Preparation method of long-chain oligonucleotide

Abstract

The invention discloses a preparation method of long-chain oligonucleotide, and relates to the technical field of synthesis of long-chain oligonucleotide. According to the method, the desiliconization reaction is directly carried out after ammonolysis, ammonia removal is not needed, the quenching reaction is directly carried out after the desiliconization reaction, the steps of sample centrifugal concentration, drying, ethanol precipitation and the like are not needed, and the long-chain oligonucleotide is obtained through purification. By adopting a solid-phase synthesis method, the method has the technical advantages of high synthesis automation degree and short synthesis period on the whole, and the preparation process of the long-chain oligonucleotide is high in efficiency and fast in output. In addition, hydrophobic chromatography is arranged to tolerate a desiliconized crude product system, so that the loss of consumables is reduced, the nucleic acid synthesis cost is reduced, and large-scale synthesis of long-chain oligonucleotides is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of long-chain oligonucleotide synthesis, and in particular to a method for preparing long-chain oligonucleotide. Background Art

[0002] Medium- and short-chain oligonucleotides represented by ASO and siRNA generally refer to oligonucleotides of 25 nt, which are currently the hot research topics for nucleic acid drugs. Long-chain oligonucleotides represented by sgRNA are generally 100 nt or longer, and are mainly used for gene editing CRISPR therapy, and are often used in the treatment of rare diseases. Due to the long length of long-chain oligonucleotides, in addition to the general solid-phase synthesis method, they can also be prepared by enzyme ligation.

[0003] The main process of solid phase synthesis is to first deprotect the dimethoxytrityl (DMT) of the hydroxyl group on the carrier on a solid phase synthesizer to obtain free hydroxyl groups, and then react the phosphoramidite monomer and the hydroxyl group from which DMT was removed under the catalysis of an activator to form a new phosphite triester to achieve chain elongation. Subsequently, the unstable trivalent phosphorus is oxidized to pentavalent phosphorus under the action of an oxidant or a sulfurizing agent, and acetic anhydride is used to cap the uncoupled 5'-OH, and the cycle is repeated to finally obtain an oligonucleotide of the target sequence. The oligonucleotide fragment is then cut from the solid phase resin with concentrated ammonia water, and the oligonucleotide is obtained by deprotection and purification.

[0004] The main process of the enzyme ligation method is to first prepare relatively pure short-chain oligonucleotide fragments through solid-phase synthesis, then connect the fragments under the action of DNA splint and T4 RNA ligase to obtain crude long-chain oligonucleotides, and then purify them to prepare high-purity long-chain oligonucleotide products.

[0005] The traditional solid-phase synthesis method for synthesizing long-chain oligonucleotides represented by sgRNA requires the use of 2'-O-TBDMS monomers. After the solid-phase synthesis is completed, fluorine-containing reagents (such as TBAF / THF, TEA·3HF) are often required for additional desilication reactions. Currently, this reaction often requires aminolysis followed by ammonia removal, and additional ethanol precipitation and filtration and drying steps are required after the desilication reaction, which has the disadvantages of long cycles and large losses.

[0006] The enzyme ligation method requires the use of multiple short RNA fragments and DNA splints, all of which need to be purified and have a long cycle. The T4 RNA ligase used is relatively expensive, the conditions are relatively harsh, and the enzyme ligation effect is poor when encountering special modified sequences. Therefore, the universality of the enzyme ligation method is not strong.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The object of the present invention is to provide a method for preparing long-chain oligonucleotides to solve the above-mentioned shortcomings of long post-synthesis treatment and purification cycle in solid phase synthesis of long-chain oligonucleotides.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a method for preparing a long-chain oligonucleotide, comprising the following steps:

[0011] S1: contacting the solid phase support bound with nucleotides with a deprotection reagent to remove the protecting group on the 5'-OH of the nucleotides;

[0012] S2: adding monomer and activator to carry out coupling reaction;

[0013] S3: oxidation or sulfurization of the coupling product;

[0014] S4: Capping reaction: adding capping reagent to carry out capping reaction;

[0015] S5: Repeat S1-S4 until a long-chain oligonucleotide is synthesized;

[0016] S6: cyanoethyl removal reaction;

[0017] S7: Ammonolysis and desiliconization reaction:

