Method for preparing Cemdisiran

Abstract

The present invention provides a method for preparing Cemdisiran. Cemdisiran is siRNA, which is composed of a sense strand and an antisense strand through complementary pairing; the preparation method includes: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, using RNA ligase to catalyze the connection between the sense strand substrates and the antisense strand substrates with a phosphodiester bond to obtain a sense strand and an antisense strand, thereby obtaining Cemdisiran; the sense strand substrate can form a sense strand, and the antisense strand substrate can form an antisense strand. Compared with the preparation of Cemdisiran by chemical synthesis, the product obtained by the preparation method of the present application has a higher purity, less impurities, and mild reaction conditions, which is convenient for industrialized scale-up production.

Description

Technical Field

[0001] The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing Cemdisiran. Background Art

[0002] Small interfering RNA (siRNA) is a double-stranded RNA with a length of 19-25 nt. After entering the cell, siRNA can dissociate into single strands, in which the positive strand can specifically bind to the messenger RNA (mRNA) of the target gene through base matching, inducing a series of effects, and finally degrading the mRNA of the target gene, preventing the translation of mRNA to achieve the effect of hindering the expression of the target gene. In recent years, the research and development of siRNA drugs has received extensive attention. siRNA drugs act on mRNA, and the druggable targets are significantly larger than traditional small molecule drugs whose action sites are proteins; in addition, siRNA drugs can act on new targets by changing the sequence, and the research and development time is relatively short.

[0003] Cemdisiran is a siRNA-type double-stranded RNA drug developed by Alnylam and Regeneron, and is currently in Phase 3 clinical trials. Cemdisiran can be used to improve the urine protein (proteinuria) value of adult patients with immunoglobulin A nephropathy (IgA nephropathy, IgAN). IgA nephropathy is the main cause of chronic kidney disease and renal failure. It is a chronic, progressive inflammatory disease associated with kidney damage. This disease affects about 2.5 people out of 100,000 people each year, especially those around 30 and 40 years old. The number of patients worldwide is about 200,000.

[0004] The existing synthesis method of Cemdisiran is chemical solid phase synthesis, using a solid phase carrier such as controlled pore glass beads (CPG) or polystyrene resin, and cyclic synthesis through the phosphoramidite triester method to extend the oligonucleotide chain from the 3' to 5' direction. After the synthesis cycle is completed, the Cemdisiran chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain a pure product. The above-mentioned synthesis process requires an expensive nucleic acid synthesizer, and the scale of synthesis is limited by the scale of the synthesis instrument. The scale of the batch process is in the kg level. Increasing the synthesis flux requires adding more instruments or more batches, and the cost of scale expansion is very high. In addition, the solid phase synthesis method is a cyclic method, and the yield of the synthesis decreases with the increase of the synthesis chain length, and the impurities generated in the synthesis process, such as impurities with one more nucleotide than the target sequence (N+1 impurities) or impurities with one less nucleotide than the target sequence (N-1 impurities), will also increase with the increase of the synthesis chain length, and the final content of N+1 and N-1 impurities is 1-3%. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for preparing Cemdisiran, so as to solve the problem of low purity of Cemdisiran prepared in the prior art.

[0006] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a method for preparing Cemdisiran is provided, wherein Cemdisiran is siRNA, and is composed of a sense strand and an antisense strand through complementary pairing; the method comprises: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, wherein the sense strand substrate can form a sense strand, and the antisense strand substrate can form an antisense strand; the sense strand substrate and the antisense strand substrate are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate and the antisense strand substrate are not connected to each other, so as to form a double-stranded nucleotide structure containing a nick; using RNA ligase to connect the bases at both ends of the nick by phosphodiester bonds to form Cemdisiran; the bases at both ends of the nick are the 5' end and the 3' end of different substrates, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Cemdisiran; the RNA ligase is selected from RNA ligase family 1 or RNA ligase family 2; the RNA ligase comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; or an enzyme that has 70% or more identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 6 and has the activity of catalyzing the formation of the phosphodiester bond.

[0007] Furthermore, the sense chain substrate and the antisense chain substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method; preferably, the sense chain is the nucleic acid sequence shown in SEQ ID NO: 14, and the antisense chain is the nucleic acid sequence shown in SEQ ID NO: 15; preferably, the sense chain is composed of 2 or more sense chain substrates, and the length of the sense chain substrate is 3-18nt, more preferably 4-16nt; preferably, the antisense chain is composed of 2 or more antisense chain substrates, and the length of the antisense chain substrate is 3-22nt, more preferably 8-13nt.

