Method for preparing and purifying nucleic acid single strand

Abstract

The invention provides a method for synthesizing a nucleic acid single strand, which comprises the following steps: i) providing nucleoside, the nucleoside comprises a protecting group at the 5 '-position, the protecting group at the 5'-position of the nucleoside is removed, and free 5 '-hydroxyl is generated, ii) adding an activator and nucleoside in a molar ratio of 1.5: 1 or more, the activator is 5-(benzylthio)-1H-tetrazole (BTT), the added nucleoside respectively comprises a phosphorous acid group and a protecting group at the 3'-position and the 5 '-position, and the molar ratio of the phosphorous acid group to the protecting group at the 5'-position to the protecting group at the 5 '-position to the protecting group at the 5'-position is 1.5: 1 or more. The method comprises the following steps: (1) adding a phosphorous acid group at the 3'position of the nucleoside into the nucleoside, reacting the 5 '-hydroxyl of the nucleoside in the previous step with the phosphorous acid group at the 3' position of the added nucleoside to generate a phosphorous acid triester bond, oxidizing the phosphorous acid triester bond into a phosphotriester bond, and acetylating the free 5 '-hydroxyl which does not react with the added nucleoside, (2) removing the protecting group at the 5' position of the nucleoside added in the step (2) to generate the free 5 '-hydroxyl, and (3) removing the protecting group at the 3' position of the added nucleoside to generate the free 5 '-hydroxyl. Iv) repeating steps ii) and iii) to add nucleosides to the nucleic acid single strand until the last but one nucleoside is added, and v) executing step ii) to add the last nucleoside to the nucleic acid single strand.

Description

[0001] Cross-reference

[0002] This invention claims the priority of the Chinese patent application with the application number 202311731694.1 filed on December 14, 2023, the entire content of which is incorporated herein by reference. Technical field

[0003] This application relates to a nucleic acid single strand, including DNA single strand and RNA strand, especially the preparation process of RNA strand, which is particularly suitable for the large-scale (e.g., gram (g) level) preparation of long-chain nucleic acid single strands, such as long-chain RNA (e.g., RNA of 100 nt). This application also relates to a method for purifying nucleic acid single strands using hydrophobic chromatography. Background art

[0004] With the rise of technologies such as RNA vaccines and CRISPR, the applications of RNA molecules have become more diverse and received increasing attention.

[0005] RNA molecules are mainly composed of uridine (U), cytidine (C), guanosine (G), and adenosine (A). Compared with DNA molecules, RNA molecules have the problem of unstable structure due to reasons such as being basically in a single-stranded form and the free 2'-OH of the pentose being prone to attacking the phosphate group at the 3'-position, resulting in hydrolysis. Therefore, in order to keep RNA molecules stable and thus play their intended functions normally, certain chemical modifications are often required on nucleosides, including, for example, N6-methyladenosine (m 6 A), N6,2'-O-dimethyladenosine (m 6 Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m 5 C), and N4-acetylcytidine (ac 4 C), etc.

[0006] The methods for preparing RNA include plasmid production, in vitro transcription, chemical synthesis, etc. Among them, chemical synthesis is the best choice when high sequence accuracy is required or there are requirements for nucleotide modifications (especially the use of specific modified nucleosides at specific positions). In the mainstream solid-phase synthesis method, nucleoside phosphoramidite monomers are mainly used to synthesize nucleotide chains in the 3' to 5' direction, and the reactive amino and hydroxyl groups in the nucleoside phosphoramidite monomers are both protected with protecting groups to avoid unnecessary side reactions. Specifically, the synthesis process includes: 1) removing the 5'-protecting group of the nucleoside linked to the solid support; 2) adding the phosphoramidite monomer to activate its 3'-phosphate group; 3) allowing the 5'-OH of the nucleoside linked to the solid phase to attack the phosphorus in the 3'-group of the added phosphoramidite monomer to form a phosphite triester bond, completing the "coupling" of the nucleoside and the phosphoramidite; 4) oxidizing the phosphite triester to a stable phosphate triester; 5) capping and blocking the unreacted 5'-OH linked to the solid support to prevent it from participating in subsequent cycles. According to the sequence and length of the nucleotide chain to be synthesized, the above 1)-5) are cycled. After the synthesis is completed, the nucleotide chain is cleaved from the solid support, the protecting groups are removed, such as the group protecting the 2'-OH or the group protecting the free oxygen of the phosphate, and purification is carried out.

[0007] In each cycle of nucleotide chain extension, there is a possibility that the newly added phosphoramidite monomer fails to "couple" with the nucleotide linked to the solid phase as planned. The nucleotide chains in which this situation occurs will be capped and blocked to terminate the reaction and removed in the final purification step. As the length of the nucleotide chain to be synthesized increases, the number of nucleotide chains eliminated increases significantly, resulting in an exponential decrease in the yield of nucleotide chain synthesis. After the purification step, it is possible to obtain only 3-5% of the theoretical yield of RNA. In the clinical application of RNA molecules, for example, in cell therapy, the sgRNA that needs to be delivered into the body to participate in cell editing usually requires an amount at the "gram (g)" level.

[0008] The synthesizers currently used on the market, even large-scale synthesizers such as AKTA oligopilotplus100, are difficult to synthesize a large amount of 100-nt RNA molecules. In the current market, generally, the requirement for RNA at the "gram (g)" level is met by synthesizing in small amounts multiple times and finally combining them. However, the disadvantages of doing so are high cost and long preparation cycle. Summary of the Invention

[0009] The inventors of the present application achieved RNA synthesis, especially the synthesis of long RNA chains (e.g., 100 nt), with a shorter preparation cycle, lower preparation cost, and higher yield by i) optimizing the molar ratio of the activator to the phosphoramidite monomer in the RNA synthesis process, ii) selecting a suitable activator, iii) optimizing the coupling reaction time according to different nucleosides, iv) selecting a suitable purification method, v) optimizing the parameters in the purification method, etc. This method is applicable to the synthesis of ordinary RNA and also to the synthesis of highly modified RNA.

[0010] Based on the principle of solid-phase synthesis, the present application is also applicable to the large-scale synthesis of single-stranded DNA, with the only difference being the selection of nucleoside monomers with deoxyribose.

[0011] Thus, in the first aspect, the present application provides a method for preparing a single-stranded nucleic acid, which may include:

[0012] i) Providing a nucleoside that contains a protecting group at the 5'-position, removing the protecting group at the 5'-position of the nucleoside to generate a free 5'-hydroxyl group,

[0013] ii) Adding an activator and a nucleoside in a molar ratio of 1.5:1 or more. The added nucleoside contains phosphite groups at the 3'- and 5'-positions and a protecting group, such that the 5'-hydroxyl group of the nucleoside from the previous step reacts with the 3'-phosphite group of the added nucleoside in the presence of the activator to form a phosphite triester bond, then converting the phosphite triester bond to a phosphate triester bond in the presence of an oxidant, and then acetylating the free 5'-hydroxyl group that did not react with the added nucleoside to form a phosphite triester bond.

[0014] iv) Repeating steps ii) and iii) to add nucleosides to the single-stranded nucleic acid until the penultimate preset nucleoside is added, and

[0015] v) Performing step ii) to add the last nucleoside to the single-stranded nucleic acid.

[0016] The activator added in step ii) can be 5-(benzylthio)-1H-tetrazole (BTT), 5-ethylthiotetrazole (ETT), or 4,5-dicyanoimidazole (DCI). In particular, the activator added in step ii) can be 5-(benzylthio)-1H-tetrazole (BTT). The concentration of BTT can be, for example, 0.3 M.

[0017] The nucleoside provided in step i) and the nucleoside added in step ii) can be in a molar ratio of, for example, 1:1.

[0018] In step ii), the activator and the nucleoside can be added in a molar ratio of 1.5:1 or more. For example, in step ii), the activator and the nucleoside can be added in a molar ratio of 1.5:1 or more, 2.0:1 or more, 2.5:1 or more, or 3.0:1 or more, and 6.0:1 or less, 5.5:1 or less, 5.0:1 or less, or 4.5:1 or less. In some embodiments, in step ii), the activator and the nucleoside can be added in a molar ratio of 3.0:1 - 4.5:1. In some embodiments, in step ii), the activator and the nucleoside can be added in a molar ratio of 3.0:1.

[0019] When uridine / thymidine, cytidine, or adenosine is added in step ii), the reaction in which the 5'-hydroxyl group of the nucleoside obtained from the previous step reacts with the 3'-phosphite group of the added nucleoside to form a phosphite triester bond lasts for 15 minutes; when guanosine is added in step ii), the reaction in which the 5'-hydroxyl group of the nucleoside obtained from the previous step reacts with the 3'-phosphite group of the added nucleoside to form a phosphite triester bond lasts for 20 minutes.

[0020] The nucleoside provided in step i) can be a phosphoramidite monomer. The nucleoside provided in step i) can include a protecting group on each reactive amino or hydroxyl group. In the case where the nucleic acid single strand is an RNA strand, the nucleoside provided in step i) can include protecting groups at the 2'-position, 3'- and 5'-positions. In the case where the nucleic acid single strand is a DNA strand, the nucleoside provided in step i) can include protecting groups at the 3'- and 5'-positions. The nucleoside provided in step i) can include tert-butyldimethylsilyl (TBS), fluoro, methoxyethyl (MOE), locked nucleic acid (LNA), or methyl as a protecting group at the 2'-position. The nucleoside provided in step i) can include a phosphite group at the 3'-position. The phosphite group can include diisopropylamino and a protecting group, and the protecting group can be 2-cyanoethyl. The nucleoside provided in step i) can include 4,4'-dimethoxytriphenylmethyl (DMT) as a protecting group at the 5'-position. The nucleoside provided in step i) can include, but is not limited to, DMT-2'-O-TBDMS-A(Bz)-CE phosphoramidite, DMT-2'-O-TBDMS-G(iBu)-CE phosphoramidite, DMT-2'-O-TBDMS-C(Ac)-CE phosphoramidite, DMT-2'-O-TBDMS-U-CE phosphoramidite, DMT-2'-O-Me-A(Bz)-CE phosphoramidite, DMT-2'-O-Me-G(iBu)-CE phosphoramidite, DMT-2'-O-Me-C(Ac)-CE phosphoramidite, and DMT-2'-O-Me-U-CE phosphoramidite.

