A high-efficiency and low-cost method for solid-phase synthesis of oligonucleotides

Abstract

The application discloses a high-efficiency and low-cost method for synthesizing oligonucleotides in solid phase, and relates to the technical field of oligonucleotide synthesis. The steps of the method are as follows: synthesizing a solid-phase carrier synthesis column, performing first cleaning; performing twice de-capping; performing first small pressure in the first de-capping; performing second small pressure and once first large pressure in the second de-capping; performing second cleaning; performing several times of activation coupling; performing third small pressure in each activation coupling except the last activation coupling; performing fourth small pressure and once second large pressure in the last activation coupling; performing twice oxidation; performing fifth small pressure in the first oxidation; performing sixth small pressure and once third large pressure in the second oxidation; performing twice capping; performing seventh small pressure in the first capping; performing eighth small pressure and once third large pressure in the second oxidation; circulating, ammonolysis, drying, elution, and obtaining oligonucleotides.

Description

Technical Field

[0001] This invention relates to the field of oligonucleotide synthesis technology, and particularly to a method for solid-phase synthesis of oligonucleotides. Background Technology

[0002] With the rise of new nucleic acid drugs, the demand for oligonucleotides is increasing. Although the enzymatic synthesis of oligonucleotides has become increasingly sophisticated, the solid-phase phosphoramidite method will continue to be the main synthesis method for oligonucleotides for some time to come.

[0003] Currently, the solid-phase phosphoramidite method synthesizes single-chain oligonucleotides by reacting a solid support with reagents, drying / air-drying, and then reacting with the next reagent. The process includes deprotection, activation, coupling, oxidation, and capping; each of these five chemical reactions constitutes a cycle. Acetonitrile is added to wash the solid support during some steps to ensure the reagents from the previous step are thoroughly cleaned and do not affect the next reaction. Each cycle ligates one nucleotide monomer onto the solid support. Through several cycles, the nucleotide monomers are linked and immobilized on the solid support via phosphodiester bonds. The most commonly used solid support is controlled-pore glass beads (CPG). CPG covalently binds to the hydroxyl groups of the initial nucleotides through the linker compound, and the hydroxyl groups of CPG are protected with dimethoxytriphenylmethyl (DMT).

[0004] Currently, the industry generally uses specialized oligonucleotide synthesizers to prepare oligonucleotides for solid-phase synthesis. The specific reaction steps for synthesizing single-chain oligonucleotides using the conventional solid-phase phosphoramidite-triester method are as follows.

[0005] (1) Washing: The solid support is washed with a cleaning agent. The gas flow pressure operation generally requires a low pressure and waiting period before a high pressure is applied. The whole process takes 5-10 seconds.

[0006] (2) Deblocking: The protecting group DMT of the hydroxyl group on CPG is removed with a decapping agent to expose the 5' hydroxyl group for the next coupling step. The decapping process is usually set up several times.

[0007] (3) The decapping process involves gas flow pressure operation on the decapping agent and the solid support. The gas flow pressure operation generally requires starting with low pressure and waiting, repeating this several times, and then applying high pressure. The total time for this process is 20-40 seconds.

[0008] (4) Activation: Before activation, the monomer and activator are mixed and fed into the synthesis column. At this time, the activator donates a proton to the N atom of the diisopropylamine group on the 3' phosphate. The protonated diisopropylamine is a free group that forms an active intermediate, phosphoramidite tetrazolium, with the activator. An excess of activator is used in this step to ensure sufficient monomer activation.

[0009] Coupling: The phosphoramidite tetrazolium undergoes a nucleophilic reaction with the 5' hydroxyl group on CPG, resulting in coupling and the removal of the tetrazolium, thus extending the synthesized oligonucleotide chain by one.

[0010] The activation coupling process is typically set to run several times.

[0011] (5) The activation coupling process involves gas flow pressure manipulation of the activating coupling agent and the solid support. The gas flow pressure manipulation generally requires waiting and low pressure, repeating several times, waiting again, and finally applying high pressure. The total time for this process is 30-60 seconds.

[0012] (6) Oxidation: After the coupling reaction, the newly added nucleotide is linked to the oligonucleotide chain on CPG via a phosphite bond (phosphorus is trivalent). This phosphite bond is unstable and easily hydrolyzed by acids and bases. Therefore, an oxidizing agent is added to oxidize trivalent phosphorus to pentavalent phosphorus. The commonly used oxidizing agent is a tetrahydrofuran solution of iodine. The oxidation process is usually repeated several times.

[0013] (7) The oxidation process involves gas flow pressure operation between the oxidant and the solid support. Gas flow pressure operation generally requires starting with low pressure and waiting, repeating this process several times, and finally applying high pressure. The total time for this process is 30-60 seconds.

[0014] (8) Capping: To prevent unreacted 5' hydroxyl groups from continuing to participate in the reaction in subsequent cycles (the main cause of n-1 deletion fragments), a capping agent needs to be added at the end to block them. Acetylation is commonly used to block this hydroxyl group. The capping process is usually set up several times.

[0015] (9) The capping process involves gas flow pressure operation between the capping agent and the solid support. The gas flow pressure operation generally requires waiting and low pressure, repeating several times, waiting again, and finally high pressure. The total time for this process is 20-40 seconds.

[0016] (10) Cycle: Repeat the above 5 chemical reactions until the oligonucleotide chain is extended to the required length.

[0017] As can be seen, when synthesizing single-chain oligonucleotides using the solid-phase phosphoramidite-triester method described above, low pressure is often required after each important step to ensure contact and full reaction between the reagents and the carrier; high pressure is often required to completely expel the reagents from the carrier and avoid affecting the next reaction. Currently, commonly used oligonucleotide synthesizers generally employ suction or jet methods to perform these processes. These gas pressure operations are crucial steps in ensuring the quality of the synthesis. Summary of the Invention

[0018] This invention provides a highly efficient and low-cost solid-phase synthesis method for oligonucleotides, in which decapping, activation coupling, oxidation, and capping each involve at least two reactions. More importantly, this invention optimizes the gas flow pressure operation for decapping, activation coupling, oxidation, and capping, eliminating some high-pressure operations while retaining some reagents on the support for further reaction; simultaneously, it reduces the amount of reagents used and adjusts the low-pressure time to ensure that a small amount of reagent remains on the solid support surface after the final low-pressure step, continuing to participate in the reaction during machine operation, before directly spraying the second reagent reaction. Ultimately, while maintaining a high reaction success rate, this significantly reduces synthesis time and the amount of synthetic reagents required. This is specifically achieved through the following techniques.

