Preparation method for sirna

Abstract

Provided in the present invention is a preparation method for a small nucleic acid drug. Specifically, provided is a preparation method for an siRNA for inhibiting PCSK9 gene expression.

Description

siRNA preparation method Technical Field

[0001] This invention relates to the field of small nucleic acid drug synthesis, and more specifically, to a method for preparing siRNA that inhibits PCSK9 gene expression. Background Technology

[0002] Oligonucleotide therapeutics are a class of drugs based on DNA and / or RNA structures that function between genes and proteins. In recent years, significant progress has been made in the treatment of a variety of diseases through the development of natural and modified oligonucleotides. Many of these therapeutics are highly effective in treating a range of rare diseases, and oligonucleotide-based therapies targeting a broader range of disease populations are already on the market or under development. Therefore, as the application of oligonucleotide therapeutics, particularly siRNA, in the treatment of common diseases becomes increasingly widespread, the demand for siRNA is growing rapidly, necessitating large-scale manufacturing.

[0003] The phosphoramide process is currently the main technique for synthesizing oligonucleotide drugs such as siRNA, encompassing four key steps: deprotection, coupling, capping, and oxidation. While this method can produce products ranging from milligrams to kilograms, it faces challenges in synthesizing longer oligonucleotide chains due to increased impurities and low purity. Typically, oligonucleotides synthesized using the phosphoramide process do not exceed 200 nucleotides in length, as the chemical reaction efficiency, purity, and yield decrease significantly with increasing chain length. Furthermore, the process consumes large amounts of organic solvents, increasing costs and exacerbating environmental pollution and wastewater treatment difficulties. During synthesis, persistent PO / PS impurities, N+1 impurities, and N-1 impurities are generated, with their production increasing dramatically with chain length, further complicating post-processing and reducing yield. Therefore, there is a need to develop a more efficient and environmentally friendly siRNA synthesis technology. Summary of the Invention

[0004] This application provides a method for preparing siRNA, characterized in that the siRNA comprises a sense strand and an antisense strand; the sense strand is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', and the antisense strand is 5'-mA*fC*mAmAmAfAmGmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3', wherein A, U, G, and C represent nucleotides with adenine, uracil, guanine, and cytosine as bases, respectively; m indicates that the nucleotide adjacent to its right is 2'-O-methyl modified; f indicates that the nucleotide adjacent to its right is 2'-fluorine modified; * indicates that the linking group between the two groups is a phosphate thioester; and B indicates a baseless nucleotide; the 5' end of the sense strand is a hydroxyl group, and the 3' end is a UM group, the structure of which is: The antisense strand has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the method includes: (1) providing a reaction system comprising a first double-stranded substrate, a second double-stranded substrate, and an RNA ligase, wherein the first double-stranded substrate comprises a paired first positive strand substrate and a second antisense strand substrate, and the second double-stranded substrate comprises a paired second positive strand substrate and a first antisense strand substrate; (2) the first double-stranded substrate and the second double-stranded substrate form the siRNA under the catalytic conditions of the RNA ligase, wherein the RNA ligase catalyzes the ligation of the first positive strand substrate and the second positive strand substrate to form the positive strand, and catalyzes the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand; the first positive strand substrate and the second positive strand substrate can form the positive strand, and the first antisense strand substrate and the second antisense strand substrate can form the antisense strand; the length of the first positive strand substrate and the second positive strand substrate is 2-17 nt, and the length of the first antisense strand substrate and the second antisense strand substrate is 2-19 nt.

[0005] In some embodiments, the first positive substrate is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmC-3', the second positive substrate is 5'-PmUmUmUmUmGmU*UM-3', the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3', where P represents a phosphorus group.

[0006] In some embodiments, the first positive substrate is 5'-B*mA*mGmAmCmCmU-3', the second positive substrate is 5'-PfGmUfUfUfUmGmCmUmUmUmGmU*UM-3', the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3', where P represents a phosphorus group.

[0007] In some embodiments, the first sense substrate is 5'-B*mA*mGmAmCmCmUfGmU-3', the second sense substrate is 5'-PfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense substrate is 5'-mA*fC*mAmAmAfAmG-3', and the second antisense substrate is 5'-PmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3', where P represents a phosphorus group.

[0008] In some embodiments, the first positive substrate is 5'-B*mA*mGmAmCmC-3', the second positive substrate is 5'-PmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3', where P represents a phosphorus group. Attached Figure Description

[0009] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0010] Figure 1 shows a schematic diagram of the siRNA structure according to an embodiment of the present invention.

[0011] Figure 2 shows a schematic diagram of the UM and L96 groups according to an embodiment of the present invention.

[0012] Figure 3 shows the liquid chromatography detection results of the fragment mixture according to Embodiment 1 of the present invention.

[0013] Figure 4 shows the liquid chromatography detection results of the reaction solution after the enzymatic reaction according to Example 1 of the present invention.

[0014] Figure 5 shows the liquid chromatography detection results of the fragment mixture according to Embodiment 2 of the present invention.

[0015] Figure 6 shows the liquid chromatography detection results of the reaction solution after the enzymatic reaction according to Example 2 of the present invention.

[0016] Figure 7 shows the liquid chromatography detection results of the fragment mixture according to Embodiment 3 of the present invention.

[0017] Figure 8 shows the liquid chromatography detection results of the reaction solution after the enzymatic reaction according to Example 3 of the present invention.

[0018] Figure 9 shows the liquid chromatography detection results of the fragment mixture according to Embodiment 4 of the present invention.

[0019] Figure 10 shows the liquid chromatography detection results of the reaction solution after the enzymatic reaction according to Example 4 of the present invention.

[0020] Figure 11 shows the reaction conversion rates at different enzymatic reaction temperatures and times according to Example 6 of the present invention. Detailed Implementation

[0021] definition

[0022] Unless otherwise specified, the following terms and phrases used in this invention are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0023] As used herein, the term “about” is intended to indicate that the values ​​cited should not be interpreted as absolute values ​​and that measurement errors, batch-to-batch variations, and / or device-to-device variations should also be taken into account. Except where ranges of measurement errors or variations are specified herein (e.g., HPLC analysis errors are typically ±5%, high-resolution mass spectrometry errors are typically ±5 ppm, low-resolution mass spectrometry errors are typically ±0.5 Da, etc.), the term “about” indicates an approximate value that may differ by ±10%, ±5%, ±2%, or ±1% when used before numerical designations, such as temperature, time, amount, and concentration, including ranges.

[0024] As used herein, “small interfering RNA” and “siRNA” are interchangeable and function at least partially through an RNAi mechanism to regulate target nucleic acids and / or proteins encoded by the target nucleic acids. In some embodiments, the siRNA is at least partially complementary to the coding sequence of a target gene expressed in cells. In some embodiments, after delivery of the siRNA to cells expressing the gene, the siRNA is able to inhibit or block gene expression in vitro or in vivo. Typically, the siRNA contains a double-stranded region of fewer than 60, 50, 40, or 30 complementary base pairs; preferably, it contains a double-stranded region of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 complementary base pairs. In some embodiments, the sense and antisense strands of the siRNA are each independently 15-25 nucleotides long, and the length of the complementary double-stranded region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 base pairs. In some embodiments, the siRNA has a sense strand of 19 nucleotides and an antisense strand of 21 nucleotides, forming a complementary double-stranded region of 19 base pairs.

[0025] In some embodiments, siRNA may include one or more modified nucleotides. In some embodiments, the sense strand of siRNA may include one or more modified nucleotides. In some embodiments, the sense strand of siRNA may include 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 modified nucleotides. In some embodiments, the sense strand of siRNA may include 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 modified nucleotide. In some embodiments, the antisense strand of siRNA may include one or more modified nucleotides. In some schemes, the antisense strand of siRNA may include 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 modified nucleotides. In some schemes, the sense strand of siRNA may include 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 modified nucleotide. In some schemes, the sense strand of siRNA includes 19 modified nucleotides, and the antisense strand includes 21 modified nucleotides, and neither the sense strand nor the antisense strand contains native nucleotides.

[0026] In some embodiments, the modified nucleotide may include phosphate backbone modification, ribose modification, base modification, etc. In some embodiments, the modified nucleotide is a ribose-modified nucleotide. In some embodiments, the modified nucleotide is a 2'-O-methyl-modified nucleotide or a 2'-fluorine-modified nucleotide.

[0027] In some embodiments, siRNA may include one or more modified nucleoside internucleotide bonds. In some embodiments, the modified nucleoside internucleotide bonds include modified phosphate groups, such as phosphate thioesters. In some embodiments, the sense strand of siRNA includes one or more phosphate thioester nucleoside internucleotide bonds. In some embodiments, the antisense strand of siRNA includes one or more phosphate thioester nucleoside internucleotide bonds.

