Preparation method for sirna for inhibiting PCSK9 gene expression

Abstract

Provided in the present invention is a preparation method for a siRNA for inhibiting PCSK9 gene expression. The siRNA in the preparation method is a double-stranded RNA consisting of a sense strand and an antisense strand which are complementarily paired. The preparation method comprises: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, using the RNA ligase for catalyzing the sense strand substrate and the antisense strand substrate to be connected by means of a phosphodiester bond, and obtaining a sense strand and an antisense strand, so as to obtain the siRNA. The sense strand substrate can form the sense strand, and the antisense strand substrate can form the antisense strand. The sense strand is a nucleic acid sequence as shown in SEQ ID NO: 45, and the antisense strand is a nucleic acid sequence as shown as SEQ ID NO: 46. The solution solves the problem in the prior art of low purity of the siRNA, and is applicable in the field of medicine biosynthesis.

Description

Preparation method of siRNA for inhibiting PCSK9 gene expression

[0001] This application is based on the Chinese application with CN application number 202311335115.1 and application date October 16, 2023, and claims its priority. The disclosed content of the CN application is again introduced as a whole into this application. Technical Field

[0002] The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing siRNA for inhibiting PCSK9 gene expression. Background Art

[0003] Many diseases are caused by abnormal gene expression and mutations, yet these diseases are often incurable with traditional small molecule drugs. Effective gene expression intervention has become the only treatment option for these diseases. siRNA is a double-stranded RNA oligonucleotide, 19 to 21 base pairs in length. In organisms, gene silencing through siRNA-mediated RNA interference (RNAi) is an important method of gene expression regulation, specifically triggering the degradation of target mRNA and inhibiting gene expression. Following the discovery of RNAi, the technology was rapidly applied to disease treatment, ushering in a significant opportunity for the development of gene therapy. This technology utilizes small interfering RNA (siRNA) to specifically inhibit the expression of the target pathogenic gene, thereby achieving therapeutic effects. In 2018, the US FDA approved an siRNA drug for the treatment of peripheral neuropathy caused by hereditary transthyretin amyloidosis, filling a gap in the field of siRNA therapy and paving the way for its subsequent development. Currently, there are five approved siRNA therapies worldwide. With the development of siRNA drugs for the treatment of hypercholesterolemia, siRNA drugs are becoming a standard of care. siRNA drugs generally intervene at the source, offering both symptomatic and fundamental treatments. They offer advantages such as rapid target screening, high therapeutic efficiency, low toxicity, and strong specificity. Their effects can last up to six months, allowing patients to take them as little as twice a year, making them a very promising treatment option.

[0004] The synthesis of siRNA mainly relies on the phosphoramidite method to synthesize, and this method comprises 4 steps of deprotection, coupling, capping and oxidation.But along with the prolongation of chain length, chemical reaction efficiency, synthesis purity and productive rate decline seriously, the oligo length synthesized by this method generally can not exceed 200 nucleotides (nt), can use a large amount of organic solvents simultaneously in building-up process, greatly increased the cost of reaction cost and three wastes treatment, also increased the burden of environmental pollution.Simultaneously in the process of reaction, can produce the N+1 impurity and N-1 impurity that are difficult to remove, along with the generation of the increase impurity of chain length can sharply increase, increased the difficulty of aftertreatment, greatly reduced yield.Along with siRNA is used for the popularization of treating commonly encountered diseases, the demand to siRNA will greatly increase, but current synthetic method is due to equipment limitation, by larger difficulty in amplifying production, therefore need to develop a kind of synthetic method of siRNA more efficient green.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a method for preparing siRNA that inhibits PCSK9 gene expression, so as to solve the problem of low purity of such siRNA prepared in the prior art.

[0007] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for preparing siRNA for inhibiting PCSK9 gene expression is provided, wherein the siRNA is a double-stranded RNA composed of a complementary sense chain and an antisense chain; the preparation method comprises: mixing a sense chain substrate, an antisense chain substrate, and an RNA ligase, and using the RNA ligase to catalyze the connection between the sense chain substrates and the antisense chain substrates via phosphodiester bonds to obtain a sense chain and an antisense chain, thereby obtaining the siRNA; the sense chain substrates can form a sense chain, and the antisense chain substrates can form an antisense chain, the sense chain is the nucleic acid sequence shown in SEQ ID NO: 45, and the antisense chain is the nucleic acid sequence shown in SEQ ID NO: 46.

[0008] Furthermore, the 5' end of the sense chain is a hydroxyl group, the 3' end is an L96 group, the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st ribonucleotides of the sense chain have 2' methoxy modifications; the 7th and 9th ribonucleotides have 2' fluorine modifications; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate; the 11th position is a deoxyribonucleotide, which is thymine; the 5' end of the antisense chain is a hydroxyl group, the 3' end is an L96 group, ...7th and 9th ribonucleotides have 2' fluorine modifications; the 7th and 9th ribonucleotides have 2' fluorine modifications; the 7th and 7th ribonucleotides have 2' fluorine modifications; the 7th and 7th ribonucleotides have 2' fluorine modifications; the 7th and 7th ribonucleotides have 2' fluorine modifications; the 7th and 7th ribonucleotides have The antisense strand has a hydroxyl end, 2'-methoxy modifications on ribonucleotides at positions 1, 3, 7, 9, 11, 13, 15, 17, 19, 20, 21, 22, and 23, and 2'-fluorine modifications on ribonucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, 16, and 18. The 1st and 2nd ribonucleotides are linked by a phosphorothioate, the 2nd and 3rd ribonucleotides are linked by a phosphorothioate, the 21st and 22nd ribonucleotides are linked by a phosphorothioate, and the 22nd and 23rd ribonucleotides are linked by a phosphorothioate.