[0019] This invention provides a method for efficient and low-cost solid-phase synthesis of oligonucleotides, comprising the following steps:

[0020] The solid-supported synthesis column was first cleaned.

[0021] The solid-phase support synthesis column, after the first cleaning, undergoes two decapping processes; the first decapping is performed under a first low pressure; the second decapping is performed under a second low pressure and a first high pressure.

[0022] The solid-phase support synthesis column, after being decapped, is subjected to a second cleaning.

[0023] The solid-phase support synthesis column after the second cleaning is subjected to several activation couplings; each activation coupling except the last one is subjected to a third low pressure; the last activation coupling is subjected to a fourth low pressure and a second high pressure.

[0024] The solid-phase support synthesis column after activation coupling was subjected to two oxidation processes; the first oxidation was carried out at the fifth low pressure; the second oxidation was carried out at the sixth low pressure and the third high pressure.

[0025] The oxidized solid-support synthesis column was capped twice; the first capping was performed under the seventh minor pressure; the second capping was performed under the eighth minor pressure and the third major pressure.

[0026] Based on the oligonucleotide length, the cycle is repeated a corresponding number of times. After the cycle is completed, the solid-phase support synthesis column is subjected to ammonolysis, drying, and elution to obtain the oligonucleotide.

[0027] Furthermore, the method for the first small pressure is as follows: apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.3 s, wait for 4 s.

[0028] Furthermore, the method for the second small pressure is: small pressure for 0.3 s, wait for 10 s, small pressure for 0.3 s, wait for 10 s, small pressure for 0.4 s, wait for 10 s; the method for the first large pressure is: large pressure for 6 s.

[0029] Furthermore, the method for the third small pressure is as follows: wait 3 s, apply small pressure for 0.2 s, wait 10 s, apply small pressure for 0.3 s, wait 10 s, apply small pressure for 0.2 s, and wait (Wait) for 6 s.

[0030] Furthermore, the method for the fourth small pressure is: wait 3 s, apply small pressure for 0.2 s, wait 12 s, apply small pressure for 0.3 s, wait 12 s, apply small pressure for 0.3 s, wait 10 s; the method for the second large pressure is a large pressure for 6 s.

[0031] Furthermore, the method for the fifth small pressure is as follows: apply small pressure for 0.4 s, wait for 8 s, apply small pressure for 0.6 s, wait for 10 s, apply small pressure for 0.4 s, wait for 6 s.

[0032] Furthermore, the method for the sixth small pressure is: small pressure for 0.4 s, wait for 10 s, small pressure for 0.6 s, wait for 10 s, small pressure for 0.6 s, wait for 8 s; the method for the third large pressure is large pressure for 6 s.

[0033] Furthermore, the method for the seventh small pressure is as follows: wait 3 s, apply small pressure for 0.4 s, wait 6 s, apply small pressure for 0.4 s, wait 8 s, apply small pressure for 0.3 s, and wait 4 s.

[0034] Furthermore, the method for the eighth small pressure is: wait 3 s, apply small pressure for 0.4 s, wait 6 s, apply small pressure for 0.4 s, wait 8 s, apply small pressure for 0.5 s, wait 8 s; the method for the third large pressure is: apply large pressure for 6 s.

[0035] Furthermore, the first cleaning method is: cleaning the solid support synthesis column with anhydrous acetonitrile, and performing the ninth low pressure and the fourth high pressure; the second cleaning method is: cleaning the solid support synthesis column with anhydrous acetonitrile, and performing the tenth low pressure and the fifth high pressure, for a total of 2 cleanings;

[0036] Furthermore, the methods for the ninth and tenth small pressures are both small pressure for 1 second, followed by a 2-second wait; the methods for the fourth and fifth large pressures are both large pressure for 6 seconds.

[0037] Compared with the prior art, the advantages of the present invention are as follows: Compared with the current conventional synthesis procedure in which the reagents must be dried in each step of the reaction to ensure that the carrier is dry and does not participate in the next reaction, the present invention adjusts the procedure: when the same reagent is reacted, the drying process of the previous reagent can be omitted (i.e., high pressure), and the overall amount of reagent added and reaction time are reduced. By fine-tuning the low pressure (ensuring that a small amount of reagent is retained on the solid support after the final low pressure), the same synthesis efficiency and synthesis quality are achieved. Attached Figure Description

[0038] Figure 1 This is the high performance liquid chromatogram of sample 1 in Example 3.

[0039] Figure 2 This is the high performance liquid chromatogram of sample 2 in Example 3.

[0040] Figure 3 This is the high performance liquid chromatography chromatogram of sample 3 in Example 3.

[0041] Figure 4 The high performance liquid chromatography chromatogram of sample 4 in Example 3 is shown.

[0042] Figure 5 The high performance liquid chromatography chromatogram of sample 5 in Example 3 is shown.

[0043] Figure 6 The high-performance liquid chromatography (HPLC) chromatogram of sample 6 in Example 3 is shown. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] It should be noted that the "low pressure" claimed in this invention refers to providing small gas flow pressures in stages to ensure sufficient contact and reaction between the reagent and the solid support. "High pressure" refers to providing a larger gas flow pressure to completely expel the reagent from the solid support, preventing it from participating in subsequent reagent reactions. Generally, the gas flow used for both low and high pressure is an inert gas, typically argon or nitrogen.

