Preparation method of siRNA for inhibiting FXI gene expression

Abstract

The present invention provides a method for preparing siRNA for inhibiting FXI gene expression. The siRNA is Fitusiran, which is a double-stranded RNA composed 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, 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, forming a double-stranded nucleotide structure containing a nick; and using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Fitusiran. The method can solve the problem of low purity of Fitusiran prepared in the prior art, and is applicable to the field of drug biosynthesis technology.

Description

Technical Field

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

[0002] In the existing technology, diseases caused by gene expression and mutation cannot be cured by traditional small molecule drugs. Such diseases can only be treated by gene expression intervention. siRNA is a double-stranded RNA oligonucleotide with a length of 19-25nt, which can specifically bind to the mRNA of the same target gene and prevent the translation process, thereby hindering the expression of the target gene, that is, gene silencing caused by RNA interference (RNAi). The research and development of siRNA drugs has brought great development prospects to gene therapy and has been widely used in the field of disease treatment. siRNA acts on mRNA, and the druggable target is significantly larger than that of traditional small molecule drugs whose action sites are proteins, and it can act on new targets by changing the sequence. Intervene in gene expression.

[0003] Fitusiran is a gene-targeted siRNA double-stranded RNA drug, first announced in 2016, developed by Alnylam and Regeneron, and currently in Phase 3 clinical trials. Fitusiran inhibits the transcription and expression of a specific gene through a small molecule RNA fragment, reduces the expression of antithrombin, inhibits the synthesis of coagulation factors, prevents bleeding events, and enhances the body's coagulation ability. It is used to treat hemophilia.

[0004] The synthesis of Fitusiran mainly uses a solid phase carrier and is cyclically synthesized by the phosphoramidite triester method. After the synthesis cycle is completed, the Fitusiran chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain the target product. The yield of product synthesis in this preparation method decreases with the increase of the synthesis chain length. The impurities generated during 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. They are not easy to remove and the purification process is complicated. As Fitusiran is increasingly widely used in the treatment of universal hemophilia, its synthesis scale is limited by the limitations of the synthesis instrument, the cost is high, and it is difficult to scale up production. Therefore, it is necessary to develop a more efficient method for synthesizing Fitusiran. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for preparing siRNA for inhibiting the expression of FXI gene, so as to solve the problem of low purity of Fitusiran 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 siRNA for inhibiting the expression of FXI gene is provided, wherein the siRNA is Fitusiran, which is a double-stranded RNA composed 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, 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 Fitusiran;

[0007] The bases at both ends of the nick are the 5' end and 3' end of different substrates, respectively, the 5' end is a phosphate, and the 3' end is a hydroxyl; RNA ligase is used to connect the phosphate at the 5' end and the hydroxyl at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Fitusiran; the RNA ligase is selected from: one or more RNA ligases containing the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; or an enzyme that has more than 70% identity with any RNA ligase having the amino acid sequence shown in SEQ ID NO: 1 to SEQID NO: 5 and has the activity of catalyzing the formation of phosphodiester bonds.

[0008] Furthermore, the nucleotide sequence of the sense strand is the nucleotide sequence shown in SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO: 24.

[0009] Further, the sense strand substrate includes 2 or more strands, and the antisense strand substrate includes 2 or more strands; preferably, the length of the sense strand substrate is 2-19 nt, more preferably 4-17 nt, and further preferably 8-13 nt; preferably, the length of the antisense strand substrate is 2-21 nt, more preferably 4-19 nt, and further preferably 8-15 nt.

[0010] Furthermore, the sense chain and antisense chain substrates 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 form Fitusiran through base complementary pairing; preferably, the sense chain substrate and the antisense chain substrate are annealed and then mixed with RNA ligase to obtain Fitusiran.

[0011] Further, the 3' end of the first sense chain substrate and the 5' end of the second sense chain 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.

[0012] Furthermore, the nucleotide sequence of the first sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 10; preferably, the nucleotide sequence of the first antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 11.

[0013] Further, the nucleotide sequence of the first sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 14; preferably, the nucleotide sequence of the first antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 15.

