Composite group oligonucleotide synthesis carrier as well as preparation method and application thereof

Abstract

The invention relates to a composite group oligonucleotide synthesis carrier as well as a preparation method and application thereof. The composite group oligonucleotide synthesis carrier comprises a carrier and groups fixed on the carrier, the groups comprise an activator group and an oligonucleotide group, and the activator group comprises a triazole group and a derivative thereof. According to the invention, the activator group is combined with the activated phosphoramidite group to promote the phosphoramidite group to gather on the carrier, namely, the distance between the activated phosphoramidite group and oligonucleotide is shortened, so that the accuracy of the condensation reaction is improved, the reaction efficiency is improved, and finally, the synthesis efficiency is improved. Meanwhile, in a general nucleic acid synthesis method, in order to ensure full proceeding of the condensation reaction, an excessive amount of monomer reagent is generally needed to be added, and the scheme can greatly improve the accuracy of the condensation reaction, so that less monomer reagent can be allowed to be used, and the cost of the nucleic acid synthesis reaction is reduced.

Description

Technical Field

[0001] The invention relates to the field of nucleic acid synthesis, in particular to a composite group oligonucleotide synthesis carrier and a preparation method and application thereof. Background Art

[0002] Phosphoramidite chemistry is a method for chemically synthesizing nucleic acids developed in the 1980s. In contrast to biosynthesis, chemical synthesis proceeds from the 3`→5` direction of nucleic acids and consists of four main steps: detritylation (the 5`-DMT protecting group is removed from the nucleoside on the first solid support), coupling (the free 5'-OH of the nucleoside attached to the first solid support attacks the phosphorus of the incoming second nucleoside, displacing its diisopropylamino group), oxidation (the unstable phosphite triester is converted into a stable phosphotriester) and a capping step (the solid support-attached nucleoside with an unreacted 5'-OH is acetylated to prevent the extension of sequences with deletion mutations).

[0003] A key challenge in chemical DNA synthesis is the generation of DNA >300 nucleotides, which is limited by the efficiency of the extension cycle, i.e., the efficiency with which each nucleotide is incorporated into the sequence. For example, with an extension cycle efficiency of 99%, the theoretical yield of an oligonucleotide containing 120 nucleotides is about 30% (0.99^120 × 100%). The yield of the synthesis depends largely on the coupling efficiency. As the length of the oligonucleotide increases, especially when the number of oligonucleotides is greater than 240, the synthesis yield will decrease to less than 10%.

[0004] Therefore, it is necessary to provide a composite group oligonucleotide synthesis carrier and its preparation method and application. Summary of the invention

[0005] In order to improve the synthesis efficiency of chemically synthesized nucleic acids, it is necessary to provide a composite group oligonucleotide synthesis carrier and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a composite group oligonucleotide synthesis carrier, comprising a carrier and a group fixed on the carrier, wherein the group comprises an activator group and an oligonucleotide group, and the activator group comprises a triazole group and its derivatives.

[0007] After adding the phosphoramidite monomer, the conventional solid phase carrier is first "activated" (protonated) by the acidic catalyst ETT [5-(ethylthio)-1H-tetrazole], and then undergoes a condensation reaction. Due to the influence of factors such as steric hindrance, catalyst performance, and reagent flow rate, the condensation efficiency can reach up to about 99.5%, and it is difficult to maintain stability. The synthetic carrier of the present application comprises an activator group and an oligonucleotide, and the activator group comprises a triazole group and its derivatives. When it is applied to the synthesis of oligonucleotides, the carrier is first detritylated to remove the 5'-DMT protecting group on the oligonucleotide, and then the phosphoramidite monomer is added to the reaction system and activated by the acidic catalyst. The activated phosphoramidite monomer combines with the triazole group and its derivatives on the carrier to form an intermediate and is enriched on the surface of the carrier. Subsequently, the adjacent phosphoramidite group on the intermediate undergoes a condensation reaction with the oligonucleotide group so that the activated phosphoramidite group is connected to the 5'-OH of the oligonucleotide group, thereby completing the extension of the oligonucleotide chain. The present application combines the activated phosphoramidite group through an activator group to promote the latter to gather at the carrier, that is, to "pull closer" the distance between the activated phosphoramidite group and the oligonucleotide, thereby improving the accuracy of the condensation reaction, improving the reaction efficiency, and ultimately achieving an improvement in the synthesis efficiency. At the same time, in general nucleic acid synthesis methods, in order to ensure that the condensation reaction is fully carried out, it is generally necessary to add an excessive amount of monomer reagents, and this scheme can greatly improve the accuracy of the condensation reaction, so it can allow the use of less monomer reagents, thereby reducing the cost of nucleic acid synthesis reactions. In addition, the intermediate that does not react can be restored to its original state by hydrolysis of an aqueous solution of iodine in a subsequent oxidation step.