[0009] Furthermore, the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10 or SEQ ID NO: 13 to SEQ ID NO: 40; or an enzyme that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10, SEQ ID NO: 13 to SEQ ID NO: 40, and has activity in catalyzing phosphodiester bond formation.

[0010] Furthermore, the sense chain substrate includes 2 or more substrates, and the antisense chain substrate includes 2 or more substrates; preferably, the length of the sense chain substrate is 2-19 nt, more preferably 3-17 nt, and further preferably 6-15 nt; preferably, the length of the antisense chain substrate is 2-21 nt, more preferably 3-19 nt, and further preferably 6-15 nt.

[0011] Furthermore, the sense chain substrate and the antisense chain substrate each include two substrates, the sense chain substrate includes a first sense chain substrate and a second sense chain substrate, and the antisense chain substrate includes a first antisense chain substrate and a second antisense chain substrate; the preparation method includes: mixing the first sense chain substrate, the second sense chain substrate, the first antisense chain substrate and the second antisense chain substrate, using RNA ligase to catalyze the connection of the first sense chain substrate and the second sense chain substrate to form a sense chain, catalyzing the connection of the first antisense chain substrate and the second antisense chain substrate to form an antisense chain, and the sense chain and the antisense chain are complementary paired to form this siRNA; preferably, the sense chain substrate and the antisense chain substrate are annealed and then mixed with RNA ligase to obtain this siRNA.

[0012] Furthermore, the 3' end of the first sense chain substrate and the 5' end of the second sense chain substrate are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate and the 5' end of the second antisense chain substrate are connected under the catalysis of RNA ligase to form an antisense chain; preferably, the 5' end of the first sense chain substrate is a hydroxyl group and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate is a phosphate group and the 3' end is an L96 group; preferably, the 5' end of the first antisense chain substrate is a hydroxyl group and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate is a phosphate group and the 3' end is a hydroxyl group.

[0013] Furthermore, the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 41, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 42; or the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 53, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 54.

[0014] Furthermore, the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 44, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 43; or the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 56, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 55.

[0015] Furthermore, the sense chain substrate and the antisense chain substrate each include three substrates, the sense chain substrate includes a first sense chain substrate, a second sense chain substrate and a third sense chain substrate; the antisense chain substrate includes a first antisense chain substrate, a second antisense chain substrate and a third antisense chain substrate; preferably, the first sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 47; the second sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 48; the third sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 49; preferably, the first antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 52; the second antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 51; and the third antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 50.

[0016] Furthermore, the concentration of the sense chain substrate and the antisense chain substrate is 1 μm-1 M; preferably, the concentration of RNA ligase is 0.01-10 mg / mL; preferably, the reaction system formed by mixing the sense chain substrate, the antisense chain substrate and the RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT; preferably, the reaction temperature of the preparation method is 0-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 0.1-48h, more preferably 12-24h.

[0017] By applying the technical solution of the present invention and utilizing the above-mentioned preparation method, RNA ligase catalyzes the ligation of sense strand substrates to form the sense strand of this siRNA, and catalyzes the ligation of antisense strand substrates to form the antisense strand of this siRNA, thereby achieving the biosynthetic preparation of this complex siRNA with multiple modifications. Compared to preparing this siRNA by chemical synthesis, the product obtained by the preparation method of the present application has higher purity, fewer impurities, and mild reaction conditions, which facilitates industrial scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] FIG1 shows a schematic diagram of the siRNA structure according to an embodiment of the present invention.

[0020] FIG2 shows a schematic diagram of the structure of the L96 group according to an embodiment of the present invention.

[0021] FIG3 shows the results of SDS-PAGE gel electrophoresis according to Example 2 of the present invention.

[0022] FIG4 shows a diagram of liquid chromatography and mass spectrometry detection results according to Example 2 of the present invention.

[0023] FIG5 shows a diagram of liquid chromatography and mass spectrometry detection results according to Example 2 of the present invention.

[0024] FIG6 shows a diagram of liquid chromatography detection results according to Example 6 of the present invention.

[0025] FIG. 7 shows a diagram of liquid chromatography detection results according to Example 7 of the present invention. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0027] Explanation of terms:

[0028] N-1 impurity: A nucleic acid impurity that has a single base deletion compared to the target synthesized sequence.

[0029] N+1 impurities: Nucleic acid impurities with a single additional base compared to the target synthetic sequence.

[0030] As mentioned in the background art, the prior art uses chemical synthesis to prepare such siRNAs. This not only uses large amounts of organic solvents, which is inconsistent with the development of green chemistry, but also results in low product purity, high levels of impurities, and, in particular, the presence of difficult-to-remove N+1 and N-1 impurities, which affect subsequent product purification. In this application, the inventors attempted to develop a method for preparing siRNA that inhibits PCSK9 gene expression, utilizing enzyme-catalyzed synthesis of such siRNAs, and thus proposed a series of protection schemes herein.

[0031] In a first typical embodiment of the present application, a method for preparing siRNA for inhibiting PCSK9 gene expression is provided, wherein the above-mentioned siRNA is a double-stranded RNA composed of a complementary paired sense chain and an antisense chain; the preparation method comprises: mixing a sense chain substrate, an antisense chain substrate and an RNA ligase, and using the RNA ligase to catalyze the connection between the sense chain substrates and the antisense chain substrates with phosphodiester bonds to obtain a sense chain and an antisense chain, thereby obtaining such siRNA; the sense chain substrates can form a sense chain, and the antisense chain substrates can form an antisense chain; the sense chain is the nucleic acid sequence shown in SEQ ID NO: 45, and the antisense chain is the nucleic acid sequence shown in SEQ ID NO: 46.