[0008] The double-stranded RNA formed by annealing the sense strand substrate and the antisense strand substrate has 3 or more base combinations capable of complementary pairing; preferably, the ends of the double-stranded RNA are sticky ends; preferably, the length of the sticky ends is 2-8 nt.

[0009] 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 Cemdisiran.

[0010] Further, 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.

[0011] Furthermore, the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 10 or 16, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 11 or 17.

[0012] Furthermore, the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 13 or 19, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 12 or 18.

[0013] Furthermore, the sense strand substrate and the antisense strand substrate each include three substrates,

[0014] The sense strand substrate includes a first sense strand substrate, a second sense strand substrate and a third sense strand substrate;

[0015] The antisense strand substrate includes a first antisense strand substrate, a second antisense strand substrate and a third antisense strand substrate; preferably, the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 20; the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 21; the third sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 22; preferably, the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 25; the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 24; the third antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 23.

[0016] Furthermore, the concentration of the sense strand substrate and the antisense strand substrate is 0.1-4.5 mM; preferably, the concentration of RNA ligase is 0.05-0.6 mg / mL, more preferably 0.2 mg / mL; preferably, 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; preferably, the temperature of the enzyme catalyzed reaction is 0-60°C, more preferably 4-37°C; preferably, the time of the enzyme catalyzed reaction is 0.5-24 h, more preferably 16-24 h; preferably, the pH value of the enzyme catalyzed reaction is 6.0-8.5; preferably, after the enzyme catalyzed reaction, purification is carried out and Cemdisiran is obtained after freeze-drying.

[0017] By applying the technical solution of the present invention and utilizing the above-mentioned preparation method, RNA ligase is used to catalyze the connection between the sense strand substrates to form the sense strand of Cemdisiran, and to catalyze the connection between the antisense strand substrates to form the antisense strand of Cemdisiran, thereby realizing the preparation of such a complex structure and siRNA with multiple modifications by biosynthesis. Compared with the preparation of Cemdisiran by chemical synthesis, the product obtained by the preparation method of the present application has higher purity, less impurities, and mild reaction conditions, which is convenient for industrialized scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0019] Figure 1 A schematic diagram showing the synthesis of Cemdisiran using RNA ligase in the present invention is shown;

[0020] Figure 2The Urea-PAGE detection result diagram of the catalytic products of ligase 31, ligase 48 and ligase 11 in Example 1 of the present invention is shown;

[0021] Figure 3 The figure shows the HPLC detection result of ligase 48 in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] 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 in conjunction with the embodiments.

[0023] Terminology explanation:

[0024] N+1 impurity: A nucleic acid impurity that has a single nucleotide extra attached compared to the target synthetic sequence.

[0025] N-1 impurity: A nucleic acid impurity that has a single nucleotide missing compared to the target synthetic sequence.

[0026] As mentioned in the background technology, the prior art uses chemical synthesis to prepare Cemdisiran, which not only uses a large amount of organic solvents, but also does not conform to the development concept of green chemistry, and the cost of scale-up of the synthesis is very high. In addition, there are low purity and many impurities in the prepared product, especially N+1 impurities and N-1 impurities that are difficult to remove, which affect the subsequent purification of the product. In the present application, the inventors attempt to develop a method for preparing siRNA for treating immunoglobulin A nephropathy, using enzyme-catalyzed synthesis of Cemdisiran, and thus propose a series of protection schemes of the present application.

[0027] In a first typical embodiment of the present application, a method for preparing Cemdisiran is provided, wherein Cemdisiran is siRNA, and is composed of a sense strand and an antisense strand through complementary pairing; the method comprises: mixing a sense strand substrate, an antisense strand substrate and an RNA ligase, wherein the sense strand substrate can form a sense strand, and the antisense strand substrate can form an antisense strand; the sense strand substrate and the antisense strand substrate are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate and the antisense strand substrate are not connected to each other, so as to form a double-stranded nucleotide structure containing a nick; using RNA ligase to connect the bases at both ends of the nick by phosphodiester bonds to form Cemdisiran; the bases at both ends of the nick are the 5' end and the 3' end of different substrates, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; using RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Cemdisiran; the RNA ligase is selected from RNA ligase family 1 or RNA ligase family 2; the RNA ligase comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, or one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; or an enzyme having 70% or more identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 6 and having activity of catalyzing phosphodiester bond formation. The double-stranded nucleotide structure containing a nick formed by the sense strand substrate and the antisense strand substrate has a sticky end.