[0021] The nucleoside added in step ii) can be a phosphoramidite monomer. The nucleoside added in step ii) can contain a protecting group on each reactive amino or hydroxyl group. In the case where the nucleic acid single strand is an RNA strand, the nucleoside provided in step ii) can contain protecting groups at the 2'-position, 3'- and 5'-positions. In the case where the nucleic acid single strand is a DNA strand, the nucleoside provided in step ii) can contain protecting groups at the 3'- and 5'-positions. The nucleoside added in step ii) can contain tert-butyldimethylsilyl (TBS), fluoro, methoxyethyl (MOE), locked nucleic acid (LNA), or methyl as a protecting group at the 2'-position. The nucleoside added in step ii) can contain 4,4'-dimethoxytriphenylmethyl (DMT) as a protecting group at the 5'-position. The nucleoside added in step ii) can contain a phosphite group at the 3'-position. The phosphite group can contain diisopropylamino and a protecting group, which can be 2-cyanoethyl. The nucleoside added in step ii) can include, but is not limited to, DMT-2'-O-TBDMS-A(Bz)-CE phosphoramidite, DMT-2'-O-TBDMS-G(iBu)-CE phosphoramidite, DMT-2'-O-TBDMS-C(Ac)-CE phosphoramidite, DMT-2'-O-TBDMS-U-CE phosphoramidite, DMT-2'-O-Me-A(Bz)-CE phosphoramidite, DMT-2'-O-Me-G(iBu)-CE phosphoramidite, DMT-2'-O-Me-C(Ac)-CE phosphoramidite, and DMT-2'-O-Me-U-CE phosphoramidite. The nucleoside added in step ii) can be in, for example, acetonitrile. The nucleoside added in step ii) can be at a concentration of 0.1 - 0.3 M, especially 0.2 M. In some embodiments, the nucleoside added in step ii) can be at a concentration of 0.1 - 0.3 M, especially 0.2 M, in acetonitrile.

[0022] In step i) and / or step iii), the 5'-position protecting group of the nucleoside can be removed by acid treatment, for example, treatment with trichloroacetic acid or dichloroacetic acid. The amount of trichloroacetic acid or dichloroacetic acid can be controlled by observing the UV value, and the addition of trichloroacetic acid or dichloroacetic acid is stopped when the UV is lower than, for example, 500.

[0023] In step ii), the phosphite triester bond can be oxidized to an oxo-phosphate ester or a phosphorothioate ester.

[0024] In step ii), the phosphite triester bond can be converted to a phosphate triester bond by iodine in the presence of water and pyridine. For example, a 0.05 M iodine solution in pyridine / water 90 / 10 (v / v) can be used. The iodine solution in pyridine / water can be added in an amount of a 2:1 molar ratio to the nucleoside. The reaction of converting the phosphite triester bond to a phosphate triester bond by iodine in the presence of water and pyridine can last for about 2 minutes.

[0025] In step ii), bis(benzoyl) disulfide and xylanthrene hydride can be used as sulfur transfer reagents with Beaucage reagent, and the phosphoramidite method can be used to synthesize oligonucleotide phosphorothioates.

[0026] In step ii), acetylating the free 5'-hydroxyl group of the nucleoside that did not react with the added nucleoside to form a phosphite triester bond in the previous step can include forming an intermediate from acetic anhydride and N-methylimidazole in tetrahydrofuran containing pyridine, and reacting the intermediate with the 5'-OH of the nucleoside to generate an acetylated 5'-OH. The reaction for acetylating the free 5'-hydroxyl group of the nucleoside that did not react with the added nucleoside to form a phosphite triester bond in the previous step can be carried out for about 2 minutes.

[0027] The nucleoside in step i) can, in some embodiments, be linked to a solid support. For example, the nucleoside in step i) can be linked to the solid support via a 3'-position group, such as a 3'-O-linker group. The solid support can be controlled pore glass (CPG) or polystyrene (PS). In some embodiments, the nucleoside in step i) can contain a phosphite group at the 3'-position, and the surface of the solid support contains a hydroxyl group. By reacting the phosphite group on the nucleoside with the hydroxyl group on the surface of the solid support, the nucleoside in step i) is linked to the solid support. The reaction between the phosphite group on the nucleoside and the hydroxyl group on the surface of the solid support can be carried out in the presence of an activator such as BTT. In some embodiments, the method can include, prior to step i), a step of linking the nucleoside in step i) to the solid support. The nucleoside can be reacted with the surface group of the solid support at a molar ratio of about 3:1. The protecting group, such as DMT, on the surface reaction group of the solid support can be removed by acid treatment, such as treatment with trichloroacetic acid or dichloroacetic acid. In some embodiments, the method of the present application can further include, after step v), a step of cleaving the synthesized single-stranded nucleic acid from the solid support. The cleavage can be carried out, for example, by treating the solid support with concentrated ammonia water. In some embodiments, the solid support is treated with concentrated ammonia water at 65°C and 160 rpm.

[0028] In some embodiments, the surface of the solid support may contain reactive groups (such as hydroxyl groups) protected by protecting groups. Through acid treatment, such as treatment with trichloroacetic acid or dichloroacetic acid, the protecting groups on the reactive groups on the surface of the solid support are removed. Then, nucleosides are provided to the solid support at a molar ratio of nucleoside:reactive group on the surface of the solid support of about 3:1, and an activator is added at a molar ratio of activator:nucleoside of 1.5:1 or more, 2.0:1 or more, 2.5:1 or more, or 3.0:1 or more, and 6.0:1 or less, 5.5:1 or less, 5.0:1 or less, or 4.5:1 or less, especially 3.0:1 - 4.5:1. Activator BTT is added to carry out the linking reaction between the nucleoside and the reactive group on the surface of the solid support.

[0029] In some embodiments, the solid support may contain 900 μmol or more of groups capable of linking with nucleosides. In some embodiments, the solid support may contain 900 μmol or more of groups capable of linking with nucleosides. The nucleoside in step i) can be obtained by providing 2700 μmol of nucleosides and 12150 μmol of BTT to the solid support.

[0030] In some embodiments, the solid support may contain 1200 μmol or more of groups capable of linking with nucleosides. In some embodiments, the solid support may contain 1200 μmol or more of groups capable of linking with nucleosides. The nucleoside in step i) can be obtained by providing 3600 μmol of nucleosides and 16200 μmol of BTT to the solid support.

[0031] The method of the present application may further include the step of removing the protecting group at the 2'-position of the nucleoside (in the case of synthesizing an RNA strand), the protecting group on the 3'-phosphite / phosphate group, and / or the protecting group on the base as needed. The protecting group at the 2'-position (in the case of synthesizing an RNA strand), the protecting group on the 3'-phosphite / phosphate group, or the protecting group on the base can be removed by treatment under basic conditions, such as adding hot concentrated ammonia water. The protecting group on the base may include, but is not limited to, N(6)-benzoyl, N(4)-benzoyl, and N(2)-isobutyryl. The protecting group at the 2'-position may include, but is not limited to, tert-butyldimethylsilyl (TBS). The protecting group on the 3'-phosphite / phosphate group may be 2-cyanoethyl. In some embodiments, the protecting group at the 2'-position, such as tert-butyldimethylsilyl (TBS), is removed by using a solution containing DMSO, triethylamine trihydrofluoride, and triethylamine.

[0032] The method of the present application can be carried out in a nucleic acid synthesizer. In particular, the method of the present application can be carried out in a large-scale nucleic acid synthesizer, for example, it can be carried out in a nucleic acid synthesizer with a synthesis scale of more than 50 μmol. In some embodiments, the method of the present application is carried out in an AKTA oligopilot plus synthesizer, such as an AKTA OLIGOPILOT 10 / 100 synthesizer.

[0033] The length of the single-stranded nucleic acid to be synthesized by the method of the present application can be 10 nt or more, 20 nt or more, 30 nt or more, 40 nt or more, 50 nt or more, 60 nt or more, 70 nt or more, 80 nt or more, 90 nt or more, or 100 nt or more. In some embodiments, the length of the single-stranded nucleic acid to be synthesized by the method of the present application can be 80 nt or more, particularly 90 nt or more, particularly 100 nt - 200 nt. In some embodiments, the length of the single-stranded nucleic acid to be synthesized by the method of the present application can be 100 nt.

[0034] The single-stranded nucleic acid can be an RNA strand or a DNA single strand, particularly an RNA strand. For example, the RNA strand can be a small nucleic acid, and the small nucleic acid includes, but is not limited to, oligonucleotide molecules such as small interfering nucleic acid (siRNA), antisense nucleic acid (ASO), microRNA (miRNA), and aptamer (Aptamer); the RNA strand can be an RNA strand used as sgRNA, which can be a single-stranded sgRNA or a double-stranded form of crRNA and tracrRNA.

[0035] In some embodiments, the single-stranded nucleic acid can be an RNA strand, and the method can include: i) providing a nucleoside that contains protecting groups at the 2'-position and 5'-position, removing the protecting group at the 5'-position of the nucleoside to generate a free 5'-hydroxyl group; ii) adding an activator and the nucleoside in a molar ratio of 1.5:1 or more, where the activator is 5-(benzylthio)-1H-tetrazole (BTT), and the added nucleoside contains protecting groups, a phosphite group, and a protecting group at the 2'-position, 3'-position, and 5'-position respectively, such that the 5'-hydroxyl group of the nucleoside from the previous step reacts with the phosphite group at the 3'-position of the added nucleoside in the presence of the activator to form a phosphite triester bond, converting the phosphite triester bond to a phosphate triester bond by oxidation, and acetylating the free 5'-hydroxyl group of the nucleoside from the previous step that did not react with the added nucleoside to form a phosphite triester bond; iii) removing the protecting group at the 5'-position of the nucleoside added in step ii) to generate a free 5'-hydroxyl group; iv) repeating steps ii) and iii) to add nucleosides to the RNA strand until the penultimate nucleoside is added; and v) performing step ii) to add the last nucleoside to the RNA strand.

[0036] The method of the present application may further include a step of purifying the synthesized single-stranded nucleic acid after step v). For example, the synthesized single-stranded nucleic acid can be purified by hydrophobic chromatography, reverse-phase HPLC, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, or polyacrylamide gel purification. In some embodiments, the synthesized single-stranded nucleic acid contains a hydrophobic group, and the method may include subjecting the synthesized single-stranded nucleic acid to hydrophobic chromatography. The hydrophobic group in the synthesized single-stranded nucleic acid can be 4,4'-dimethoxytrityl (DMT).

[0037] In some embodiments, the method of the present application may include, after step v), a step of cleaving the synthesized single-stranded nucleic acid from the solid-phase support and subjecting the cleaved single-stranded nucleic acid to hydrophobic chromatography. In some embodiments, the method of the present application may include, after step v), a step of cleaving the synthesized single-stranded nucleic acid from the solid-phase support, removing some or all of the protecting groups other than the protecting group at the 5'-position of the last nucleoside as needed, and subjecting the single-stranded nucleic acid to hydrophobic chromatography. The single-stranded nucleic acid subjected to hydrophobic chromatography may contain a hydrophobic group, such as 4,4'-dimethoxytrityl (DMT) at the 5'-position of the last nucleoside. Some or all of the protecting groups other than the protecting group at the 5'-position of the last nucleoside, such as the protecting group at the 2'-position, the protecting group on the 3'-phosphite / phosphate group, or the protecting group on the base, can be removed by treatment under basic conditions, such as adding hot concentrated ammonia water.