[0028] In some embodiments, the ends of the siRNA (e.g., the 3' end, 5' end, 3' end, and 5' end of the sense strand, the antisense strand, the 3' end and 5' end of the antisense substrate, the 3' end and 5' end of the sense substrate, the 3' end and 5' end of the antisense substrate) may include modifying groups, including but not limited to phosphorus (P) groups (e.g., phosphate groups ((HO)2(O=)PO-), thiophosphate groups ((HO)2(S=)PO-), vinylphosphonic acid groups ((HO)2(O=)P-CH=CH-)), nucleotide analogs (e.g., non-basic nucleotides (B), such as trans non-basic nucleotides, 2'-deoxy-non-basic nucleotides, trans 2'-deoxy-non-basic nucleotides, etc.), ligand groups (e.g., GalNAc ligand groups, such as UM groups, L96 groups), etc. In some embodiments, the 5' end of the sense strand of the siRNA includes a non-basic nucleotide. In some embodiments, the 3' end of the sense strand of the siRNA includes a GalNAc ligand group (e.g., a UM group, an L96 group). In some embodiments, the 5' end of the sense strand substrate includes a phosphate group. In some embodiments, the 5' end of the antisense strand substrate includes a phosphate group.

[0029] It should be noted that, unless otherwise stated in this application, terminal groups are not counted when calculating the length of the siRNA strand (e.g., the sense or antisense strand). For example, the sequence 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3' in this document is 19 nucleotides long, excluding the 5' B group and the 5' UM group; the sequence 5'-PmUmUmUmUmGmU*UM-3' in this document is 6 nucleotides long, excluding the 5' P group and the 5' UM group.

[0030] As used herein, A, U, G, and C represent nucleotides with adenine, uracil, guanine, and cytosine as bases, respectively. m indicates that the nucleotide adjacent to it on its right is 2'-O-methyl modified, for example, mA represents 2'-O-methyl modified adenosine, mU represents 2'-O-methyl modified uridine, mG represents 2'-O-methyl modified guanosine, and mC represents 2'-O-methyl modified cytidine. f indicates that the nucleotide adjacent to it on its right is 2'-fluorine modified, for example, fA represents 2'-fluorine modified adenosine, fU represents 2'-fluorine modified uridine, fG represents 2'-fluorine modified guanosine, and fC represents 2'-fluorine modified cytidine. * indicates that the linking group between the two groups is a thiophosphate ester, B indicates a baseless nucleotide (e.g., a 2'-deoxy-baseless nucleotide), and P represents a phosphorus group (e.g., a phosphate group).

[0031] As used in this article, UM represents a GalNAc ligand with the following structure:

[0032] As used in this article, L96 represents a GalNAc ligand with the following structure:

[0033] In some embodiments, the GalNAc ligand (e.g., the UM group, L96 group) is linked to the siRNA (e.g., the positive strand of the siRNA) via a phosphate ester bond. In some embodiments, the GalNAc ligand (e.g., the UM group, L96 group) is linked to the siRNA (e.g., the positive strand of the siRNA) via a phosphate thioester bond. In some embodiments, the GalNAc ligand (e.g., the UM group, L96 group) is linked to the 3' end of the positive strand of the siRNA via a phosphate thioester bond.

[0034] siRNA preparation method

[0035] This article provides a method for preparing siRNA, characterized in that the siRNA comprises a sense strand and an antisense strand; the sense strand is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3' (RNA-1), and the antisense strand is 5'-mA*fC*mAmAmAfAmGmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-2), wherein A, U, G, and C represent nucleotides with adenine, uracil, guanine, and cytosine as bases, respectively; m indicates that the nucleotide adjacent to its right is modified with a 2'-O-methyl group; f indicates that the nucleotide adjacent to its right is modified with a 2'-fluorine group; * indicates that the linking group between the two groups is a phosphate thioester; and B indicates a baseless nucleotide; the 5' end of the sense strand is a hydroxyl group, and the 3' end is a UM group, the structure of which is as follows: The antisense strand has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the method includes: (1) providing a reaction system comprising a first double-stranded substrate, a second double-stranded substrate, and an RNA ligase, wherein the first double-stranded substrate comprises a paired first positive strand substrate and a second antisense strand substrate, and the second double-stranded substrate comprises a paired second positive strand substrate and a first antisense strand substrate; (2) the first double-stranded substrate and the second double-stranded substrate form the siRNA under the catalytic conditions of the RNA ligase, wherein the RNA ligase catalyzes the ligation of the first positive strand substrate and the second positive strand substrate to form the positive strand, and catalyzes the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand; the first positive strand substrate and the second positive strand substrate can form the positive strand, and the first antisense strand substrate and the second antisense strand substrate can form the antisense strand; the length of the first positive strand substrate and the second positive strand substrate is 2-17 nucleotides (nt), and the length of the first antisense strand substrate and the second antisense strand substrate is 2-19 nt.

[0036] In some embodiments, the method further includes: mixing the first sense substrate and the second antisense substrate to pair the first sense substrate and the second antisense substrate to form the first double-stranded substrate, and mixing the second sense substrate and the first antisense substrate to pair the second sense substrate and the first antisense substrate to form the second double-stranded substrate, and then mixing the first double-stranded substrate, the second double-stranded substrate and the RNA ligase to form the reaction system.

[0037] In some embodiments, the method further includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate, such that the first sense substrate and the second antisense substrate pair up to form the first double-stranded substrate, and such that the second sense substrate and the first antisense substrate pair up to form the second double-stranded substrate, and then mixing with the RNA ligase to form the reaction system.

[0038] In some embodiments, the method further includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, the second antisense substrate, and the RNA ligase, such that the first sense substrate and the second antisense substrate pair to form the first double-stranded substrate, and the second sense substrate and the first antisense substrate pair to form the second double-stranded substrate, thereby forming the reaction system.

[0039] In this document, "annealing" refers to a reaction in which a first sense substrate, a second sense substrate, a first antisense substrate, and a second sense substrate are mixed to form a complex, which is stabilized by hydrogen bonding between nucleotide bases. The resulting complex comprises a first double-stranded substrate containing paired first and second antisense substrates, and a second double-stranded substrate containing paired second and first antisense substrates. Hydrogen bonding can occur in a manner similar to Watson-Crick base pairing. Annealing can be performed at room temperature (e.g., 15-30°C, 20-25°C) or at a specific temperature. In some embodiments, annealing lasts for 10-30 minutes, e.g., 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. Annealing can also be performed by heating and holding the temperature for a period of time followed by cooling to room temperature or a specific temperature. In some embodiments, annealing is performed by heating to about 50°C or higher (e.g., about 60°C or higher, about 70°C or higher, about 80°C or higher, or about 90°C or higher) and maintaining the temperature for about 5 minutes or more (e.g., about 10 minutes or more, about 15 minutes or more, or about 20 minutes or more, e.g., about 5-10 minutes, about 5-15 minutes, or about 5-20 minutes). In some embodiments, a first positive chain substrate and a second positive chain substrate, a second positive chain substrate and a first antisense chain substrate are respectively mixed and annealed to form a first double-chain substrate and a second double-chain substrate. In some embodiments, a first positive chain substrate, a second positive chain substrate, a first antisense chain substrate and a second positive chain substrate are co-mixed and annealed to form a first double-chain substrate and a second double-chain substrate.

[0040] In some embodiments, the reaction system contains essentially no or no first sense substrate, second sense substrate, first antisense substrate, and second sense substrate in single-chain form.

[0041] In some embodiments, the RNA ligase is any enzyme with RNA ligation function. In some embodiments, the RNA ligase is selected from one or more enzymes of the RNA ligase family RnI 1, RnI 2, RnI 3, and RnI 5. In some embodiments, the RNA ligase is selected from the T4 RNA ligase family RnI 1. In some embodiments, the RNA ligase is selected from the T4 RNA ligase family RnI 2. In some embodiments, the RNA ligase can be a free RNA ligase or an immobilized RNA ligase. In some embodiments, the RNA ligase is a commercially available ligase. In some embodiments, the RNA ligase is a laboratory-prepared ligase. In some embodiments, the RNA ligase is derived from microorganisms such as viruses, archaea, bacteria, and fungi.

[0042] In some embodiments, the 3' end of the first positive strand substrate and the 5' end of the second positive strand substrate are ligated under the catalysis of the RNA ligase to form the positive strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated under the catalysis of the RNA ligase to form the antisense strand. In some embodiments, the formed positive and antisense strands are paired, thereby the first and second double-stranded substrates are ligated under the catalysis of the RNA ligase to form the siRNA.