[0018] The solid phase carrier is placed in an ammonolysis reagent for reaction, and each 1 umol of the solid phase carrier is contacted with 53.5-58.9 mmol or 1-1.1 ml of the ammonolysis reagent, and the reaction is carried out in a water bath at 35°C-45°C for 14-20 hours. After filtering, the filtrate is washed with an alcohol solution, and DMSO is added to the solution at a ratio of 1-1.5 ml of DMSO per 1 umol of the solid phase carrier, and a reducing agent is added at a ratio of 1.25-1.5 ml or 7.7-9.2 mmol of the reducing agent, and the pH is adjusted to 8.0-9.0, and the reaction is carried out in a water bath at 50°C±0.5°C for 4-4.5 hours; the reaction is quenched;

[0019] The reducing agent is selected from fluorine-containing reagents;

[0020] S8: Quenching reaction: quenching the reaction with Tris-HCl;

[0021] S9: Purification: The reaction solution after quenching the reaction is loaded onto a hydrophobic chromatography column for hydrophobic chromatography.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a method for preparing long-chain oligonucleotides. In the method, a desiliconization reaction is directly performed after aminolysis without the need for ammonia removal. After the desiliconization reaction, there is no need for steps such as sample centrifugation concentration, drying and ethanol precipitation, and a quenching reaction can be directly performed to obtain the long-chain oligonucleotides through purification.

[0024] The present invention adopts a solid phase synthesis method, which has the technical advantages of high degree of synthesis automation and short synthesis cycle. The preparation process of long-chain oligonucleotides is efficient and has fast output. In addition, the hydrophobic chromatography is set to tolerate the crude product system after desiliconization, reduce the loss of consumables, reduce the cost of nucleic acid synthesis, and contribute to the large-scale synthesis of long-chain oligonucleotides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 A roadmap for the synthetic preparation of sgRNA;

[0027] Figure 2 The effect of different pH on the purity of DMTON in the one-pot desiliconization reaction of sgRNA;

[0028] Figure 3 This is a comparison chart of the results of crude sgRNA before and after HIC purification;

[0029] Figure 4 This is the HPLC chart of the final pure sgRNA. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] In a first aspect, the present invention provides a method for preparing a long-chain oligonucleotide, comprising the following steps:

[0032] S1: contacting the solid phase support bound with nucleotides with a deprotection reagent to remove the protecting group on the 5'-OH of the nucleotides; the reaction equation is as follows:

[0033]

[0034] S2: Add monomer and activator to carry out coupling reaction; the reaction equation is as follows:

[0035]

[0036] S3: The coupling product is subjected to oxidation or sulfurization reaction; the reaction equation is as follows:

[0037]

[0038] S4: Capping reaction: Add capping reagent to carry out capping reaction; the reaction equation is as follows:

[0039]

[0040] S5: Repeat S1-S4 until a long-chain oligonucleotide is synthesized;

[0041] S6: Removal of cyanoethyl group; the reaction equation is as follows:

[0042]

[0043] S7: Ammonolysis and desiliconization reaction: place the solid phase carrier in an ammonolysis reagent for reaction, contact each 1umol of the solid phase carrier with 1-1.1ml of the ammonolysis reagent or 53.5-58.9mmol, react in a water bath at 35℃-45℃ for 14-20 hours, filter, take the filtrate and rinse with an alcohol solution, add DMSO at a ratio of 1-1.5ml DMSO per 1umol of the solution, add a reducing agent at a ratio of 1.25-1.5ml reducing agent per 1umol of the solution or 7.7-9.2mmol, adjust the pH to 8.0-9.0, react in a water bath at 50℃±0.5℃ for 4-4.5h; quench the reaction;

[0044] The reducing agent is selected from fluorine-containing reagents;

[0045] The reaction equation is as follows:

[0046]

[0047] S8: Quenching reaction: quenching the reaction with Tris-HCl;

[0048] S9: Purification: The reaction solution after quenching the reaction is loaded onto a hydrophobic chromatography column for hydrophobic chromatography.