[0028] The sense strand substrate and the antisense strand substrate can be chemically synthesized by a solid phase method or a liquid phase method.

[0029] 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, and the difference from the sense strand is that there is a nick between the sense strand substrates and they are not connected by a phosphodiester bond. Similarly, RNA ligase is used to connect two or more antisense strand substrates by a phosphodiester bond to obtain the antisense strand of Cemdisiran.

[0030] In the above preparation method, the sense strand substrate, the antisense strand substrate and the RNA ligase are mixed together, and Cemdisiran is directly prepared in a one-pot method. In the one-pot ligation, the sense strand substrate and the antisense strand substrate can undergo base complementary pairing to form a double-stranded structure, and the RNA ligase recognizes the double-stranded structure and connects the nicks in the double-stranded structure, thereby preparing the target product Cemdisiran.

[0031] Any method capable of RNA synthesis is applicable to the present application. In a preferred embodiment, the sense strand substrate and the antisense strand substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method. In a preferred embodiment, the sense strand is the nucleic acid sequence shown in SEQ ID NO: 14, and the antisense strand is the nucleic acid sequence shown in SEQ ID NO: 15.

[0032] SEQ ID NO: 14:

[0033] AmsAmsGfCmAfAmGfAmUfAfUfUmUfUmUmAfUmAfAmUmAm.

[0034] SEQ ID NO: 15:

[0035] UmsAfsUfUmAfUmAmAfAmAfAmUmAmUfCmUfUmGfCmUmUmsUmsUmdTdT.

[0036] In the present application, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide. f indicates 2' fluorine modification of the ribonucleotide, s in "sAm", "sGf" and the like indicates thio modification of the 5' phosphate of the ribonucleotide, and d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide.

[0037] The sense strand of Cemdisiran is 21nt long, and the antisense strand is 25nt long. The sense strand / antisense strand is divided into several sense strand / antisense strand substrates of different lengths according to the connection efficiency, and connected by enzyme-catalyzed reaction, so as to obtain a product with high purity. In a preferred embodiment, the sense strand is composed of 2 or more sense strand substrates, and the length of the sense strand substrate is 3-18nt, more preferably 4-16nt; preferably, the antisense strand is composed of 2 or more antisense strand substrates, and the length of the antisense strand substrate is 3-22nt, more preferably 8-13nt.

[0038] When Cemdisiran is synthesized using a one-pot method, Figure 1 As shown, the sense strand substrate and the antisense strand substrate can undergo base pairing to form a double-stranded structure, and RNA ligase recognizes the double-stranded structure with a nick formed by the complementary pairing of the substrates, thereby connecting the sense strand substrate and the antisense strand substrate to obtain Cemdisiran. In a preferred embodiment, the double-stranded RNA formed by annealing the sense strand substrate and the antisense strand substrate has 3 or more base combinations that can be complementary to each other. In a preferred embodiment, the ends of the double-stranded RNA are sticky ends; the length of the sticky ends is 2-8nt.

[0039] Any RNA ligase that can recognize a double-stranded structure with a nick formed by complementary pairing of substrates and catalyze the formation of a phosphodiester bond between a phosphate group and a hydroxyl group is suitable for the present application. In a preferred embodiment, the RNA ligase is selected from RNA ligase family 1 or RNA ligase family 2.

[0040] In a preferred embodiment, the RNA ligase includes one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; or an enzyme having more than 70% identity with any RNA ligase shown in SEQ ID NO: 1-SEQ ID NO: 6, including but not limited to 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) and having activity in catalyzing phosphodiester bond formation. Among them, the RNA ligase of RNA ligase family 1 includes SEQ ID NO: 4 or SEQ ID NO: 6, and the RNA ligase of RNA ligase family 2 includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 5.