[0038] For hydrophobic chromatography, a hydrophilic polymethacrylate can be used as the packing material, and the surface of the hydrophilic polymethacrylate may contain a hydrophobic group. The hydrophobic group can be phenyl or butyl, especially phenyl. The particle size of the packing material can be 30 - 80 μm, especially 60 μm. In some embodiments, hydrophobic chromatography can use a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 32 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 35 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 60 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 80 μm, or a hydrophilic polymethacrylate with a butyl group on the surface and a particle size of 32 μm. In some embodiments, hydrophobic chromatography can use a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 60 μm as the packing material.

[0039] Hydrophobic chromatography can use a buffer containing (NH4)2SO4, Na2SO4, or NaCl as the mobile phase, especially a buffer containing (NH4)2SO4 or Na2SO4. In particular, hydrophobic chromatography can use a buffer containing (NH4)2SO4 as the mobile phase, especially a PBS or Tris solution containing (NH4)2SO4. In some embodiments, hydrophobic chromatography uses a PBS solution containing 0.4 M (NH4)2SO4 as the initial mobile phase and gradually reduces the concentration of (NH4)2SO4 to 0 during the purification process.

[0040] The method of the present application can further include steps of ultrafiltration desalting and / or high performance liquid chromatography after hydrophobic chromatography.

[0041] In one embodiment, the method of the present application can include, after step v), cleaving the synthesized single-stranded nucleic acid from the solid support, removing some or all of the protecting groups except the 5'-position protecting group on the last nucleoside as needed, and performing high performance liquid chromatography on the single-stranded nucleic acid.

[0042] In one embodiment, the method of the present application can include, after step v), cleaving the synthesized single-stranded nucleic acid from the solid support, removing some or all of the protecting groups except the 5'-position protecting group on the last nucleoside as needed, and performing hydrophobic chromatography, ultrafiltration desalting, and high performance liquid chromatography on the single-stranded nucleic acid.

[0043] In some embodiments, the method of the present application is a method for preparing gram-scale single-stranded nucleic acids, especially gram-scale purified single-stranded nucleic acids, especially gram-scale purified long-chain nucleic acids.

[0044] In some embodiments, the method of the present application is a method for preparing gram-scale RNA chains, especially gram-scale purified RNA chains.

[0045] In a second aspect, the present application can provide a method for purifying a single-stranded nucleic acid, including performing hydrophobic chromatography on the single-stranded nucleic acid, wherein the single-stranded nucleic acid contains a hydrophobic group.

[0046] The single-stranded nucleic acid can be a chemically synthesized nucleic acid, such as a nucleic acid synthesized by the phosphoramidite method, especially a nucleic acid synthesized by the solid-phase phosphoramidite method, especially a crude synthetic product containing the single-stranded nucleic acid. In some embodiments, the single-stranded nucleic acid can be a single-stranded nucleic acid synthesized by the method of the present application, especially a crude synthetic product containing the single-stranded nucleic acid.

[0047] The hydrophobic group contained in the single-stranded nucleic acid can be 4,4'-dimethoxytrityl (DMT). When the single-stranded nucleic acid is synthesized by the method of the present application, the hydrophobic group contained in the single-stranded nucleic acid can be the hydrophobic protecting group at the 5'-position of the last nucleoside, such as 4,4'-dimethoxytrityl (DMT).

[0048] Hydrophobic chromatography can use a hydrophilic polymethacrylate as the packing material, where the surface of the hydrophilic polymethacrylate contains hydrophobic groups. The hydrophobic groups can be phenyl or butyl, especially phenyl. The particle size of the packing material is 30 - 80 μm, especially 60 μm. In some embodiments, hydrophobic chromatography can use a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 32 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 35 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 60 μm, a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 80 μm, or a hydrophilic polymethacrylate with a butyl group on the surface and a particle size of 32 μm. In some embodiments, hydrophobic chromatography can use a hydrophilic polymethacrylate with a phenyl group on the surface and a particle size of 60 μm as the packing material.

[0049] Hydrophobic chromatography can use a buffer solution of (NH4)2SO4, Na2SO4 or NaCl as the mobile phase, especially a buffer solution of (NH4)2SO4 or Na2SO4. In particular, hydrophobic chromatography can use a buffer solution of (NH4)2SO4 as the mobile phase, especially a PBS or Tris solution of (NH4)2SO4. In some embodiments, hydrophobic chromatography uses a PBS solution of 0.4 M (NH4)2SO4 as the initial mobile phase and gradually reduces the concentration of (NH4)2SO4 to 0 during the purification process.

[0050] The purification method of the present application can further include steps of ultrafiltration desalting and / or high performance liquid chromatography after hydrophobic chromatography.

[0051] The RNA synthesis method and purification method of the present application solve the technical problem that it is difficult to achieve large-scale synthesis of long nucleic acid chains, especially long-chain RNA. In particular, the synthesis method of the present application improves the synthesis yield, shortening the production time for obtaining gram-level nucleic acid single strands such as RNA. The purification method of the present application enables more truncated nucleic acid single strands that have not completed the full-chain synthesis to be quickly and effectively removed, reducing the cost, increasing the purity, and shortening the purification time. At the same time, the method of the present application is also applicable to the large-scale production of nucleic acid broken chains such as short-chain RNA.

[0052] In particular, when the solid support used contains more than 900 μmol, especially more than 1200 μmol of groups capable of linking to nucleosides, the nucleic acid single-strand synthesis method of the present application, combined with hydrophobic chromatography, ultrafiltration desalting and HPLC, can obtain gram-level purified long nucleic acid single strands, such as long-chain RNA.

[0053] Based on the following specific descriptions and examples, other features and advantages of the current disclosure will become more apparent. The specific descriptions and examples should not be construed as restrictive. The content of all documents, Genbank records, patents, and published patent applications cited in this application are expressly incorporated herein by reference. Description of the Drawings

[0054] The following specific descriptions are given by way of example and are not intended to limit the present invention to the specific embodiments described. It can be better understood in conjunction with the drawings.

[0055] Figure 1 The HPLC peak charts of the synthetic products are shown when using a molar ratio of 1.5:1 (activator: phosphoramidite monomer) and a coupling reaction time of 15 min (A), using a molar ratio of 1.5:1 (activator: phosphoramidite monomer) and a coupling reaction time of 10 min (B), and using a molar ratio of 3:1 (activator: phosphoramidite monomer) and a coupling reaction time of 15 min (C).

[0056] Figure 2 The elution peak chart (A) during hydrophobic chromatography and the HPLC peak chart (B) after hydrophobic chromatography are shown.

[0057] Figure 3 The HPLC peak chart of the long-chain RNA synthesized by the method of this application after hydrophobic chromatography and ultrafiltration desalting is shown. Detailed Description of the Invention

[0058] The terms used herein, unless otherwise specified, have the ordinary meanings in dictionaries, textbooks, technical reference books, or the meanings commonly understood by those skilled in the art. The following descriptions of some terms are only for the purpose of facilitating the understanding of this application and are not intended to specifically limit these terms, unless otherwise specified.

[0059] "Nucleoside" is a component of DNA and RNA, which contains ribose (for RNA) or deoxyribose (for DNA) and a base. Ribose is usually a pentose sugar, and the carbon atom at the 1'-position forms a glycosidic bond with the nitrogen atom in the pyrimidine molecule or the nitrogen atom in the purine molecule, generating an N-glycoside, that is, the furan riboside of pyrimidine or purine, which is called ribonucleoside. A glycosidic bond is formed between the carbon atom at the 1'-position in 2-deoxyribose and the nitrogen atom in the pyrimidine molecule or the nitrogen atom in the purine molecule, becoming the furan deoxyriboside of pyrimidine or purine, which is called deoxyribonucleoside. Common nucleosides include uridine (uracil-1-β-D-ribofuranoside), adenosine (adenine-9-β-D-ribofuranoside), cytidine (cytosine-1-β-D-ribofuranoside), guanosine (guanine-9-β-D-ribofuranoside), and thymidine (thymine-1-β-D-2'-deoxyribofuranoside).

[0060] The "nucleoside" in this application includes the above-mentioned natural nucleoside molecules and their derivatives, including nucleosides, nucleotides, and phosphoramidites containing unnatural sugars or unnatural bases. In this application, when related to the synthesis of RNA, the nucleoside only refers to the nucleoside related to RNA composed of ribose, including uridine, adenosine, cytidine, and guanosine, while when related to the synthesis of DNA, it refers to the nucleoside related to DNA composed of deoxyribose.

[0061] "Nucleotide" refers to a molecule composed of a nucleoside and a phosphate group, which may contain a hydroxyl group at the 5'-position and a phosphate group at the 3'-position, or a hydroxyl group at the 3'-position and a phosphate group at the 5'-position. Correspondingly, "phosphoramidite" refers to a molecule composed of a nucleoside and a phosphite group, and the reactive hydroxyl group and amino group of the nucleoside and the phosphite group contain protecting groups, usually containing a phosphite group at the 3'-position and a protecting group protecting the free hydroxyl group at the 5'-position.

[0062] "Protecting group" refers to a group used to protect the reactive hydroxyl group or amino group on the nucleoside (phosphoramidite) from unexpected reactions. These protecting groups can be removed when needed. During the synthesis of RNA by the phosphoramidite method, the hydrogen in the 2'-OH of the nucleoside can be replaced by a protecting group such as methyl or TBS, the hydrogen in the 5'-OH can be replaced by a protecting group such as DMT, and the hydrogen of the hydroxyl group of the phosphite group at the 3'-position can be replaced by, for example, 2-cyanoethyl.

[0063] The "activator" in this application refers to a compound that can make the phosphite group in a state capable of reacting with a reactive hydroxyl group (especially the 5'-hydroxyl group of a nucleoside), and in particular, a compound that can protonate the diisopropylamino group on the phosphite group, thereby enabling the phosphite group to react with a reactive hydroxyl group (especially the 5'-hydroxyl group of a nucleoside). The activator can be an organic acid, such as ETT (5-ethylthiotetrazole), DCI (4,5-dicyanoimidazole), BTT (5-(benzylthio)-1H-tetrazole).

[0064] "Hydrophobic chromatography" or "HIC" is a process in which the hydrophobicity on the surface of a molecule (such as an RNA molecule) binds to a hydrophobic carrier (such as a packing material) at a high salt concentration and is eluted one by one according to different hydrophobicities during elution with gradually decreasing salt concentration.