[0049] The solid phase synthesis of sgRNA adopts the DMT-ON mode. The present invention directly performs desiliconization reaction after aminolysis, without the need for ammonia removal, thereby improving the efficiency of the preparation process. The purpose of the vacuum ammonia removal step after the original aminolysis reaction is to remove ammonia and water, and to avoid the influence of the fluorine-containing reagent under acidic conditions on the 5'-O-DMT protecting group in the desiliconization reaction caused by the lack of ammonia removal; if ammonia is not removed, 5'-O-DMT is sensitive to acid, and natural RNA will produce chain breaks due to the phosphate exchange reaction at positions 2 and 3 under alkaline conditions, which will cause the 5'-O-DMT protecting group to be partially missing, which is not conducive to the stability of the 5'-O-DMT protecting group and the stability of natural RNA. However, the present invention does not need to perform ammonia removal and water removal steps separately, and adjusts the pH, and performs a water desiliconization reaction under pH conditions of 8.0-9.0, thereby improving the purity of the long-chain oligonucleotide-DMT product, and facilitating the subsequent purification of the long-chain oligonucleotide-DMT product of the target purity by means of hydrophobic properties with the DMT group through hydrophobic chromatography.

[0050] After the desiliconization reaction, there is no need for sample centrifugation concentration, drying and ethanol precipitation steps, and the quenching reaction can be directly carried out to obtain long-chain oligonucleotides after purification. After the desiliconization reaction, Tris-HCl reagent is usually added to quench the reaction. The crude product contains a large amount of DMSO, TEA, HF, ammonia and Tris-HCl. The system is relatively complex, and some reagents have certain corrosiveness, such as HF and DMSO. The usual process is to add ethanol precipitation after adding high concentration of NaAc, but the subsequent static precipitation, centrifugation, filtration and washing operation cycle is long. In the ultrafiltration desalination process, the crude product system will greatly reduce the life of the membrane package. The C18 filler in the reverse phase purification system is also poor in tolerance to fluorine-containing reagents, and often causes sample flow through in a high salt system. The present invention uses HIC (hydrophobic chromatography) purification, which is characterized in that the HIC filler can meet the characteristics of high salt loading and low salt elution. The crude product after the desiliconization reaction can be directly loaded for HIC purification without special treatment, and the sample flow through phenomenon does not occur. The filler can also tolerate the crude product system, which greatly saves the preparation cycle. Since hydrophobic chromatography can tolerate the crude product system after desiliconization, it reduces the loss of consumables, lowers the cost of nucleic acid synthesis, and facilitates the large-scale synthesis of long-chain oligonucleotides.

[0051] The present invention adopts a solid phase synthesis method, which has the technical advantages of high degree of synthesis automation and short synthesis cycle. The preparation process of long-chain oligonucleotides is efficient and has fast output.

[0052] The deprotection reagent in step S1 can be 3% dichloroacetic acid / toluene, 10% dichloroacetic acid / toluene, 3% dichloroacetic acid / dichloromethane, 3% trichloroacetic acid / dichloromethane, preferably 3% dichloroacetic acid / toluene, and those skilled in the art can adjust the setting as needed.

[0053] In step S2, during coupling, an activator is added, and the activator used can be 0.6M ETT (5-ethylthiotetrazolyl) / acetonitrile solution, 0.3M BTT (5-benzylthiotetrazolyl) / acetonitrile solution, 1.0M DCI (4,5-dicyanoimidazole) / acetonitrile solution, preferably 0.3M BTT / acetonitrile solution. In one embodiment, the activator is selected from tetrazole, 5-ethylthiotetrazolyl, dicyanoimidazole or pyridinium salt; the activator is selected from any one of 5-ethylthiotetrazolyl acetonitrile solution and 4,5-dicyanoimidazole acetonitrile solution.

[0054] In step S3, the oxidant used in the oxidation reaction is a 50 mM iodine aqueous solution, which is not limited.

[0055] In step S4, a capping reagent is added in the capping reaction, and the capping reagent includes but is not limited to acetic anhydride, N-methylimidazole and other reagents.

[0056] Step S6: Removal of cyanoethyl group reaction, wherein the cyanoethyl group is removed by using a diethylamine acetonitrile solution or a triethylamine acetonitrile solution; preferably, the solid phase support is treated with a 20% to 50% diethylamine acetonitrile solution or a triethylamine acetonitrile solution for 10 to 120 minutes. For example, a 20% diethylamine / acetonitrile solution is used for 10 minutes to remove the cyanoethyl group.

[0057] In step S7, during the ammonolysis and desiliconization reactions: the solid phase carrier is placed in an ammonolysis reagent for reaction, and each 1 umol of the solid phase carrier is contacted with 1-1.1 ml or 53.5, 54 mmol, 55 mmol, 56 mmol, 57 mmol, 58 mmol or 58.9 mmol of the ammonolysis reagent, and the reaction is carried out in a water bath at 35°C-40°C, 35°C-42°C, or 35°C-45°C for 14-15h, 14-16h, 15-18h, or 18-20h.