[0041] SEQ ID NO: 1: (ligase 48, Escherichia phage JN02)

[0042] MFKKYSSLENHYNSKFIEKLYTNGLTTGVWVAREKIHGTNFSLIIERDNVTCAKRTGPILPAEDFYGYEIVLKKYDKAIKAVQEVMESISTSVPVSYQVFGEFAGGGIQKGVDYGEKDFYVFDIIINTESDDTYYMSDYEMQDFCNTFGFKMAPMLGRGTFDSLIMIP NDLDSVLAAYNSTASEDLVEANNCVFDANVIGDNTAEGYVLKPCFPKWLSNGTRVAIKCKNSKFSEKKKSDKPVKTQVPLTEIDKNLLDVLACYVTLNRVNNVISKIGTVTPKDFGKVMGLTVQDILEETSREGIVLTSSDNPNLVKKELVRMVQDVLRPAWIELVS.

[0043] SEQ ID NO: 2: (Ligase 25, Vibrio phage NT-1)

[0044] MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF。

[0045] SEQ ID NO: 3: (Ligase 26, Escherichia phage AR1)

[0046] MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY。

[0047] SEQ ID NO: 4: (Ligase 31, Vibrio phage VH12019)

[0048] MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAHADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA。

[0049] SEQ ID NO: 5: (Ligase 41, Vibrio phage VH7D)

[0050] MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATAHEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV。

[0051] SEQ ID NO: 6: (Ligase 42, Escherichia phage JN02)

[0052] MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY。

[0053] SEQ ID NO: 7: (Ligase 11, Thermococcus)

[0054] MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD。

[0055] SEQ ID NO: 8: (Ligase 20, Archaea)

[0056] MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH。

[0057] SEQ ID NO: 9: (Ligase 32, bacteria)

[0058] MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD。

[0059] Identity in this application refers to the "identity" between amino acid sequences or nucleic acid sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the 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.

[0060] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (for example, 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 have a high probability of having the same active site, active pocket, active mechanism, protein structure, etc. as the protein provided by the sequence in a).

[0061] 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).

[0062] Substitution and replacement rules. Generally speaking, amino acids with similar properties will have similar effects after replacement. For example, conservative amino acid replacements may occur in the above homologous proteins. "Conservative amino acid replacements" include but are not limited to:

[0063] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0064] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0065] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0066] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0067] A person 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.

[0068] In this application, only the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 6, or an enzyme with more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 6, can be used to catalyze the formation of a phosphodiester bond between the substrates in this application to obtain the product Cemdisiran. In the relevant experiments of this application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 6 that can synthesize Cemdisiran by screening from a large number of enzymes (50 species). However, the negative results that accounted for a large proportion (up to 70%) in the experiment showed that most RNA ligases were difficult to catalyze the synthesis of Cemdisiran, including but not limited to the RNA ligases shown in SEQ ID NO: 7 to SEQ ID NO: 9. In this application specification, only SEQ ID NO: 7 to SEQ ID NO: 9 are used as examples to reflect this type of RNA ligase that has no catalytic activity for the synthesis of Cemdisiran.

[0069] 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 the antisense chain are complementary paired to form Cemdisiran.

[0070] In a preferred embodiment, 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.

[0071] In a preferred embodiment, the first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 10 or 16, and the second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 11 or 17.

[0072] In a preferred embodiment, the first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 13 or 19, and the second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 12 or 18.

[0073] Cemdisiran can be prepared using the above preparation method and the substrates shown in SEQ ID NOs: 10-13 or SEQ ID NOs: 16-19. However, it should be noted that the selection of substrates is not limited to the substrates shown in SEQ ID NOs: 10-13 or SEQ ID NOs: 16-19. All substrates that can be combined to form a sense chain and an antisense chain can be applied to the above preparation method. The above preparation method is suitable for the preparation of Cemdisiran but is not limited to the difference in substrate connection positions. The above preparation has a good connection effect for the connection of the sense chain sequence and the antisense chain sequence of Cemdisiran. The number of sense chain substrates or antisense chain substrates includes but is not limited to 2, 3, 4 or even more.

[0074] SEQ ID NO: 10:

[0075] AmsAmsGfCmAfAmGfAmUfAfUfUmUfUmUmAf.

[0076] SEQ ID NO: 11:

[0077] UmAfAmUmAm.

[0078] SEQ ID NO: 12:

[0079] UfCmUfUmGfCmUmUmsUmsUmdTdT.

[0080] SEQ ID NO: 13:

[0081] UmsAfsUfUmAfUmAmAfAmAfAmUmAm.