[0014] Further, 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 nucleotide sequence of the first sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the second sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the third sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 19; preferably, the nucleotide sequence of the first antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 22; the nucleotide sequence of the second antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 20.

[0015] Furthermore, the concentrations of the sense chain substrate and the antisense chain substrate are independently selected from 0.1-4.5 mM; 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 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48h, more preferably 12-24h.

[0016] By applying the technical solution of the present invention and utilizing the above-mentioned preparation method, under the catalysis of RNA ligase, the sense strand substrates are connected to form the Fitusiran sense strand, and the antisense strand substrates are connected to form the Fitusiran antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method for preparing Fitusiran by chemical synthesis, the product obtained by the preparation method of the present application has high purity, few impurities, a simple preparation process, mild reaction conditions, low organic reagent dosage, reduced production costs, and is easy to achieve scale-up industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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:

[0018] Figure 1 A schematic diagram of the structure of L96 according to an embodiment of the present invention is shown.

[0019] Figure 2 A schematic diagram of a substrate reaction according to Example 1 of the present invention is shown.

[0020] Figure 3The figure shows the gel electrophoresis result according to Example 1 of the present invention.

[0021] Figure 4 The HPLC test result diagram according to Example 2 of the present invention is shown. 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 a chemical synthesis method for the preparation of Fitusiran, which is not only complicated and costly, but also produces a large number of N+1 and N-1 impurities, which affect the subsequent purification of the product. In this application, the inventor attempts to develop a method for preparing siRNA that inhibits the expression of the FXI gene, and a method for preparing Fitusiran using enzyme catalysis.

[0027] In a first typical embodiment of the present application, a method for preparing siRNA for inhibiting FXI gene expression is provided, wherein the siRNA is Fitusiran, which is a double-stranded RNA composed of a complementary sense strand and an antisense strand; the preparation method comprises:

[0028] The preparation method comprises:

[0029] 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, forming 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 Fitusiran;

[0030] The bases at both ends of the nick are the 5' end and 3' end of different substrates, respectively, the 5' end is a phosphate, and the 3' end is a hydroxyl; RNA ligase is used to connect the phosphate at the 5' end and the hydroxyl at the 3' end upstream and downstream of the nick to form a phosphodiester bond to obtain Fitusiran; the RNA ligase is selected from: one or more RNA ligases of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQID NO: 5; or an enzyme that has more than 70% identity with any RNA ligase of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has the activity of catalyzing the formation of the above-mentioned phosphodiester bond.

[0031] The sense strand substrate and antisense strand substrate can be synthesized by solid phase chemical synthesis or liquid phase chemical synthesis.

[0032] In the above preparation method, the sense strand substrate is a nucleotide sequence that can form two or more sense strands, that is, 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, the antisense strand substrate and the antisense strand have the above characteristics. Two or more sense strand substrates or antisense strand substrates are connected by a phosphodiester bond using RNA ligase to obtain the sense strand and antisense strand of Fitusiran.

[0033] In the present application, the sense chain substrate, the antisense chain substrate and the RNA ligase are mixed to prepare Fitusiran. The sense chain substrate and the antisense chain substrate are connected by hydrogen bonds formed by base complementarity to form a double-stranded nucleotide structure containing a sticky end; the double-stranded nucleotide containing the sticky end is further connected with other substrates through base complementary pairing, wherein the head and tail bases of the sense chain substrate and the antisense chain substrate are not connected to each other, and a double-stranded nucleotide structure containing a nick can be formed; finally, the nicks are connected by phosphodiester bonds using RNA ligase to obtain the target product Fitusiran. Preferably, the sense chain substrate and the antisense chain substrate are annealed and then mixed with RNA ligase to obtain Fitusiran.

[0034] In the above preparation method, the sense chain substrate and the antisense chain substrate can be mixed and annealed first, and the sense chain substrate and the antisense chain substrate can form a double-stranded RNA structure through base complementary pairing, and there are nicks between different substrates in the double-stranded RNA structure. Then, the annealed reaction system is mixed with RNA ligase, and the RNA ligase is used to connect the phosphate groups and hydroxyl groups on both sides of the nick with phosphodiester bonds to repair the nick, thereby obtaining the target product Fitusiran with a complete double-stranded structure. In a preferred embodiment, the nucleotide sequence of the sense chain is the nucleotide sequence shown in SEQ ID NO: 26, and the nucleotide sequence of the antisense chain is the nucleotide sequence shown in SEQ ID NO: 27.