[0032] In this application, siRNA refers to a double-stranded RNA sequence with a sense strand as SEQ ID NO: 45 and an antisense strand as SEQ ID NO: 46. In the above preparation method, the sense strand substrate is two or more nucleotide sequences that can form a sense strand. That is, the multiple nucleotide sequences of the sense strand substrate can be spliced ​​to form a sequence identical to the sense strand sequence, but differ from the sense strand in that the sense strand substrate contains a nick and is not connected by a phosphodiester bond. Similarly, RNA ligase is used to connect the two or more antisense strand substrates by phosphodiester bonds to obtain the antisense strand of the siRNA.

[0033] In the above preparation method, the sense chain substrate, the antisense chain substrate and the RNA ligase are mixed together, and the siRNA is directly prepared in a one-pot method. In the one-pot connection, the sense chain substrate and the antisense chain substrate can undergo base complementary pairing to form a double-stranded structure. After the RNA ligase recognizes the double-stranded structure, it connects the nicks in the double-stranded structure, thereby preparing the target product, the siRNA. The structure of the target product siRNA is shown in Figure 1. The siRNA prepared by the above preparation method includes the nucleic acid shown in Figure 1, and also includes nucleic acids bound to ions, including but not limited to their sodium salt form. The ions bound to the nucleic acid do not affect the progress of the above preparation method, and the binding of ions and nucleic acids can proceed spontaneously in the reaction system.

[0034] In a preferred embodiment, the 5' end of the sense strand is a hydroxyl group, the 3' end is an L96 group, the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st ribonucleotides of the sense strand have 2' methoxy modifications; the 7th and 9th ribonucleotides have 2' fluorine modifications; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate; the 11th position is a deoxyribonucleotide, which is thymine; the 5' end of the antisense strand is a hydroxyl group , the 3' end is a hydroxyl group, the 1st, 3rd, 7th, 9th, 11th and 13th, 15th, 17th, 19th, 20th, 21st, 22nd and 23rd ribonucleotides of the antisense strand have 2' methoxy modifications, the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th and 18th ribonucleotides have 2' fluorine modifications, the 1st and 2nd ribonucleotides are linked by phosphorothioate, the 2nd and 3rd ribonucleotides are linked by phosphorothioate, the 21st and 22nd ribonucleotides are linked by phosphorothioate, and the 22nd and 23rd ribonucleotides are linked by phosphorothioate.

[0035] In a preferred embodiment, the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10 or SEQ ID NO: 13 to SEQ ID NO: 40; or an enzyme that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10, SEQ ID NO: 13 to SEQ ID NO: 40, and has activity in catalyzing phosphodiester bond formation.

[0036] The aforementioned RNA ligases are all capable of recognizing a nicked double-stranded structure formed by complementary substrate pairing, thereby catalyzing the formation of a phosphodiester bond between a phosphate group and a hydroxyl group. RNA ligases exhibit varying activities with respect to different substrates. The aforementioned RNA ligase, screened from a large number of RNA ligases in the aforementioned preparation method, is capable of ligating and preparing siRNAs whose sense strand is the nucleic acid sequence set forth in SEQ ID NO: 45 and whose antisense strand is the nucleic acid sequence set forth in SEQ ID NO: 46.

[0037] SEQ ID NO: 1 (E1, Salmonella enterica):

[0038] SEQ ID NO: 2(E2, YersiniaphagevB_YepM_ZN18):

[0039] SEQ ID NO: 3 (E3, Shigella phage pSs-1):

[0040] SEQ ID NO:4(E4,Bacteriophage,T4Rnl2):

[0041] SEQ ID NO:5(E5,Bacteriophage T7,T7 D NA):

[0042] SEQ ID NO:6(E6,Bacteriophage):

[0043] SEQ ID NO:7(E7,Klebsiella phage KP27):

[0044] SEQ ID NO:8(E8,Vibrio phage phi-ST2):

[0045] SEQ ID NO:9(E9,Klebsiella phage KP15):

[0046] SEQ ID NO:10(E10,Vibrio phage JN02):

[0047] SEQ ID NO:11(E11,Bacteriophage RB69):

[0048] SEQ ID NO:12(E12,Aeromonas virus Aeh1):

[0049] SEQ ID NO:13(E13,Vibrio phage JS98):

[0050] SEQ ID NO:14(E14,Vibrio phage):

[0051] SEQ ID NO:15(E15,Acidobacteriabacterium):

[0052] SEQ ID NO:16(E16,Vibrio phage nt-1 rnlA):

[0053] SEQ ID NO:17(E17,Streptomyces phage):

[0054] SEQ ID NO:18(E18,Morganella phage vB_MmoM_MP1):

[0055] SEQ ID NO:19(E19,Flavobacterium terrae):

[0056] SEQ ID NO:20(E20,Klebsiella phage vB_Kpn_F48):

[0057] SEQ ID NO:21(E21,Escherichia phage EcS1):

[0058] SEQ ID NO:22(E22,Acinetobacter phage AbTZA1):

[0059] SEQ ID NO:23(E23,Klebsiella phage KMI11):

[0060] SEQ ID NO:24(E24,Yasminevirus sp.GU-2018):

[0061] SEQ ID NO:25(E25,Enterobacter phage vB_EclM_CIP9):

[0062] SEQ ID NO:26(E26,Escherichia phage CJ20):

[0063] SEQ ID NO:27(E27,Cronobacter phage A24):

[0064] SEQ ID NO:28(E28,Rotaria sp.Silwood1):

[0065] SEQ ID NO:29(E29,Acinetobacter phage Ac42):

[0066] SEQ ID NO:30(E30,Helcococcus kunzii ATCC 51366):