[0082] SEQ ID NO: 16:

[0083] AmsAmsGfCmAfAmGfAmUfAfUfUmUf.

[0084] SEQ ID NO: 17:

[0085] UmUmAfUmAfAmUmAm.

[0086] SEQ ID NO: 18:

[0087] UmAmUfCmUfUmGfCmUmUmsUmsUmdTdT.

[0088] SEQ ID NO: 19:

[0089] UmsAfsUfUmAfUmAmAfAmAfAm.

[0090] 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: 20; the second sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 21; the third sense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 22; preferably, the first antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 25; the second antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 24; the third antisense chain substrate is the nucleic acid sequence shown in SEQ ID NO: 23.

[0091] SEQ ID NO: 20:

[0092] AmsAmsGfCmAfAmGf.

[0093] SEQ ID NO: 21:

[0094] AmUfAfUfUmUfUmUmAfUm.

[0095] SEQ ID NO: 22:

[0096] AfAmUmAm.

[0097] SEQ ID NO: 23:

[0098] GfCmUmUmsUmsUmdTdT.

[0099] SEQ ID NO: 24:

[0100] AmAfAmUmAmUfCmUfUm.

[0101] SEQ ID NO: 25:

[0102] UmsAfsUfUmAfUmAmAf.

[0103] In a preferred embodiment, the concentration of the sense strand substrate and the antisense strand substrate is 0.1-4.5 mM; preferably, the concentration of RNA ligase is 0.05-0.6 mg / mL, more preferably 0.2 mg / mL; preferably, 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; preferably, the temperature of the enzyme catalyzed reaction is 0-60°C, more preferably 4-37°C; preferably, the time of the enzyme catalyzed reaction is 0.5-24 h, more preferably 16-24 h; preferably, the pH value of the enzyme catalyzed reaction is 6.0-8.5; preferably, after the enzyme catalyzed reaction, purification is carried out and Cemdisiran is obtained after freeze-drying.

[0104] The concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are each selected from, including but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 or 4.5 mM; the reaction temperature of the preparation method includes but is not limited to 10, 15, 16, 20, 25, 30, 35 or 40°C; the reaction time of the preparation method includes but is not limited to 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45 or 48 h.

[0105] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0106] Example 1

[0107] The following four single-stranded RNA fragments were designed based on the sequence of Cemdisiran:

[0108]

[0109] The above four single-stranded RNA fragments were prepared by solid phase synthesis. Among them, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, s in "sAm", "sGf" and other writings represents thio modification of the 5' phosphate of the ribonucleotide, d before A, C, G or T represents that the nucleotide is a deoxyribonucleotide, L96 is an N-acetylgalactosamine group (GalNAc), as shown in the following formula, and the leftmost wavy line of the L96 protective group represents that the group is connected to the last nucleotide of the sense chain.

[0110]

[0111] Cemdisiran-1 has 2' methoxy modifications on ribonucleotides at positions 1, 2, 4, 6, 8, 12, 14 and 15; 2' fluorine modifications on ribonucleotides at positions 3, 5, 7, 9, 10, 11, 13 and 16; and thio modifications on the 5' phosphates of ribonucleotides at positions 2 and 3.

[0112] Cemdisiran-2 has 2' methoxy modification on the 1st, 3rd, 4th and 5th ribonucleotides and 2' fluorine modification on the 2nd ribonucleotide.

[0113] Cemdisiran-3 has 2' methoxy modification on the 2nd, 4th, 6th, 7th, 8th, 9th and 10th ribonucleotides; 2' fluorine modification on the 1st, 3rd and 5th ribonucleotides; thio modification on the 5' phosphate of the 9th and 10th ribonucleotides; and thymine (T) on the 11th and 12th deoxyribonucleotides.

[0114] Cemdisiran-4 has 2' methoxy modifications on ribonucleotides at positions 1, 4, 6, 7, 9, 11, 12, and 13, 2' fluorine modifications on ribonucleotides at positions 2, 3, 5, 8, and 10, and thio modifications on the 5' phosphates of ribonucleotides at positions 2 and 3.

[0115] The sense strand of the prepared Cemdisiran is 5'-AmsAmsGfCmAfAmGfAmUfAfUfUmUfUmUmAfUmAfAmUmAm (SEQ ID NO: 14)-L96-3', and the antisense strand is 5'-UmsAfsUfUmAfUmAmAfAmAfAmUmAmUfCmUfUmGfCmUmUmsUmsUmdTdT-3' (SEQ ID NO: 15).