[0058] Add DMSO at a ratio of 1-1.5 ml DMSO per 1 umol solution, add reducing agent at a ratio of 1.25-1.3 ml reducing agent or 1.25-1.4 ml reducing agent or 7.7-8 mmol, 7.7-9 mmol per 1 umol solution, adjust the pH to 8.0-9.0, react in a water bath at 50°C±0.5°C for 4-4.5 hours; quench the reaction.

[0059] For the preparation of long-chain oligonucleotides taking sgRNA as an example, Unylinker CPG 1000A solid phase carrier or Unylinker CPG 2000A solid phase carrier may be used, preferably Unylinker CPG 2000A solid phase carrier.

[0060] In a preferred embodiment of the present invention, in step S9, the chromatographic column used in the hydrophobic chromatography is a Phenyl-15L chromatographic column with a specification of 4.6 mm × 250 mm, and the mobile phase A is a high salt system, which includes 0-20 mM phosphate, 1.5-3 M sodium chloride and 0-20% acetonitrile, and the pH is 9.2 ± 0.2, and the mobile phase B is a low salt system, which includes 0-20 mM phosphate and 0-20% acetonitrile, and the pH is 9.2 ± 0.2. HIC filler can meet the characteristics of high salt loading and low salt elution. The DMT group in the long-chain oligonucleotide-5'-O-DMT has good hydrophobic properties and interacts with the hydrophobic chromatography column, so that the long-chain oligonucleotide is bound to the hydrophobic chromatography column, and the impurities such as DMSO, TEA, HF, ammonia and Tris-HCl in the sample are eluted under the mobile phase with high salt concentration, and further eluted through the low-salt mobile phase to obtain the crude long-chain oligonucleotide.

[0061] In a preferred embodiment of the present invention, the reaction solution after quenching the reaction is diluted with mobile phase A to a conductivity of >120mS / cm before purification, and then filtered and loaded with a flow rate of 150cm / h, and the chromatographic peak at 260nm is monitored; 100% mobile phase A is used for elution until UV260nm is <50mAu, and then gradient elution is performed, and the elution program of the gradient elution is 0-100%, mobile phase B.

[0062] Under the above elution procedure, a better purification effect is achieved.

[0063] In a preferred embodiment of the present invention, the reaction solution after quenching the reaction is diluted and filtered with a 0.22 um filter membrane to remove impurities in the reaction solution.

[0064] In a preferred embodiment of the present invention, in step S7, the fluorine-containing reagent is triethylamine trihydrofluoride; the reagent for adjusting pH is selected from ammonia water or triethylamine; and the filtrate is rinsed with 50% ethanol.

[0065] In a preferred embodiment of the present invention, after step S9, DMT acid hydrolysis is further included: contacting 80% acetic acid solution with the eluate obtained by hydrophobic chromatography in S9, pH 4.0-4.2, and reacting at room temperature for 1-2.5 hours. The 5'-O-DMT protecting group is removed by acid hydrolysis, thereby obtaining the target long-chain oligonucleotide.

[0066] In a preferred embodiment of the present invention, DMT acid hydrolysis further includes reverse phase purification, and reverse phase purification includes the following steps: first adjusting the pH of the DMT acid hydrolysis reaction solution to 7.0±0.5, then diluting the DMT acid hydrolysis reaction solution, filtering, and loading the filtrate onto a reverse phase chromatography column for elution. Reverse chromatography can easily obtain a high purity sgRNA pure product.

[0067] In a preferred embodiment of the present invention, the sample flow rate is 150 cm / h, the chromatographic peak at 290 nm is monitored, and the following elution gradient is used for elution: 0-20% mobile phase B, 1CV; 20-80% mobile phase B, 60CV, and fractions are collected when UV290nm>10mAu, and collection is stopped when UV290nm<10mAu.

[0068] CV refers to the volume of a unit chromatographic column, and 1CV is 1 chromatographic column volume.

[0069] In a preferred embodiment of the present invention, the conditions for reverse phase purification include: using a C18 purification chromatography column with a size of 4.6 mm×150 mm, mobile phase A is 0.1 M DBAA (di-n-butylamine acetate), and mobile phase B is acetonitrile.

