Synthesis process for oligonucleotide primer

Abstract

The present invention belongs to the technical field of nucleic acid synthesis. Disclosed is a synthesis process for an oligonucleotide primer. The synthesis process comprises the following steps: step 1, adding dried chloromethylated polystyrene resin to toluene, after swelling, adding p-diaminoazobenzene, and performing a reaction at 70℃ under stirring, so as to obtain microspheres; and loading a protection group on the surface of the microspheres to obtain a solid-phase support; step 2, synthesizing a primer: completing the synthesis of the primer on the solid-phase support, so as to obtain a primer crude product, and performing an ammonolysis reaction: putting the solid-phase support containing the primer crude product into an ammonolysis solution and performing ammonolysis, the ammonolysis being carried out alternately under ultraviolet light and visible light; and step 3, purifying the primer.

Description

A synthesis process of oligonucleotide primers Technical Field

[0001] The invention belongs to the technical field of nucleic acid synthesis, and particularly relates to a synthesis process of an oligonucleotide primer. Background Art

[0002] Currently, oligoDNA synthesis generally uses the solid-phase phosphoramidite triester method. This involves immobilizing DNA on a solid support and then, through a four-step reaction cycle: deprotection, coupling, capping, and oxidation, linking nucleotide monomers one by one to complete the synthesis of the DNA chain. This method offers the advantages of high efficiency, rapid coupling, and relatively stable starting reactants.

[0003] Features of this method:

[0004] The first nucleotide is 3' fixed to the solid support;

[0005] Each nucleotide is linked together in sequence;

[0006] The direction of DNA synthesis is 3'→5';

[0007] The synthesis is carried out in a hydrophobic environment.

[0008] After all synthesis cycles are completed, a crude target DNA fragment is obtained. This fragment undergoes aminolysis and is then purified by various methods. Aminolysis involves a cleavage step, chemically cleaving the synthesized oligonucleotide from the solid support (CPG). Fresh, concentrated ammonia is often used to cleave the ester bond between the linker compound and the initial nucleoside on the CPG.

[0009] The main issues with ammonolysis include long reaction times (16 hours at 55°C and 8 hours at 65°C), low production efficiency, and the high temperature and pressure required for the reaction. While gas-phase ammonolysis offers advantages such as rapid, efficient, and simple operation, it also has disadvantages such as high equipment requirements, the use of toxic gases, potential safety hazards, environmental pollution, and significant structural damage to the primer.

[0010] Summary of the Invention

[0011] The purpose of the present invention is to provide a synthesis process of oligonucleotide primers to solve the problem of slow aminolysis reaction rate.

[0012] The purpose of the present invention can be achieved through the following technical solutions:

[0013] A process for synthesizing an oligonucleotide primer comprises the following steps:

[0014] In the first step, dry chloromethylated polystyrene resin is added to toluene, and after swelling, p-diaminoazobenzene is added and stirred at 70°C. After the reaction is completed, ethanol extraction, washing, and drying are performed to obtain microspheres; protective groups are loaded on the surface of the microspheres to obtain a solid phase carrier;

[0015] Step 2: Primer synthesis: Primer synthesis is completed on a solid support to obtain a crude primer product; aminolysis reaction: The solid support containing the crude primer product is placed in an aminolysis solution for aminolysis; the aminolysis is carried out under alternating ultraviolet light and visible light;

[0016] Step 3: Purify the primers.

[0017] Furthermore, the ammonolysis solution includes an organic amine, an alcohol, and an alkaline aqueous solution; the mass ratio of the organic amine, alcohol, and alkaline aqueous solution is 5:1:0.1; the organic amine includes diethylenetriamine and tris(2-aminoethyl)amine; the alcohol includes methanol and ethanol; and the alkaline aqueous solution is 1 mol / L sodium hydroxide and 1 mol / L potassium hydroxide aqueous solution. The organic amine in the ammonolysis solution is milder, and the addition of an appropriate amount of alkaline aqueous solution to the ammonolysis solution provides sufficient hydroxide ions, which not only increases the reaction rate but also avoids the residual problem when conventional ammonia water is used as the ammonolysis solution.

[0018] Furthermore, ultraviolet light and visible light are alternately irradiated: ultraviolet light (UV<366 nm) for 10 min; visible light (visible light>400 nm) for 10 min.