[0116] The four single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture, which was annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzyme-catalyzed ligation reaction. The reaction conditions were as follows: 100 μM substrate fragment, 10 eq ATP, 100 eq MgCl2, 10 eq DTT, and a final concentration of 0.2 mg / mL RNA ligase were added in sequence in a 10 uL reactor, 1911 V 50 mM Tris-HCl pH 7.5, and reacted at 16°C for 16 hours. After the reaction, the protein was inactivated by heating at 80°C for 5 min, and the supernatant was obtained by centrifugation. The Urea-PAGE detection results were as follows: Figure 2 shown. Figure 2Lane M represents the RNA molecule standard (marker), lane 1 represents the reaction system of ligase 31, lane 2 represents the reaction system of ligase 48, and lane 3 represents the reaction system of ligase 11. The yield is estimated by the grayscale analysis results of the product bands in the Urea-PAGE results. The final yield results are shown in the following table:

[0117]

[0118] In Example 1, the yield is estimated based on the grayscale analysis results of the target band in the Urea-PAGE results, "None" means that no target product band is detected, "++" means that the yield is 25-50% (excluding the endpoint value of 50%), "+++" means that the yield is 50-90%, and ++++ means that the yield is >90%.

[0119] The grayscale data of the product and substrate are obtained by grayscale analysis of the Urea-PAGE gel electrophoresis result image. The yield calculation formula in the embodiment is: yield = product grayscale data / (product grayscale data + substrate grayscale data).

[0120] Activity screening of six RNA ligases revealed that ligases 48, 25 and 31 had the best ligation effects and could convert most substrates into Cemdisiran.

[0121] Example 2

[0122] Select ligase 48 with good reaction activity, use the annealed substrate fragment for enzyme-catalyzed ligation reaction, the reaction conditions are as follows: add substrate fragment 800 μM, ATP 4eq, MgCl2 12.5eq, DTT1.25 eq, the above RNA ligase with a final concentration of 0.2 mg / mL in a 50 uL reactor, 239 V 50 mM Tris-HCl pH 7.5, react at 16℃ for 16 h. After the reaction, heat at 80℃ for 5 min to inactivate the protein, centrifuge and take the supernatant. Submit for HPLC and LC-MS detection, the HPLC result of ligase 48 is shown in the following example. Figure 3 shown.

[0123] The yield is measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample, and the results are shown in the following table:

[0124]

[0125] In Example 2, the yield was calculated based on the statistical results of the target peak in the HPLC results, and ++++ indicated a yield of >90%.

[0126] The molecular weight of the sense chain product was identified by LC-MS as 8677.95, and the molecular weight of the antisense chain product was 8096.11. The theoretical values ​​of the sense chain product were 8677.94±8, and the theoretical values ​​of the antisense chain product were 8096.11±8, indicating that Cemdisiran was generated by ligation by ligase 48.

[0127] Comparative Example 1

[0128] The average yield of the full-length Cemdisiran product synthesized using solid phase synthesis was 32.6%, and the total proportion of N+1 and N-1 impurities was 1.47%.

[0129] The yield of the Cemdisiran product produced by the enzymatic ligation method involved in the present invention is 87.48% in the enzymatic synthesis step, and the average solid phase synthesis yield of the substrate used is 39.2%. Multiplying them together, the yield of the overall process is 34.3%, which is higher than the average yield of the full-length Cemdisiran product synthesized by solid phase, and the total proportion of N+1 and N-1 impurities in the Cemdisiran product synthesized by the enzymatic method is 0.4%, which is lower than the proportion of such impurities in the full-length Cemdisiran product synthesized by solid phase.