[0070] In a preferred embodiment of the present invention, the ammonolysis reagent is selected from any one of the following: concentrated aqueous ammonia, AMA (ammonia methylamine solution), 4:1 (v / v) aqueous ammonia ethanol solution, EAMA (ethanol AMA solution), and a mixed solution of diethylenetriamine with an alkaline aqueous solution and an alcohol.

[0071] In a preferred embodiment of the present invention, the alkaline aqueous solution is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, beryllium hydroxide, magnesium hydroxide, calcium hydroxide solution and a combination thereof, and the alcohol is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol and a combination thereof; wherein the aminolysis buffer is a mixed solution of alcohol and water in a volume ratio of 1:5-80.

[0072] In a preferred embodiment of the present invention, the monomer is a ribonucleotide monomer with 2'-O-TBDMS, which protects 2'-OH.

[0073] In a preferred embodiment of the present invention, the monomer is selected from a natural RNA monomer with 2'-O-TBDMS and / or a natural RNA monomer with 2'-O-TBDMS and nucleotide modifications.

[0074] In a preferred embodiment of the present invention, the nucleotide modification is selected from one or more of ribose modification, base modification and phosphate backbone modification;

[0075] In a preferred embodiment of the present invention, the modification is selected from one or more of fluoro modification, thio modification, 2-methoxy modification, cholesterol modification, and LNA modification.

[0076] The sequence may be all-natural RNA, or may be modified with three thiophosphates and 2'-OMe at the 5' and 3' ends respectively and the middle 94 nt being a natural RNA sequence, but a sequence with too low a natural RNA ratio shall not be used.

[0077] Taking into account the difficulty in preparing long-chain oligonucleotides, a segmented synthesis strategy was adopted for solid-phase synthesis, that is, the 1-50nt segment was the first segment, and 5-fold equivalents of monomers were used for coupling; the 50-80nt segment was the second segment, and 6-fold equivalents of monomers were used for coupling; the 80-100nt segment was the third segment, and 6-fold equivalents of monomers were used for coupling and two couplings were performed.

[0078] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0079] Example 1

[0080] This example provides a method for preparing a long-chain oligonucleotide (sgRNA), and the fitted sequence is as follows: 5′-Ams Gms Ums rC rC rU rC rArU rC rC rC rU rC rA rA rG rC rG rU rU rU rUrArG rC rU rArG rU rArA rU rArG rC rArArG rU rU rArA rArA rU rArA rG rGrC rU rA rG rU rC rC rG rU rU rArU rC rA rArC rU rU rG rA rA rArArA rG rU rG rC rA rC rG rU rC rG rU rG rC rUs Ums Ums Um-3', where "m" represents 2'-OMe modification, "s" represents phosphorothioate modification, and "r" represents natural RNA and uses 2'-O-TBDMS protecting group. The sequence is characterized by three 2'-OMe modifications and phosphorothioate modifications at the 5'-end and 3'-end, respectively, in order to enhance the stability of the nucleic acid. In addition, the 2-amino group of guanine phosphoramidite (rG or Gm) uses dmf (dimethylformamide) protecting group, cytosine phosphoramidite monomer (rC) uses Ac (acetyl) protecting group, and adenine phosphoramidite monomer (rA and Am) uses Bz (benzoyl) protecting group.

[0081] In this embodiment, the synthesis of long-chain oligonucleotides using sgRNA as an example adopts a standard solid-phase synthesis method, namely, removal of the DMT protecting group, coupling, oxidation / sulfurization, capping, and then removal of the cyanoethyl group and then aminolysis and cleavage of the carrier for deprotection, followed by filtering the carrier and direct desiliconization reaction. Among them, the solid phase carrier uses the Unylinker CPG 2000A of the Chemgenes manufacturer, the synthesizer uses the OligoPilot plus of the GE manufacturer, and the synthesis column uses a 6.3ml fixed steel column (bottom diameter 2cm, column height 2cm). After fully loading the solid phase carrier, the synthesis scale is 57umol. For the roadmap of the synthesis preparation method of sgRNA, please refer to Figure 1 shown.