[0046] Oligonucleotide synthesizers from different manufacturers, using the solid-phase phosphoramidite trimer method, generally include both "low-pressure" and "high-pressure" functions. The only difference lies in the airflow pressure at these two levels. However, regardless of the pressure setting, the function and purpose are the same, and it does not affect the final synthesis efficiency.

[0047] The yibo-48 synthesizer used in the following embodiments of the present invention employs a jet blowing method ("small blowing" and "large blowing").

[0048] The reagent and consumable information used in some embodiments of the present invention is shown in Table 1.

[0049] Table 1. Reagent and Consumable Information

[0050]

[0051]

[0052] In some embodiments of the present invention, the information on the synthesized DNA single strands and RNA single strands is shown in Table 2 below.

[0053] Table 2 Information on DNA and RNA single strands

[0054]

[0055]

[0056] Comparative Example 1

[0057] For single-stranded DNA, the conventional solid-phase synthesis method for oligonucleotides provided in this comparative example has the following specific steps:

[0058] 1. First cleaning: Same as step 1 in Example 1.

[0059] 2. Two-stage hat removal treatment

[0060] (1) Use 200 μl of decapping agent to decap the solid-supported synthesis column sample. Repeat the decapping treatment twice.

[0061] (2) For each decapping process, the decapping agent and the solid support synthesis column sample were subjected to a blowing treatment. Specifically, the blowing was performed for 0.3 s, followed by a wait of 10 s; the blowing was performed for 0.3 s, followed by a wait of 10 s; the blowing was performed for 0.4 s, followed by a wait of 10 s; and the blowing was performed for 6 s. The total time for the airflow pressure operation was 37 s.

[0062] 3. Second cleaning: Same as step 3 in Example 1.

[0063] 4. Two activation coupling treatments

[0064] (1) The solid-supported synthesis column sample was activated and coupled using 50 μl monomer and 60 μl activator. The activation and coupling treatment was repeated twice.

[0065] (2) For each activation coupling treatment, the monomer, activator, and solid support synthesis column sample were subjected to a blow-drain treatment. Specifically, the process was: wait 3 s, pdrain 0.2 s, wait 10 s, pdrain 0.3 s, wait 10 s, pdrain 0.3 s, wait 10 s, and hdrain 6 s. The total time for the gas flow pressure operation was 39.8 s.

[0066] 5. Two oxidation treatments

[0067] (1) The solid-supported synthesis column sample was oxidized with 80 μl of oxidant. The oxidation treatment was repeated twice.

[0068] (2) For each oxidation treatment, the oxidant and the solid support synthesis column sample were subjected to a blowing treatment. Specifically, the blowing was 0.4 s followed by a wait of 10 s, the blowing was 0.6 s followed by a wait of 10 s, the blowing was 0.6 s followed by a wait of 8 s, and the blowing was 6 s. The total time for the gas flow pressure operation was 35.6 s.

[0069] 6. Double capping treatment

[0070] (1) The solid-support synthesis column sample was capped with 60 μl of capping agent A and 60 μl of capping agent B. The capping treatment was repeated twice.

[0071] (2) For each capping process, the capping agent and the solid support synthesis column sample were subjected to a spraying treatment. Specifically, the process was: wait 3 s, pdrian 0.4 s, wait 6 s, pdrian 0.4 s, wait 8 s, pdrian 0.5 s, wait 8 s, and hdrain 6 s. The total time for the airflow pressure operation was 32.3 s.

[0072] 7. Third Wash:

[0073] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0074] (2) Perform gas flow pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow (Pdrian) for 1 s, wait for 2 s, and then perform a heavy blow (Hdrain) for 6 s. The total gas flow pressure operation time is 9 s.

[0075] 8. Determine the number of cycles for steps (2)-(7) above based on the length of the DNA single strand.

[0076] 9. Ammonolysis: After the synthesis is completed, the synthesis support is placed in an ammonolysis apparatus and ammonolysed at 90℃ and 80 psi for 2 h.

[0077] 10. Drying: After ammonolysis is completed, place the synthesis carrier in an oven at 60°C and dry for 10 min.

[0078] 11. Elution: Add 200 μl of acetonitrile to the synthesis vector, wash and dry under vacuum, repeat once. Add 80% acetonitrile to the synthesis vector, wash and dry under vacuum, repeat once. Add 200 μl of elution buffer to the synthesis vector and elute into a 96-well collection plate, repeat once. The oligonucleotides are then eluted into the collection plate.

[0079] 12. Mass spectrometry detection: After filtering an appropriate amount of sample, the purity of the sample was obtained by mass spectrometry detection as shown in Table 3 below. It can be seen that the purity of all 12 samples is within the normal range.

[0080] Table 3. Sample purity results detected by mass spectrometry

[0081]

[0082] The time and reagent usage for decapping, activation coupling, oxidation, and capping treatments in this comparative example are shown in Table 4 below.

[0083] Table 4. Reagent usage and time for one cycle of Comparative Example 1

[0084]

[0085] Comparative Example 2

[0086] For the six 21nt RNA single strands in Table 2 above, the conventional solid-phase synthesis method for oligonucleotides provided in this comparative example is as follows:

[0087] 1. First cleaning: Same as step 1 in Example 3.

[0088] 2. Two-stage deblocking process

[0089] (1) Use 200 μl of decapping agent to decap the solid-supported synthesis column sample. Repeat the decapping treatment twice.

[0090] (2) For each decapping process, the decapping agent and the solid support synthesis column sample were subjected to a blowing treatment. Specifically, the blowing was performed for 0.3 s, followed by a wait of 10 s; the blowing was performed for 0.3 s, followed by a wait of 10 s; the blowing was performed for 0.4 s, followed by a wait of 10 s; and the blowing was performed for 6 s. The total time for the airflow pressure operation was 37 s.

[0091] 3. Second Wash: Same as step 3 in Example 3.

[0092] 4. Four-stage activation coupling (Couple) treatment

[0093] (1) The solid-supported synthesis column sample was activated and coupled using 50 μl monomer and 60 μl activator. The activation and coupling treatment was repeated 4 times.