[0067] SEQ ID NO:31(E31,Vibrio phage CHOED):

[0068] SEQ ID NO:32(E32,Cronobacter phage S13):

[0069] SEQ ID NO:33(E33,Thecamonas trahens ATCC 50062):

[0070] SEQ ID NO:34(E34,Acinetobacter phage vB_AbaM_phiAbaA1):

[0071] SEQ ID NO:35(E35,Klebsiella phage Mineola):

[0072] SEQ ID NO:36(E36,Enterococcus phage 9181):

[0073] SEQ ID NO:37(E37,Aeromonas phage Ahp1_CNU-2021):

[0074] SEQ ID NO:38(E38,Megavirus chiliensis):

[0075] SEQ ID NO: 39 (E39, Acanthamoeba polyphaga moumouvirus):

[0076] SEQ ID NO: 40 (E40, Salmonella phage S16):

[0077] Identity as used herein refers to the "identity" between amino acid sequences or nucleic acid sequences, i.e., the total ratio of identical amino acid residues or nucleotides in an amino acid sequence or nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0078] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, or even 99.9% or more) identity and the same function, whose active sites, active pockets, active mechanisms, protein structures, etc. are most likely the same as the proteins provided by the sequence in a).

[0079] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0080] Substitution and replacement rules generally state that amino acids with similar properties will have similar effects after substitution. For example, conservative amino acid substitutions may occur in the homologous proteins mentioned above. "Conservative amino acid substitutions" include but are not limited to:

[0081] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0082] Substitution of bulky hydrophobic amino acids (Phe, Tyr, Trp) with other bulky hydrophobic amino acids;

[0083] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0084] Amino acids with polar and uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar and uncharged side chains.

[0085] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0086] In a preferred embodiment, the sense chain substrate includes 2 or more substrates, and the antisense chain substrate includes 2 or more substrates; preferably, the length of the sense chain substrate is 2-19 nt, more preferably 3-17 nt, and further preferably 6-15 nt; preferably, the length of the antisense chain substrate is 2-21 nt, more preferably 3-19 nt, and further preferably 6-15 nt.

[0087] In a preferred embodiment, the sense chain substrate and the antisense chain substrate each include two substrates, the sense chain substrate includes a first sense chain substrate and a second sense chain substrate, and the antisense chain substrate includes a first antisense chain substrate and a second antisense chain substrate; the preparation method includes: mixing the first sense chain substrate, the second sense chain substrate, the first antisense chain substrate, and the second antisense chain substrate, using RNA ligase to catalyze the connection of the first sense chain substrate and the second sense chain substrate to form a sense chain, catalyzing the connection of the first antisense chain substrate and the second antisense chain substrate to form an antisense chain, and the sense chain and antisense chain are complementary paired to form this siRNA; preferably, the sense chain substrate and the antisense chain substrate are annealed and then mixed with RNA ligase to obtain this siRNA.

[0088] In the above preparation method, the sense strand substrate and the antisense strand substrate can be first mixed and annealed. The sense strand substrate and the antisense strand substrate can form a double-stranded RNA structure through base complementary pairing between the two strands. The double-stranded RNA structure contains nicks between the different substrates. The annealed reaction system is then mixed with RNA ligase, which uses RNA ligase to connect the phosphate and hydroxyl groups on both sides of the nick through phosphodiester bonds, repairing the nick, thereby obtaining the target product, such as siRNA, with a complete double-stranded structure.

[0089] In a preferred embodiment, the 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are connected under the catalysis of RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are connected under the catalysis of RNA ligase to form an antisense strand; preferably, in the above preparation method, the 5' end of the first sense strand substrate is a 5' protecting group and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate is a phosphate group and the 3' end is an L96 group (N-acetylgalactosamine group derivative); the 5' end of the first antisense strand substrate is a hydroxyl group and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate is a phosphate group and the 3' end is a hydroxyl group. The structure of the above L96 group is shown in Figure 2.

[0090] In a preferred embodiment, the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 41, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 42; or the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 53, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 54.

[0091] In a preferred embodiment, the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 44, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 43; or the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 56, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 55.

[0092] This siRNA can be prepared using the above preparation method and the substrates shown in SEQ ID NOs: 41-44 or 53-56. However, it should be noted that the selection of substrates is not limited to the substrates shown in SEQ ID NOs: 41-44 or 53-56. Any substrate that can be combined to form a sense strand and an antisense strand can be used in the above preparation method. The above preparation method is suitable for the preparation of this siRNA but is not limited to the difference in substrate connection position. The above preparation has a good connection effect for the connection of the sense strand sequence and the antisense strand sequence of this siRNA. The number of sense strand substrates or antisense strand substrates includes but is not limited to 2, 3, 4 or even more.

[0093] In a preferred embodiment, the sense chain substrate and the antisense chain substrate both include three substrates, the sense chain substrate includes a first sense chain substrate, a second sense chain substrate and a third sense chain substrate; the antisense chain substrate includes a first antisense chain substrate, a second antisense chain substrate and a third antisense chain substrate; preferably, the first sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 47; the second sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 48; the third sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 49; preferably, the first antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 52; the second antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 51; and the third antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 50.

[0094] In a preferred embodiment, the concentration of the sense strand substrate and the antisense strand substrate is 1 μM-1 M; preferably, the concentration of RNA ligase is 0.01-10 mg / mL; preferably, the reaction system formed by mixing the sense strand substrate, the antisense strand substrate and the RNA ligase further includes ATP, Tris-HCl, MgCl2 and DTT; preferably, the reaction temperature of the preparation method is 0-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 0.1-48h, more preferably 12-24h.

