time was 120 s; the purity result was as follows: 90.9%.

[0098] Comparative Example 4

[0099] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 2, and the reaction time was 150 s; the purity result was as follows: 90.7%.

[0100] Comparative Example 5

[0101] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 2, and the reaction time was 180 s; the purity result was as follows: 89.6%.

[0102] Comparative Example 6

[0103] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 3, and the reaction time was 120 s; the purity result was as follows: 88.7%.

[0104] Comparative Example 7

[0105] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 3, and the reaction time was 150 s; the purity result was as follows: 87.9%.

[0106] Comparative Example 8

[0107] The same nucleic acid was used as in Example 1, except that the deprotection reaction conditions were changed: the number of deprotection reactions in the nucleic acid synthesis step was 3, and the reaction time was 180 s; the purity result was as follows: 87.7%.

[0108] Table 9 Deprotection reaction times and reaction times for nucleic acid synthesis in Comparative Examples 1-8

[0109]

[0110]

[0111] By finding the embodiments and comparative examples, when other conditions remain unchanged, changing the reaction times, it is found that increasing the deprotection times provides more opportunities to remove impurities in the reaction and reduce accumulated impurities. In multi-step synthesis, impurities may be introduced in each step. Increasing the deprotection times can remove these impurities at an earlier stage to avoid their accumulation in subsequent steps. And increasing the reaction time allows the reagent to fully react with a specific protecting group in the deprotection step. When the number of times is 3 or the time is long, there is also a risk of reduced purity, and the production time is long and inefficient, so in order to reduce costs and improve production efficiency, the most suitable flow rate number and time are selected. Therefore, the present invention introduces the concept of long-chain synthesis by short-chain synthesis. Since the protecting group may not be completely removed in the long-chain synthesis, the subsequent reaction is hindered, affecting the synthesis efficiency and product purity. Therefore, the present invention performs 3-segment synthesis on the long chain. As the length of the synthesized chain is longer, different flow rates, reaction times and time are used in three sections to achieve the optimal method, reduce production costs, etc.

[0112] Example 4

[0113] 1. Target nucleic acid DNA synthesis

[0114] The DNA sequence is shown in SEQ ID NO. 2. The specific DNA sequence is 3'-TGGCT GAGCC ACGGT GAAAAAGTTC AACTA TTGCC TGATC GGAAT TGGCT GAGCC ACGGT GAAAA AGTTC AACTA TTGCC TGATCGGAAT-5' DNA length is 90 nt.

[0115] 2. Synthesis Method

[0116] This example provides the same synthesis method as Example 1, except that the first nucleic acid segment is deprotected once and the flow rate is 12.6 mL / min; the second nucleic acid segment is deprotected once and the flow rate is 15.7 mL / min; the third nucleic acid segment is deprotected twice and the flow rate is 18.9 mL / min. The number of reactions and reaction times are shown in Table 1, and the reaction flow rates and reagents are shown in Tables 2 and 3.

[0117] The specific steps are as follows:

[0118] The present invention synthesizes and purifies the long-chain DNA nucleic acid of the present invention according to the following steps:

[0119] 2.1 Step 1: Synthesis of long-chain DNA

[0120] (1) According to the method of synthesizing DNA nucleic acid by solid phase phosphoramidite triester method, the synthesis conditions of DNA nucleic acid synthesizer are adjusted, and long-chain DNA nucleic acid is synthesized using Bio-Oligo series nucleic acid synthesizer;

[0121] (2) Prepare the deprotecting agent, activating coupling agent, oxidizing agent and capping agent used in the synthesis. The reagents are all from Suzhou Kelema Biotechnology Co., Ltd., wherein the deprotecting agent is a 3% mixed solvent of dichloroacetic acid (DCA) and dichloromethane (DCM), and the amount used each time is 39.25 mL; the activating coupling agent is a mixed solution of 0.3 M benzotriazole, 0.5% N-methylimidazole, and 99.5% anhydrous acetonitrile, and the amount used each time is 4.5 mL; the oxidizing agent is a mixed solution of 0.05 M iodine-pyridine / water (v / v=9 / 1), and the amount used each time is 4.8 mL; the capping agent CAP A is a 5% phenoxyacetic anhydride solution, and the amount used each time is 6.3 mL; the capping agent CAP B is a solution of methyl acetimidate / pyridine / anhydrous acetonitrile (v / v / v=2 / 3 / 5), and the amount used each time is 6.3 mL. The capping agents CAPA and CAP B are automatically added to the instrument by the machine at the same time. Dissolve 5 g of DNA monomer in 25 mL of anhydrous acetonitrile and protect with nitrogen. Use 1.58 mL each time. Load all the above synthesis reagents onto a DNA nucleic acid synthesizer.