[0130] 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 is used to catalyze the connection between the sense chain substrates to form the sense chain of Cemdisiran, and catalyze the connection between the antisense chain substrates to form the antisense chain of Cemdisiran, thereby realizing the preparation of such a complex structure and siRNA with multiple modifications by biosynthesis. Compared with the preparation of Cemdisiran by chemical synthesis, the product obtained by the preparation method of the present application has higher purity, less impurities (the content of N+1 and N-1 impurities <0.5%), and the subsequent final product purification pressure is small, and the reaction conditions are mild, and a large amount of organic reagents are not used, which can reduce production costs and facilitate industrial scale-up production.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing Cemdisiran, characterized in that: The Cemdisiran is a siRNA, which is composed of a sense strand and an antisense strand through complementary pairing; The method comprises: Mix the sense strand substrate, antisense strand substrate and RNA ligase. Wherein, the sense strand substrate can constitute the sense strand, and the antisense strand substrate can constitute the antisense strand; Part of the sense strand substrate and part of the antisense strand substrate are connected by hydrogen bonds formed by base complementarity to form a double-stranded nucleotide structure with sticky ends; The double-stranded nucleotide structure containing sticky ends continues to connect with the remaining part of the sense strand substrate fragment and the remaining part of the antisense strand substrate fragment through hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate and the antisense strand substrate are not connected to each other, forming a double-stranded nucleotide structure containing a nick; Using the RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Cemdisiran; The bases at both ends of the notch are the 5' end and 3' end of different substrates, respectively, the 5' end is a phosphate group, and the 3' end is a hydroxyl group; Using the RNA ligase to connect the phosphate group at the 5' end and the hydroxyl group at the 3' end upstream and downstream of the nick to form the phosphodiester bond, thereby obtaining Cemdisiran; The RNA ligase is selected from RNA ligase family 1 or RNA ligase family 2; The RNA ligase is one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; The sense strand is composed of 2 or 3 sense strand substrates, and the length of the sense strand substrate is 3-18 nt; The antisense strand is composed of 2 or 3 antisense strand substrates, and the length of the antisense strand substrate is 3-22 nt.

2. The method according to claim 1, characterized in that The sense strand substrate and the antisense strand substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method.

3. The method according to claim 2, characterized in that The double-stranded RNA formed by annealing the sense strand substrate and the antisense strand substrate has 3 or more base combinations capable of complementary pairing.

4. The method according to claim 2, 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 connection of the first sense strand substrate and the second sense strand substrate to form the sense strand, catalyzing the connection of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand, and the sense strand and the antisense strand complementarily pair to form Cemdisiran.

5. The method according to claim 4, 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; 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.

6. The method according to claim 5, characterized in that The first sense strand substrate is a nucleic acid sequence shown in SEQ ID NO: 10 or 16, and the second sense strand substrate is a nucleic acid sequence shown in SEQ ID NO: 11 or 17; SEQ ID NO: 10: AmsAmsGfCmAfAmGfAmUfAfUfUmUfUmUmAf; SEQ ID NO: 11: UmAfAmUmAm; SEQ ID NO: 16: AmsAmsGfCmAfAmGfAmUfAfUfUmUf; SEQ ID NO: 17: UmUmAfUmAfAmUmAm; The first sense strand substrate shown in SEQ ID NO: 10 has 2' methoxy modification on ribonucleotides at positions 1, 2, 4, 6, 8, 12, 14 and 15; 2' fluorine modification on ribonucleotides at positions 3, 5, 7, 9, 10, 11, 13 and 16; and thio modification on the 5' phosphate of ribonucleotides at positions 2 and 3; The first sense strand substrate shown in SEQ ID NO: 16 has 2' methoxy modification on ribonucleotides at positions 1, 2, 4, 6, 8 and 12; 2' fluorine modification on ribonucleotides at positions 3, 5, 7, 9, 10, 11 and 13; and thio modification on the 5' phosphate of ribonucleotides at positions 2 and 3; The second sense strand substrate shown in SEQ ID NO: 11 has 2' methoxy modification on the 1st, 3rd, 4th and 5th ribonucleotides, and 2' fluorine modification on the 2nd ribonucleotide; The second sense strand substrate shown in SEQ ID NO: 17 has 2' methoxy modification on the 1st, 2nd, 4th, 6th, 7th and 8th ribonucleotides, and 2' fluorine modification on the 3rd and 5th ribonucleotides.