[0095] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0096] Example 1 Purification and preparation of RNA ligase

[0097] The gene capable of expressing RNA ligase was constructed into the pET28a vector and transformed into the Escherichia coli BL21 (DE3) expression strain. The expression strain was inoculated into 5 mL LB medium (containing 50 μg / mL) at a 0.1% inoculum and cultured at 37°C for 16 h. The resulting culture was transferred to 500 mL LB medium at a 1% inoculum and cultured at 37°C until the OD 600 =0.6, 0.1mM IPTG was added and induced at 20°C for 16h. The resulting induced culture was centrifuged to obtain bacterial sludge. The bacterial sludge was resuspended in lysis buffer (50mM Tris-Cl pH8.0, 0-500mM NaCl, 10% glycerol) to a final bacterial concentration of 10%, and the crude enzyme solution was obtained after ultrasonic disruption and centrifugation. 10mM imidazole was added to the crude enzyme solution, and the enzyme solution obtained after filtration with a 0.22μm filter membrane was purified with a nickel column. After desalting and replacing the solution, it was purified again with a strong anion column. The obtained enzyme solution was desalted and replaced with the solution and stored in 30% glycerol.

[0098] Example 2

[0099] Add substrates 1-4 to a clean reagent bottle to prepare a reaction system to a final volume of 100 μL. The sequences of substrates 1-4 are shown in Table 1. The concentration of RNA fragments 1-4 (i.e., substrates 1-4) was 50 μM, the ATP concentration was 1 mM, the Tris-HCl (pH 7.5 @ 25°C) concentration was 50 mM, the MgCl2 concentration was 10 mM, and the DTT concentration was 1 mM. Commercial ligases T4 RNA2 (E4), T7 DNA ligase (E5), or other ligases (E1-E3, E6-E16) were added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. The resulting reaction system was inactivated by heating at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. The supernatant was analyzed by SDS-PAGE. The SDS-PAGE results are shown in Figure 3. The results showed that some ligases could catalyze the synthesis of the final product. The theoretical molecular weight of the sense chain was 8636.95±1, and the theoretical molecular weight of the antisense chain was 7694.20±1. Mass spectrometry identification showed that the molecular weight was consistent with the final product. The liquid chromatography and mass spectrometry detection results are shown in Figures 4 and 5, where Figure 4 is a liquid chromatography diagram of this siRNA and a mass spectrometry result diagram of the siRNA sense chain, and Figure 5 is a liquid chromatography diagram of this siRNA and a mass spectrometry result diagram of the siRNA antisense chain.

[0100] In Figure 3, lane 1 is a reaction system catalyzed by E1, lane 2 is a nucleic acid molecular weight standard, lane 3 is a reaction system catalyzed by E2, lane 4 is a reaction system catalyzed by E3, lane 5 is a reaction system catalyzed by E4, lane 6 is a reaction system catalyzed by E5, lane 7 is a reaction system catalyzed by E6, lane 8 is a reaction system catalyzed by E7, lane 9 is a reaction system catalyzed by E8, lane 10 is a negative control reaction system without added enzyme, lane 11 is a reaction system catalyzed by E9, lane 12 is a reaction system catalyzed by E10, lane 13 is a reaction system catalyzed by E11, lane 14 is a reaction system catalyzed by E12, lane 15 is a reaction system catalyzed by E13, lane 16 is a reaction system catalyzed by E14, lane 17 is a reaction system catalyzed by E15, and lane 18 is a reaction system catalyzed by E16.

[0101] Table 1

[0102] Wherein, m after A, C, G or U indicates 2'-methoxy modification of the ribonucleotide, f indicates 2'-fluorine modification of the ribonucleotide, s indicates phosphorothioate linkage, d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide, and L96 is the N-acetylgalactosamine group derivative shown in FIG2 .

[0103] Substrate 1 has 2' methoxy modifications on the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th and 14th ribonucleotides; 2' fluorine modifications on the 7th and 9th ribonucleotides; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate; the 11th position is a deoxyribonucleotide, which is thymine (T).

[0104] The 1st to 7th ribonucleotides of substrate 2 all have 2' methoxy modifications.

[0105] Substrate 3 has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 7, 8, 9 and 10, 2' fluorine modifications on ribonucleotides at positions 1, 3 and 5, a phosphorothioate linkage between ribonucleotides at positions 8 and 9, and a phosphorothioate linkage between ribonucleotides at positions 9 and 10.

[0106] Substrate 4 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 7, 9, 11 and 13, 2' fluorine modifications on ribonucleotides at positions 2, 4, 5, 6, 8, 10 and 12, a phosphorothioate linkage between ribonucleotides at positions 1 and 2, and a phosphorothioate linkage between ribonucleotides at positions 2 and 3.

[0107] The sense strand (SS) of the prepared siRNA is 5'-CmsUmsAmGmAmCmCfUmGfUmdTUmUmGmCmUmUmUmUmGmUm-3' (SEQ ID NO: 45).

[0108] The antisense strand (AS) is 5'-AmsCfsAmAfAfAfGmCfAmAfAmAfCmAfGmGfUmCfUmAmGmsAmsAm-3' (SEQ ID NO: 46).

[0109] The 5' end of the sense chain is a hydroxyl group, the 3' end is an L96 group, and the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st ribonucleotides of the sense chain have 2' methoxy modifications; the 7th and 9th ribonucleotides have 2' fluorine modifications; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate; the 11th position is a deoxyribonucleotide, which is thymine (T).