[0008] Furthermore, the triazole group and its derivatives contain at least one of a carbon group, an oxime group and an amide group. The above groups can combine with the activated phosphoramidite to form an intermediate, and the intermediate can further produce a condensation reaction to transfer the activated phosphoramidite group to the 5'-OH end of the oligonucleotide group.

[0009] Furthermore, the raw materials for preparing the activator group include the following compounds, , , , , , , , or Furthermore, the raw material for preparing the activator group is or The above compounds have less steric hindrance and higher binding effect, which is beneficial to promote the condensation reaction.

[0010] Further, the oligonucleotide group is a protected nucleoside or an unprotected nucleoside. Further, the protected nucleoside is a DMTrO-protected nucleoside.

[0011] Further, the carrier is at least one of CPG, PS resin, PAN resin, PMA resin, silica gel and cellulose membrane. Further, the carrier is an amino CPG carrier. Amine CPG is a commercial solid phase carrier that is easy to obtain. Amine groups are easy to react with groups such as silanol, acetyl, and hydroxyl, so amino groups are usually used to modify solid phase carriers such as CPG, PS microspheres, PAN resin microspheres, PMA resin microspheres, and cellulose membranes. In theory, conventional polar groups can also be used to connect solid phase carriers.

[0012] The second aspect of the present application provides a method for preparing the above-mentioned carrier, comprising the following steps: connecting the activator group and the oligonucleotide group on the carrier.

[0013] The above preparation method can obtain a carrier comprising an activator group and an oligonucleotide group.

[0014] Further, the amino CPG resin is washed with anhydrous acetonitrile and then dried; dichloromethane is added to the first reaction container, the dried amino CPG resin is added under stirring, and then 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine, N,N'-dicyclohexylcarboximide and 4-dimethylaminopyridine are added, and the mixture is stirred in water at 20-30°C for 16-30 hours, filtered, washed with dichloromethane and dried; dimethyl imide solution is added to the second reaction container, the amino CPG resin is added under stirring, and then the preparation raw material containing the activator group, N,N'-dicyclohexylcarboximide and 4-dimethylaminopyridine are added, and the mixture is stirred in water at 20-30°C for 16-30 hours, filtered, washed with dichloromethane and dried. Furthermore, the preparation raw material is at least one of N,N'-dicyclohexylcarbodiimide and N,N'-dicyclohexylcarbodiimide.

[0015] The third aspect of the present application provides a method for synthesizing nucleic acid, which uses the above-mentioned vector to synthesize nucleic acid.

[0016] Furthermore, the carrier is placed on a synthesizer, the condensation reaction time is set to 30-60 seconds, the phosphoramidite monomer concentration is 0.1-0.4 mol / L, the oxidizing agent is iodine water or organic solution, and after the chain extension reaction is completed, the reaction product is cut off from the carrier for recovery.

[0017] The above synthesis method can improve the synthesis efficiency of nucleic acid and reduce the usage of phosphoramidite monomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of a method for preparing a carrier according to an embodiment.

[0019] Figure 2 Schematic diagram of the carrier of Example 1.

[0020] Figure 3 Schematic diagram of the carrier of Example 4.

[0021] Figure 4 Schematic diagram of the carrier of Comparative Example 1.

[0022] Figure 5 The chromatograms of Example 4 and Comparative Example 1 during the synthesis of 42mer oligonucleotides are shown.

[0023] Figure 6 The chromatograms of Example 4 and Comparative Example 1 during the synthesis of 84mer oligonucleotides are shown. DETAILED DESCRIPTION

[0024] For ease of understanding of the application, the application will be described more fully below. The application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the application more thorough and comprehensive.