[0082] S1: The DMT protection group removal reaction is controlled by UV, the monitoring wavelength is 400nm, the integration starts when the starting UV>500mAu, and the integration ends and the reaction stops when the ending UV<500mAu. The reaction equation for removing the DMT protection group on the 5'-OH of the nucleotide is as follows:

[0083]

[0084] S2: 0.2M phosphoramidite monomer acetonitrile solution and 0.3M BTT (benzylthiotetrazolyl) acetonitrile solution were used in the coupling reaction, and the reaction volume mixing ratio of phosphoramidite monomer acetonitrile solution and BTT (benzylthiotetrazolyl) acetonitrile solution was 3:7. According to the staged synthesis control strategy, based on the amount of solid phase support at the start of the reaction, 1-50nt was synthesized according to 5eq of monomer reaction equivalents, cycle time 12min, 51-80nt was synthesized according to 6eq of monomer reaction equivalents, cycle time 15min, 81-100nt was synthesized according to 6eq of monomer reaction equivalents, cycle time 12min and two coupling strategies.

[0085] The coupling reaction equation is as follows:

[0086]

[0087] S3: The oxidation reaction was performed using 50 mM iodine aqueous solution, with an iodine equivalent of 5 eq and a reaction time of 2.5 minutes. The sulfidation reaction was performed using 0.2 M XH (hydrogenated xanthocyanin) pyridine solution, with an XH equivalent of 10 eq and a reaction time of 6 minutes.

[0088] The oxidation and sulfidation reaction equations are as follows:

[0089]

[0090] S4: The capping reaction was performed with a reagent volume of 0.5 column volume (CV) and a reaction time of 0.5 min.

[0091] The capping reaction equation is as follows:

[0092]

[0093] S5: Repeat S1-S4, after synthesizing to 100 nt, use DMT-ON mode to retain the 5'-O-DMT protecting group.

[0094] S6: Decyanoethylation reaction: Use 20% DEA (diethylamine) acetonitrile solution, 4CV reagent volume and 10 minutes reaction time to perform cyanoethylation reaction, and finally obtain a solid phase carrier with a full-length sequence.

[0095] The decyanoethylation reaction equation is as follows:

[0096]

[0097] S7: Ammonolysis and desiliconization reaction:

[0098] The reaction equation is as follows:

[0099]

[0100] The ammonolysis and desiliconization reaction used in this embodiment is a one-pot reaction, that is, the solid phase carrier is immersed in 25% concentrated ammonia water for reaction, the solid phase carrier is filtered, and the filtrate is directly subjected to desiliconization reaction without additional vacuum concentration and ammonia and water removal steps.

[0101] In this embodiment, the ammonolysis reagent is 25% concentrated ammonia water. In addition, the ammonolysis reagent may also include AMA (ammonia methylamine solution), 4:1 (v / v) ammonia ethanol solution, EAMA (ethanol AMA solution), etc.

[0102] The specific aminolysis reaction process is as follows: 25% concentrated ammonia water is used. 1 ml of concentrated ammonia water is added to 1 umol of the solid phase carrier, and the solid phase carrier is placed in a 45°C water bath for 16 hours, and then the solid phase carrier is returned to room temperature, and the solid phase carrier is filtered using a 0.22 um hydrophilic filter membrane, and then the solid phase carrier is washed three times with 50% ethanol, and the washing liquid is combined and fixed to 2.5 ml.

[0103] Desiliconization reaction:

[0104] Directly add DMSO to the elution solution at a ratio of 1umol solid phase support to 1ml DMSO, then add TEA·3HF at a ratio of 1umol solid phase support to 1.25ml TEA·3HF (7.7mmol), monitor the pH to be acidic, then add 25% concentrated ammonia water to the solution to adjust the pH back to 9.0. Place the desiliconization reaction in a 50℃ water bath for 4h and then take it out.

[0105] S8: 1 M Tris-HCl (pH=8.0) was added to quench the reaction in an ice-water bath to obtain a crude product solution.

[0106] S9: Purification

[0107] Using HIC (hydrophobic chromatography) purification method, the crude product system with complex components can be directly loaded onto the sample without the need for ethanol precipitation step.

[0108] HIC purification used a Phenyl-15L chromatography column (specification: 4.6mm×250mm), mobile phase A used a high salt system of 10mM phosphate + 3M sodium chloride + 10% acetonitrile (pH=9.2±0.2), and mobile phase B used a low salt system of 10mM phosphate + 10% acetonitrile (pH=9.2±0.2). Before purification, the crude product system was diluted with mobile phase A to a conductivity of >120mS / cm, and then filtered with a 0.22um filter membrane. The flow rate was 150cm / h, and UV was used to monitor three wavelengths of 290nm, 260nm and 208nm. At the beginning, 100% mobile phase A was used for elution. After the DMF-OFF impurities of the failed sequence were eluted (UV260nm<50mAu), a gradient of 0-100% B (15min) was set to elute the DMT-ON component with low salt. Figure 3 A comparison of the results before and after HIC purification is shown.