[0110] The 5' end of the antisense chain is a hydroxyl group, the 3' end is a hydroxyl group, the 1st, 3rd, 7th, 9th, 11th and 13th, 15th, 17th, 19th, 20th, 21st, 22nd and 23rd ribonucleotides of the antisense chain have 2' methoxy modifications, the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th and 18th ribonucleotides have 2' fluorine modifications, the 1st and 2nd ribonucleotides are linked by phosphorothioate, the 2nd and 3rd ribonucleotides are linked by phosphorothioate, the 21st and 22nd ribonucleotides are linked by phosphorothioate, and the 22nd and 23rd ribonucleotides are linked by phosphorothioate.

[0111] Example 3

[0112] Substrates 1-4 were added to a clean reagent bottle to prepare a reaction system to a final volume of 100 μL. The concentration of RNA fragments 1-4 was 200 μM, ATP was 1 mM, Tris-HCl (pH 7.5 @ 25°C) was 50 mM, MgCl₂ was 10 mM, and DTT was 1 mM. Commercial ligases (T4 RNA₂, T7 DNA ligase, or one or more of the aforementioned RNA ligases) were added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. The resulting reaction system was inactivated by heating at 80°C for 5 min, and the precipitate was removed by centrifugation at 12,000 rpm. To facilitate quantification, a UPLC method was developed to accurately monitor the reaction status, and the supernatant was analyzed by UPLC. The results are shown in Table 2, demonstrating that some ligases were able to catalyze the synthesis of the final product.

[0113] Table 2

[0114] For the expression of activity, "-" indicates that the product system purity is 0.1-5% (excluding the endpoint value of 5%), "+" indicates that the product system purity is 5%-20% (excluding the endpoint value of 20%), "++" indicates that the product system purity is 20%-30% (excluding the endpoint value of 30%), "+++" indicates that the product system purity is 30%-50%, and "×" indicates that no product is generated. In this application, "product system purity" means the ratio of the target product to the total components in the reaction system (target product + residual substrate) in the reaction system, which can be calculated by the area under the curve of different components in the liquid chromatography results.

[0115] Example 4

[0116] Substrates 1-4 were added to a clean reagent vial to prepare a reaction system to a final volume of 100 μL. RNA fragments 1-4 were prepared at the same concentration (50 μM to 1000 μM), ATP was 1 mM, Tris-HCl (pH 7.5 @ 25°C) was 50 mM, MgCl₂ was 10 mM, and DTT was 1 mM. Ligase was added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 hours. The resulting reaction system was inactivated by heating at 80°C for 5 minutes, the precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by UPLC. The results, shown in Table 3, show that the optimal ligase catalyzed substrate concentrations up to 1 mM and significantly outperformed the commercial enzyme T4 RNA ligase₂.

[0117] Table 3

[0118] For the expression of activity, "-" indicates that the purity of the product system is 0.1-5%, "+" indicates that the purity of the product system is 5%-20% (excluding the endpoint value of 5%), "++" indicates that the purity of the product system is 20%-30% (excluding the endpoint value of 20%), "+++" indicates that the purity of the product system is 30%-50% (excluding the endpoint value of 30%), and "×" indicates that no product is generated.

[0119] Example 5

[0120] Substrates 1-4 were added to a clean reagent bottle to prepare a reaction system to a final volume of 100 mL. The concentration of RNA fragments 1-4 was 800 μM, the concentration of ATP was 2 mM, the concentration of Tris-HCl (pH 7.5 @ 25°C) was 50 mM, the concentration of MgCl2 was 10 mM, and the concentration of DTT was 1 mM. Ligase was added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. An aliquot of the resulting reaction system was inactivated by heating at 80°C for 5 min to inactivate the ligase. The precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by UPLC, which showed complete catalysis. The reaction system was heat inactivated and centrifuged at 12,000 rpm for 30 min to remove the ligase. The reaction system was purified by ion column purification, desalted, and lyophilized to obtain the final product. The final product was assayed for water content, residual endotoxin, residual enzyme, residual solvent, residual metal, and residual host protein. Weighing yielded 1.4 g of product, with an 80% yield and 9% water content. The product had denaturation purity of 96% and native purity of 96%. Residual host protein was 0.2 ppm, enzyme residue was <6 ppm, and metal residues were as follows: Ni: 7 ppm; Cr: 3 ppm; Fe: 43 ppm; Ti: 4 ppm; Mn: 1 ppm; Na: 31169 ppm; As: 0.5%; Cd: 0.5%; Hg: 0.5%; Pb: 0.5%; all tests met the quality standards for parenteral pharmaceuticals. The endotoxin residue was 9 EU / mg. Because the procedure was performed in a non-clean environment, which could have potentially contaminated the product with endotoxins, the product was endotoxin-free in a clean environment. The final endotoxin residue was 0.2 EU / mg, meeting the quality standards for siRNA pharmaceuticals. This demonstrates that the enzymatic synthesis of this siRNA pharmaceutical is feasible, with extremely high product yield and a simple process.

[0121] Example 6

[0122] Substrates 5-10 (sequences of substrates 5-10 are shown in Table 4) were added to a clean reagent vial. The reaction system was prepared to a final volume of 200 μL with a substrate concentration of 500 μM, 1 mM ATP, 50 mM Tris-HCl (pH 7.5 @ 25°C), 10 mM MgCl2, and 1 mM DTT. Ligase was added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. The resulting reaction system was inactivated by heating at 80°C for 5 min to inactivate the ligase. The precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by UPLC. The results are shown in Table 5. The results show that the product was formed.

[0123] Table 4

[0124] wherein m after A, C, G or U indicates a 2'-methoxy modification of the ribonucleotide, f indicates a 2'-fluorine modification of the ribonucleotide, s indicates a phosphorothioate bond, d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide, and L96 represents an N-acetylgalactosamine group derivative.

[0125] The substrate 5 has 2' methoxy modifications on the 1st, 2nd, 3rd, 4th, 5th, 6th and 8th ribonucleotides; the 7th ribonucleotide has a 2' fluorine modification; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate.