[0094] (2) For each activation coupling treatment, the monomer, activator, and solid support synthesis column sample were subjected to a blow-dry treatment. Specifically, the process was: wait 3 s, pdrian 0.2 s, wait 12 s, pdrian 0.3 s, wait 12 s, pdrian 0.3 s, wait 10 s, and hdrain 6 s. The total time for the gas flow pressure operation was 43.8 s.

[0095] 5. Two oxidation treatments

[0096] (1) The solid-supported synthesis column sample was oxidized with 80 μl of oxidant. The oxidation treatment was repeated twice.

[0097] (2) For each oxidation treatment, the oxidant and the solid support synthesis column sample were subjected to a blowing treatment. Specifically, the blowing was 0.4 s followed by a wait of 10 s, the blowing was 0.6 s followed by a wait of 10 s, the blowing was 0.6 s followed by a wait of 8 s, and the blowing was 6 s. The total time for the gas flow pressure operation was 35.6 s.

[0098] 6. Double capping treatment

[0099] (1) Cap the solid-supported synthesis column sample with 60 μl of capping agent A and 60 μl of capping agent B. Repeat the capping treatment once.

[0100] (2) For each capping process, the capping agent and the solid support synthesis column sample were subjected to a spraying treatment. Specifically, the process was: wait 3 s, pdrian 0.4 s, wait 6 s, pdrian 0.4 s, wait 8 s, pdrian 0.5 s, wait 8 s, and hdrain 6 s. The total time for the airflow pressure operation was 32.3 s.

[0101] 7. Third Wash:

[0102] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0103] (2) Perform gas flow pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow (Pdrian) for 1 s, wait for 2 s, and then perform a heavy blow (Hdrain) for 6 s. The total gas flow pressure operation time is 9 s.

[0104] 8. Determine the number of cycles for steps (2)-(7) above based on the length of the RNA single strand.

[0105] 9. Ammonolysis: Transfer the synthesized solid support to a 2 ml threaded tube and add 500 μL of ice-cold ammonia solution. Ammonolyze in a 65°C water bath for 2 hours to remove the protecting groups on the bases.

[0106] 10. Concentration and freeze-drying: Transfer the ammonolysis solution to a 2ml centrifuge tube and concentrate and freeze-dry.

[0107] 11. Removal of TBDMS protecting groups. Add 50 μL of dimethyl sulfoxide to the lyophilized centrifuge tube, heat in a 65°C water bath for 10 min, then add 200 μL of triethylamine trifluoroacetate, heat in a 65°C water bath for 2 h to remove the TBDMS protecting groups.

[0108] 12. Freeze-drying after precipitation. After the TBDMS removal reaction is complete, add 100 μL of 3 mol / L NaAc solution (pH 4.2) to a centrifuge tube, then add 1500 μL of ice-cold ethanol, and incubate at -80°C for 1 hour. Then centrifuge at 12000 rpm for 10 minutes. Discard the supernatant and concentrate to freeze-dry the sample precipitate.

[0109] 13. HPLC Analysis. After dissolving the sample, HPLC quantitative analysis was performed, as shown in Table 5 below.

[0110] Table 5. Purity Results of High Performance Liquid Chromatography

[0111]

[0112] The time and reagent usage for decapping, activation coupling, oxidation, and capping treatments in this comparative example are shown in Table 6 below.

[0113] Table 6. Reagent usage and time for one cycle in Comparative Example 2

[0114]

[0115] Example 1: Synthesis of a 20 nt DNA single strand

[0116] For the 12 DNA single strands with a length of 20 nt in Table 2 above, compared with Comparative Example 1, the solid-phase synthesis method of oligonucleotides used in this embodiment has eliminated the large blowing operation in each cycle of steps (3), (5), (6), and (7) during the first decapping, first activation coupling, first oxidation, and first capping, and reduced the amount of reagents used in each step; the large blowing operation is retained in the second decapping, second activation coupling, second oxidation, and second capping. Steps (1), (2), and (4) of this embodiment are the same as those of Comparative Example 1, that is, the large blowing operation is retained.

[0117] Since the large blowing operation was removed during the first cycle, the small blowing time in this embodiment needs to be adjusted accordingly to ensure that a small amount of reagent has not completely permeated into the carrier at the end.

[0118] The specific DNA single-strand synthesis steps in this embodiment are as follows:

[0119] 1. First Wash

[0120] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0121] (2) Perform gas flow pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow (Pdrian) for 1 s, wait for 2 s, and then perform a heavy blow (Hdrain) for 6 s. The total gas flow pressure operation time is 9 s.

[0122] 2. Two-stage deblocking process

[0123] (1) The solid-support synthesis column sample was decapped using a decapping agent. The decapping process was repeated twice. The first time, 160 μl of decapping agent was used (80% of the amount of decapping agent used in Comparative Example 1), and the second time, 200 μl of decapping agent was used.

[0124] (2) The first decapping treatment involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process was: pdrian for 0.3 s, wait for 10 s, pdrian for 0.3 s, wait for 10 s, pdrian for 0.3 s, wait for 4 s. The total time for the gas pressure operation was 24.9 s.

[0125] The second decapping process involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process consisted of: a small pdrain (0.3 s), a wait (10 s), a small pdrain (0.3 s), a wait (10 s), a small pdrain (0.4 s), a wait (10 s), and a large pdrain (6 s). The total time for the airflow pressure operation was 37 s.

[0126] 3. Second Wash

[0127] (1) The solid support synthesis column sample after decapping was cleaned with 240 μl of anhydrous acetonitrile, and the cleaning was performed twice.

[0128] (2) After each cleaning, a blowing process is performed. Specifically, a light blowing (Pdrian) is applied for 1 second, followed by a wait of 2 seconds and then a heavy blowing (Hdrian) for 6 seconds. The total time for the airflow pressure operation is 9 seconds.

[0129] 4. Two-stage activation coupling treatment

[0130] (1) The solid-supported synthesis column sample was activated and coupled using monomers and activators. The activation and coupling treatment was repeated twice. The first time, 40 μl of monomer (80% of the amount of monomer used in Comparative Example 1) and 48 μl of activator (80% of the amount of activator used in Comparative Example 1) were used. The second time, 50 μl of monomer and 60 μl of activator were used.