[0065] The phosphoramidite method is the gold standard for chemical synthesis of RNA or DNA, which adds nucleosides in sequence in the 3' to 5' direction. Adding each nucleoside undergoes the steps of i) deprotecting the group, ii) coupling, iii) oxidizing, and iv) capping. The most critical step is coupling, that is, the 5'-OH of the nucleoside reacts with the 3'-phosphite group of another nucleoside to connect the two nucleosides, thereby extending the nucleic acid single strand. However, this step is not 100% successful. The nucleic acid single strand that fails to add a new nucleoside will exit the subsequent synthesis but still remain in the reaction system, thus exponentially reducing the subsequent coupling efficiency. Therefore, the yield of RNA synthesis largely depends on the coupling efficiency. Theoretically, assuming the initial coupling efficiency of the reaction is 90%, after 50 cycles, the proportion of the target full-length sequence is about 0.52%. Even if the initial coupling efficiency can reach 98.5%, after 100 cycles, the proportion of the target full-length sequence will also drop to about 22.1%.

[0066] Therefore, in the face of the current demand for "gram (g)" long-chain RNA, the field can only adopt the method of multiple small-scale syntheses and combine the products obtained in several syntheses. This inevitably causes problems such as a long preparation cycle and high costs.

[0067] The inventors of this application designed an optimized synthesis process by screening activators, adjusting the molar ratio of the activator to the phosphoramidite monomer, and / or optimizing the time of each "coupling" step, so that the proportion of the target nucleic acid single strand in the synthesis product is relatively high.

[0068] In addition, by retaining the 5'-position protecting group such as DMT of the last nucleoside in the nucleic acid single strand and taking advantage of its hydrophobicity, the inventor rapidly separates the nucleic acid single strand with DMT from the short nucleic acid chain with 5'-hydroxy acetylation in hydrophobic chromatography. The separation efficiency and throughput are much higher than those of traditional high-performance liquid chromatography, shortening the purification cycle and saving costs. The inventor also screened out the combination with the highest purification efficiency, recovery efficiency and without damaging the stability of RNA molecules by using different fillers and mobile phases. Through hydrophobic chromatography, the purity of the RNA synthesis product can reach about 40%.

[0069] The phosphoramidite method usually uses a solid-phase carrier as the medium, which is convenient for the addition of nucleosides. Specifically, usually the first nucleoside is connected to the solid-phase carrier via a 3'-position group, and then the required nucleosides are added one by one to the nucleoside connected to the solid-phase carrier to achieve the extension of the nucleic acid chain. Connecting the nucleoside to the solid-phase carrier can conveniently introduce the required materials into the carrier and wash away the unreacted or excess materials after the reaction. It is also possible to choose to carry out the phosphoramidite synthesis method in solution, but its convenience and synthesis efficiency are not as good as the solid-phase method.

[0070] The solid-phase carrier usually needs to have 1) chemical stability, that is, except for the initial bonding with the group or linker structure on the nucleoside, the solid-phase carrier neither participates in nor is affected by the subsequent synthesis reaction; 2) good swelling property in some solvents; 3) certain mechanical strength, that is, good anti-abrasion and anti-extrusion properties. Once the carrier particles become powder, it is very difficult to perform the rapid filtration operation. The solid-phase carriers widely used in the solid-phase synthesis method include two types of carriers: controlled-pore glass (CPG) and polystyrene (PS). The CPG carrier has the advantages of good mechanical properties, no swelling, controllable pore size, etc., but it is difficult to achieve a high loading amount (<100 μmol / g). The PS carrier has the advantage of a high loading amount (350 μmol / g) and can be used as an application for large-scale synthesis of oligonucleotide carriers, but it has the disadvantages of a very high swelling property and consuming a large amount of solvent. Specifically, the controlled-pore glass (CPG) is made of silica. There are many irregular pores inside the CPG sphere, and it has pore size stability. Due to space reasons, the CPG with a large pore size is suitable for the synthesis of long fragments, and the CPG with a short pore size is suitable for the synthesis of short fragments. CPG products with a pore size are suitable for the synthesis of oligonucleotides <35 nt, such as therapeutic oligonucleotides, and the synthesis loading amount can be as high as 100 μmol / g. CPG products with a pore size are suitable for the synthesis of oligonucleotides >35 nt or highly modified oligonucleotides. CPG products with pore sizes are suitable for the synthesis of oligonucleotides with a scale of more than 80 nt, and the synthesis loading is usually 10 - 20 μmol / g. Polystyrene-based carriers are composed of beads with diameters of 20 - 150 μm. Monodisperse polystyrene microspheres are crosslinked using a crosslinking agent, usually divinylbenzene, and the crosslinking degree is between 1% and 2%. In this crosslinking degree range, the resin has good swelling properties in DMF and DCM, and is a three-dimensional network structure in terms of sterics, allowing reactant molecules to move freely inside the resin.

[0071] The surface of the solid-phase carrier may contain reactive groups, such as hydroxyl groups, which are protected by protecting groups and are in an inactive state. Before the synthesis of a single-stranded nucleic acid such as an RNA strand begins, it can be treated, for example, with an acid such as trichloroacetic acid or dichloroacetic acid, to remove the protecting groups on the reactive groups on the surface of the solid-phase carrier, such as DMT, so that the reactive group is in a state where it can react with the group on the nucleoside. For example, the reactive group on the surface of the solid-phase carrier can be linked to the phosphite group on the nucleoside in the presence of an activator such as BTT. In some embodiments, the surface of the solid-phase carrier contains reactive groups (such as hydroxyl groups) protected by protecting groups. By acid treatment, such as using trichloroacetic acid or dichloroacetic acid treatment, to remove the protecting groups on the reactive groups on the surface of the solid-phase carrier, and then nucleoside is provided to the solid-phase carrier at a molar ratio of nucleoside:reactive group on the surface of the solid-phase carrier of about 3:1, and the activator is added at a molar ratio of activator:nucleoside of 1.5:1 or more, 2.0:1 or more, 2.5:1 or more, or 3.0:1 or more, and 6.0:1 or less, 5.5:1 or less, 5.0:1 or less, or 4.5:1 or less, especially 3.0:1 - 4.5:1, to carry out the linking reaction between the nucleoside and the reactive group on the surface of the solid-phase carrier.

[0072] There are also some solid-phase carriers on the market whose surfaces contain nucleosides, so that the connection between the first nucleoside and the solid-phase carrier can be omitted and the coupling between nucleosides can be directly carried out.

[0073] In the examples of the present application, a solid-phase carrier with reactive groups (protected by protecting groups) on the surface is used. However, using a solid-phase carrier with nucleosides on the surface, g-level purified single-stranded nucleic acids, especially long RNA strands, especially 100-nt long RNA strands, can also be obtained by the method of the present application.

[0074] The phosphoramidite monomers used in the synthesis method have a structure similar to that of natural nucleotides, except that they contain a phosphite group at the 3'-position instead of a phosphate group, and they contain protecting groups at the reactive hydroxyl or amino groups of natural nucleotides, so that unnecessary reactions do not occur in subsequent synthesis reactions. For example, the phosphoramidite monomers can contain a DMT group at the 5'-position, which replaces the hydrogen in the 5'-hydroxyl group of natural nucleosides, can contain a 2-cyanoethyl protecting group in the 3'-phosphite group, which replaces the hydrogen in the hydroxyl group of the phosphite group, and can contain a methyl or TBS at the 3'-position, which replaces the hydrogen in the 3'-hydroxyl group of natural nucleosides. In addition, the bases on natural nucleosides, such as adenine, cytosine, guanine, etc., all contain exocyclic primary amino groups, and the phosphoramidite monomers can also contain benzene ring-containing protecting groups at these positions. The structures of exemplary phosphoramidite monomers are shown in Table 1.

[0075] Table 1. Names and structural formulas of exemplary phosphoramidite monomers

[0076]

[0077]

[0078] In the phosphoramidite synthesis method, first, the first nucleoside (such as a phosphoramidite monomer) is connected to the solid support via the 3'-position, and its 5'-protecting group is removed to generate a reactive 5'-hydroxyl group. Specifically, the 5'-protecting group can be removed by treatment under acidic conditions, such as treatment with trichloroacetic acid or dichloroacetic acid, to remove the 5'-protecting group such as DMT. At this time, only this 5'-hydroxyl group on the nucleoside is reactive.

[0079] Thereafter, the nucleoside to be added (such as phosphoramidite monomer) and the activator are added to the nucleoside linked to the solid support at a certain ratio. The activator can be an organic acid, which functions to protonate the diisopropylamino group on the 3'-phosphite group, thereby reacting with the 5'-hydroxyl group of the nucleoside linked to the solid support to form a phosphite triester bond. The organic acids that can be used as activators include, but are not limited to, ETT (5-ethylthiotetrazole), DCI (4,5-dicyanoimidazole), and BTT (5-(benzylthio)-1H-tetrazole). When adding BTT and phosphoramidite monomer to the nucleoside linked to the solid support at a molar ratio of activator:phosphoramidite monomer of 1.5:1, BTT shows better synthesis effect than DCI. When using DCI, when synthesizing to 30 nt, since the amount of DMT removed is too low, the instrument cannot continue the synthesis; while when using BTT, a target peak is detected in HPLC. When BTT is selected and the molar ratio of BTT to phosphoramidite monomer is further adjusted, it is found that a molar ratio above 3:1, combined with a coupling reaction time of 15 min, can not only result in the appearance of a target peak in HPLC, but also make MS 100% correct. When the molar ratio of BTT:phosphoramidite monomer is further increased, the above effect is not significantly improved. Therefore, BTT and phosphoramidite monomer can be added within a molar ratio range above 3:1, especially 3:1 - 4.5:1. In terms of cost, adding BTT and phosphoramidite monomer at a molar ratio of 3:1 is sufficient. When the molar ratio of the activator to the phosphoramidite monomer is less than 1.5:1, even if BTT is selected as the activator, the synthesis effect is not satisfactory, and at least it is impossible to obtain g-level purified long nucleic acid single strands such as RNA strands according to the method of this application.

[0080] The phosphite triester bond formed by the reaction of the newly added nucleoside with the nucleoside linked to the solid support is unstable and can be oxidized to a stable phosphate triester. For example, the oxidation of the phosphite triester can be achieved with iodine in the presence of water and pyridine. The product is a phosphate triester, and a 2-cyanoethyl protecting group remains on its free oxygen.

[0081] For the nucleoside linked to the solid support that has not "coupled" with the newly added nucleoside, its unreacted 5'-hydroxyl group can be capped and blocked, and will no longer participate in the subsequent "coupling", that is, the length of the nucleic acid single strand will no longer change, and it will be removed in the purification step. In the capping reaction, acetic anhydride and N-methylimidazole can react to form an intermediate in the solvent tetrahydrofuran containing a small amount of pyridine, and this intermediate is used to acetylate the target hydroxyl group. The role of pyridine is to maintain a basic pH, thereby preventing free acetate / acetic acid from removing DMT from the phosphoramidite monomer. The capping step can be carried out after the step of oxidizing the phosphite triester, preferably after the step of oxidizing the phosphite triester.