[0126] The substrate 6 has 2' methoxy modifications on the 2nd, 4th, 5th, 6th, 7th and 8th ribonucleotides; the 1st ribonucleotide has a 2' fluorine modification; the 3rd position is a deoxyribonucleotide, which is thymine (T).

[0127] Substrate 7 has 2' methoxy modifications on ribonucleotides 1, 2, 3, 4, and 5.

[0128] The substrate 8 has 2' methoxy modifications on the 1st, 3rd, 4th, 5th, 6th and 7th ribonucleotides; the 2nd ribonucleotide has a 2' fluorine modification; the 5th and 6th ribonucleotides are linked by phosphorothioate, and the 6th and 7th ribonucleotides are linked by phosphorothioate.

[0129] Substrate 9 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, and 7; and 2' fluorine modifications on ribonucleotides at positions 2, 4, 6, and 8.

[0130] The substrate 10 has 2' methoxy modifications on the 1st, 3rd and 7th ribonucleotides; 2' fluorine modifications on the 2nd, 4th, 5th, 6th and 8th ribonucleotides; a phosphorothioate linkage between the 1st and 2nd ribonucleotides, and a phosphorothioate linkage between the 2nd and 3rd ribonucleotides.

[0131] Table 5

[0132] For the expression of activity, "-" indicates that the purity of the product system is 0.1-5%, "+" indicates that the purity of the product system is 5%-20% (excluding the endpoint value of 5%), "++" indicates that the purity of the product system is 20%-30% (excluding the endpoint value of 20%), "+++" indicates that the purity of the product system is 30%-50% (excluding the endpoint value of 30%), and "×" indicates that no product is generated.

[0133] The UPLC detection spectra of the reaction system without enzyme and catalysis using partial enzymes are shown in Figure 6.

[0134] Example 7

[0135] Substrates 11-14 (sequences of substrates 11-14 are shown in Table 6) were added to a clean reagent vial. The reaction system was prepared to a final volume of 200 μL with a substrate concentration of 500 μM, 1 mM ATP, 50 mM Tris-HCl (pH 7.5 @ 25°C), 10 mM MgCl2, and 1 mM DTT. Ligase was added to a concentration of 0.2 mg / mL. The reaction system was incubated at 16°C for 16 h. The resulting reaction system was inactivated by heating at 80°C for 5 min to inactivate the ligase. The precipitate was removed by centrifugation at 12,000 rpm, and the supernatant was analyzed by UPLC. The results are shown in Table 7. The results show that the product was formed.

[0136] Table 6

[0137] wherein m after A, C, G or U indicates a 2'-methoxy modification of the ribonucleotide, f indicates a 2'-fluorine modification of the ribonucleotide, s indicates a phosphorothioate bond, d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide, and L96 represents an N-acetylgalactosamine group derivative.

[0138] The substrate 11 has 2' methoxy modifications on the 1st, 2nd, 3rd, 4th, 5th, 6th and 8th ribonucleotides; the 7th ribonucleotide has a 2' fluorine modification; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate.

[0139] The substrate 12 has 2' methoxy modifications on the 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th ribonucleotides; the 1st ribonucleotide has a 2' fluorine modification; the 3rd position is a deoxyribonucleotide, which is thymine (T).

[0140] Substrate 13 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 11, 12, 13, 14 and 15, 2' fluorine modifications on ribonucleotides at positions 2, 4, 5, 6, 8 and 10, a phosphorothioate linkage between ribonucleotides at positions 13 and 14, and a phosphorothioate linkage between ribonucleotides at positions 14 and 15.

[0141] Substrate 14 has 2' methoxy modifications on the 1st, 3rd and 7th ribonucleotides, 2' fluorine modifications on the 2nd, 4th, 5th, 6th and 8th ribonucleotides, a phosphorothioate linkage between the 1st and 2nd ribonucleotides, and a phosphorothioate linkage between the 2nd and 3rd ribonucleotides.

[0142] Table 7

[0143] For the expression of activity, "-" indicates that the purity of the product system is 0.1-5% (excluding the endpoint value of 5%), "+" indicates that the purity of the product system is 5%-20% (excluding the endpoint value of 20%), "++" indicates that the purity of the product system is 20%-30% (excluding the endpoint value of 30%), "+++" indicates that the purity of the product system is 30%-50%, and "×" indicates that no product is generated.

[0144] The chromatograms of the enzyme-free control system and the reaction system catalyzed by some enzymes detected by UPLC are shown in Figure 7.

[0145] Comparative Example 1

[0146] The target siRNA was synthesized using a conventional solid-phase method (solid-phase synthesizer). The product was tested for purity and impurities by mass spectrometry. Comparison was made with target siRNA synthesized enzymatically using E-7 enzyme at the same concentration. The enzymatically synthesized product was found to have higher purity. Purity data is shown in Table 8.

[0147] Table 8

[0148] Furthermore, UPLC and mass spectrometry were used to detect the impurity content. The results showed that the N-1 and N+1 impurities in the target siRNA synthesized by enzymatic synthesis were much lower than the impurity content of the target siRNA synthesized by solid-phase synthesis. The impurity content results are shown in Table 9. This shows that the enzymatic synthesis of target siRNA is more advantageous.

[0149] Table 9

[0150] ND means not detected.

[0151] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the preparation method of the present application, RNA ligase catalyzes the connection between the sense chain substrates to form the sense chain of the target siRNA, and catalyzes the connection between the antisense chain substrates to form the antisense chain of the target siRNA, thereby realizing the preparation of such a complex structure with multiple modifications by biosynthesis. Compared with the preparation of target siRNA by chemical synthesis, the product obtained by the preparation method of the present application has higher purity, less impurities, and less pressure for subsequent final product purification. In addition, the reaction conditions are mild and do not use a large amount of organic reagents, which can reduce production costs and facilitate industrial scale-up production.