[0131] (2) The first activation coupling treatment involved spraying the monomer, activator, and solid support synthesis column sample. Specifically, the process was: wait 3 s, pdrian 0.2 s, wait 10 s, pdrian 0.3 s, wait 10 s, pdrian 0.2 s, and wait 4 s. The total time for the gas pressure operation was 27.7 s.

[0132] The second activation coupling treatment involved spraying the monomer, activator, and solid-supported synthesis column sample. Specifically, the process consisted of a 3-second wait, a 0.2-second pdrian, a 10-second wait, a 0.3-second pdrian, a 10-second wait, a 0.3-second pdrian, a 10-second wait, and a 6-second high-pressure pdrian. The total time for this gas flow pressure operation was 39.8 seconds.

[0133] 5. Two-stage oxidation (Oxi) treatment

[0134] (1) The solid-supported synthesis column sample was oxidized using an oxidant. The oxidation process was repeated twice. The first time, 60 μl of oxidant was used (75% of the amount of oxidant used in Comparative Example 1), and the second time, 80 μl of oxidant was used.

[0135] (2) The first oxidation treatment involved spraying the oxidant and the solid support synthesis column sample. Specifically, the spraying was performed as follows: 0.4 s pdrian followed by 8 s wait; 0.6 s pdrian followed by 10 s wait; and 0.4 s pdrian followed by 6 s wait. The total time for the gas pressure operation was 24.8 s.

[0136] The second oxidation treatment involved spraying the oxidant and the solid-phase support synthesis column sample. Specifically, the process was: pdrian for 0.4 s, wait for 10 s; pdrian for 0.6 s, wait for 10 s; pdrian for 0.6 s, wait for 8 s; and hdrian for 6 s. The total time for the gas flow pressure operation was 35.6 s.

[0137] 6. Double Cap Processing

[0138] (1) The solid-support synthesis column samples were capped using capping agent A and capping agent B. The capping treatment was repeated twice. The first time, 45 μl of capping agent A (75% of the amount of capping agent A used in Comparative Example 1) and 45 μl of capping agent B (75% of the amount of capping agent B used in Comparative Example 1) were used. The second time, 60 μl of capping agent A and 60 μl of capping agent B were used.

[0139] (2) First capping treatment: The capping agent and the solid support synthesis column sample were subjected to a spray treatment. Specifically, the process was: wait 3 s, pdrian 0.4 s, wait 6 s, pdrian 0.4 s, wait 8 s, pdrian 0.3 s, and wait 4 s. The total time for the gas pressure operation was 22.1 s.

[0140] The second capping process involved spraying the capping agent and the solid support synthesis column sample. Specifically, the process consisted of a 3-second wait, a 0.4-second pdrian, a 6-second wait, a 0.4-second pdrian, a 8-second wait, a 0.5-second pdrian, a 8-second wait, and a 6-second hdrian. The total time for this airflow pressure operation was 32.3 seconds.

[0141] 7. Third Wash:

[0142] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0143] (2) Perform gas flow pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow (Pdrian) for 1 s, wait for 2 s, and then perform a heavy blow (Hdrain) for 6 s. The total gas flow pressure operation time is 9 s.

[0144] 8. Determine the number of cycles for steps (2)-(7) above based on the length of the DNA single strand.

[0145] 9. Ammonolysis: After the synthesis is completed, the synthesis support is placed in an ammonolysis apparatus and ammonolysed at 90℃ and 80 psi for 2 h.

[0146] 10. Drying: After ammonolysis is completed, place the synthesis carrier in an oven at 60°C and dry for 10 min.

[0147] 11. Elution: Add 200 μl of acetonitrile to the synthesis carrier, wash and dry under vacuum, repeat once. Add 80% acetonitrile to the synthesis carrier, wash and dry under vacuum, repeat once. Add 200 μl of elution buffer to the synthesis carrier and elute into a 96-well deep-plate, repeat once. The oligonucleotides are then eluted into the deep-plate.

[0148] 12. Mass spectrometry detection: After filtering an appropriate amount of solid-phase support synthesis column sample, the purity of the sample was detected by mass spectrometry as shown in Table 7 below. It can be seen that the purity of all 12 samples is within the normal range.

[0149] Table 7. Sample purity results detected by mass spectrometry

[0150]

[0151] As shown in Table 8 below, when synthesizing 20 nt DNA single strands using solid-phase synthesis in this embodiment, compared with Comparative Example 1, one cycle of this embodiment saves approximately 13.9% of the time and 10-12.5% ​​of the reagents. Furthermore, the sample purity remains normal. This indicates that this method meets the requirements for short-chain DNA synthesis.

[0152] Table 8. Reagent and time savings for one cycle in Example 1

[0153]

[0154] Example 2: Synthesis of a 60 nt DNA single strand

[0155] For the 12 DNA single strands with a length of 20nt in Table 2 above, compared with Comparative Example 1, the solid-phase synthesis method of oligonucleotides used in this embodiment eliminated the large blowing operation in the first cycle and reduced the amount of reagents used in each step; in the second cycle, the same airflow pressure operation and reagent amount as Comparative Example 1 were maintained.

[0156] Since the large blowing operation was removed during the first cycle, the small blowing time in this embodiment needs to be adjusted accordingly to ensure that a small amount of reagent has not completely permeated into the carrier at the end.

[0157] The specific DNA single-strand synthesis steps in this embodiment are as follows:

[0158] 1. First Wash: Same as step 2 in Example 1.

[0159] 2. Two-stage deblocking process

[0160] (1) The solid-support synthesis column sample was decapped using a decapping agent. The decapping process was repeated twice. The first time, 160 μl of decapping agent was used (80% of the amount of decapping agent used in Comparative Example 1), and the second time, 200 μl of decapping agent was used.