[0082] At this point, under the premise of successful "coupling", a new nucleoside has been added to the first nucleoside attached to the solid support. After that, this cycle is repeated, and nucleosides (phosphoramidite monomers) are gradually added until all nucleosides (phosphoramidite monomers) are added. The last nucleoside (phosphoramidite monomer) can either remove the 5'-position protecting group or retain the 5'-position protecting group such as DMT and then remove it after the purification step. After that, the synthesized products, including single-stranded nucleic acids with a preset length and sequence, and single-stranded nucleic acids that have not completed the synthesis of the preset length, can be cleaved from the solid support to generate free 3'-hydroxyl groups. For example, the cleavage can be carried out by treating the solid support with, for example, concentrated ammonia water. In some embodiments, the solid support is treated with concentrated ammonia water at 65°C and 160 rpm.

[0083] After the cleavage is completed, the remaining part or all of the protecting groups on the single-stranded nucleic acid can be removed as needed by treatment under basic conditions, such as treatment with concentrated ammonia water, especially hot concentrated ammonia water. This is a step to remove protecting groups such as 2'-position protecting groups, protecting groups on 3'-position phosphite / phosphate groups, and / or protecting groups on bases. Protecting groups on bases can include, but are not limited to, N(6)-benzoyl, N(4)-benzoyl, and N(2)-isobutyryl. 2'-position protecting groups can include, but are not limited to, tert-butyldimethylsilyl (TBS), fluoro, MOE, LNA, or methyl. Generally, only TBS needs to be removed, and the other several groups can be retained in the final product. The protecting group on the 3'-position phosphite / phosphate group can be 2-cyanoethyl. In some embodiments, the 2'-position protecting group, such as tert-butyldimethylsilyl (TBS), can be removed by using a solution containing DMSO, triethylamine trihydrofluoride, and triethylamine.

[0084] Although the synthesis of the nucleic acid strand of the present application, such as an RNA strand, is completed on a specific synthesizer, the inventors believe that by selecting the activator and optimizing the molar ratio of the activator to the phosphoramidite monomer, the same synthesis effect can be achieved on any synthesizer. Coupled with the application of HIC, nucleic acid long chains, such as RNA long chains, can be prepared at the gram level.

[0085] As described above, on the nucleic acid single strand that has completed the synthesis of the preset length, the last added nucleoside can retain its 5'-position protecting group such as DMT, while for the nucleic acid single strand that has not completed the synthesis of the preset length, its 5'-position protecting group has been removed and acetylated. Since protecting groups such as DMT have certain hydrophobicity, this difference can be utilized to rapidly separate the nucleic acid single strand that has completed the synthesis of the preset length by using, for example, hydrophobic interaction chromatography (HIC). Compared with HPLC, the operation of HIC is simpler and the throughput is larger. In addition, HIC can also remove low-molecular substances such as ammonium hydroxide, N-1 impurities, N+1 impurities, P=O impurities, cyanoethylation (CNEt) impurities, abasic impurities, etc. In particular, if there are other hydrophobic protecting groups on the nucleic acid single strand in addition to DMT, they need to be removed before HIC.

[0086] For HIC, the packing material and the mobile phase are two important parameters. By using different packing materials in the column, it was found that using hydrophilic modified microspheres with a particle size of 60 μm and phenyl groups coupled on the surface as the packing material, the purity and recovery rate of nucleic acid chains such as RNA were both ideal. After determining the column packing material, the mobile phase of HIC was also selected. The results showed that the purification effect of (NH4)2SO4 was similar to that of Na2SO4 and both were better than that of NaCl, and when using 1.0 M (NH4)2SO4 and 1.0 M Na2SO4, the separation effect between the target nucleic acid chain with a DMT group such as an RNA chain and the impurities without DMT was better. Considering the purity and recovery rate, 0.5 M (NH4)2SO4 and 1.0 M Na2SO4 were selected. In addition, the RNA sample with DMT was placed in PBS or Tris with 0.5 M (NH4)2SO4, or PBS or Tris with 1.0 M Na2SO4 to observe the stability of nucleic acid single strands such as RNA molecules. The results showed that after 3 h of placing in PBS with 0.5 M (NH4)2SO4, the DMT protecting group slightly fell off, while after 1 h of placing in PBS with 0.5 M Na2SO4, the DMT protecting group was observed to fall off. Therefore, the PBS or Tris solution of (NH4)2SO4 was selected as the initial mobile phase and its concentration was gradually reduced during the HIC process. The nucleic acid chain sample obtained by HIC such as an RNA sample can be treated with acid to remove the DMT on the last nucleoside to generate a 5'-hydroxyl group.

[0087] The nucleic acid single strand synthesized by the phosphoramidite method such as an RNA chain can also be purified by other methods, such as hydrophobic interaction chromatography, reverse-phase HPLC, ion exchange chromatography, size exclusion chromatography, affinity chromatography, or polyacrylamide gel, etc. In particular, after HIC, the nucleic acid chain sample such as an RNA sample can also be further purified by other purification methods.

[0088] The optimized HIC in this application is applicable not only to the single-stranded nucleic acids synthesized by the method of this application, but also to other single-stranded nucleic acids synthesized by the phosphoramidite method, as long as the protecting group carried on the last nucleoside has sufficient hydrophobicity to enable its separation from impurities.

[0089] When the solid support contains 1200 μmol of groups capable of binding to nucleosides, such as hydroxyl groups, according to the synthesis method of this application, approximately 366.0 μmol (about 12.0 g) of crude 100-nt RNA can be obtained.

[0090] When the solid support contains 900 μmol of groups capable of binding to nucleosides, such as hydroxyl groups, according to the synthesis method of this application, approximately 292.0 μmol (9.64 g) of crude 100-nt RNA can be obtained. After these crude RNAs are subjected to HIC, ultrafiltration, and HPLC, the amount of RNA obtained is approximately 931.0 mg, which is extremely close to the gram level.

[0091] Based on the above data, it can be seen that according to the synthesis and purification method of this application, when the solid support contains more than 900 μmol of groups capable of binding to nucleosides, such as hydroxyl groups, pure long single-stranded nucleic acids at the "gram" level can be obtained, especially pure long single-stranded nucleic acids of 100 nt.

[0092] Hereinafter, specific examples will be combined to further elaborate on the content of this application. The examples are given only by way of illustration and do not limit the protection scope of this application.

[0093] Example

[0094] Example 1. Optimization of RNA synthesis conditions

[0095] Using DMT-2'-O-TBDMS-A(Bz)-CE phosphoramidite monomer (rA, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-TBDMS-G(iBu)-CE phosphoramidite monomer (rG, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-TBDMS-C(Ac)-CE phosphoramidite monomer (rC, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-TBDMS-U-CE phosphoramidite monomer (rU, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-Me-A(Bz)-CE phosphoramidite monomer (mA, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-Me-G(iBu)-CE phosphoramidite monomer (mG, Shanghai Zhaowei Technology Development Co., Ltd.), DMT-2'-O-Me-C(Ac)-CE phosphoramidite monomer (mC, Shanghai Zhaowei Technology Development Co., Ltd.), and DMT-2'-O-Me-U-CE phosphoramidite monomer (mU, Shanghai Zhaowei Technology Development Co., Ltd.) as raw materials for RNA synthesis, 100-nt RNA was synthesized using an AKTA oligopilot plus synthesizer (AKTA OLIGOPILOT 10 / 100, Cytiva).

[0096] Calculating the usage amount of the solid-phase carrier based on the synthesis scale (600 μmol) / carrier loading (30 μmol / g), 20 g of the solid-phase carrier (CPG2000A, Chemgene) was weighed using an electronic balance, so that the total amount of hydroxyl groups with protecting groups on the surface of the carrier that can be connected to the nucleoside monomers was 600 μmol. The 60-mm synthesis column was rinsed 2-3 times with acetonitrile and dried, and the weighed 20 g of the solid-phase carrier was promptly added to the synthesis column, and the carrier and the piston rod were wetted with acetonitrile. The wetted piston rod was pressed into the synthesis column, and the synthesis column was fixed to ensure that the carrier in the synthesis column was flat.

[0097] The synthesizer was turned on, and the instrument control program was started. The instrument pressure was normal, and there was no liquid leakage in all pipelines. In the sequence option of the synthesizer, the required synthesis sequence was manually input, and the first 70 nt and the last 30 nt were set separately. The reaction program was set according to the synthesis steps of deprotection - coupling - oxidation - capping, with a total of 100 cycles.

[0098] In step 1) deprotection of each cycle, 3% dichloroacetic acid (DCA) was used to remove the DMT protecting group on the hydroxyl group on the surface of the solid-phase carrier or the 5'-DMT on the phosphoramidite monomer attached to the solid-phase carrier. The amount of the deprotection reagent dichloroacetic acid (DCA) solution was controlled according to the UV value of the instrument during the reaction. When the instrument UV was lower than 500, the instrument stopped adding the deprotection reagent dichloroacetic acid (DCA) solution and entered the cleaning step.

[0099] In the coupling reaction of step 2), the following six different conditions are set.

[0100] ① Add 9 ml of 0.25 M activator DCI (Shanghai Lingjiang Industrial Development Co., Ltd.) and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 1.25:1. In the presence of the activator, carry out the coupling reaction between the 3'-position of the phosphoramidite monomer and the hydroxyl group on the surface of the solid support or the 5'-hydroxyl group on the phosphoramidite monomer attached to the solid support. The coupling reaction time for each phosphoramidite monomer is 10 min.

[0101] ② Add 9 ml of 0.3 M activator BTT (Anhui Shilian) and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 1.5:1. The coupling reaction time for each phosphoramidite monomer is 10 min.

[0102] ③ Add 9 ml of 0.3 M activator BTT and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 1.5:1. The coupling reaction time for each phosphoramidite monomer is 15 min.

[0103] ④ Add 18 ml of 0.3 M activator BTT and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 3:1. The coupling reaction time for each phosphoramidite monomer is 15 min.

[0104] ⑤ Add 18 ml of 0.3 M activator BTT and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 3:1. The coupling reaction time for A, C, and U phosphoramidite monomers is 15 min, and the coupling reaction time for G phosphoramidite monomer is 20 min.

[0105] ⑥ Add 27 ml of 0.3 M activator BTT and 9 ml of 0.2 mol / l phosphoramidite monomer dissolved in acetonitrile at a molar ratio of 4.5:1. The coupling reaction time for A, C, and U phosphoramidite monomers is 15 min, and the coupling reaction time for G phosphoramidite monomer is 20 min.

[0106] In the oxidation of step 3), add 60 ml of 0.05 M iodine solution pyridine / water 90 / 10 (v / v) as the oxidant to the solid support and react for 2 min.

[0107] In the capping of step 4), add 120 ml of cap A / B mixture (Anhui Shilian Special Solvents Co., Ltd.) to the synthesis column containing the solid support and react for 1.5 min.