[0025] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0027] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0028] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0029] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0030] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0031] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0032] The "particles" mentioned in this application, or materials with a defined particle size distribution, are not necessarily spherical in shape, but may be irregular, primary particles or secondary particles. The particle size of irregular particles is the average of their maximum diameter and minimum diameter.

[0033] Example 1: This example provides a solid phase carrier and its application in nucleic acid synthesis.

[0034] Synthesis of solid phase support: Step 1. Prepare 1 g of amino CPG resin (pore size: 200 nm, loading capacity: 20 umol / g, Chemgenes, product number: N-5100-20, the product number corresponding to the pore size of 100 nm is N-5100-10, and the product number corresponding to the pore size of 50 nm is N-5100-05). Wash 3 times with anhydrous acetonitrile and then dry in a vacuum oven.

[0035] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine, 10 mg of N,N'-dicyclohexylcarboximide, and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 20 °C for 24 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0036] Step 3. Add 2 ml of dimethylformamide solution to a 100 ml round-bottom flask, add 30 mg of the dried Step 2 resin to the flask while stirring, and then add 0.10mmol, N,N'-dicyclohexylcarboximide: 160mg, 4-dimethylaminopyridine: 26mg. Heat in a water bath at 20℃ and stir for 24 hours. After filtering, wash with dimethylformamide 3 times, then wash with dichloromethane 3 times, and finally dry in a vacuum oven.

[0037] The schematic diagram of the final vector structure is shown in Figure 2 .

[0038] Example 2: This example provides a solid phase carrier and its application in nucleic acid synthesis.

[0039] Synthesis of solid phase support: Step 1. Amine CPG resin (pore size: 50 nm, loading capacity: 30 umol / g, Chemgenes, product number: N-5100-05): 1 g, washed with anhydrous acetonitrile 3 times, and then dried in a vacuum oven.

[0040] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine, 10 mg of N,N'-dicyclohexylcarboximide, and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 30 °C for 16 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0041] Step 3. Add 2 ml of dimethylformamide solution to a 100 ml round-bottom flask, add 30 mg of the dried Step 2 resin to the flask while stirring, and then add 0.15mmol, N,N'-dicyclohexylcarboximide: 160mg, 4-dimethylaminopyridine: 26mg. Stir and react in a water bath at 30°C for 16 hours. After filtering, wash with dimethylformamide 3 times, then wash with dichloromethane 3 times, and finally dry in a vacuum oven.

[0042] The schematic diagram of the final vector structure is shown in Figure 2 .

[0043] Example 3: This example provides a solid phase carrier and its application in nucleic acid synthesis.

[0044] Synthesis of solid phase support: Step 1. Amino CPG resin (pore size: 100 nm, loading capacity: 25 umol / g, Chemgenes, product number: N-5100-10): 1 g, washed with anhydrous acetonitrile 3 times, and then dried in a vacuum oven.

[0045] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine, 10 mg of N,N'-dicyclohexylcarboximide, and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 20 °C for 24 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0046] Step 3. Add 2 ml of dimethylformamide solution to a 100 ml round-bottom flask, add 30 mg of the dried Step 2 resin to the flask while stirring, and then add 0.12mmol, N,N'-dicyclohexylcarboximide: 160mg, 4-dimethylaminopyridine: 26mg. Stir and react in a water bath at 20 ℃ for 24 hours. After filtering, wash with dimethylformamide 3 times, then wash with dichloromethane 3 times, and finally dry in a vacuum oven.

[0047] Example 4: This example provides a solid phase carrier and its application in nucleic acid synthesis.

[0048] Step 1. Amine CPG resin (pore size: 200 nm, loading capacity: 20 umol / g, Chemgenes, product number: N-5100-20): 1 g, washed with anhydrous acetonitrile 3 times, and then dried in a vacuum oven.

[0049] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, and then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine; 10 mg of N,N'-dicyclohexylcarboximide; and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 20 °C for 24 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0050] Step 3. Add 2 ml of dimethylformamide solution to a 100 ml round-bottom flask, add 30 mg of the dried Step 2 resin to the flask while stirring, and then add 0.15mmol, N,N'-dicyclohexylcarboximide: 160mg, 4-dimethylaminopyridine: 26mg. Heat in a water bath at 20°C and stir for 24 hours. After filtering, wash with dimethylformamide 3 times, then wash with dichloromethane 3 times, and finally dry in a vacuum oven.