[0161] (2) The first decapping treatment involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process was: 0.3 s blowing followed by a 10 s wait, 0.3 s blowing followed by a 12 s wait, and 0.3 s blowing followed by a 4 s wait. The total time for the airflow pressure operation was 26.9 s.

[0162] The second decapping process involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process consisted of a light spray for 0.3 s, followed by a 10-s wait; a light spray for 0.3 s, followed by a 10-s wait; a light spray for 0.4 s, followed by a 10-s wait; and a heavy spray for 6 s. The total time for the gas flow pressure operation was 37 s.

[0163] 3. Second Wash: Same as step 4 in Example 1.

[0164] 4. Two-stage activation coupling treatment

[0165] (1) The solid-phase support synthesis column sample was activated and coupled using monomers and activators. The activation and coupling treatment was repeated twice. The first time, 42 μl of monomer (84% of the monomer amount in Comparative Example 1) and 50 μl of activator (approximately 83.33% of the activator amount in Comparative Example 1) were used. The second time, 50 μl of monomer and 60 μl of activator were used.

[0166] (2) After the first activation coupling treatment, the monomer, activator and solid support synthesis column sample were subjected to a blow-blowing treatment. Specifically, wait 3 s, blow lightly for 0.2 s, wait 12 s, blow lightly for 0.3 s, wait 10 s, blow lightly for 0.2 s, wait 6 s. The total time for the gas flow pressure operation was 31.7 s.

[0167] After the second activation coupling treatment, the monomer, activator, and solid support synthesis column sample were subjected to a blow-blowing treatment. Specifically, the process was as follows: wait 3 s, blow lightly for 0.2 s, wait 10 s, blow lightly for 0.3 s, wait 10 s, blow lightly for 0.3 s, wait 10 s, and blow heavily for 6 s. The total time for the gas flow pressure operation was 39.8 s.

[0168] 5. Two-stage oxidation (Oxi) treatment

[0169] (1) The solid-supported synthesis column sample was oxidized using an oxidant. The oxidation process was repeated twice. The first time, 60 μl of oxidant was used (75% of the amount of oxidant used in Comparative Example 1), and the second time, 80 μl of oxidant was used.

[0170] (2) The first oxidation treatment involved spraying the oxidant and the solid support synthesis column sample. Specifically, the process was: 0.4 s purge followed by an 8 s wait, 0.6 s purge followed by a 10 s wait, and then 0.4 s purge followed by a 6 s wait. The total time for the gas pressure operation was 24.8 s.

[0171] The second oxidation treatment involved spraying the oxidant and the solid-phase support synthesis column sample. Specifically, the process consisted of a light spray for 0.4 s, followed by a 10 s wait; a light spray for 0.6 s, followed by a 10 s wait; a light spray for 0.6 s, followed by an 8 s wait; and a heavy spray for 6 s. The total time for the gas flow pressure operation was 35.6 s.

[0172] 6. Double Cap Processing

[0173] (1) The solid-support synthesis column samples were capped using capping agent A and capping agent B. The capping treatment was repeated twice. The first time, 45 μl of capping agent A (75% of the amount of capping agent A used in Comparative Example 1) and 45 μl of capping agent B (75% of the amount of capping agent B used in Comparative Example 1) were used. The second time, 60 μl of capping agent A and 60 μl of capping agent B were used.

[0174] (2) First capping treatment: The capping agent and the solid support synthesis column sample were sprayed. Specifically, wait 3 s, then blow lightly for 0.4 s, wait 6 s, blow lightly for 0.4 s, wait 8 s, blow lightly for 0.3 s, and wait 4 s. The total time for the air pressure operation was 22.1 s.

[0175] The second capping treatment involved spraying the capping agent and the solid support synthesis column sample. Specifically, the process was: wait 3 seconds, apply a small purge for 0.4 seconds, wait 6 seconds, apply a small purge for 0.4 seconds, wait 8 seconds, apply a small purge for 0.5 seconds, wait 8 seconds, and then apply a large purge for 6 seconds. The total time for the gas flow pressure operation was 32.3 seconds.

[0176] 7. Third Wash:

[0177] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0178] (2) Perform gas pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow for 1 s, wait for 2 s, and then perform a strong blow for 6 s. The total time for the gas pressure operation is 9 s.

[0179] 8. Determine the number of cycles for steps (2)-(7) above based on the length of the DNA single strand.

[0180] 9. Ammonolysis: After the synthesis is completed, the synthesis support is placed in an ammonolysis apparatus and ammonolysed at 90℃ and 80 psi for 2 h.

[0181] 10. Drying: After ammonolysis is completed, place the synthesis carrier in an oven at 60°C and dry for 10 min.

[0182] 11. Elution: Add 200 μl of acetonitrile to the synthesis carrier, wash and dry under vacuum, repeat once. Add 80% acetonitrile to the synthesis carrier, wash and dry under vacuum, repeat once. Add 200 μl of elution buffer to the synthesis carrier and elute into a 96-well deep-plate, repeat once. The oligonucleotides are then eluted into the deep-plate.

[0183] 12. Mass spectrometry detection: After filtering an appropriate amount of sample, the purity of the sample was obtained by mass spectrometry detection as shown in Table 9 below. It can be seen that the purity of all 12 samples is within the normal range.

[0184] Table 9. Sample purity results detected by mass spectrometry

[0185]

[0186] As shown in Table 10 below, when synthesizing 20 nt DNA single strands using solid-phase synthesis in this embodiment, compared with Comparative Example 1, one cycle of this embodiment saves approximately 12.0% of the time and 8-12.5% ​​of the reagents. Furthermore, the sample purity remains normal. This indicates that this method meets the requirements for short-chain DNA synthesis.

[0187] Table 10. Reagent and time savings for one cycle in Example 2

[0188]

[0189] Example 3

[0190] For the six 21nt RNA single strands in Table 2 above, the solid-phase synthesis method of oligonucleotides used in this embodiment is slightly different from that in Examples 1 and 2.