[0108] The above steps 1)-4) are continuously cycled 100 times to complete the synthesis of RNA. An additional step 1) is added to remove the DMT on the last added phosphoramidite monomer.

[0109] After the synthesis is completed, the carrier in the synthesis column is dried with argon. After drying, the piston rod is removed, and the dried carrier is transferred to a weighing pan and weighed using an electronic balance.

[0110] Take 1.0 g of the dried solid-phase carriers treated under the above different conditions and put them into pressure-resistant bottles. Add 15 ml of ammonolysis reagent to each pressure-resistant bottle and react at 65 °C and 160 rpm for 35 min. After ammonolysis, place the pressure-resistant bottles in a refrigerator at -20 ± 5 °C and let them stand for 10 min. Slowly open each pressure-resistant bottle, filter using a 0.22-μm disposable sterile filter, wash the pressure-resistant bottles and the solid-phase carriers with 3 ml of ammonolysis reagent, and filter again. Transfer the obtained filtrate evenly to a 250-ml centrifuge bottle, add 1 / 9V (V is the volume of the filtrate) of 3M sodium acetate solution and 1V (V is the total volume of the filtrate + 3M sodium acetate solution) of isopropanol to the bottle, shake well, and place it in a refrigerator at -20 ± 5 °C for more than 45 min. After centrifugation, dry the precipitate, then add 5.0 ml of DMSO (ALADDIN) and 2.0 ml of triethylamine trihydrofluoride in sequence, and react at 65 °C and 160 rpm for 3 h to remove the 2'-protecting group TBDMS. Perform isopropanol precipitation again, remove the supernatant, and dry the precipitate to obtain the crude RNA.

[0111] Dissolve 6 ml of water in each sample. After complete dissolution, filter using a 0.22-μm disposable sterile filter, and wash the inner wall of the centrifuge bottle 2-3 times with 4 ml of water. Then pour the washing solution into the filter for filtration.

[0112] Take the filtered crude product dissolution solution and perform quantitative analysis using an enzyme-linked immunosorbent assay reader. The amount of the crude product in each sample is about 4.5 μmol (about 150 mg). Take 5 μL of the crude product dissolution solution, add 195 μL of injection water, and perform HPLC analysis. Determine whether the above synthesis steps are successful according to the elution position of the target product in HPCL at about 2.5 min. Then, take another 5 μL of the crude product dissolution solution, add 195 μL of injection water for MS qualitative analysis, and detect whether the actual molecular weight is consistent with the theoretical molecular weight according to the MS results.

[0113] The results of HPLC and MS showed that when the above coupling condition ① was used for synthesis, when the synthesis reached 30 nt, the UV value of DMT deprotection on the instrument was lower than the set threshold, and the signal acquisition stopped because the signal could not be continuously collected; when the above coupling conditions ② - ⑥ were used, the target peak could be detected by HPLC, indicating the synthesis of the 100 nt target product. More specifically, when coupling conditions ② and ③ were used, although the HPLC target peak around 2.5 min could be obtained, it might be due to the too small amount (too low purity) of the target strand, resulting in impurity interference during MS detection and the main peak could not be identified; when coupling conditions ④ - ⑥ were used, the HPLC target peak around 2.5 min could be obtained, and subsequent MS analysis showed that the synthesized sequence was 100% correct. Figure 1 (A - C) respectively show the HPLC analysis results of the crude products obtained under coupling conditions ②, ③, and ④.

[0114] The inventors also tried to synthesize long RNA strands under the condition of increasing the molar ratio of DCI to phosphoramidite monomer, but the effect was not ideal.

[0115] From the above, it can be seen that: i) As an activator, BTT can complete the synthesis of 100 nt RNA, and the effect may be better than that of DCI under specific ratios; ii) The molar ratio of the activator to the phosphoramidite monomer has a great influence on RNA synthesis. When the molar ratio of the activator:phosphoramidite monomer ≥ 1.5:1, the acquisition of the target product can be detected. As the molar ratio of the activator:phosphoramidite monomer increases, the proportion of the target main peak becomes more and more obvious. There are very obvious main peaks within the range of 3:1 - 4.5:1, and the MS is 100% correct.

[0116] Example 2. Optimization of RNA hydrophobic interaction chromatography (HIC) conditions

[0117] Using an ion purification device (Sepax, SCG - 100V2), using NaCl buffer, and selecting different chromatographic column packings, hydrophobic interaction chromatography (HIC) was performed on the RNA sample to enrich the target RNA molecules, remove the capped and blocked RNA strands, as well as some other impurities. The specific conditions are shown in Table 2.

[0118] The RNA sample used was purchased from Genscript, with a length of 86 nt, and the last phosphoramidite monomer retained the 5'-DMT protecting group.

[0119] Specifically, the following buffers were prepared.

[0120] Buffer A: 20 mM PBS, pH = 9.0 ± 0.1.

[0121] Buffer B: 2 M NaCl.

[0122] Buffer C: 4 M NaCl.

[0123] Buffer D: Water for injection.

[0124] First, equilibrate the chromatographic column (7.7 * 100 mm, 60 μm, Suzhou NanoMicro Biotech Co., Ltd.). After confirming that the chromatographic column is equilibrated, take 4 ml (0.5 μmol) of the RNA sample, add an appropriate amount of Buffer C to the RNA sample to make the NaCl concentration the same as that in Buffer B. After the chromatographic column is equilibrated until the conductivity is stable, perform sample loading. During the sample loading process, observe whether there is an absorption at UV260 on the computer screen. When UV260 > 50 mAU, collect the pre-peak of the molecules without the DMT group. After the sample loading is completed, the instrument enters the first stage of DMT OFF elution, and the molecules without the DMT group (including the capped nucleic acid chains) will be eluted. During this process, the UV260 absorption will gradually decrease. Then, enter the second stage of DMT OFF elution. During this process, the UV260 absorption is always relatively low (about 300 mAU). Then, enter the DMT ON target elution stage, that is, elute the molecules with the DMT group (i.e., the synthesized target nucleic acid chain). When UV260 > 600 mAU, collect the DMT ON target sample until UV260 < 50 mAU and then stop collection.

[0125] Take 5 μl of the receiving solution obtained under different chromatographic column packing conditions, add 195 μl of water, and perform HPLC purity analysis. The specific parameters of the HPLC analysis method are: 5 - 40% buffer (5% 100 mM TEAA + 95% ACN), 8 min.

[0126] Table 2. Settings and results of different chromatographic column packings and mobile phases

[0127]

[0128] From the results in Table 2, it can be seen that the best comprehensive effect is obtained by using the phenyl - 60 μm chromatographic column packing. Therefore, phenyl - 60 μm is selected as the chromatographic column packing.

[0129] Next, according to the operation of Example 1, synthesize 100 nt RNA using coupling condition ④, and finally retain the 5'-DMT protecting group of the last added phosphoramidite monomer without performing an additional deprotection step.

[0130] Take 6 g of the dry solid support containing the above RNA synthesis product and transfer it to a pressure-resistant bottle. Add 90 ml of ammonolysis reagent to the pressure-resistant bottle and react at 65 °C and 160 rpm for 35 min. After the ammonolysis is completed, place the pressure-resistant bottle in a refrigerator at -20 ± 5 °C and let it stand for 10 min. Slowly open each pressure-resistant bottle, filter using a 0.22-μm disposable sterile filter, wash the pressure-resistant bottle and the solid support with 18.0 mL of ammonolysis reagent, and filter again. Transfer the obtained filtrate evenly to a 250-ml centrifuge bottle, add 1 / 9 V (V is the volume of the filtrate) of 3 M sodium acetate solution and 1 V (V is the total volume of the filtrate + 3 M sodium acetate solution) of isopropanol to the bottle, shake well, and place it in a refrigerator at -20 ± 5 °C for at least 45 min. After centrifugation, dry the precipitate, then add 30.0 mL of DMSO (ALADDIN) and 12.0 mL of triethylamine trihydrofluoride in sequence, and react at 65 °C and 160 rpm for 3 h to remove the 2'-protecting group TBDMS. Perform isopropanol precipitation again, remove the supernatant, and dry the precipitate to obtain the crude RNA.

[0131] Add 60 ml of TEAA + TEA buffer (pH = 11 ± 0.5), perform the filtration step in Example 1, take the filtered crude product dissolution solution and perform quantitative analysis using an enzyme-linked immunosorbent assay reader to obtain approximately 22 μmol (726 mg) of crude RNA.

[0132] After that, prepare the following buffers.

[0133] Buffer A: 20 mM PBS, pH = 9.0 ± 0.1.

[0134] Buffer B: NaCl, (NH4)2SO4, or Na2SO4 in Buffer A, pH = 9.0 ± 0.1, as shown in Table 3 specifically.

[0135] Buffer C: NaCl, (NH4)2SO4, or Na2SO4 in 100 mM PBS, pH = 9.0 ± 0.1.

[0136] Buffer D: Water for injection.

[0137] First, balance various chromatographic columns (7.7 * 100 mm, 60 μm, Suzhou NanoMicro Biotech Co., Ltd.). After confirming that the chromatographic column is balanced, take 4 ml (0.5 μmol) of the sample, add an appropriate amount of buffer C to the sample to make the salt concentration in the solution the same as that in buffer B, and gently shake to mix evenly. After the chromatographic column is balanced until the conductivity is stable, perform sample loading. During the sample loading process, observe whether there is an absorption at UV260 on the computer screen. When UV260 > 50 mAU, collect the front impurity peak of the molecules without the DMT group. After the sample loading is completed, the instrument enters the first stage of DMT OFF elution, and the molecules without the DMT group (including the capped nucleic acid chains) will be eluted. During this process, the UV260 absorption will gradually decrease. Then, enter the second stage of DMT OFF elution. During this process, the UV260 absorption is always relatively low (about 300 mAU). Then, enter the DMT ON target elution stage, that is, elute the molecules with the DMT group (i.e., the synthesized target nucleic acid chain). When UV260 > 600 mAU, collect the DMT ON target sample until UV260 < 50 mAU and then stop collecting.

[0138] Add an appropriate amount of 30% glacial acetic acid to the receiving solution obtained under different mobile phase conditions and let it stand overnight at room temperature to remove the DMT protecting group from the hydroxyl group. Then, take 5 μl of the sample, add 195 μl of water, and perform HPLC purity analysis. The specific parameters of the HPLC analysis method are: 5 - 40% buffer (5% 100 mM TEAA + 95% ACN), 8 min.

[0139] Table 3. Settings of different mobile phase components and concentrations 1 and results

[0140]

[0141]

[0142] As shown in Table 3, after comparing different mobile phases, it was found that the purification effect of (NH4)2SO4 was similar to that of Na2SO4, but better than that of NaCl. When using 0.5 M (NH4)2SO4 and 1.0 M Na2SO4, the results of purity and recovery rate were relatively ideal.