[0043] In some embodiments, the 5' end of the first positive substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second positive substrate is a phosphate group, and the 3' end is a UM group. In some embodiments, the 5' end of the first antisense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense substrate is a phosphate group, and the 3' end is a hydroxyl group.

[0044] In some embodiments, the 5' end of the first positive substrate is a hydroxyl group and the 3' end is a phosphate group; the 5' end of the second positive substrate is a hydroxyl group and the 3' end is a UM group. In some embodiments, the 5' end of the first antisense substrate is a hydroxyl group and the 3' end is a phosphate group; the 5' end of the second antisense substrate is a hydroxyl group and the 3' end is a hydroxyl group.

[0045] In some embodiments, the 5' end of the first sense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense substrate is a phosphate group, and the 3' end is a UM group; and the 5' end of the first antisense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense substrate is a phosphate group, and the 3' end is a hydroxyl group.

[0046] In some embodiments, the lengths of the first and second positive chain substrates are 2-17 nt, or 3-16 nt, or 4-15 nt, or 5-14 nt, or 6-13 nt, or 7-12 nt, or 8-11 nt, or 9-10 nt. In some embodiments, the lengths of the first and second antisense chain substrates are 2-19 nt, or 3-18 nt, or 4-17 nt, or 5-16 nt, or 6-15 nt, or 7-14 nt, or 8-13 nt, or 9-12 nt, or 10-11 nt.

[0047] In some embodiments, the lengths of the first and second justice chain substrates are 2nt and 17nt, 3nt and 16nt, 4nt and 15nt, 5nt and 14nt, 6nt and 13nt, 7nt and 12nt, 8nt and 11nt, 9nt and 10nt, 10nt and 9nt, 11nt and 8nt, 12nt and 7nt, 13nt and 6nt, 14nt and 5nt, 15nt and 4nt, 16nt and 3nt, or 17nt and 2nt. In some embodiments, the lengths of the first antisense substrate and the second antisense substrate are 2nt and 19nt, 3nt and 18nt, 4nt and 17nt, 5nt and 16nt, 6nt and 15nt, 7nt and 14nt, 8nt and 13nt, 9nt and 12nt, 10nt and 11nt, 11nt and 10nt, 12nt and 9nt, 13nt and 8nt, 14nt and 7nt, 15nt and 6nt, 16nt and 5nt, 17nt and 4nt, 18nt and 3nt, or 19nt and 2nt.

[0048] In some embodiments, the length of the first justice chain substrate is 13 nt and the length of the second justice chain substrate is 6 nt. In some embodiments, the length of the first justice chain substrate is 6 nt and the length of the second justice chain substrate is 13 nt. In some embodiments, the length of the first justice chain substrate is 8 nt and the length of the second justice chain substrate is 11 nt. In some embodiments, the length of the first justice chain substrate is 5 nt and the length of the second justice chain substrate is 14 nt.

[0049] In some embodiments, the length of the first antisense substrate is 9 nt and the length of the second antisense substrate is 12 nt. In some embodiments, the length of the first antisense substrate is 7 nt and the length of the second antisense substrate is 14 nt.

[0050] In some embodiments, the length of the first positive chain substrate is 13 nt, the length of the second positive chain substrate is 6 nt, the length of the first negative chain substrate is 9 nt, and the length of the second negative chain substrate is 12 nt. In some embodiments, the length of the first positive chain substrate is 6 nt, the length of the second positive chain substrate is 13 nt, the length of the first negative chain substrate is 9 nt, and the length of the second negative chain substrate is 12 nt. In some embodiments, the length of the first positive chain substrate is 8 nt, the length of the second positive chain substrate is 11 nt, the length of the first negative chain substrate is 7 nt, and the length of the second negative chain substrate is 14 nt. In some embodiments, the length of the first positive chain substrate is 5 nt, the length of the second positive chain substrate is 14 nt, the length of the first negative chain substrate is 9 nt, and the length of the second negative chain substrate is 12 nt.

[0051] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmC-3' (RNA-3), the second sense substrate is 5'-PmUmUmUmUmGmU*UM-3' (RNA-4), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3' (RNA-11), and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-12).

[0052] In some embodiments, the first sense substrate is 5'-B*mA*mGmAmCmCmU-OH-3' (RNA-5), the second sense substrate is 5'-PfGmUfUfUfUmGmCmUmUmUmGmU*UM-3' (RNA-6), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-OH-3' (RNA-11), and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-12).

[0053] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCmUfGmU-3' (RNA-7), the second sense substrate is 5'-PfUfUfUmGmCmUmUmUmUmGmU*UM-3' (RNA-8), the first antisense substrate is 5'-mA*fC*mAmAmAfAmG-3' (RNA-13), and the second antisense substrate is 5'-PmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-14).

[0054] In some embodiments, the first sense substrate is 5'-B*mA*mGmAmCmC-3' (RNA-9), the second sense substrate is 5'-PmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3' (RNA-10), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3' (RNA-11), and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-12).

[0055] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCP-3' (RNA-15), the second sense substrate is 5'-mUmUmUmUmGmU*UM-3' (RNA-16), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmAP-3' (RNA-23), and the second antisense substrate is 5'-mAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-24).

[0056] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCmUP-3' (RNA-17), the second sense substrate is 5'-fGmUfUfUfUmGmCmUmUmUmGmU*UM-3' (RNA-18), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmAP-3' (RNA-23), and the second antisense substrate is 5'-mAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-24).

[0057] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCmUfGmUP-3' (RNA-19), the second sense substrate is 5'-fUfUfUmGmCmUmUmUmUmGmU*UM-3' (RNA-20), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGP-3' (RNA-25), and the second antisense substrate is 5'-mCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-26).

[0058] In some implementations, the first sense substrate is 5'-B*mA*mGmAmCmCP-3' (RNA-21), the second sense substrate is 5'-mUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3' (RNA-22), the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmAP-3' (RNA-23), and the second antisense substrate is 5'-mAmAmAmCfAmGfGmUmCmU*mA*mG-3' (RNA-24).

[0059] In some embodiments, the phosphorus group is a phosphate group. In some embodiments, the abasic nucleotide is a 2'-deoxyabasic nucleotide. In some embodiments, the phosphorus group is a phosphate group, and the abasic nucleotide is a 2'-deoxyabasic nucleotide.

[0060] In some embodiments, the concentrations of the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate in the reaction system are approximately 0.1 mM to approximately 10 mM, respectively. In some embodiments, the concentrations of the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate in the reaction system may also be approximately 0.2 mM to approximately 10 mM, approximately 0.3 mM to approximately 10 mM, approximately 0.4 mM to approximately 10 mM, approximately 0.5 mM to approximately 10 mM, approximately 0.6 mM to approximately 10 mM, approximately 0.7 mM to approximately 10 mM, approximately 0.8 mM to approximately 10 mM, approximately 0.9 mM to approximately 10 mM, approximately 1 mM to approximately 10 mM, 1.2 mM to approximately 10 mM, 1.5 mM to approximately 10 mM, 2 mM to approximately 10 mM, 2.5 mM to approximately 10 mM, 3 mM to approximately 10 mM, 3.5 mM to approximately 10 mM, 4 mM to approximately 10 mM, 4.5 mM to approximately 10 mM, 5 mM to approximately 10 mM, 5 mM to approximately 10 mM, 4 mM to approximately 10 mM, 5 ... mM to about 10mM, 6mM to about 10mM, 7mM to about 10mM, 8mM to about 10mM, 9mM to about 10mM, 0.2mM to about 9mM, about 0.3mM to about 9mM, about 0.4mM to about 9mM, about 0.5mM to about 9mM, about 0.6mM to about 9mM, about 0.7mM to about 9mM, about 0.8mM to about 9mM, about 0.9mM mM to about 9mM, about 1mM to about 9mM, 1.2mM to about 9mM, 1.5mM to about 9mM, 2mM to about 9mM, 2.5mM to about 9mM, 3mM to about 9mM, 3.5mM to about 9mM, 4mM to about 9mM, 4.5mM to about 9mM, 5mM to about 9mM, 6mM to about 9mM, 7mM to about 9mM, or 8mM to about 9mM. In some embodiments, the concentrations of the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate in the reaction system may also be 0.2 mM to about 0.5 mM, about 0.5 mM to about 1 mM, about 0.5 mM to about 1.5 mM, about 1 mM to about 1.5 mM, about 1 mM to about 2 mM, about 1 mM to about 2.5 mM, 1.5 mM to about 2 mM, 1.5 mM to about 2.5 mM, about 1.5 mM to about 3 mM, about 2 mM to about 2.5 mM, about 2 mM to about 3 mM, or about 2 mM to about 3.5 mM.