[0051] The schematic diagram of the final vector structure is shown in Figure 3 .

[0052] Example 5: This example provides a solid phase carrier and its application in nucleic acid synthesis.

[0053] Step 1. Amino CPG resin (pore size: 100 nm, loading capacity: 25 umol / g, Chemgenes, product number: N-5100-10): 1 g, washed with anhydrous acetonitrile 3 times, and then dried in a vacuum oven.

[0054] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, and then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine; 10 mg of N,N'-dicyclohexylcarboximide; and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 20 °C for 24 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0055] Step 3. Add 2 ml of dimethylformamide solution to a 100 ml round-bottom flask, add 30 mg of the dried Step 2 resin to the flask while stirring, and then add 0.15mmol, N,N'-dicyclohexylcarboximide: 160mg, 4-dimethylaminopyridine: 26mg. Heat in a water bath at 20°C and stir for 24 hours. After filtering, wash with dimethylformamide 3 times, then wash with dichloromethane 3 times, and finally dry in a vacuum oven.

[0056] The schematic diagram of the final vector structure is shown in Figure 3 .

[0057] Comparative Example 1: This comparative example provides a solid phase carrier and its application in nucleic acid synthesis.

[0058] Step 1. Amino CPG resin (pore size: 200 nm, loading capacity: 20 umol / g, Chemgenes, product number: N-5100-20): 1 g, washed with anhydrous acetonitrile three times, and then dried in a vacuum oven.

[0059] Step 2. Add 20 ml of dichloromethane to a 100 ml round-bottom flask, and then add 100 mg of the dried amino CPG resin to the flask while stirring. Add 8.3 mg of 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine; 10 mg of N,N'-dicyclohexylcarbodiimide; and 1.7 mg of 4-dimethylaminopyridine to the flask. Stir and react in a water bath at 20°C for 24 hours. After filtering, wash with dichloromethane three times, and then dry in a vacuum oven.

[0060] Step 3. Add 10 ml of pyridine-acetic anhydride solution to a 100 ml round-bottom flask, add 30 mg of dried Step 2 resin to the flask under stirring, and then add 30 mg of 4-dimethylaminopyridine. Heat in a water bath at 20°C and stir for 2 hours. After filtering, wash with dichloromethane 3 times, and then dry in a vacuum oven.

[0061] The schematic diagram of the final vector structure is shown in Figure 4 .

[0062] The vectors of the above-mentioned Examples and Comparative Examples were used for nucleic acid synthesis.

[0063] The steps for nucleic acid synthesis are as follows: Synthesis method of 40mer oligonucleotide: 1. Load the solid phase carrier 1 and the composite carrier 2 with a loading capacity of 0.5 umol onto the automatic synthesizer respectively.

[0064] 2. The condensation reaction time is set to 30 seconds, and the phosphoramidite monomer concentration is 0.2 mol. 3. Oxidizing agent used: 0.2 mol I2 in tetrahydrofuran / pyridine / water solution.

[0065] 4. After the chain extension reaction is completed, use concentrated ammonia water at 60°C for 3 hours to cut the oligonucleotide product from the solid phase support.

[0066] 5. The obtained product was purified and analyzed by HPLC.

[0067] Synthesis method of 84mer oligonucleotide: 1. Load the solid phase carrier 1 and the composite carrier 2 with a loading capacity of 0.5 umol onto the automatic synthesizer respectively.

[0068] 2. The condensation reaction time is set to 60 seconds, and the phosphoramidite monomer concentration is 0.2 mol. 3. Oxidizing agent used: 0.2 mol I2 in tetrahydrofuran / pyridine / water solution.

[0069] 4. After the chain extension reaction is completed, use concentrated ammonia water at 60°C for 3 hours to cut the oligonucleotide product from the solid phase support.

[0070] 5. The obtained product was purified and analyzed by HPLC.

[0071] The structures of nucleic acid synthesis are recorded in Table 1, where the synthesis efficiency refers to the purity of the final product.

[0072] Table 1 Data of nucleic acid synthesis of Examples 1-5 and Comparative Example 1.