[0122] (3) Weigh 1.5 g of solid phase carrier CPG and use a 6.3 mL synthesis column; load it onto the column base of the synthesizer.

[0123] (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 the synthesis.

[0124] (5) During the synthesis process, check the reagent dosage and equipment operating status until the synthesis is completed.

[0125] 2.2 Step 2: Ammonolysis

[0126] (1) After the synthesis, the CPG powder connected with nucleic acid was placed in 20 mL of concentrated ammonia water and heated to 65° C. for 12 h. After the reaction, it was cooled to room temperature.

[0127] (2) The turbid liquid after the above aminolysis is filtered to obtain a concentrated ammonia solution containing nucleic acid, which is then concentrated to a dry powder state, and 1 mL of dimethyl sulfoxide (DMSO) is added to completely dissolve the nucleic acid. After dissolution, 1.5 mL of triethylamine trihydrofluoride is added and heated to 65° C. for 2.5 hours to obtain a crude nucleic acid with the 2'-end silicon-based protecting group (TBS / TOM) removed.

[0128] (3) Alcohol precipitation. Add 35 mL of isopropanol to the crude nucleic acid obtained in step 2, place in a -20°C refrigerator, wait for 2 hours, and then take out. Centrifuge at 12,000 r / min to obtain a white nucleic acid solid.

[0129] 2.3 Step 3: Detection

[0130] 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 high-purity long-chain nucleic acids were purified.

[0131] 3. Synthesis Results and Purification

[0132] The synthesis parameters were used to synthesize a 90 nt DNA long chain, as shown in Table 10 below.

[0133] Table 10 90nt DNA chain synthesis results

[0134]

[0135]

[0136] Test graph such as Figure 3 , perform chromatographic determination and the chromatographic results are as follows Figure 4-5 As shown, the test results show that according to this synthesis parameter, a 90 nt DNA long chain is synthesized, and the purity of the crude product is 64.2%.

[0137] Comparative Example 9

[0138] The same nucleic acid was used as in Example 4, except that the deprotection reaction conditions were changed: the synthesis was not divided into 3 stages, the flow rate was maintained at 12.6 mL / min, and the number of deprotection times was not increased. The purity result was as follows: 23.08%.

[0139] Comparative Example 10

[0140] 1. Target nucleic acid DNA synthesis

[0141] The target DNA is the sequence shown in SEQ ID NO. 3, specifically 3'-TAAGG CTAGT CCGTT ATCAA CTTGAAAAAG TGGCA CCGAG TCGGT TAAGG CTAGT CCGTT ATCAA CTTGA AAAAG TGGCA CCGAG TCGGTCCCATGTGCC-5', and the length of the DNA is 100 nt.

[0142] 2. Synthesis method is the same as in Example 4

[0143] 3. Synthesis Results and Purification

[0144] 100 nt DNA long chain synthesis was performed using these synthesis parameters, as shown in Table 11.

[0145] Table 11 100nt DNA chain synthesis results

[0146]

[0147] The test results show that according to this synthesis parameter, the 100nt DNA long chain is synthesized with a crude product purity of 35.2%.

[0148] Example 5

[0149] 1. Target RNA Synthesis

[0150] The target RNA is the sequence shown in SEQ ID NO.4, the RNA sequence is 3'-AAGUU AAAAU AAGGC UAGUCCGUUA UCAAC UUGAA AAAGU GGCAC CGAGU CGGUG CUUUU-5', and the RNA length is 60 nt.

[0151] 2. Synthesis method is the same as in Example 4

[0152] 3. Synthesis Results and Purification

[0153] 60nt RNA long chain synthesis was performed using these synthesis parameters, as shown in Table 12 below.

[0154] Table 12 60nt RNA chain synthesis results

[0155]

[0156] Experimental results analysis

[0157] This study aims to improve the purity of the synthesized product by improving the deprotection step of the solid phase phosphoramidite triester method for synthesizing long nucleic acid chains. The experiment is divided into multiple examples and comparative examples, and nucleic acid chains of different lengths are synthesized. The number and time of deprotection reactions that can improve the purity of the synthesis are unexpectedly discovered.