7. The method according to claim 5, characterized in that The first antisense strand substrate is a nucleic acid sequence shown in SEQ ID NO: 13 or 19, and the second antisense strand substrate is a nucleic acid sequence shown in SEQ ID NO: 12 or 18; SEQ ID NO: 12: UfCmUfUmGfCmUmUmsUmsUmdTdT; SEQ ID NO: 13: UmsAfsUfUmAfUmAmAfAmAfAmUmAm; SEQ ID NO: 18: UmAmUfCmUfUmGfCmUmUmsUmsUmdTdT; SEQ ID NO: 19: UmsAfsUfUmAfUmAmAfAmAfAm; The second antisense strand substrate shown in SEQ ID NO: 12 has 2' methoxy modification on the 2nd, 4th, 6th, 7th, 8th, 9th and 10th ribonucleotides; has 2' fluorine modification on the 1st, 3rd and 5th ribonucleotides; has thio modification on the 5' phosphate of the 9th and 10th ribonucleotides; and the 11th and 12th ribonucleotides are deoxyribonucleotides, which are thymine T; The first antisense strand substrate shown in SEQ ID NO: 13 has 2' methoxy modification on ribonucleotides at positions 1, 4, 6, 7, 9, 11, 12 and 13, 2' fluorine modification on ribonucleotides at positions 2, 3, 5, 8 and 10, and thio modification on 5' phosphate of ribonucleotides at positions 2 and 3; The second antisense strand substrate shown in SEQ ID NO: 18 has 2' methoxy modification on the 1st, 2nd, 4th, 6th, 8th, 9th, 10th, 11th and 12th ribonucleotides; has 2' fluorine modification on the 3rd, 5th and 7th ribonucleotides; has thio modification on the 5' phosphate of the 11th and 12th ribonucleotides; and the 13th and 14th ribonucleotides are deoxyribonucleotides, which are thymine T; The first antisense strand substrate shown in SEQ ID NO: 19 has 2' methoxy modification on ribonucleotides at positions 1, 4, 6, 7, 9 and 11, 2' fluorine modification on ribonucleotides at positions 2, 3, 5, 8 and 10, and thio modification on the 5' phosphate of ribonucleotides at positions 2 and 3.

8. The method according to claim 2, 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.

9. The method according to claim 8, characterized in that The first sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 20; The second sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 21; The third sense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 22; SEQ ID NO: 20: AmsAmsGfCmAfAmGf; SEQ ID NO: 21: AmUfAfUfUmUfUmUmAfUm; SEQ ID NO: 22: AfAmUmAm; The first sense strand substrate has 2' methoxy modification on the 1st, 2nd, 4th and 6th ribonucleotides; 2' fluorine modification on the 3rd, 5th and 7th ribonucleotides; and thio modification on the 5' phosphate of the 2nd and 3rd ribonucleotides; The second sense strand substrate has 2' methoxy modification on the 1st, 5th, 7th, 8th and 10th ribonucleotides; and 2' fluorine modification on the 2nd, 3rd, 4th, 6th and 9th ribonucleotides; The third sense strand substrate has 2' methoxy modification on the 2nd, 3rd and 4th ribonucleotides; and 2' fluorine modification on the 1st ribonucleotide.

10. The method according to claim 8, characterized in that The first antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 25; The second antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 24; The third antisense strand substrate is the nucleic acid sequence shown in SEQ ID NO: 23; SEQ ID NO: 23: GfCmUmUmsUmsUmdTdT; SEQ ID NO: 24: AmAfAmUmAmUfCmUfUm; SEQ ID NO: 25: UmsAfsUfUmAfUmAmAf; The first antisense strand substrate has 2' methoxy modification on the 1st, 4th, 6th and 7th ribonucleotides; 2' fluorine modification on the 2nd, 3rd, 5th and 8th ribonucleotides; and thio modification on the 5' phosphate of the 2nd and 3rd ribonucleotides; The second antisense strand substrate has 2' methoxy modification on the 1st, 3rd, 4th, 5th, 7th, and 9th ribonucleotides; and 2' fluorine modification on the 2nd, 6th, and 8th ribonucleotides; The third antisense strand substrate has 2' methoxy modification on the 2nd, 3rd, 4th, 5th and 6th ribonucleotides; 2' fluorine modification on the 1st ribonucleotide; thio modification on the 5' phosphate of the 5th and 6th ribonucleotides; and deoxyribonucleotides at the 7th and 8th positions are thymine T.

11. The method according to any one of claims 1 to 3, characterized in that: The concentration of the sense strand substrate and the antisense strand substrate is 0.1-4.5 mM.

12. The method according to any one of claims 1 to 3, characterized in that The concentration of the RNA ligase is 0.05-0.6 mg / mL.

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