[0061] In some embodiments, the concentrations of the first double-chain substrate and the second double-chain substrate in the reaction system are about 0.1 mM to about 10 mM, respectively. In some embodiments, the concentrations of the first double-chain substrate and the second double-chain substrate in the reaction system may also be approximately 0.2 mM to approximately 10 mM, approximately 0.3 mM to approximately 10 mM, approximately 0.4 mM to approximately 10 mM, approximately 0.5 mM to approximately 10 mM, approximately 0.6 mM to approximately 10 mM, approximately 0.7 mM to approximately 10 mM, approximately 0.8 mM to approximately 10 mM, approximately 0.9 mM to approximately 10 mM, approximately 1 mM to approximately 10 mM, 1.2 mM to approximately 10 mM, 1.5 mM to approximately 10 mM, 2 mM to approximately 10 mM, 2.5 mM to approximately 10 mM, 3 mM to approximately 10 mM, 3.5 mM to approximately 10 mM, 4 mM to approximately 10 mM, 4.5 mM to approximately 10 mM, 5 mM to approximately 10 mM, 6 mM, etc. M to about 10mM, 7mM to about 10mM, 8mM to about 10mM, 9mM to about 10mM, 0.2mM to about 9mM, about 0.3mM to about 9mM, about 0.4mM to about 9mM, about 0.5mM to about 9mM, about 0.6mM to about 9mM, about 0.7mM to about 9mM, about 0.8mM to about 9mM, about 0.9mM to about 9mM, about 1mM to about 9mM, 1.2mM to about 9mM, 1.5mM to about 9mM, 2mM to about 9mM, 2.5mM to about 9mM, 3mM to about 9mM, 3.5mM to about 9mM, 4mM to about 9mM, 4.5mM to about 9mM, 5mM to about 9mM, 6mM to about 9mM, 7mM to about 9mM, or 8mM to about 9mM. In some embodiments, the concentrations of the first double-chain substrate and the second double-chain substrate in the reaction system may also be 0.2 mM to about 0.5 mM, about 0.5 mM to about 1 mM, about 0.5 mM to about 1.5 mM, about 1 mM to about 1.5 mM, about 1 mM to about 2 mM, about 1 mM to about 2.5 mM, 1.5 mM to about 2 mM, 1.5 mM to about 2.5 mM, about 1.5 mM to about 3 mM, about 2 mM to about 2.5 mM, about 2 mM to about 3 mM, or about 2 mM to about 3.5 mM.

[0062] In some embodiments, the concentration of the RNA ligase in the reaction system is from about 1 μg / mL to about 200 μg / mL. In some embodiments, the concentration of the RNA ligase in the reaction system may also be from about 2 μg / mL to about 200 μg / mL, from about 3 μg / mL to about 200 μg / mL, from about 4 μg / mL to about 200 μg / mL, from about 5 μg / mL to about 200 μg / mL, from about 6 μg / mL to about 200 μg / mL, from about 7 μg / mL to about 200 μg / mL, or from about 8 μg / mL to about 200 μg / mL. mL, about 9 μg / mL to about 200 μg / mL, about 10 μg / mL to about 200 μg / mL, about 15 μg / mL to about 200 μg / mL, about 20 μg / mL to about 200 μg / mL, about 25 μg / mL to about 200 μg / mL, about 30 μg / mL to about 200 μg / mL, about 35 μg / mL to about 200 μg / mL, about 40 μg / mL to about 200 μg / mL, about 45 5 μg / mL to about 200 μg / mL, 6 about 0 μg / mL to about 200 μg / mL, 70 μg / mL to about 200 μg / mL, 80 μg / mL to about 200 μg / mL, 90 μg / mL to about 200 μg / mL, 100 μg / mL to about 200 μg / mL, 110 μg / mL to about 200 μg / mL, 120 μg / mL g / mL to about 200 μg / mL, about 130 μg / mL to about 200 μg / mL, about 140 μg / mL to about 200 μg / mL, about 150 μg / mL to about 200 μg / mL, about 160 μg / mL to about 200 μg / mL, about 170 μg / mL to about 200 μg / mL, about 180 μg / mL to about 200 μg / mL, or about 190 μg / mL to about 200 μg / mL, etc. In some embodiments, the concentration of the RNA ligase in the reaction system may also be about 1-5 μg / mL, about 5-10 μg / mL, about 10-20 μg / mL, about 15-23 μg / mL, about 25-35 μg / mL, about 30-50 μg / mL, about 40-60 μg / mL, about 60-90 μg / mL, about 90-110 μg / mL, or about 110-130 μg / mL.

[0063] In some embodiments, the reaction system further includes ATP, magnesium acetate, and DTT.

[0064] In some embodiments, the concentration of ATP in the reaction system is about 1-10 mM. In some embodiments, the concentration of ATP in the reaction system may also be about 1-10 mM, about 2-10 mM, about 3-10 mM, about 4-10 mM, about 5-10 mM, about 6-10 mM, about 7-10 mM, about 8-10 mM, about 9-10 mM, about 1-9 mM, about 2-9 mM, about 3-9 mM, about 4-9 mM, about 5-9 mM, about 6-9 mM, about 7-9 mM, about 8-9 mM, about 1-8 mM, about 2-8 mM, or about 3-8 mM. Approximately 4-8 mM, approximately 5-8 mM, approximately 6-8 mM, approximately 7-8 mM, approximately 1-7 mM, approximately 2-7 mM, approximately 3-7 mM, approximately 4-7 mM, approximately 5-7 mM, approximately 6-7 mM, approximately 1-6 mM, approximately 2-6 mM, approximately 3-6 mM, approximately 4-6 mM, approximately 5-6 mM, approximately 1-5 mM, approximately 2-5 mM, approximately 3-5 mM, approximately 4-5 mM, approximately 1-4 mM, approximately 2-4 mM, approximately 3-4 mM, approximately 1-3 mM, approximately 2-3 mM, or approximately 1-2 mM. In some embodiments, the concentration of ATP in the reaction system is approximately 3-7 mM.

[0065] In some embodiments, the concentration of magnesium acetate in the reaction system is about 5-20 mM. In some embodiments, the concentration of magnesium acetate in the reaction system may also be about 6-20 mM, about 7-20 mM, about 8-20 mM, about 9-20 mM, about 10-20 mM, about 11-20 mM, about 12-20 mM, about 13-20 mM, about 14-20 mM, about 15-20 mM, about 16-20 mM, about 17-20 mM, about 18-20 mM, about 19-20 mM, about 5-19 mM, about 6-19 mM, about 7-19 mM, about 8-19 mM, about 9-19 mM, about 10-19 mM, about 11-19 mM, about 12-19 mM, about 13-19 mM, about 14-19 mM, about 15- 19mM, approximately 16-19mM, approximately 17-19mM, approximately 18-19mM, approximately 5-18mM, approximately 6-18mM, approximately 7-18mM, approximately 8-18mM, approximately 9-18mM, approximately 10-18mM, approximately 11-18mM, approximately 12-18mM, approximately 13-18mM, approximately 14-18mM, approximately 15-18mM, approximately 16-18mM, approximately 17-18mM, approximately 5-17mM, approximately 6-17mM, approximately 7-17mM, approximately 8-17mM, approximately 9-17mM, approximately 10-17mM, approximately 11-17mM, approximately 12-17mM, approximately 13-17mM, approximately 14-17mM, approximately 15-17mM, approximately 16 -17mM, approximately 5-16mM, approximately 6-16mM, approximately 7-16mM, approximately 8-16mM, approximately 9-16mM, approximately 10-16mM, approximately 11-16mM, approximately 12-16mM, approximately 13-16mM, approximately 14-16mM, approximately 15-16mM, approximately 5-15mM, approximately 6-15mM, approximately 7-15mM, approximately 8-15mM, approximately 9-15mM, approximately 10-15mM, approximately 11-15mM, approximately 12-15mM, approximately 13-15mM, approximately 14-15mM, approximately 5-14mM, approximately 6-14mM, approximately 7-14mM, approximately 8-14mM, approximately 9-14mM, approximately 10-14mM, approximately 11-14m M, approximately 12-14 mm, approximately 13-14 mm, approximately 5-13 mm, approximately 6-13 mm, approximately 7-13 mm, approximately 8-13 mm, approximately 9-13 mm, approximately 10-13 mm, approximately 11-13 mm, approximately 12-13 mm, approximately 5-12 mm, approximately 6-12 mm, approximately 7-12 mm, approximately 8-12 mm, approximately 9-12 mm, approximately 10-12 mm, approximately 11-12 mm, approximately 5-11 mm, approximately 6-11 mm, approximately 7-11 mm, approximately 8-11 mm, approximately 9-11 mm, approximately 10-11 mm, approximately 5-10 mm, approximately 6-10 mm, approximately 7-10 mm, approximately 8-10 mm, approximately 9-10 mm, approximately 5-9 mmApproximately 6-9 mM, approximately 7-9 mM, approximately 8-9 mM, approximately 5-8 mM, approximately 6-8 mM, approximately 7-8 mM, approximately 5-7 mM, approximately 6-7 mM, or approximately 5-6 mM. In some embodiments, the concentration of magnesium acetate in the reaction system is approximately 8-15 mM.