[0019] Furthermore, the preparation method of the solid phase carrier containing nucleic acid is a solid phase phosphoramidite method, and the specific steps are as follows:

[0020] A deprotection reaction is performed on the protective group on the surface of the solid phase carrier using a deprotection agent; a nucleic acid fragment with a protective group is coupled with a hydroxyl group to obtain a conjugate; an oxidation reaction is performed on the conjugate to obtain an oxidation product; and a capping reaction is performed on the oxidation product to obtain a solid phase carrier containing a crude primer product.

[0021] Furthermore, the purification includes HPLC purification, OPC purification, and desalting purification.

[0022] Furthermore, the protecting group is 4,4′-dimethoxytrityl. (The steps for loading the protecting group on the microsphere surface to obtain a solid phase carrier refer to the coupling of aminomethyl polystyrene carrier and spacer arm in Liang Hong, Li Peng, Zhang Jing, and Su Zhiguo. Preparation and performance characterization of polystyrene matrix RNA solid phase organic synthesis carrier [J]. Chinese Journal of Process Engineering, 2009, 9(04): 770-775.)

[0023] Furthermore, the chlorine content of the chloromethylated polystyrene resin is 4 meq / gm; and the particle size of the chloromethylated polystyrene resin is 50-60 μm.

[0024] Furthermore, the mass ratio of the chloromethylated polystyrene resin to p-diaminoazobenzene is 12:0.8-1.

[0025] Furthermore, the ammonolysis temperature is 50-65°C.

[0026] Beneficial effects of the present invention:

[0027] The present invention provides a synthesis process for oligonucleotide primers, wherein the amino process involved is more efficient, the aminolysis conditions are milder, error sequences are less likely to be generated during the primer synthesis process, and the product purity is relatively high.

[0028] The oligonucleotide primer synthesis process of the present invention utilizes solid-phase synthesis, in which an azo structure is introduced into the solid support. Using a self-made solid support, combined with ultraviolet light, improves the efficiency of the aminolysis process. Under light conditions, the covalent bond structure of the azobenzene in the solid support changes from trans to cis. This isomerization generates a torsional force that increases the efficiency of primer detachment from the solid support, thereby shortening the aminolysis process and improving its efficiency. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0030] Example 1

[0031] A process for synthesizing an oligonucleotide primer comprises the following steps:

[0032] The first step is to add 6g of dry chloromethylated polystyrene resin (Merrifield resin for peptide synthesis - imported Tulsion CMB-200) to 100mL of toluene, swell for 12h, add 0.5g of p-diaminoazobenzene, stir and react at 70°C for 24h, extract with anhydrous ethanol, then wash with 0.1mol / L sodium hydroxide solution and water until there is no chloride ion, and vacuum dry at 60°C to obtain microspheres; the chlorine content of the chloromethylated polystyrene resin is 4meq / gm; the particle size of the chloromethylated polystyrene resin is 50-60μm; the mass ratio of the chloromethylated polystyrene resin to p-diaminoazobenzene is 12:0.8.

[0033] A protective group is loaded on the surface of the microsphere to obtain a solid phase carrier; the protective group is 4,4′-dimethoxytrityl (the steps of loading the protective group on the surface of the microsphere to obtain a solid phase carrier refer to the coupling of aminomethyl polystyrene carrier and spacer arm in Liang Hong; Li Peng; Zhang Jing; Su Zhiguo. Preparation and performance characterization of polystyrene matrix RNA solid phase organic synthesis carrier [J]. Chinese Journal of Process Engineering, 2009, 9(04): 770-775.);

[0034] Step 2: Primer synthesis: Primer synthesis is completed on a solid support, and a deprotection agent is used to deprotect the protective groups on the surface of the solid support. A nucleic acid fragment with a protective group is coupled with a hydroxyl group to obtain a conjugate. The conjugate is oxidized to obtain an oxidation product. The oxidation product is capped to obtain a solid support containing the crude primer product. Ammonolysis: The solid support containing the crude primer product is placed in an aminolysis solution for aminolysis. The aminolysis is carried out under alternating ultraviolet and visible light conditions: ultraviolet light (UV < 366 nm) is irradiated for 10 minutes, and visible light (visible light > 400 nm) is irradiated for 10 minutes. The aminolysis temperature is 50°C, and the aminolysis solution comprises an organic amine, an alcohol, and an alkaline aqueous solution. The mass ratio of the organic amine, alcohol, and alkaline aqueous solution is 5:1:0.1. The organic amine is diethylenetriamine; the alcohol is ethanol; and the alkaline aqueous solution is 1 mol / L sodium hydroxide.