[0109] S10: DMT acid hydrolysis and reverse phase secondary purification to obtain pure sgRNA. DMT acid hydrolysis uses 80% acetic acid solution. After adding 80% acetic acid to the HIC purified and piped solution, adjust the pH to 4.0-4.2. After reacting at room temperature for 2 hours and confirming the completion of the reaction by HPLC, add 1M NaOH solution to adjust the pH to 7.0, and then the crude product is reverse-phase purified. The reverse phase purification method uses a universal C18 purification column (specification 4.6mm×150mm), mobile phase A is 0.1M DBAA (di-n-butylamine acetate), and mobile phase B is acetonitrile. The crude product solution of DMT acid hydrolysis and pH adjustment is diluted with 0.5 times the volume of mobile phase A, filtered through 0.22um, and directly loaded with the sample, the flow rate is 150cm / h, and UV monitoring is 290nm, 280nm and 260nm. The gradient was set to 0-20% B (1CV), 20-80% B (60CV), and fractions were collected when UV290nm>10mAu. The fraction per tube was 0.2CV. Collection was stopped when UV290nm<10mAu. The final pure product was obtained after HPLC confirmation. Figure 4 The HPLC chart of the final pure product shows a purity of 88.9%.

[0110] Example 2

[0111] Compared with Example 1, the only difference is that step S7 is different. In this embodiment, 25% concentrated ammonia water is added to the solution to adjust the pH value back to 8.

[0112] Comparative Example 1

[0113] Compared with Example 1, the only difference is that, in step S7, 25% concentrated ammonia water is added to the solution to adjust the pH value to 7.

[0114] Comparative Example 2

[0115] Compared with Example 1, the only difference is that, in step S7, 25% concentrated ammonia water is added to the solution to adjust the pH value to 6.

[0116] Comparative Example 3

[0117] To prepare long-chain oligonucleotides according to the aminolysis followed by ethanol precipitation process, it is necessary to add 3M sodium acetate and 5 times the volume of ethanol after quenching the reduction reaction, and let it stand overnight at 2-8°C. The next day, take it out and centrifuge it for several hours. After washing the precipitate several times, the solid crude product can be obtained before the next step of purification.

[0118] This method for preparing long-chain oligonucleotides requires at least 1 day more preparation cycle than the preparation process provided in Example 1 of the present invention.

[0119] Experimental Example 1

[0120] This experimental example studies the stability of the crude solution at different pH values ​​(6.0, 7.0, 8.0 and 9.0) at -20°C. It is found that the purity of the crude DMT-ON is 45.9% at pH = 9.0, 33.7% at pH = 8.0, 14.2% at pH = 7.0, and almost 0 at pH = 6.0. Figure 2 Therefore, the present invention uses the condition of pH=9.0 to carry out the desiliconization reaction in water.

[0121] In summary, the present invention uses pH = 9.0 to carry out the desiliconization reaction in a one-pot method, which is based on comprehensive consideration of the stability of the 5'-O-DMT protecting group and the stability of natural RNA, and is obtained by studying and screening the stability of 5'-O-DMT and the purity of crude products at different pH. 5'-O-DMT is sensitive to acid, and natural RNA will produce chain breaks due to the phosphate exchange reaction at positions 2 and 3 under alkaline conditions. Affecting the stability of long-chain oligonucleotide-5'-O-DMT.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a long-chain oligonucleotide, characterized in that: It includes the following steps: S1: contacting the solid phase support bound with nucleotides with a deprotection reagent to remove the protecting group on the 5'-OH of the nucleotides; S2: adding monomer and activator to carry out coupling reaction; S3: oxidation or sulfurization of the coupling product; S4: Capping reaction: adding capping reagent to carry out capping reaction; S5: Repeat S1-S4 until a long-chain oligonucleotide is synthesized; S6: cyanoethyl removal reaction; S7: Ammonolysis and desiliconization reaction: place the solid phase carrier in an ammonolysis reagent for reaction, contact each 1umol of the solid phase carrier with 53.5-58.9mmol or 1-1.1ml of the ammonolysis reagent, react in a water bath at 35℃-45℃ for 14-20 hours, filter, take the filtrate and rinse with an alcohol solution, add DMSO at a ratio of 1-1.5ml DMSO per 1umol of the solid phase carrier, add a reducing agent at a ratio of 1.25-1.5ml or 7.7-9.2mmol of the reducing agent to the solution, adjust the pH to 8.0-9.0, react in a water bath at 50℃±0.5℃ for 4-4.5h; quench the reaction; The reducing agent is selected from fluorine-containing reagents; S8: Quenching reaction: quenching the reaction with Tris-HCl; S9： Purification: The reaction solution after quenching the reaction is loaded onto a hydrophobic chromatography column for hydrophobic chromatography.