[0066] In some embodiments, the concentration of DTT in the reaction system is about 0.5-2 mM. In some embodiments, the concentration of DTT in the reaction system may also be about 0.5-1.8 mM, about 0.5-1.5 mM, about 0.5-1.2 mM, about 0.5-1 mM, about 0.5-0.8 mM, about 0.8-2 mM, about 0.8-1.8 mM, about 0.8-1.5 mM, about 0.8-1.2 mM, about 0.8-1 mM, about 1-2 mM, about 1-1.8 mM, about 1-1.5 mM, about 1-1.2 mM, about 1.2-2 mM, about 1.2-1.8 mM, about 1.2-1.5 mM, about 1.5-2 mM, about 1.5-1.8 mM, or about 1.8-2 mM. In some embodiments, the concentration of magnesium acetate in the reaction system is about 0.8-1.2 mM.

[0067] In some embodiments, the reaction system is carried out at a reaction temperature of about 20 to about 40°C. In some embodiments, the reaction temperature is about 25 to about 40°C, about 30 to about 40°C, about 30 to about 39°C, about 30 to about 38°C, about 30 to about 37°C, about 30 to about 36°C, about 30 to about 35°C, about 30 to about 34°C, or about 31 to about 34°C. In some embodiments, the reaction temperature is about 20°C, about 22°C, about 24°C, about 26°C, about 28°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 38°C, or about 40°C. In some embodiments, the reaction temperature is about 33°C.

[0068] In some embodiments, the reaction time of the reaction system is from about 0.1 h to about 30 h. In some embodiments, the reaction time is from about 0.5 h to about 30 h, from about 1 h to about 30 h, from about 1 h to about 25 h, from about 2 h to about 25 h, from about 3 h to about 25 h, from about 4 h to about 25 h, from about 5 h to about 25 h, or from about 5 h to about 20 h. In some embodiments, the reaction time is approximately 0.1 h, approximately 0.2 h, approximately 0.3 h, approximately 0.4 h, approximately 0.5 h, approximately 0.6 h, approximately 0.7 h, approximately 0.8 h, approximately 0.9 h, approximately 1 h, approximately 1.5 h, approximately 2 h, approximately 2.5 h, approximately 3 h, approximately 3.5 h, approximately 4 h, approximately 4.5 h, approximately 5 h, approximately 6 h, approximately 7 h, approximately 8 h, approximately 9 h, approximately 10 h, approximately 12 h, approximately 14 h, approximately 16 h, approximately 18 h, approximately 20 h, approximately 22 h, approximately 24 h, approximately 26 h, approximately 28 h, or approximately 30 h. Those skilled in the art will understand that the actual reaction time can also be adjusted according to the reaction scale, reaction temperature, and reaction state, and the reaction status can be monitored using conventional methods in the art, such as HPLC.

[0069] In some embodiments, the preparation method provided herein can generate a positive and / or negative strand from a positive and / or negative substrate, respectively, with a conversion efficiency of not less than 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The conversion efficiency can be measured by methods known in the art, such as the method described in Example 1 of this application.

[0070] In some embodiments, the moisture content of the siRNA product obtained by the preparation method provided herein is not higher than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or 0.5%. In some embodiments, the analytical purity of the siRNA product is not lower than 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example, by denaturing ion-pair reversed-phase chromatography (denaturing IPRP), non-denaturing ion-pair reversed-phase chromatography (non-denaturing IPRP), or ion exchange chromatography (IEX). In some embodiments, the Cd residue of the siRNA product is not higher than 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm, or 0.01 ppm; the Pb residue is not higher than 20 ppm, 19 ppm, 18 ppm, 17 ppm, 16 ppm, 15 ppm, 14 ppm, 13 ppm, 12 ppm, 11 ppm, 10 ppm, or 5 ppm; and the As residue is not high. The concentrations of particulate matter (PMT) at 0.50 ppm, 0.49 ppm, 0.48 ppm, 0.47 ppm, 0.46 ppm, 0.45 ppm, 0.44 ppm, 0.43 ppm, 0.42 ppm, 0.41 ppm, 0.40 ppm, or 0.20 ppm are specified; the residual amount of mercury (Hg) is not higher than 0.15 ppm, 0.14 ppm, 0.13 ppm, 0.12 ppm, 0.11 ppm, 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, or 0.05 ppm are specified. PM, 0.04ppm, 0.03ppm, 0.02ppm, or 0.01ppm; Co residue not exceeding 0.20ppm, 0.19ppm, 0.18ppm, 0.17ppm, 0.16ppm, 0.15ppm, 0.14ppm, 0.13ppm, 0.12ppm, 0.11ppm, 0.1ppm, 0.09ppm, 0.08ppm, 0.07ppm, 0.06ppm, or 0.05ppm; V residue not exceeding 0.50ppm, 0.40 ppm, 0.30ppm, 0.25ppm, 0.20ppm, 0.15ppm, or 0.10ppm; Ni residue not exceeding 1ppm, 0.90ppm, 0.80ppm, 0.70ppm, 0.60ppm, 0.50ppm, 0.40ppm, 0.30ppm, 0.20ppm, or 0.10ppm; Li residue not exceeding 10.0ppm, 9.0ppm, 8.0ppm, 7.5ppm, 7.0ppm, 6.5ppm, 6.0ppm, or 5ppm.5 ppm or 5.0 ppm; Sb residue not exceeding 3.5 ppm, 3 ppm, 2.9 ppm, 2.8 ppm, 2.7 ppm, 2.6 ppm, 2.5 ppm, 2.4 ppm, 2.3 ppm, 2.2 ppm, 2.1 ppm, 2 ppm, 1.5 ppm, or 1 ppm; Cu residue not exceeding 11.0 ppm, 10.0 ppm, 9.0 ppm, 8.5 ppm, 8.0 ppm, 7.5 ppm, 7.0 ppm, 6.5 ppm, 6.0 ppm, 5.5 ppm, or 5.0 ppm; Enzyme residue not exceeding 10 ppm, 9 ppm, 8 ppm, 7 ppm, 6 ppm, 5 ppm, 4 ppm, 3 ppm, 2.5 ppm, or 2 ppm. The concentrations of particulate matter (PM2.5), 1.5 ppm, 1 ppm, 0.9 ppm, 0.8 ppm, 0.7 ppm, 0.6 ppm, 0.5 ppm, 0.4 ppm, 0.3 ppm, 0.2 ppm, or 0.1 ppm are specified. Endotoxin residues are not higher than 0.2 EU / mg, 0.15 EU / mg, 0.1 EU / mg, 0.09 EU / mg, 0.08 EU / mg, 0.07 EU / mg, 0.06 EU / mg, or 0.05 EU / mg are specified. Microbial limits are not higher than 15 CFU / g, 14 CFU / g, 13 CFU / g, 12 CFU / g, 11 CFU / g, 10 CFU / g, 9 CFU / g, 8 CFU / g, 7 CFU / g, 6 CFU / g, or 5 CFU / g are specified.

[0071] The following RNA sequences are provided in this article.

[0072] The following RNA sequences are also provided in this article.

[0073] This application provides the following non-limiting implementation methods.

[0074] Implementation Method 1. A method for preparing siRNA, characterized in that the siRNA comprises a sense strand and an antisense strand; the sense strand is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', and the antisense strand is 5'-mA*fC*mAmAmAfAmGmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3', wherein A, U, G, and C represent nucleotides with adenine, uracil, guanine, and cytosine as bases, respectively; m indicates that the nucleotide adjacent to it on the right is modified with a 2'-O-methyl group; f indicates that the nucleotide adjacent to it on the right is modified with a 2'-fluorine group; * indicates that the linking group between the two groups is a phosphate thioester; and B indicates a baseless nucleotide; the 5' end of the sense strand is a hydroxyl group, and the 3' end is a UM group, the structure of which is: The antisense strand has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the method includes: mixing a first positive strand substrate, a second positive strand substrate, a first antisense strand substrate, a second antisense strand substrate, and an RNA ligase to form a reaction system, wherein the RNA ligase catalyzes the ligation of the first positive strand substrate and the second positive strand substrate to form the positive strand, and catalyzes the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand, the positive strand and the antisense strand complementaryly pairing to form the siRNA; the first positive strand substrate and the second positive strand substrate can form the positive strand, and the first antisense strand substrate and the second antisense strand substrate can form the antisense strand; the length of the first positive strand substrate and the second positive strand substrate is 2-17 nucleotides (nt), and the length of the first antisense strand substrate and the second antisense strand substrate is 2-19 nt.