[0035] Step 3: HPLC purification of primers.

[0036] Example 2

[0037] A process for synthesizing an oligonucleotide primer comprises the following steps:

[0038] In the first step, 6 g of dry chloromethylated polystyrene resin (Merrifield resin for peptide synthesis - imported Tulsion CMB-200) was added to 100 mL of toluene, swollen for 12 hours, and then 0.5 g of p-diaminoazobenzene was added. The mixture was stirred and reacted at 70°C for 24 hours. After the reaction, it was extracted with anhydrous ethanol, and then washed with 0.1 mol / L sodium hydroxide solution and water until there was no chloride ion, and vacuum dried at 60°C to obtain microspheres; the chlorine content of the chloromethylated polystyrene resin was 4 meq / gm; the particle size of the chloromethylated polystyrene resin was 50-60 μm; the mass ratio of the chloromethylated polystyrene resin to p-diaminoazobenzene was 12:0.9.

[0039] A protective group is loaded on the surface of the microsphere to obtain a solid phase carrier; the protective group is 4,4′-dimethoxytrityl (the steps of loading the protective group on the surface of the microsphere to obtain a solid phase carrier refer to the coupling of aminomethyl polystyrene carrier and spacer arm in Liang Hong; Li Peng; Zhang Jing; Su Zhiguo. Preparation and performance characterization of polystyrene matrix RNA solid phase organic synthesis carrier [J]. Chinese Journal of Process Engineering, 2009, 9(04): 770-775.);

[0040] Step 2: Primer synthesis: Primer synthesis is completed on a solid support, and a deprotection agent is used to deprotect the protective groups on the surface of the solid support. A nucleic acid fragment with a protective group is coupled with a hydroxyl group to obtain a conjugate. The conjugate is oxidized to obtain an oxidation product. The oxidation product is capped to obtain a solid support containing the crude primer product. Ammonolysis: The solid support containing the crude primer product is placed in an aminolysis solution for aminolysis. The aminolysis is carried out under alternating ultraviolet and visible light conditions: ultraviolet light (UV < 366 nm) is irradiated for 10 minutes, and visible light (visible light > 400 nm) is irradiated for 10 minutes. The aminolysis temperature is 55°C, and the aminolysis solution comprises an organic amine, an alcohol, and an alkaline aqueous solution. The mass ratio of the organic amine, alcohol, and alkaline aqueous solution is 5:1:0.1. The organic amine is diethylenetriamine; the alcohol is ethanol; and the alkaline aqueous solution is 1 mol / L sodium hydroxide.

[0041] Step 3: HPLC purification of primers.

[0042] Example 3

[0043] A process for synthesizing an oligonucleotide primer comprises the following steps:

[0044] In the first step, 6 g of dry chloromethylated polystyrene resin (Merrifield resin for peptide synthesis - imported Tulsion CMB-200) was added to 100 mL of toluene. After swelling for 12 hours, 0.5 g of p-diaminoazobenzene was added and stirred at 70°C for 24 hours. After the reaction, it was extracted with anhydrous ethanol, and then washed with 0.1 mol / L sodium hydroxide solution and water until there was no chloride ion, and dried in vacuo at 60°C to obtain microspheres; the chlorine content of the chloromethylated polystyrene resin was 4 meq / gm; the particle size of the chloromethylated polystyrene resin was 50-60 μm; the mass ratio of the chloromethylated polystyrene resin to p-diaminoazobenzene was 12:1.