[0191] In this embodiment, the large blowing operation was removed during the first decapping, oxidation, and capping treatments, as well as the first, second, and third activation coupling treatments, and the amount of reagents used in each step was reduced. During the second decapping, oxidation, and capping treatments, as well as the fourth activation coupling treatment, the same airflow pressure and reagent amount as in Comparative Example 1 were maintained.

[0192] Since the large blowing operation was removed during the first cycle, the small blowing time in this embodiment needs to be adjusted accordingly to ensure that a small amount of reagent has not completely permeated into the carrier at the end.

[0193] The specific steps for RNA single-strand synthesis in this embodiment are as follows:

[0194] 1. First Wash: Same as step 2 in Example 1.

[0195] 2. Two-stage deblocking process

[0196] (1) The solid-support synthesis column sample was decapped using a decapping agent. The decapping process was repeated twice. The first time, 160 μl of decapping agent was used (80% of the amount of decapping agent used in Comparative Example 2), and the second time, 200 μl of decapping agent was used.

[0197] (2) The first decapping treatment involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process was: 0.3 s gentle blowing followed by a 10 s wait, 0.3 s gentle blowing followed by a 12 s wait, and 0.3 s gentle blowing followed by a 4 s wait. The total time for the airflow pressure operation was 26.9 s.

[0198] The second decapping process involved spraying the decapping agent and the solid support synthesis column sample. Specifically, the process consisted of a light spray for 0.3 seconds, followed by a 10-second wait; a light spray for 0.3 seconds, followed by a 10-second wait; a light spray for 0.4 seconds, followed by a 10-second wait; and a heavy spray for 6 seconds. The total time for the gas flow pressure operation was 37 seconds.

[0199] 3. Second Wash: Same as step 4 in Example 1.

[0200] 4. Four-stage activation coupling (Couple) treatment

[0201] (1) The solid-phase support synthesis column sample was activated and coupled using monomers and activators. The activation and coupling treatment was repeated 4 times. The first, second and third times, 42 μl of monomer (84% of the amount of monomer used in Comparative Example 2) and 50 μl of activator (approximately 83.33% of the amount of activator used in Comparative Example 2) were used. The fourth time, 50 μl of monomer and 60 μl of activator were used.

[0202] (2) The first, second, and third activation coupling treatments were performed on the monomer, activator, and solid support synthesis column samples by spraying. Specifically, the process was as follows: wait 3 s, spray lightly for 0.2 s, wait 12 s, spray lightly for 0.3 s, wait 12 s, spray lightly for 0.2 s, and wait 6 s. The total time for the gas pressure operation was 33.7 s.

[0203] The fourth activation coupling treatment involved spraying the monomer, activator, and solid-supported synthesis column sample. Specifically, the process consisted of a 3-second wait, a 0.2-second gentle spray, a 12-second wait, a 0.3-second gentle spray, a 12-second wait, a 0.3-second gentle spray, a 10-second wait, and a 6-second strong spray. The total time for this gas flow pressure operation was 43.8 seconds.

[0204] 5. Two-stage oxidation (Oxi) treatment

[0205] (1) The solid-supported synthesis column sample was oxidized using an oxidant. The oxidation process was repeated twice. The first time, 60 μl of oxidant was used (75% of the amount of oxidant used in Comparative Example 2), and the second time, 80 μl of oxidant was used.

[0206] (2) The first oxidation treatment involves blowing the oxidant and the solid support synthesis column sample. Specifically, the blowing is performed for 0.4 s, followed by an 8 s wait; then the blowing is performed for 0.6 s, followed by an 10 s wait; and finally the blowing is performed for 0.4 s, followed by an 6 s wait. The total time for the gas flow pressure operation is 24.8 s.

[0207] The second oxidation treatment involved spraying the oxidant and the solid-phase support synthesis column sample. Specifically, the process consisted of a light spray for 0.4 s, followed by a 10 s wait; a light spray for 0.6 s, followed by a 10 s wait; a light spray for 0.6 s, followed by an 8 s wait; and a heavy spray for 6 s. The total time for the gas flow pressure operation was 35.6 s.

[0208] 6. Double Cap Processing

[0209] (1) The solid-support synthesis column samples were capped using capping agent A and capping agent B. The capping treatment was repeated twice. The first time, 45 μl of capping agent A (75% of the amount of capping agent A used in Comparative Example 2) and 45 μl of capping agent B (75% of the amount of capping agent B used in Comparative Example 2) were used. The second time, 60 μl of capping agent B and 60 μl of capping agent B were used.

[0210] (2) First capping treatment: The capping agent and the solid support synthesis column sample were subjected to a spraying treatment. Specifically, wait 3s, spray lightly for 0.4s, wait 6s, spray lightly for 0.4s, wait 8s, spray lightly for 0.3s, and wait 4s. The total time for the gas flow pressure operation was 22.1s.

[0211] The second capping treatment involved spraying the capping agent and the solid support synthesis column sample. Specifically, the process consisted of a 3-second wait, a 0.4-second gentle blow, a 6-second wait, a 0.4-second gentle blow, an 8-second wait, a 0.5-second gentle blow, an 8-second wait, and a 6-second strong blow. The total time for the gas flow pressure operation was 32.3 seconds.

[0212] 7. Third Wash:

[0213] (1) Clean the solid-support synthesis column sample with 200 μl of anhydrous acetonitrile;

[0214] (2) Perform gas pressure operation on the cleaned solid support synthesis column sample. Specifically, perform a light blow for 1 second, wait for 2 seconds, and then perform a strong blow for 6 seconds. The total time for the gas pressure operation is 9 seconds.

[0215] 8. Determine the number of cycles for steps (2)-(7) above based on the length of the RNA single strand.

[0216] 9. Ammonolysis: Transfer the synthesized solid support to a 2 ml threaded tube and add 500 μL of icy ammonia solution. Incubate at 65°C in a water bath for 2 hours to remove protecting groups from the bases.

[0217] 10. Concentration and freeze-drying: Transfer the ammonolysis solution to a 2ml centrifuge tube and concentrate and freeze-dry.