[0075] Implementation Method 2. The method according to Implementation Method 1, characterized in that the first sense substrate, the second sense substrate, the first antisense substrate and the second antisense substrate are mixed and annealed, and then mixed with the RNA ligase to form the reaction system.

[0076] Implementation Method 3. The method according to Implementation Method 1 or 2, characterized in that the 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are ligated under the catalysis of the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated under the catalysis of the RNA ligase to form the antisense strand.

[0077] Implementation Method 4. The method according to any one of Implementation Methods 1 to 3, characterized in that: the 5' end of the first positive substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second positive substrate is a phosphate group, and the 3' end is a UM group; and the 5' end of the first antisense substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense substrate is a phosphate group, and the 3' end is a hydroxyl group.

[0078] Implementation Method 5. The method according to any one of Implementation Methods 1 to 4, characterized in that: (a) the length of the first justice chain substrate is 13nt and the length of the second justice chain substrate is 6nt; or (b) the length of the first justice chain substrate is 6nt and the length of the second justice chain substrate is 13nt; or (c) the length of the first justice chain substrate is 8nt and the length of the second justice chain substrate is 11nt; or (d) the length of the first justice chain substrate is 5nt and the length of the second justice chain substrate is 14nt.

[0079] Implementation 6. The method according to any one of Implementation 1 to 5, characterized in that (a) the length of the first antisense substrate is 9nt and the length of the second antisense substrate is 12nt; or (b) the length of the first antisense substrate is 7nt and the length of the second antisense substrate is 14nt.

[0080] Implementation Method 7. The method according to any one of Implementation Methods 1 to 6, characterized in that: (a) the length of the first positive chain substrate is 13nt, the length of the second positive chain substrate is 6nt, the length of the first negative chain substrate is 9nt, and the length of the second negative chain substrate is 12nt; or (b) the length of the first positive chain substrate is 6nt, the length of the second positive chain substrate is 13nt, the length of the first negative chain substrate is 9nt, and the length of the second negative chain substrate is 12nt; or (c) the length of the first positive chain substrate is 8nt, the length of the second positive chain substrate is 11nt, the length of the first negative chain substrate is 7nt, and the length of the second negative chain substrate is 14nt; or (d) the length of the first positive chain substrate is 5nt, the length of the second positive chain substrate is 14nt, the length of the first negative chain substrate is 9nt, and the length of the second negative chain substrate is 12nt.

[0081] Implementation Method 8. The method according to any one of Implementation Methods 1 to 7, characterized in that: (a) the first positive chain substrate is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmC-3', the second positive chain substrate is 5'-PmUmUmUmUmGmU*UM-3', the first negative chain substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second negative chain substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; or (b) The first justice chain substrate is 5'-B*mA*mGmAmCmCmU-3', the second justice chain substrate is 5'-PfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense chain substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; or (c) The first justice chain substrate is 5'-B*mA*mGmAmCmCmUfGmU-3', the second justice chain substrate is 5'-PfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmG-3', and the second antisense chain substrate is 5'-PmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; Alternatively (d) the first sense substrate is 5'-B*mA*mGmAmCmC-3', the second sense substrate is 5'-PmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; where P represents a phosphorus group.

[0082] Embodiment 9. The method according to Embodiment 8, wherein the phosphorus group is a phosphate group and the abasic nucleotide is a 2'-deoxyabasic nucleotide.

[0083] Implementation Method 10. The method according to any one of Implementation Methods 1 to 9, characterized in that, in the reaction system, the concentrations of the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate are approximately 0.1 mM to approximately 10 mM, respectively; and the concentration of the RNA ligase is approximately 1 μg / mL to approximately 200 μg / mL.

[0084] Implementation Method 11. The preparation method according to any one of Implementation Methods 1 to 10, characterized in that the reaction system further includes ATP, magnesium acetate and DTT.

[0085] Embodiment 12. The preparation method according to any one of Embodiments 1 to 11, characterized in that the reaction system is carried out at a reaction temperature of about 20 to about 40°C for a reaction time of about 0.1 h to about 30 h.

[0086] Example

[0087] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0088] Example 1

[0089] The substrate fragments are prepared using solid-phase synthesis processes known in the art, which mainly include solid-phase synthesis, cleavage, purification (optional), and ultrafiltration. For the synthesis of substrate fragments without N-acetylgalactosamine (GalNAc) ligands, the starting vector contains the first nucleotide. For the synthesis of substrate fragments containing GalNAc ligands, the starting vector contains the GalNAc ligand.

[0090] Preparation of the reaction system: Substrate fragments RNA-3 and RNA-12 were mixed to form system A, and RNA-4 and RNA-11 were mixed to form system B. The substrate sequences are shown in Table 1. Both mixtures were annealed (heated to 90℃, maintained for 5-20 minutes, then cooled to room temperature) to form complementary double strands. Then, systems A and B were mixed thoroughly, and an appropriate amount of purified water was added to make a final reaction volume of 20 μL. The substrate concentration was 0.5–5 mM. After mixing the four substrate fragments, the mixture was stirred at room temperature for 10–60 min. Samples of the reaction system were then analyzed using denaturing ion-pair reversed-phase high-performance liquid chromatography-mass spectrometry (IPRP-HPLC-MS). The results are shown in Figure 3. Other reagents were added to the reaction system to maintain the following concentrations: magnesium acetate (5-20 mM), ATP (3-10 mM), and dithiothreitol (DTT) (0.5-2 mM). A 5% (v / v) commercial ligase was added to maintain an enzyme concentration of 20-100 μg / mL. The reaction was incubated at 37°C for at least 2 hours. After the reaction, ethylenediaminetetraacetic acid (EDTA) was added to the reaction solution, and a sample was diluted for denaturing IPRP-HPLC-MS analysis. The results showed that the ligase catalyzed the synthesis of the target siRNA. The theoretical molecular weight of the positive strand of the target siRNA was 8221.2 ± 4, and the theoretical molecular weight of the negative strand was 7084.0 ± 4. Mass spectrometry results showed that the molecular weights matched those of the target siRNA. The liquid chromatography results are shown in Figure 4, indicating that the substrate fragment had been converted into the positive and negative strands of the target product.

[0091] The sense strand (SS) of the target siRNA obtained was 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3'(RNA-1).

[0092] The antisense strand (SS) of the target siRNA obtained was 5'-mA*fC*mAmAmAfAmGmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3'(RNA-2).

[0093] The conversion efficiency is calculated using the following formula: Conversion efficiency = (Sum of peak areas of generated products ÷ Sum of peak areas of products and remaining substrate) × 100%

[0094] The method successfully prepared the target siRNA with a conversion efficiency of 94.31%. Table 1

[0095] Example 2

[0096] Substrate fragments RNA-5, RNA-6, RNA-11, and RNA-12 were synthesized using solid-phase synthesis methods known in the art. Reaction systems were then prepared: RNA-5 and RNA-12 were mixed to form system A, and RNA-6 and RNA-11 were mixed to form system B. The substrate sequences are shown in Table 2. Both mixtures were annealed (heated to 90°C, maintained for 5-20 minutes, then cooled to room temperature) to form complementary double strands. Systems A and B were then thoroughly mixed, and an appropriate amount of purified water was added to bring the final volume to 20 μL. The substrate concentration was 0.5–5 mM. After mixing the four substrate fragments, the mixture was stirred at room temperature for 10–60 minutes. Samples of the reaction system were then analyzed by denaturing IPRP-HPLC-MS. The results are shown in Figure 5. Other reagents were added to the reaction system to maintain magnesium acetate concentration at 5-20 mM, ATP concentration at 3-10 mM, and DTT concentration at 0.5-2 mM. A commercial ligase was added at a volume percentage of 5%, bringing the enzyme concentration to 20 μg / mL-100 μg / mL. The reaction was incubated at 37℃ for at least 2 hours. After the reaction, EDTA was added to the reaction solution, and a sample was diluted for denaturing IPRP-HPLC-MS analysis. The results showed that the ligase catalyzed the synthesis of the target siRNA. Mass spectrometry results showed that the molecular weight matched that of the target siRNA. HPLC analysis results (Figure 6) indicate that the fragment has been converted into the sense and antisense strands of the target siRNA. The method successfully prepared the target siRNA with a conversion efficiency of 93.00%. Table 2