[0143] The RNA sample with DMT was placed in PBS or Tris containing 0.5 M (NH4)2SO4, or PBS or Tris containing 1.0 M Na2SO4, and the stability of the RNA molecules was observed. It was found that the DMT protecting group was slightly detached 3 h after being placed in PBS containing 0.5 M (NH4)2SO4, while the detachment of the DMT protecting group was observed 1 h after being placed in PBS containing 1.0 M Na2SO4. Based on the above experimental results, the PBS solution containing (NH4)2SO4 was selected as the mobile phase buffer.

[0144] Next, phenyl-60 μm was selected as the chromatographic column packing, and the PBS solution containing 0.4 M (NH4)2SO4 was used as the mobile phase buffer. 30 g of the dry solid support containing 100 nt RNA synthesized under coupling condition ④ in Example 2 was taken and subjected to ammonolysis treatment under the conditions described in Example 2. The specific operation was as follows: the obtained 30 g of dry solid support was divided into 3 portions and transferred to 3 pressure-resistant bottles respectively, so that each pressure-resistant bottle contained 10.0 g of dry solid support.

[0145] Add 150.0 ml of ammonolysis reagent to each pressure-resistant bottle, and react at 65 °C and 160 rpm for 35 min to cleave the synthesized nucleic acid chain from the solid-phase support. After ammonolysis, place the 3 pressure-resistant bottles in a refrigerator at -20 ± 5 °C and let stand for 10 min. Slowly open each pressure-resistant bottle, filter using a 0.22-μm disposable sterile filter, wash the pressure-resistant bottle and the solid-phase support with 30.0 mL of ammonolysis reagent, and filter again. Transfer the obtained filtrate evenly to a 250-ml centrifuge bottle, add 1 / 9V (V is the volume of the filtrate) of 3M sodium acetate solution and 1V (V is the total volume of the filtrate + 3M sodium acetate solution) of isopropanol to the centrifuge bottle, shake well, and place it in a refrigerator at -20 ± 5 °C for more than 45 min. Take out the sample, place it in a Beckman vertical low-temperature high-speed centrifuge, centrifuge at 4 °C and 8000 rpm, and it can be centrifuged in multiple times (≤3 times) within a centrifugation time of 10 - 30 min until the supernatant is clear and the sample is completely precipitated. Remove the supernatant, add 100.0 mL of absolute ethanol, shake well to disperse the precipitate, place the sample in a Beckman vertical low-temperature high-speed centrifuge, centrifuge at 4 °C and 8000 rpm, and it can be centrifuged in multiple times (≤3 times) within a centrifugation time of 10 - 30 min until the supernatant is clear and the sample is completely precipitated. After drying the precipitate, add 50.0 mL of DMSO (ALADDIN) and 20.0 mL of triethylamine trihydrofluoride in sequence, and react at 65 °C and 160 rpm for 3 h to remove the 2'-protecting group TBDMS. Perform isopropanol precipitation again, remove the supernatant and dry it to obtain the crude RNA. Add 300 ml of TEAA + TEA buffer (pH = 11 ± 0.5), perform the filtration step in Example 1, and take the filtered crude product dissolution solution for quantitative analysis using an enzyme-labeling instrument to obtain approximately 110 μmol of the crude product. Then perform HIC purification on the obtained crude product, and the purification gradient is: equilibrate the chromatographic column with 100% buffer B; 100 - 0% buffer B, 70 min. The prepared buffers are as follows:

[0146] Buffer A: 20 mM PBS, pH = 9.0 ± 0.1;

[0147] Buffer B: 0.4 M (NH4)2SO4 in buffer A, pH = 9.0 ± 0.1;

[0148] Buffer C: 2.0 M (NH4)2SO4 in 100 mM PBS, pH = 9.0 ± 0.1;

[0149] Buffer D: water for injection.

[0150] After confirming that the chromatographic column (50*250mm, 60μm) is balanced, take 100 ml of the sample, and add 25 ml of buffer C to the sample according to 25% of the sample volume, and gently shake to mix well. After the chromatographic column is balanced until the conductivity is stable, perform sample loading. During the sample loading process, observe whether there is an absorption of UV260 on the computer screen. When UV260 > 50 mAU, collect the pre-impurity peaks of the molecules without the DMT group. After the sample loading is completed, the instrument enters the first stage of DMT OFF elution, and the molecules without the DMT group (including the capped nucleic acid chains) will be eluted. During this process, the UV260 absorption will gradually decrease. Then, enter the second stage of DMT OFF elution. During this process, the UV260 absorption is always relatively low (about 300 mAU). Then, enter the DMT ON target elution stage, that is, elute the molecules with the DMT group (i.e., the synthesized target nucleic acid chain). When UV260 > 600 mAU, collect the DMT ON target sample until UV260 < 50 mAU and then stop collecting.

[0151] Then, add an appropriate amount of 30% glacial acetic acid to the sample purified by HIC and leave it at room temperature overnight to remove the DMT protecting group from the hydroxyl group. Take 5 μl of the sample and add 195 μl of water for HPLC analysis. The specific parameters of the HPLC analysis method are: 10 - 18% buffer B (5% 100 mM TEAA + 95% ACN), 8 min.

[0152] Figure 2 (A) shows the impurity peaks and target peak diagrams displayed by the instrument during the HIC purification process, Figure 2 (B) shows the HPLC chromatogram of the sample after HIC purification. The purity of the RNA sample after HIC purification is approximately 52.0%.

[0153] Example 3. Ultrafiltration desalting of RNA

[0154] Take about 60 μmol of the sample obtained in the last part of Example 2 after the hydrophobic chromatography step and removal of DMT, and use a tangential flow ultrafiltration system (AKTA Flux6, Cytiva) to perform ultrafiltration on it to remove the detached DMT, acetic acid small molecules, and salts.

[0155] Select the conductivity mode in the instrument and set the qualified conductivity value to 60.00 μs / cm.

[0156] Connect the sample from the injection port, click on the main page, change both the makeup water pump PU01 and the circulation pump PU02 to the automatic mode, click on the operation page, only check the washing and filtration option in the automatic operation column on the right side of the process treatment, and click start. Manually adjust the opening degree of the PCV01 valve step by step to 50.00% - 40.00%. During the adjustment process, the transmembrane pressure (TMP) needs to be maintained at 0.10 - 0.50 bar.

[0157] After sample loading is completed, sterile injection water (Shandong Hualu Pharmaceutical Co., Ltd.) is introduced through the injection port, and the inner wall of the bottle is rinsed 2 - 3 times in portions by rotating a wash bottle until the conductivity is less than 60.00 μs / cm for desalination.

[0158] After the conductivity is less than 60.00 μs / cm, sample volume concentration is carried out. Finally, for a 220 ml sample, the concentration is 4.0 μmol / 15.0 mL. Change both the makeup water pump PU01 and the circulation pump PU02 to manual mode. Place a new liquid storage bottle at the permeate end, and gradually adjust the manual opening of the PCV01 valve to 50.00% - 40.00%. During the adjustment process, the transmembrane pressure (TMP) should be maintained at 0.10 - 0.50 bar.

[0159] Click on the main page, manually set the opening of the PCV01 valve to 100.00%, and set the low limit during the operation of WI01 to 0 KG. Change both the makeup water pump PU01 and the circulation pump PU02 to manual mode, adjust the three-way valve to the collection mode, manually turn on the PU02 circulation pump, and collect the sample using a 500 mL liquid storage bottle.

[0160] Example 4. HPLC purification of RNA

[0161] Use a Waters ultra-high performance liquid chromatograph (Acquity UPLC H-Class, Waters) to further purify the desalted sample obtained in Example 3 by HPLC.

[0162] First, prepare the following buffers.

[0163] Buffer A: 100 mM TEAA (pH = 8.5)

[0164] Buffer B: 5% Buffer A + 95% ACN

[0165] Buffer C: 100% acetonitrile

[0166] Buffer D: 90% ACN

[0167] After the instrument self-check is completed, open the MassLynx software, and perform sample injection preparation after all modules are normally connected online.

[0168] Perform HPLC purification using 8 - 14% of Buffer B for 140 min.

[0169] The purified sample was subjected to MS detection to screen out the target product. Then, HPLC analysis was performed on the screened target product. Specifically, 5 μl of the purified sample was mixed with 195 μl of water for HPLC analysis. The HPLC analysis detection gradient was 10 - 18% for 5 min. The HPLC detection result showed that the purity of the purified sample was 91.77%, as Figure 3 shown.

[0170] Example 5. Large-scale synthesis of RNA

[0171] According to the operation of Example 1, 100 nt RNA synthesis was carried out according to coupling condition ⑥, that is, BTT and phosphoramidite monomer were introduced at a molar ratio of 4.5:1 in the coupling step, the coupling time of guanosine was set to 20 min, the coupling time of other nucleosides was set to 15 min, and the DMT on the last phosphoramidite monomer was removed.

[0172] Specifically, 40 g of solid support (CPG2000A, Chemgene) was weighed; in the coupling step, 54 ml of 0.3 M activator BTT and 18 ml of 0.2 M phosphoramidite monomer dissolved in acetonitrile were introduced into the synthesis column, the coupling time of guanosine was set to 20 min, and the coupling time of other nucleosides was 15 min; in the oxidation step, 120 ml of 0.05 M iodine pyridine / water 90 / 10 (v / v) was added to the solid support and reacted for 2 min; in the capping step, 262.5 ml of cap A / B was added to the solid support and reacted for 2 min, and a total of 100 cycles were carried out. After the last cycle, the DMT on the last phosphoramidite monomer was removed.

[0173] After the synthesis reaction was completed, the carrier was dried to obtain 95.30 g of solid support containing the synthesis product.

[0174] 90.0 g of the obtained dried carrier was taken out and divided into 9 portions, which were respectively transferred into pressure-resistant bottles so that each pressure-resistant bottle contained 10.0 g of dried carrier (containing the synthesis product).

[0175] Add 150.0 ml of ammonolysis reagent to each pressure-resistant bottle, and react under the conditions of 65 °C and 160 rpm for 35 min to cleave the synthesized nucleic acid chain from the solid-phase carrier. After ammonolysis, place 9 pressure-resistant bottles in a refrigerator at -20 ± 5 °C and let stand for 10 min. Slowly open each pressure-resistant bottle, filter using a 0.22-μm disposable sterile filter, wash the pressure-resistant bottle and the solid-phase carrier with 30.0 ml of ammonolysis reagent, and filter again. Transfer the obtained filtrate evenly to a 250-ml centrifuge bottle, add 1 / 9V (V is the volume of the filtrate) of 3M sodium acetate solution and 1V (V is the total volume of the filtrate + 3M sodium acetate solution) of isopropanol to the bottle, shake well, and place it in a refrigerator at -20 ± 5 °C for at least 45 min. After centrifugation, dry the precipitate, then add 50.0 ml of DMSO (ALADDIN) and 20.0 ml of triethylamine trihydrofluoride in sequence, and react at 65 °C and 160 rpm for 3 h to remove the 2'-protecting group TBDMS. Perform isopropanol precipitation again, remove the supernatant, and dry the precipitate to obtain the crude RNA. Add 60.0 ml of water for dissolution. After complete dissolution, filter using a 0.22-μm disposable sterile filter, and wash the wall of the centrifuge bottle 2 - 3 times with 30.0 ml of water, then pour the washing solution into the filter for filtration.