[0218] 11. Removal of TBDMS protecting groups. Add 50 μL of dimethyl sulfoxide to the lyophilized centrifuge tube, heat in a 65°C water bath for 10 min, then add 200 μL of triethylamine trifluoroacetate, heat in a 65°C water bath for 2 h to remove the TBDMS protecting groups.

[0219] 12. Freeze-drying after precipitation. After the TBDMS removal reaction is complete, add 100 μL of 3 mol / L NaAc solution (pH 4.2) to a centrifuge tube, then add 1500 μL of ice-cold ethanol, and incubate at -80°C for 1 hour. Then centrifuge at 12000 rpm for 10 minutes. Discard the supernatant and concentrate to freeze-dry the sample precipitate.

[0220] 13. HPLC Analysis. After dissolving the sample, HPLC quantitative analysis was performed. As shown in Table 11 below, the target peak area for RNA in the sample was within the normal range. This demonstrates that this method is also applicable to RNA synthesis.

[0221] Table 11 Sample purity results detected by mass spectrometry

[0222]

[0223] As shown in Table 12 below, in this embodiment, when synthesizing 21nt RNA single strands using solid-phase synthesis, compared to Comparative Example 2, one cycle of this embodiment saves approximately 14.6% of the time and 10-12.5% ​​of the reagents. Furthermore, the sample purity remains normal. This indicates that this method meets the requirements for short-chain DNA synthesis.

[0224] Table 12. Reagent and time savings for one cycle in Example 3

[0225]

[0226] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for efficient and low-cost solid-phase synthesis of oligonucleotides, characterized in that, Includes the following steps: The solid-supported synthesis column was first cleaned. The solid-phase support synthesis column, after the first cleaning, undergoes two decapping processes; the first decapping is performed under a first low pressure; the second decapping is performed under a second low pressure and a first high pressure. The solid-phase support synthesis column, after being decapped, is subjected to a second cleaning. The solid-phase support synthesis column after the second cleaning is subjected to several activation couplings; each activation coupling except the last one is subjected to a third low pressure; the last activation coupling is subjected to a fourth low pressure and a second high pressure. The solid-phase support synthesis column after activation coupling was subjected to two oxidation processes; the first oxidation was carried out at the fifth low pressure; the second oxidation was carried out at the sixth low pressure and the third high pressure. The oxidized solid-support synthesis column was capped twice; after the first capping, a seventh minor pressure was applied; after the second capping, an eighth minor pressure and a third major pressure were applied. Based on the oligonucleotide length, the cycle is repeated a corresponding number of times. After the cycle is completed, the solid-phase support synthesis column is subjected to ammonolysis, drying, and elution to obtain the oligonucleotide.

2. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The method for the first small pressure is as follows: apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.3 s, wait for 4 s.

3. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The second small pressure method is as follows: apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.3 s, wait for 10 s, apply small pressure for 0.4 s, wait for 10 s; The method for the first maximum pressure is: maximum pressure for 6 seconds.

4. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, When the oligonucleotide is a single strand of DNA with a length ≤21nt, the method for the third small pressure is as follows: wait 3 s, apply small pressure for 0.2 s, wait 10 s, apply small pressure for 0.3 s, wait 10 s, apply small pressure for 0.2 s, and wait (Wait) 4 s; When the oligonucleotide is a single strand of DNA with a length ≥60nt, the method for the third small pressure is as follows: wait 3 s, apply small pressure for 0.2 s, wait 12 s, apply small pressure for 0.3 s, wait 10 s, apply small pressure for 0.2 s, and wait (Wait) for 6 s; When the oligonucleotide is a single strand of RNA, the third small pressure method is as follows: wait 3 s, apply small pressure for 0.2 s, wait 12 s, apply small pressure for 0.3 s, wait 12 s, apply small pressure for 0.2 s, and wait (Wait) 6 s.

5. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, When the oligonucleotide is a single strand of DNA, the method for the fourth small pressure is as follows: wait 3 s, apply small pressure for 0.2 s, wait 10 s, apply small pressure for 0.3 s, wait 10 s, apply small pressure for 0.3 s, wait 10 s; When the oligonucleotide is a single strand of RNA, the method for the fourth small pressure is as follows: wait 3 s, apply small pressure for 0.2 s, wait 12 s, apply small pressure for 0.3 s, wait 12 s, apply small pressure for 0.3 s, and wait 10 s; The second method of applying maximum pressure is 6 seconds.

6. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The method for the fifth small pressure is as follows: apply small pressure for 0.4 s, wait for 8 s, apply small pressure for 0.6 s, wait for 10 s, apply small pressure for 0.4 s, wait for 6 s.

7. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The method for the sixth small pressure is as follows: apply small pressure for 0.4 s, wait for 10 s, apply small pressure for 0.6 s, wait for 10 s, apply small pressure for 0.6 s, wait for 8 s; The third method of applying maximum pressure is 6 seconds.

8. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The method for the seventh small pressure is as follows: wait 3 s, apply small pressure for 0.4 s, wait 6 s, apply small pressure for 0.4 s, wait 8 s, apply small pressure for 0.3 s, and wait 4 s.

9. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The method for the eighth small pressure is as follows: wait 3 s, apply small pressure for 0.4 s, wait 6 s, apply small pressure for 0.4 s, wait 8 s, apply small pressure for 0.5 s, wait 8 s; The third method of applying maximum pressure is: applying maximum pressure for 6 seconds.

10. The method for efficient and low-cost solid-phase synthesis of oligonucleotides according to claim 1, characterized in that, The first cleaning method is as follows: the solid support synthesis column is cleaned with anhydrous acetonitrile, and the ninth small pressure and the fourth large pressure are performed; The second cleaning method is as follows: the solid support synthesis column is cleaned with anhydrous acetonitrile, and the tenth minimum pressure and the fifth maximum pressure are performed, for a total of 2 cleanings; Furthermore, the methods for the ninth and tenth small pressures are both 1 second of small pressure followed by a 2-second wait; the methods for the fourth and fifth large pressures are both 6 seconds of large pressure.

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