[0097] Example 3

[0098] Substrate fragments RNA-7, RNA-8, RNA-13, and RNA-14 were synthesized using solid-phase synthesis methods known in the art. Reaction systems were then prepared: RNA-7 and RNA-14 were mixed to form system A, and RNA-8 and RNA-13 were mixed to form system B. The substrate sequences are shown in Table 3. Both mixtures were annealed (heated to 90°C, maintained for 5-20 minutes, then cooled to room temperature) to form complementary double strands. Systems A and B were then thoroughly mixed, and an appropriate amount of purified water was added to bring the final volume to 20 μL. The substrate concentration was 0.5–5 mM. After mixing the four substrate fragments, the mixture was stirred at room temperature for 10–60 minutes. Samples of the reaction system were then analyzed by denaturing IPRP-HPLC-MS. The results are shown in Figure 7. Other reagents were added to the reaction system to maintain magnesium acetate concentration at 5-20 mM, ATP concentration at 3-10 mM, and DTT concentration at 0.5-2 mM. A commercial RNA ligase was added at a volume percentage of 5%, bringing the enzyme concentration to 20 μg / mL-100 μg / mL. The reaction was incubated at 37°C for at least 2 hours. After the reaction, EDTA was added to the reaction solution, and a sample was diluted for denaturing IPRP-HPLC-MS analysis. The results showed that the ligase catalyzed the synthesis of the target siRNA. Mass spectrometry results showed that the molecular weight matched that of the target siRNA. HPLC analysis results (Figure 8) indicate that the fragment has been converted into the sense and antisense strands of the target siRNA. The method successfully prepared the target siRNA with a conversion efficiency of 76.76%. Table 3

[0099] Example 4

[0100] Substrate fragments RNA-9, RNA-10, RNA-11, and RNA-12 were synthesized using solid-phase synthesis methods known in the art. Reaction systems were then prepared: RNA-9 and RNA-12 were mixed to form system A, and RNA-10 and RNA-11 were mixed to form system B. The substrate sequences are shown in Table 4. Both mixtures were annealed (heated to 90°C, maintained for 5-20 minutes, then cooled to room temperature) to form complementary double strands. Systems A and B were then thoroughly mixed, and an appropriate amount of purified water was added to bring the final volume to 20 μL. The substrate concentration was 0.5–5 mM. After mixing the four substrate fragments, the mixture was stirred at room temperature for 10–60 minutes. Samples of the reaction system were then analyzed by denaturing IPRP-HPLC-MS. The results are shown in Figure 9. Other reagents were added to the reaction system to maintain magnesium acetate concentration at 5-20 mM, ATP concentration at 3-10 mM, and DTT concentration at 0.5-2 mM. A commercial ligase was added at a volume percentage of 5%, bringing the enzyme concentration to 20 μg / mL-100 μg / mL. The reaction was carried out at 37℃ for at least 2 hours. After the reaction, EDTA was added to the reaction solution, and a sample was diluted for denaturing IPRP-HPLC-MS analysis. The results showed that the ligase catalyzed the synthesis of the target siRNA. Mass spectrometry results showed that the molecular weight matched that of the target siRNA. HPLC analysis results (Figure 10) indicated that the fragment had been converted into the sense and antisense strands of the target siRNA. The method successfully prepared the target siRNA with a conversion efficiency of 93.16%. Table 4

[0101] Example 5: Optimization of Enzyme Addition Amount

[0102] Preparation of the reaction system: Substrate fragments RNA-3 and RNA-12 were mixed to form system A, and RNA-4 and RNA-11 were mixed to form system B. The substrate sequences are shown in Table 1. Both mixtures were annealed (heated to 90℃, maintained for 5-20 minutes, then cooled to room temperature) to form complementary double strands. Then, systems A and B were thoroughly mixed, and an appropriate amount of purified water was added to make a final volume of 40 μL. The substrate concentration was 0.5–5 mM. After mixing the four substrate fragments, the mixture was stirred at room temperature for 10–60 min. Other reaction reagents were added to maintain the following concentrations: magnesium acetate at 5–20 mM, ATP at 3–10 mM, and DTT at 0.5–2 mM. Commercial ligase was added at a volume percentage of 1.25% to maintain an enzyme concentration of 5 μg / mL–25 μg / mL. The reaction system was incubated at 37℃ for at least 2 hours. After the reaction, EDTA was added to the reaction solution, and a sample was diluted for denaturing IPRP-HPLC-MS analysis. LCMS results showed that after 4 hours of reaction at 37°C, the product accounted for more than 90% of the reaction solution. By reducing the amount of enzyme, the ligation could be completed in 4 hours at 37°C. No byproducts or product degradation were observed in the reaction solution after 4 hours of reaction, and the reaction solution was stable.

[0103] Example 6: Optimization of Reaction Temperature

[0104] Preparation of the reaction system: Mix substrate fragments RNA-3 and RNA-12 to form system A, and mix RNA-4 and RNA-11 to form system B. Anneal both mixtures separately (at room temperature for 5-30 minutes) to form complementary double strands. Then mix system A and system B thoroughly and prepare 5 parallel aliquots. Add an appropriate amount of purified water to each aliquot to make the final volume 40 μL. The substrate concentration is 0.5-5 mM. After mixing the 4 substrate fragments, stir at room temperature for 10-60 min. Continue to add other reaction reagents to the reaction system to maintain the magnesium acetate concentration at 5-20 mM, the ATP concentration at 3-10 mM, and the DTT concentration at 0.5-2 mM. Add 1.25% by volume of commercial ligase to make the enzyme concentration 5 μg / mL-25 μg / mL. Five parallel reaction systems were reacted at 30℃, 31℃, 32℃, 33℃, and 34℃, respectively. Samples were taken every 1 hour for LCMS analysis. The results showed that the conversion rate reached over 90% at all reaction temperatures from 2 to 4 hours, with the fastest reaction rate at 33℃. The conversion rates at different temperatures with reaction time are shown in Figure 11.

[0105] Example 7

[0106] Four substrate fragments (RNA-3, RNA-4, RNA-11, and RNA-12) obtained from solid-phase synthesis were purified and ultrafiltered to obtain substrate fragments with higher purity. The purified fragments were mixed and stirred at room temperature for 10-25 min at a concentration of 0.5-5 mM. Other reagents were added to maintain the following concentrations: magnesium acetate (5-20 mM), ATP (3-10 mM), and DTT (0.5-2 mM). 1.25% (v / v) of commercial ligase was added to maintain an enzyme concentration of 5-25 μg / mL. The reaction was incubated at 30-37°C for at least 4 h. After the reaction, EDTA was added to the reaction solution, and samples were diluted for analysis. LCMS results showed that, compared to unpurified substrate fragments, ligation of the purified substrates effectively improved the purity of the crude product. Crude product purity data are shown in Table 5. Table 5

[0107] Furthermore, UPLC and mass spectrometry were used to detect the impurity content. The results showed that the P=O (incomplete thiolation) and P=S (oxidative thiolation) impurities of the target siRNA synthesized after fragment purification were significantly lower than those of the target siRNA synthesized after unpurified fragment enzymatic ligation. The impurity content results are shown in Table 6. This indicates that enzymatic synthesis of target siRNA after fragment purification is more advantageous. Table 6

[0108] Example 8

[0109] Four RNA substrate fragments were obtained through solid-phase synthesis. Aqueous solutions of RNA-3, RNA-12, RNA-4, and RNA-11 were added to a glass reactor and stirred at room temperature for 10-60 min. 2-4 L of buffer solution was added, followed by DTT and ATP solutions. Then, 300-500 mL of commercial ligase was added, and after thorough mixing, purified water was added to bring the final volume to 20-40 L. The resulting concentrations were 0.5-5 mM for the substrate fragments, 0.5-2 mM for DTT, 3-10 mM for ATP, and 5 μg / mL to 25 μg / mL for the enzyme. The reaction system was incubated at 33°C for at least 4 h. EDTA was added to terminate the reaction. After the reaction, the product was purified using reverse-phase chromatography, desalted by ultrafiltration, and lyophilized to obtain 1021 g of final product. The solid final product was tested for moisture, purity, enzyme residue, solvent residue, metal residue, microorganisms, and endotoxins. The product has a moisture content of 1.7%. Denatured ion-pair reversed-phase chromatography (denatured IPRP) purity is 96.9%, non-denatured ion-pair reversed-phase chromatography (non-denatured IPRP) purity is 98.6%, and ion-exchange chromatography (IEX) purity is 98.7%. Metal residues are: Cd < 0.06 ppm, Pb < 0.15 ppm, As < 0.45 ppm, Hg < 0.09 ppm, Co < 0.15 ppm, V < 0.30 ppm, Ni < 0.60 ppm, Li < 7.5 ppm, Sb < 2.7 ppm, Cu < 9.0 ppm. Enzyme residue is < 100 ppm, endotoxin residue is < 0.25 EU / mg, and the microbial limit is < 100 CFU / g. All these meet the quality standards for siRNA drugs, indicating that the enzymatic synthesis route for this siRNA drug structure is feasible, with high product purity and an extremely simple process.