[0176] Take all the filtered crude product dissolution solution and perform quantitative analysis with an enzyme-labeling instrument. The amount of the crude product obtained is 366.0 μmol (about 12.0 g).

[0177] Take 5.0 μL of the crude product dissolution solution, add 195.0 μL of injection water, perform HPLC purification and MS analysis under the conditions of 8 - 14% buffer B and 140 min in Example 4, and perform quantitative analysis with an enzyme-labeling instrument. The peak position of the target product in HPCL is about 2.5 min. The purification recovery rate of HPLC is 8.04%, and the mass of about 12 g of the crude product after purification is about 964.6 mg.

[0178] Example 6. Large-scale synthesis and HIC of RNA

[0179] According to the operation of Example 1, perform 100-nt RNA synthesis under coupling condition ⑥, that is, introduce BTT and phosphoramidite monomer at a molar ratio of 4.5:1, set the coupling time of guanosine to 20 min, and the coupling time of other nucleosides to 15 min, and retain the DMT on the last phosphoramidite monomer.

[0180] Specifically, weigh 30.0 g of solid support (CPG2000A) using a balance. In the coupling step, introduce 40.5 ml of 0.3 M activator BTT and 13.5 ml of phosphoramidite monomer dissolved in acetonitrile into the synthesis column. Set the coupling time for guanosine to 20 min and the coupling time for other nucleosides to 15 min. In the oxidation step, add 90 ml of 0.05 M iodine pyridine / water 90 / 10 (v / v) to the solid support and react for 2 min. In the capping step, add 195 ml of cap A / B to the solid support and react for 2 min, and perform a total of 100 cycles. After the last cycle, retain the DMT on the last phosphoramidite monomer.

[0181] After the synthesis is completed, dry the support to obtain 67.48 g of solid support containing the synthetic product.

[0182] Take out 66.0 g of the obtained dried support and divide it into 6 portions, each portion being 11.0 g, and transfer them to pressure-resistant bottles respectively. Add 165.0 ml of ammonolysis reagent to each pressure-resistant bottle. After ammonolysis, place 9 pressure-resistant bottles in a refrigerator at -20 ± 5 °C and let stand for 10 min. Slowly open each pressure-resistant bottle, filter using a 0.22 μm disposable sterile filter, wash the pressure-resistant bottle and the solid support with 30.0 mL of ammonolysis reagent, and filter again. Transfer the obtained filtrate evenly to a 250 ml centrifuge bottle, add 1 / 9 V (V is the volume of the filtrate) of 3 M sodium acetate solution and 1 V (V is the total volume of the filtrate + 3 M sodium acetate solution) of isopropanol to the bottle, shake well, and place it in a refrigerator at -20 ± 5 °C for at least 45 min. After centrifugation, dry the precipitate, then add 50.0 mL of DMSO (ALADDIN) and 20.0 mL of triethylamine trihydrofluoride in sequence, and react at 65 °C and 160 rpm for 3 h to remove the 2'-protecting group TBDMS. Perform isopropanol precipitation again, remove the supernatant, and dry the precipitate to obtain the crude RNA. Add 60.0 ml of freshly prepared TEAA + TEA buffer (pH = 11.0 ± 0.5) for dissolution. After complete dissolution, filter using a 0.22 μm disposable sterile filter, and rinse the centrifuge bottle wall 2 - 3 times with 30.0 mL of TEAA + TEA buffer, and pour the washing solution into the filter for filtration.

[0183] Take all the filtered crude product dissolution solution and perform quantitative analysis using an enzyme-labeled instrument to obtain a total of 292.0 μmol (9.64 g) of crude RNA.

[0184] According to the description of Example 2, phenyl-60μm was selected as the chromatographic column packing material, and PBS solution containing 0.4 mol / L (NH4)2SO4 was used as buffer B for HIC purification. The purification recovery rate of HIC measured by an enzyme-linked immunosorbent assay was 41.46%, that is, the amount of RNA after HIC was about 121.0 μmol, 3.99 g, and the volume of the solution was about 1000 ml.

[0185] Then, 50 ml of 30% glacial acetic acid was added to the 1000 ml solution to remove DMT from the HIC purified sample. Subsequently, using a tangential flow ultrafiltration system (AKTA Flux6, Cytiva), the sample was ultrafiltered according to the operation of Example 3. After the conductivity was less than 60.00 μs / cm, the sample was concentrated to obtain 450.0 mL with a concentration of 4.0 μmol / 15.0 mL. Finally, the collected sample was purified by HPLC, and the purification yield of HPLC was 23.31%, and the amount of RNA obtained was about 931.0 mg.

[0186] Although the present application has been described in conjunction with one or more embodiments, it should be understood that the present application is not limited to these embodiments. The description in the present application is intended to cover all variations and equivalents, which are all included in the spirit and scope of the appended claims. All documents cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for preparing a single-stranded nucleic acid, comprising: i) providing a nucleoside, the nucleoside comprising a protecting group at the 5' position, removing the protecting group at the 5' position of the nucleoside to generate a free 5' hydroxyl group, ii) adding an activator and a nucleoside at a molar ratio of 1.5:1 or more, wherein the added nucleoside comprises a phosphite group and a protecting group at the 3' position and the 5' position, respectively, so that the 5' hydroxyl group of the nucleoside from the previous step reacts with the 3' phosphite group of the added nucleoside in the presence of the activator to form a phosphite triester bond, and then converting the phosphite triester bond into a phosphotriester bond in the presence of an oxidant, and then acetylating the free 5' hydroxyl group that has not reacted with the added nucleoside to form a phosphite triester bond, iii) removing the 5' protecting group of the nucleoside added in step ii) to generate a free 5' hydroxyl group, iv) repeating step ii) and step iii) to add nucleosides to the nucleic acid single strand until the preset penultimate nucleoside is added, and v) performing step ii), adding the last nucleoside to the nucleic acid single strand, wherein the nucleoside in step i) is connected to a solid phase carrier, The activating agent is 5-(benzylthio)-1H-tetrazole (BTT), 5-ethylthiotetrazole (ETT), or 4,5-dicyanoimidazole (DCI).

2. The method of claim 1, wherein: In step ii), the activator and the nucleoside are added in a molar ratio of 1.5:1 to 6.0:

1.

3. The method of claim 2, wherein: In step ii), the activator and the nucleoside are added in a molar ratio of 3.0:1-4.5:

1.

4. The method of claim 1, further comprising the step of performing step iii) after step v).

5. The method according to claim 1, further comprising the step of cutting the synthesized single-stranded nucleic acid from the solid support after step v).

6. The method of claim 5, further comprising the step of subjecting the nucleic acid strand to high performance liquid chromatography after performing step iii) and cleaving the synthesized nucleic acid strand from the solid support.

7. The method of claim 6, wherein the 5' protecting group of the nucleoside in step i) is a hydrophobic group, and the method further comprises a step of subjecting the cleaved nucleic acid single strand to hydrophobic chromatography after the step of cleaving the synthesized nucleic acid single strand from the solid phase support.

8. The method of claim 7, wherein the protecting group at the 5' position of the nucleoside in step i) is 4,4'-dimethoxytrityl (DMT).

9. The method of claim 7, wherein the hydrophobic chromatography uses hydrophilic polymethyl methacrylate as filler, wherein the filler surface is modified with different hydrophobic groups.

10. The method according to claim 9, wherein the hydrophobic group is phenyl or butyl, and the particle size of the filler is 30-80 μm.

11. The method of claim 10, wherein the filler is selected from hydrophilic polymethyl methacrylate with a surface modified with phenyl and a particle size of 60 μm, hydrophilic polymethyl methacrylate with a surface modified with phenyl and a particle size of 32 μm, hydrophilic polymethyl methacrylate with a surface modified with phenyl and a particle size of 35 μm, hydrophilic polymethyl methacrylate with a surface modified with phenyl and a particle size of 80 μm, and hydrophilic polymethyl methacrylate with a surface modified with butyl and a particle size of 32 μm.

12. The method of claim 9, wherein the hydrophobic chromatography uses PBS or Tris solution containing (NH4)2SO4, Na2SO4, or NaCl as the mobile phase.

13. The method of claim 1, wherein the nucleoside provided in step i) and the nucleoside added in step ii) are in a molar ratio of 1:

1.

14. The method of claim 1, wherein: When guanosine is added in step ii), the reaction of the 5'hydroxyl group of the nucleoside obtained in the previous step with the 3'-phosphite group of the added nucleoside to form a phosphite triester bond continues for 20 minutes. When nucleosides other than guanosine are added, the reaction of the 5'hydroxyl group of the nucleoside obtained in the previous step with the 3'-phosphite group of the added nucleoside to form a phosphite triester bond continues for 15 minutes.

15. The method according to claim 7, further comprising the steps of ultrafiltration desalting, and / or high performance liquid chromatography after the hydrophobic chromatography step.

16. The method of claim 1 can also include the step of connecting the nucleoside in step i) to a solid phase carrier before step i), wherein the surface of the solid phase carrier contains more than 900 μmol of groups that can be connected to the nucleoside, and the nucleoside is provided in a molar ratio of 3:1 to the surface groups of the solid phase carrier, so that the nucleoside reacts with the groups of the solid phase carrier. The method according to claim 1 , wherein the length of the single-stranded nucleic acid is 80 nt to 200 nt. The method of claim 1 , wherein the single-stranded nucleic acid is an RNA strand.

19. A method for purifying a single-stranded nucleic acid, comprising subjecting the single-stranded nucleic acid to hydrophobic chromatography, wherein the single-stranded nucleic acid comprises a hydrophobic group.

20. The method of claim 19, wherein the hydrophobic group is 4,4'-dimethoxytrityl (DMT).

21. The method of claim 19, wherein the single-stranded nucleic acid is prepared by the method of any one of claims 1-18.

22. The method of claim 19, wherein the hydrophobic chromatography uses hydrophilic polymethyl methacrylate with a particle size of 30-80 μm as filler, wherein the surface of the filler is modified with different hydrophobic groups.

23. The method according to claim 22, wherein the filler is hydrophilic polymethyl methacrylate with a particle size of 60 μm and surface modified with phenyl groups.

24. The method of claim 19, wherein the hydrophobic chromatography uses (NH4)2SO4 in PBS or Tris as the mobile phase.

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