[0152] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing siRNA for inhibiting PCSK9 gene expression, characterized in that: The siRNA is a double-stranded RNA consisting of a complementary sense strand and an antisense strand; The preparation method comprises: Mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, and using the RNA ligase to catalyze the connection between the sense strand substrates and the antisense strand substrates through phosphodiester bonds to obtain the sense strand and the antisense strand, thereby obtaining the siRNA; The sense strand substrate can constitute the sense strand, and the antisense strand substrate can constitute the antisense strand; The sense strand is the nucleic acid sequence shown in SEQ ID NO:45, and the antisense strand is the nucleic acid sequence shown in SEQ ID NO:

46.

2. The preparation method according to claim 1, characterized in that: The 5' end of the sense strand is a hydroxyl group, the 3' end is an L96 group, the 1st, 2nd, 3rd, 4th, 5th, 6th, 8th, 10th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st ribonucleotides of the sense strand have 2' methoxy modifications; the 7th and 9th ribonucleotides have 2' fluorine modifications; the 1st and 2nd ribonucleotides are linked by phosphorothioate, and the 2nd and 3rd ribonucleotides are linked by phosphorothioate; the 11th position is a deoxyribonucleotide, which is thymine; The 5' end of the antisense chain is a hydroxyl group, the 3' end is a hydroxyl group, the 1st, 3rd, 7th, 9th, 11th, 13th, 15th, 17th, 19th, 20th, 21st, 22nd and 23rd ribonucleotides of the antisense chain have 2' methoxy modifications, the 2nd, 4th, 5th, 6th, 8th, 10th, 12th, 14th, 16th and 18th ribonucleotides have 2' fluorine modifications, the 1st and 2nd ribonucleotides are connected by phosphorothioate, the 2nd and 3rd ribonucleotides are connected by phosphorothioate, the 21st and 22nd ribonucleotides are connected by phosphorothioate, and the 22nd and 23rd ribonucleotides are connected by phosphorothioate.

3. The preparation method according to claim 1, characterized in that: The RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10 or SEQ ID NO: 13 to SEQ ID NO: 40; or An enzyme that has 70% or more identity with any one of the RNA ligases shown in SEQ ID NO: 4 to SEQ ID NO: 10 and SEQ ID NO: 13 to SEQ ID NO: 40 and has an activity of catalyzing the formation of the phosphodiester bond.

4. The preparation method according to any one of claims 1 to 3, characterized in that The sense strand substrate includes 2 or more substrates, and the antisense strand substrate includes 2 or more substrates.

5. The preparation method according to claim 4, characterized in that: The length of the sense strand substrate is 2-19 nt, and the length of the antisense strand substrate is 2-21 nt.

6. The preparation method according to claim 4, characterized in that: The sense strand substrate and the antisense strand substrate each include two substrates, the sense strand substrate includes a first sense strand substrate and a second sense strand substrate, and the antisense strand substrate includes a first antisense strand substrate and a second antisense strand substrate; The preparation method comprises: mixing the first sense strand substrate, the second sense strand substrate, the first antisense strand substrate and the second antisense strand substrate, using RNA ligase to catalyze the ligation of the first sense strand substrate and the second antisense strand substrate; The two sense strand substrates are connected to form the sense strand, and the first antisense strand substrate and the second antisense strand substrate are catalyzed to connect to form the antisense strand. The sense strand and the antisense strand are complementary and paired to form the siRNA.

7. The preparation method according to claim 6, characterized in that: The sense strand substrate and the antisense strand substrate are annealed and then mixed with the RNA ligase to obtain the siRNA.

8. The preparation method according to claim 6, characterized in that: The 3' end of the first sense strand substrate and the 5' end of the second sense strand substrate are connected 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 connected under the catalysis of the RNA ligase to form the antisense strand.

9. The preparation method according to claim 8, characterized in that: The 5' end of the first sense strand substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate is a phosphate group, and the 3' end is an L96 group; The 5' end of the first antisense strand substrate is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense strand substrate is a phosphate group, and the 3' end is a hydroxyl group.

10. The preparation method according to claim 8 or 9, characterized in that: The first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 41, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 42; or The first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO:53, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO:

54.

11. The preparation method according to claim 8 or 9, characterized in that: The first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 44, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 43; or The first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO:56, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO:

55.

12. The preparation method according to claim 4, characterized in that: The sense strand substrate and the antisense strand substrate both include three substrates. The sense strand substrate comprises a first sense strand substrate, a second sense strand substrate and a third sense strand substrate; The antisense strand substrate includes a first antisense strand substrate, a second antisense strand substrate and a third antisense strand substrate.

13. The preparation method according to claim 12, characterized in that: The first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 47; The second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 48; The third sense strand substrate is the nucleic acid sequence shown in SEQ ID NO:

49.

14. The preparation method according to claim 12, characterized in that: The first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 52; The second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 51; The third antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO:

50.

15. The preparation method according to any one of claims 1 to 3, characterized in that: The concentrations of the sense strand substrate and the antisense strand substrate are both 1 μM-1M; The concentration of the RNA ligase is 0.01-10 mg / mL.

16. The preparation method according to claim 15, characterized in that: The reaction system formed by mixing the sense strand substrate, the antisense strand substrate and the RNA ligase also includes ATP, Tris-HCl, MgCl2 and DTT.

17. The preparation method according to claim 15, characterized in that: The reaction temperature of the preparation method is 0-40°C. The reaction time of the preparation method is 0.1-48 h.

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