[0073]

[0074] According to the data in Table 1 and Figure 5-Figure 6 The results show that the synthesis efficiency (i.e., the purity of the final product) of Examples 1-5 at 84mer is significantly higher than that of Comparative Example 1. This is because the conventional solid phase carrier is first "activated" (protonated) by the acidic catalyst ETT [5-(ethylthio)-1H-tetrazole] after adding the phosphoramidite monomer, and then undergoes a condensation reaction. Due to the influence of factors such as steric hindrance, catalyst performance, and reagent flow rate, the condensation efficiency can reach a maximum of about 99.5%, and it is difficult to maintain stability. The synthetic carrier of the present application comprises an activator group and an oligonucleotide, and the activator group comprises a triazole group and its derivatives. When it is applied to the synthesis of oligonucleotides, the carrier is first detritylated to remove the 5'-DMT protecting group on the oligonucleotide, and then a phosphoramidite monomer is added to the reaction system and activated by an acidic catalyst. The activated phosphoramidite monomer is combined with the triazole group and its derivatives on the carrier to form an intermediate and enriched on the carrier surface, and then the adjacent phosphoramidite group on the intermediate undergoes a condensation reaction with the oligonucleotide group so that the activated phosphoramidite group is connected to the 5'-OH of the oligonucleotide group, thereby completing the extension of the oligonucleotide chain. The present application combines the activated phosphoramidite group with the activated phosphoramidite group through the activator group to promote the latter to gather at the carrier, that is, "pulling closer" the distance between the activated phosphoramidite group and the oligonucleotide, thereby improving the accuracy of the condensation reaction, improving the reaction efficiency, and finally achieving the improvement of the synthesis efficiency. At the same time, in general nucleic acid synthesis methods, in order to ensure the full progress of the condensation reaction, it is generally necessary to add an excess of monomer reagents, while this scheme can greatly improve the accuracy of the condensation reaction, thereby allowing the use of fewer monomer reagents, thereby reducing the cost of the nucleic acid synthesis reaction.

[0075] Example 4 uses a preferred compound as a raw material for preparing the activator group, which has less steric hindrance and has a better binding effect with phosphoramidite, and can promote the condensation reaction.

[0076] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0077] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite group oligonucleotide synthesis carrier, characterized in that: The invention comprises a carrier and a group fixed on the carrier, wherein the group comprises an activator group and an oligonucleotide group, and the activator group comprises a triazole group and a derivative thereof.

2. The carrier according to claim 1, characterized in that The triazole group and its derivatives include a triazole group substituted with a hydroxyl group.

3. The carrier according to claim 1, characterized in that The raw materials for preparing the activator group include the following compounds , , , , , , , or At least one of .

4. The carrier according to claim 1, characterized in that The oligonucleotide group is a protected nucleoside or an unprotected nucleoside.

5. The carrier according to claim 4, characterized in that The protected nucleoside is a DMTrO protected nucleoside.

6. The carrier according to claim 1, characterized in that The carrier is at least one of CPG, PS resin, PAN resin, PMA resin, silica gel and cellulose membrane.

7. The method for preparing a carrier according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: connecting the activator group and the oligonucleotide group on the carrier.

8. The method for preparing a carrier according to claim 7, characterized in that: The amino CPG resin is washed with anhydrous acetonitrile and then dried; dichloromethane is added to the first reaction container, and the dried amino CPG resin is added under stirring, and then 5'-O-(4,4'-dimethoxytrityl)-3'-O-succinylthymidine, N,N'-dicyclohexylcarboximide and 4-dimethylaminopyridine are added, and the mixture is stirred in water at 20-30°C for 16-30 hours, filtered, washed with dichloromethane and dried; dimethyl imide solution is added to the second reaction container, and the amino CPG resin is added under stirring, and then the preparation raw material containing the activator group, N,N'-dicyclohexylcarboximide and 4-dimethylaminopyridine are added, and the mixture is stirred in water at 20-30°C for 16-30 hours, filtered, washed with dichloromethane and dried.

9. The method for preparing a carrier according to claim 8, characterized in that: The preparation raw material is at least one of N,N'-dicyclohexylcarbodiimide and N,N'-dicyclohexylcarbodiimide.

10. A method for synthesizing nucleic acid, characterized in that: The method comprises the following steps: synthesizing nucleic acid using the vector described in any one of claims 1 to 6.

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