[0158] Wherein, in embodiment 1 to 3, the DNA chain of 16 bases (nt) is synthesized.By changing the flow velocity of deprotection reaction (respectively 12.6mL / min, 15.7mL / min and 18.9mL / min), it is found that with the increase of flow velocity, product purity changes less, and the flow velocity increase in the preliminary analysis short chain makes the protecting group be removed faster, but when the flow velocity is increased to 18.9mL / min, the possible reason is that the deprotection reagent feed time is constant, the flow velocity is accelerated, and the feed increases, which may cause the purine bases (adenine and guanine) on the DNA chain to fall off, resulting in the damage of the DNA chain and the incompleteness of the sequence, so the purity is slightly reduced.In embodiment 4, the DNA chain synthesized to 90nt is expanded.During the synthesis process, the nucleic acid long chain is divided into three sections, and each section adopts different deprotection conditions respectively.Results show that the crude product purity reaches 64.2%.Meanwhile, we utilize traditional synthesis method to synthesize and find that the crude product purity is only 23.08%. This further confirms the importance of segmented synthesis and optimized deprotection steps for improving the purity of long-chain nucleic acid synthesis. In Comparative Example 10, we continued to use the same method as Example 4 for the synthesis of a 100nt DNA chain. The success of the synthesis process demonstrates the applicability and effectiveness of our method in the synthesis of longer-chain nucleic acids.

[0159] Example 5: For the synthesis of 60nt RNA chains, we used the same method as DNA synthesis. The results showed that the purity of the crude product was 41.7%, which indicates that there are additional challenges in the RNA synthesis process.

[0160] Conclusion: By systematically optimizing the number of reactions and flow rate of the deprotection step, we successfully improved the purity of long-chain nucleic acid synthesis. The experimental results show that increasing the number of deprotection reactions and appropriately increasing the flow rate are effective strategies to improve synthesis efficiency and product purity. In addition, the concept of segmented synthesis provides a new perspective for the efficient synthesis of long-chain nucleic acids. The method of this study not only improves the efficiency and purity of nucleic acid synthesis, but also provides important technical support for the preparation of nucleic acid drugs.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the solution. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents based on the understanding of the present solution without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method, characterized in that: The long nucleic acid chain is synthesized in 1-3 segments, the length of the first segment of nucleic acid synthesized is 1-30 bases, the length of the second segment of nucleic acid synthesized is 31-60 bases; the length of the third segment of nucleic acid synthesized is more than 61 bases; The synthesis steps of the first nucleic acid are deprotection once, each time for 120 s, activation coupling twice, each time for 360 s, oxidation once, each time for 180 s, and capping once, each time for 60 s; The synthesis steps of the second nucleic acid are deprotection once, each time for 150 s, activation coupling twice, each time for 360 s, oxidation once, each time for 180 s, and capping once, each time for 60 s; The synthesis steps of the third nucleic acid are deprotection twice, each time for 150 seconds, activation coupling twice, each time for 360 seconds, oxidation once, each time for 180 seconds, and capping once, each time for 60 seconds.

2. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The nucleic acid is DNA.

3. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The nucleic acid has a length of 16-100 nt.

4. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The deprotection was performed using a 3% mixed solvent of dichloroacetic acid and dichloromethane.

5. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The reaction flow rate of deprotection in the synthesis step of the first nucleic acid is 12.6 mL / min; the reaction flow rate of deprotection in the synthesis step of the second nucleic acid is 15.7 mL / min; the reaction flow rate of deprotection in the synthesis step of the third nucleic acid is 18.9 mL / min.

6. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The activation coupling uses a mixed solution of 0.3 M benzotriazole, 0.5% N-methylimidazole, and 99.5% anhydrous acetonitrile.

7. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The oxidation used a 0.05 M iodine-pyridine aqueous solution.

8. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The capping agent used CAP A which is 5% phenoxyacetic anhydride, and CAP B which is methyl acetimidate / pyridine / anhydrous acetonitrile, with a volume ratio of 20:30:

50.

9. The method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to claim 1, characterized in that: The reaction flow rate of the activation coupling was 15.7 mL / min; the reaction flow rate of the oxidation was 4 mL / min; and the reaction flow rate of the capping was 6.3 mL / min.

10. Use of the method for improving the purity of long-chain nucleic acid synthesized by solid phase phosphoramidite triester method according to any one of claims 1 to 9 in the preparation of nucleic acid drugs.

Images