[0045] A protective group is loaded on the surface of the microsphere to obtain a solid phase carrier; the protective group is 4,4′-dimethoxytrityl (the steps of loading the protective group on the surface of the microsphere to obtain a solid phase carrier refer to the coupling of aminomethyl polystyrene carrier and spacer arm in Liang Hong; Li Peng; Zhang Jing; Su Zhiguo. Preparation and performance characterization of polystyrene matrix RNA solid phase organic synthesis carrier [J]. Chinese Journal of Process Engineering, 2009, 9(04): 770-775.);

[0046] Step 2: Primer synthesis: Primer synthesis is completed on a solid support, and a deprotection reaction is performed on the protective groups on the surface of the solid support using a deprotecting agent; a nucleic acid fragment with a protective group is coupled with a hydroxyl group to obtain a conjugate; the conjugate is oxidized to obtain an oxidation product; the oxidation product is capped to obtain a solid support containing the crude primer product; aminolysis reaction: the solid support containing the crude primer product is placed in an aminolysis solution and aminolysis is performed; the aminolysis is performed under alternating ultraviolet and visible light, with ultraviolet light (UV < 366nm) irradiation for 10 minutes and visible light (visible light > 400nm) irradiation for 10 minutes. The aminolysis temperature is 65°C, and the aminolysis solution includes an organic amine, an alcohol, and an alkaline aqueous solution; the mass ratio of the organic amine, alcohol, and alkaline aqueous solution is 5:1:0.1; the organic amine is diethylenetriamine; the alcohol is ethanol; and the alkaline aqueous solution is 1 mol / L sodium hydroxide.

[0047] Step 3: HPLC purification of primers.

[0048] Comparative Example 1

[0049] Compared with Example 3, this comparative example uses a CPG solid phase carrier (loading capacity of 35 μmmol / g) in the primer synthesis step.

[0050] Comparative Example 2

[0051] Compared with Comparative Example 1, this comparative example adopts concentrated ammonia water (concentration of 26%) for the aminolysis reaction step.

[0052] Performance tests were performed on Examples 1 to 3 and Comparative Examples 1 to 2, and statistical analysis was performed on the aminolysis times in the Examples and Comparative Examples.

[0053] Mass spectrometry analysis was performed using an LTQ-XL mass spectrometer; purity analysis was performed using a Shimadzu LC-MS-2020 high performance liquid chromatograph. The results are shown in Table 1:

[0054] Table 1

[0055] As can be seen from Table 1, the product obtained according to the method provided in the present invention has high purity, and the ammonolysis process is more efficient and the ammonolysis conditions are milder.

[0056] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0057] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A synthesis process of an oligonucleotide primer, characterized in that, It includes the following steps: First step: Add dry chloromethylated polystyrene resin into toluene. After swelling, add p-diaminoazobenzene and stir for reaction. After the reaction ends, perform extraction, washing, and drying to obtain microspheres; load a protecting group on the surface of the microspheres to obtain a solid-phase carrier; Second step: Synthesize primers: Complete the synthesis of primers on the solid-phase carrier to obtain a crude primer product; Ammonolysis reaction: Place the solid-phase carrier containing the crude primer product into an ammonolysis solution for ammonolysis; The ammonolysis is carried out under alternating ultraviolet light and visible light; Third step: Purify the primers.

2. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The ammonolysis solution includes organic amine, alcohol, and an alkaline aqueous solution; the mass ratio of the amounts of organic amine, alcohol, and alkaline aqueous solution used is 5:1:0.1; the organic amine includes diethylenetriamine and tris(2-aminoethyl)amine; the alcohol includes methanol and ethanol; the alkaline aqueous solution is a 1 mol / L sodium hydroxide or 1 mol / L potassium hydroxide aqueous solution.

3. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, Carried out under alternating ultraviolet light and visible light: Irradiate with ultraviolet light for 10 min; irradiate with visible light for 10 min.

4. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The preparation method of the solid-phase carrier containing nucleic acid is the solid-phase phosphoramidite method, and the specific steps are as follows: Perform a deprotection reaction on the protecting group on the surface of the solid-phase carrier using a deprotecting agent; couple the nucleic acid fragment with a protecting group to a hydroxyl group to obtain a conjugate; perform an oxidation reaction on the conjugate to obtain an oxidation product; perform a capping reaction on the oxidation product to obtain a solid-phase carrier containing a crude primer product.

5. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The purification includes HPLC purification, OPC purification, and desalting purification.

6. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The protecting group is 4,4′-dimethoxytrityl.

7. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that The chlorine content of the chloromethylated polystyrene resin is 4 meq / gm; the particle size of the chloromethylated polystyrene resin is 50 - 60 μm.

8. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The mass ratio of the amounts of the chloromethylated polystyrene resin and p-diaminoazobenzene used is 12:0.8 - 1.

9. The synthesis process of an oligonucleotide primer according to claim 1, characterized in that, The ammonolysis temperature is 50 - 65 °C.

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