[0110] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the preparation method of this application, RNA ligase catalyzes the ligation between sense and antisense substrates to form the sense strand of the target siRNA, and catalyzes the ligation between antisense substrates to form the antisense strand of the target siRNA, thereby realizing the preparation of this structurally complex siRNA with multiple modifications through enzymatic synthesis. Compared with the preparation of target siRNA using chemical solid-phase synthesis, the preparation method of this application yields products with higher purity, fewer impurities, and lower pressure for subsequent final product purification. Furthermore, the reaction conditions are mild, and no large amount of organic reagents are used, which can reduce production costs and facilitate industrial-scale production.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing siRNA, characterized in that, The siRNA includes a sense strand and an antisense strand; The justice chain is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3'. The antisense chain is 5'-mA*fC*mAmAmAfAmGmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3'. Where A, U, G and C represent nucleotides with adenine, uracil, guanine and cytosine as bases, respectively; m indicates that the nucleotide adjacent to it on the right is modified with 2'-O-methyl; f indicates that the nucleotide adjacent to it on the right is modified with 2'-fluorine; * indicates that the linking group between the two groups on the left and right is thiophosphate; and B indicates a baseless nucleotide. The positive chain has a hydroxyl group at its 5' end and a UM group at its 3' end. The structure of the UM group is as follows: The antisense chain has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; The method includes: (1) Provide a reaction system comprising a first double-stranded substrate, a second double-stranded substrate, and an RNA ligase. The first double-chain substrate comprises a paired first positive chain substrate and a second negative chain substrate, and the second double-chain substrate comprises a paired second positive chain substrate and a first negative chain substrate. (2) The first double-stranded substrate and the second double-stranded substrate form the siRNA under the catalytic conditions of the RNA ligase. The RNA ligase catalyzes the ligation of the first sense strand substrate and the second sense strand substrate to form the sense strand, and catalyzes the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand; The first justice chain substrate and the second justice chain substrate can form the justice chain, and the first antisense chain substrate and the second antisense chain substrate can form the antisense chain; The first and second sense substrates are 2-17 nucleotides (nt) long, and the first and second antisense substrates are 2-19 nt long.

2. The method according to claim 1, characterized in that, The method further includes: mixing the first sense substrate and the second antisense substrate to pair the first sense substrate and the second antisense substrate to form the first double-stranded substrate, and mixing the second sense substrate and the first antisense substrate to pair the second sense substrate and the first antisense substrate to form the second double-stranded substrate, and then mixing the first double-stranded substrate, the second double-stranded substrate and the RNA ligase to form the reaction system.

3. The method according to claim 1, characterized in that, The method further includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, and the second antisense substrate, such that the first sense substrate and the second antisense substrate pair up to form the first double-stranded substrate, and such that the second sense substrate and the first antisense substrate pair up to form the second double-stranded substrate, and then mixing with the RNA ligase to form the reaction system.

4. The method according to claim 1, characterized in that, The method further includes: mixing the first sense substrate, the second sense substrate, the first antisense substrate, the second antisense substrate, and the RNA ligase, such that the first sense substrate and the second antisense substrate pair to form the first double-stranded substrate, and the second sense substrate and the first antisense substrate pair to form the second double-stranded substrate, thereby forming the reaction system.

5. The method according to any one of claims 1-4, characterized in that, The 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are ligated together under the catalysis of the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are ligated together under the catalysis of the RNA ligase to form the antisense strand.

6. The method according to any one of claims 1 to 5, characterized in that, The first positive-chain substrate has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second positive-chain substrate has a phosphate group at its 5' end and a UM group at its 3' end; and The first antisense substrate has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second antisense substrate has a phosphate group at its 5' end and a hydroxyl group at its 3' end.

7. The method according to any one of claims 1 to 6, characterized in that, (a) The length of the first justice chain substrate is 13nt, and the length of the second justice chain substrate is 6nt; or (b) The length of the first justice chain substrate is 6nt, and the length of the second justice chain substrate is 13nt; or (c) The length of the first justice chain substrate is 8nt, and the length of the second justice chain substrate is 11nt; or (d) The length of the first justice chain substrate is 5nt and the length of the second justice chain substrate is 14nt.

8. The method according to any one of claims 1 to 7, characterized in that, (a) The length of the first antisense substrate is 9nt, and the length of the second antisense substrate is 12nt; or (b) The length of the first antisense substrate is 7nt and the length of the second antisense substrate is 14nt.

9. The method according to any one of claims 1 to 8, characterized in that, (a) The length of the first justice chain substrate is 13nt, the length of the second justice chain substrate is 6nt, the length of the first antisense chain substrate is 9nt, and the length of the second antisense chain substrate is 12nt; or (b) The length of the first justice chain substrate is 6nt, the length of the second justice chain substrate is 13nt, the length of the first antisense chain substrate is 9nt, and the length of the second antisense chain substrate is 12nt; or (c) The length of the first justice chain substrate is 8nt, the length of the second justice chain substrate is 11nt, the length of the first antisense chain substrate is 7nt, and the length of the second antisense chain substrate is 14nt; or (d) The length of the first justice chain substrate is 5nt, the length of the second justice chain substrate is 14nt, the length of the first antisense chain substrate is 9nt, and the length of the second antisense chain substrate is 12nt.

10. The method according to any one of claims 1 to 9, characterized in that, (a) The first justice chain substrate is 5'-B*mA*mGmAmCmCmUfGmUfUfUfUmGmC-3', the second justice chain substrate is 5'-PmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense chain substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; or (b) The first justice chain substrate is 5'-B*mA*mGmAmCmCmU-3', the second justice chain substrate is 5'-PfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense chain substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; or (c) The first justice chain substrate is 5'-B*mA*mGmAmCmCmUfGmU-3', the second justice chain substrate is 5'-PfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmG-3', and the second antisense chain substrate is 5'-PmCmAmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; or (d) The first justice chain substrate is 5'-B*mA*mGmAmCmC-3', the second justice chain substrate is 5'-PmUfGmUfUfUfUmGmCmUmUmUmUmGmU*UM-3', the first antisense chain substrate is 5'-mA*fC*mAmAmAfAmGmCmA-3', and the second antisense chain substrate is 5'-PmAmAmAmCfAmGfGmUmCmU*mA*mG-3'; Where P represents a phosphorus group.

11. The method according to claim 10, characterized in that, The phosphorus group is a phosphate group, and the abasic nucleotide is a 2'-deoxyabasic nucleotide.

12. The method according to any one of claims 1 to 11, characterized in that, In the reaction system, The concentrations of the first double-stranded substrate and the second double-stranded substrate are approximately 0.1 mM to approximately 10 mM, respectively. The concentration of the RNA ligase is approximately 1 μg / mL to approximately 200 μg / mL.

13. The preparation method according to any one of claims 1 to 12, characterized in that, The reaction system also includes ATP, magnesium acetate, and DTT.

14. The preparation method according to any one of claims 1 to 13, characterized in that, The reaction system is carried out at a reaction temperature of about 20 to about 40°C for a reaction time of about 0.1 h to about 30 h.

15. The preparation method according to any one of claims 1 to 14, characterized in that, The pairing is achieved by mixing the ingredients and then heating them to approximately 50°C or higher, maintaining this temperature for approximately 5 minutes or more, and then cooling them to room temperature.

16. The preparation method according to claim 15, characterized in that, The pairing is achieved by mixing and heating to approximately 90°C or higher, maintaining this temperature for approximately 5-20 minutes, and then cooling to room temperature.

17. The preparation method according to any one of claims 1 to 14, characterized in that, The pairing is formed by mixing and then annealing at room temperature for at least 5 minutes.

18. The preparation method according to claim 17, characterized in that, The pairing is formed by mixing and then annealing at room temperature for approximately 5-30 minutes.

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