2. The method for preparing a long-chain oligonucleotide according to claim 1, characterized in that: In step S9, the chromatographic column used for the hydrophobic chromatography is a Phenyl-15L chromatographic column with a specification of 4.6 mm×250 mm. The mobile phase A is a high salt system, which includes 0-20 mM phosphate, 1.5-3 M sodium chloride and 0-20% acetonitrile, and the pH is 9.2±0.

2. The mobile phase B is a low salt system, which includes 0-20 mM phosphate and 0-20% acetonitrile, and the pH is 9.2±0.

2.

3. The method for preparing a long-chain oligonucleotide according to claim 2, characterized in that: Before purification, the reaction solution after quenching reaction was diluted with mobile phase A to a conductivity of >120mS / cm, then filtered and loaded with sample, the flow rate was 150cm / h, and the chromatographic peak at 260nm was monitored; 100% mobile phase A was used for elution until UV260nm <50mAu, and gradient elution was performed, the elution program of gradient elution was 0-100%, mobile phase B; Preferably, the reaction solution after the quenching reaction is diluted and filtered with a 0.22 um filter membrane.

4. The method for preparing a long-chain oligonucleotide according to claim 1, characterized in that: In step S7, the fluorine-containing reagent is triethylamine trihydrofluoride; the reagent for adjusting pH is selected from ammonia water or triethylamine; Preferably, the filtrate is rinsed with 50% ethanol.

5. The method for preparing a long-chain oligonucleotide according to claim 1, characterized in that: After step S9, the process further includes acidolysis of DMT: contacting an 80% acetic acid solution with the eluate obtained by the hydrophobic chromatography of S9, with a pH of 4.0 to 4.2, and reacting at room temperature for 1 to 2.5 hours.

6. The method for preparing a long-chain oligonucleotide according to claim 5, characterized in that: The DMT acid hydrolysis further includes reverse phase purification, and the reverse phase purification includes the following steps: first adjusting the pH of the DMT acid hydrolysis reaction solution to 7.0±0.5, then diluting the DMT acid hydrolysis reaction solution, filtering, and loading the filtrate onto a reverse phase chromatography column for elution; Preferably, the sample flow rate is 150 cm / h, the chromatographic peak at 290 nm is monitored, and the following elution gradient is used for elution: 0-20% mobile phase B, 1CV; 20-80% mobile phase B, 60CV, and fractions are collected when UV290nm>10mAu, and collection is stopped when UV290nm<10mAu.

7. The method for preparing a long-chain oligonucleotide according to claim 6, characterized in that: The reverse phase purification conditions include: using a C18 purification chromatography column with a specification of 4.6 mm×150 mm, mobile phase A is 0.1 M DBAA, and mobile phase B is acetonitrile.

8. The method for preparing a long-chain oligonucleotide according to claim 1, characterized in that: The ammonolysis reagent is selected from any one of the following: concentrated aqueous ammonia, AMA (ammonia methylamine solution), 4:1 (v / v) aqueous ammonia ethanol solution, EAMA (ethanol AMA solution), and a mixed solution of diethylenetriamine, alkaline aqueous solution and alcohol.

9. The method for preparing a long-chain oligonucleotide according to claim 1, characterized in that: The monomer is a ribonucleotide monomer with 2'-O-TBDMS.

10. The method for preparing a long-chain oligonucleotide according to claim 9, characterized in that: The monomer is selected from a natural RNA monomer with 2'-O-TBDMS and / or a natural RNA monomer with 2'-O-TBDMS and nucleotide modification; Preferably, the nucleotide modification is selected from one or more of ribose modification, base modification and phosphate backbone modification; Preferably, the modification is selected from one or more of fluoro modification, thio modification, 2-methoxy modification, cholesterol modification, and LNA modification.

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