[0035] SEQ ID NO: 23:

[0036] GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96.

[0037] SEQ ID NO: 24:

[0038] UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm.

[0039] 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, and s before the ribonucleotide in "sGm", "sUm" and the like indicates thio modification of the 5' phosphate of the ribonucleotide.

[0040] The above-mentioned RNA ligases are all able to recognize the double-stranded structure with nicks formed by complementary pairing of substrates, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

[0041] SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

[0042] MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF。

[0043] SEQ ID NO:2(Ligase 26,Escherichia phage AR1):

[0044] MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY。

[0045] SEQ ID NO:3(Ligase 31,Vibrio phage VH12019):

[0046] MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAHADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA。

[0047] SEQ ID NO:4(Ligase 41,Vibrio phage VH7D):

[0048] MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATAHEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV。

[0049] SEQ ID NO:5(Ligase 42,Escherichia phage JN02):

[0050] MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNT GEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.

[0051] Identity in this application refers to the "identity" between amino acid sequences or nucleotide sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0052] 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).

[0053] As used herein, the 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).

[0054] 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:

[0055] hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0056] Substitution of hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) by other hydrophobic amino acids with bulky side chains;

[0057] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0058] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0059] 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.

[0060] Fitusiran has a complex secondary structure and has different specificities for binding to different enzymes. In this application, only the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, or an enzyme with more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, can be used to catalyze the formation of a phosphodiester bond between the phosphate group and the hydroxyl group of the substrate in this application to obtain the product Fitusiran. In the relevant experiments of this application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5 that can synthesize Fitusiran by screening from a large number of enzymes. However, the negative results that accounted for a large proportion in the experiment showed that most RNA ligases were difficult to catalyze the synthesis of Fitusiran, including but not limited to the RNA ligases shown in SEQ ID NO: 6 to SEQ ID NO: 8. In this application specification, only SEQ ID NO: 6 to SEQ ID NO: 8 are used as examples to reflect this type of RNA ligase that has no catalytic activity in the synthesis of Fitusiran.

[0061] SEQ ID NO: 6 (Ligase 11, Thermococcus):

[0062] MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAE EYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.

[0063] SEQ ID NO: 7 (Ligase 20, Archaea):

[0064] MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

[0065] SEQ ID NO:8(Ligase 32,bacteria):

[0066] MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFFSKKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAE EYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETYKMVSHFERMGLNIHIHDDIELGNGYWKITFKRVYDDATKEIRELWNGHAFVD。

[0067] 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 2-19nt, more preferably 4-17nt, and further preferably 8-13nt; preferably, the length of the antisense chain substrate is 2-21nt, more preferably 4-19nt, and further preferably 8-15nt.

[0068] 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 form the above-mentioned Fitusiran through base complementary pairing.

[0069] 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, in the above preparation method, 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; 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.

[0070] In a preferred embodiment, the nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 10. Preferably, the nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 11.

[0071] In a preferred embodiment, the nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 14. Preferably, the nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 15.

[0072] Fitusiran can be prepared using the above preparation method and a substrate containing a nucleotide sequence shown in SEQ ID NOs: 9-: 12 or SEQ ID NOs: 13-16. However, it should be noted that the selection of substrates is not limited to the substrates shown in SEQ ID NOs: 9-: 12 or SEQ ID NOs: 13-16. 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 Fitusiran 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 Fitusiran. The number of sense chain substrates or antisense chain substrates includes but is not limited to 2, 3, 4 or even more.

[0073] SEQ ID NO: 9: GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAf.

[0074] SEQ ID NO: 10: CmUfUmCfAmAf.

[0075] SEQ ID NO: 11: GmUfGmUfUmAfAmCfCmsAmsGm.

[0076] SEQ ID NO: 12: UmsUfsGmAfAmGfUmAfAmAfUmGm.

[0077] SEQ ID NO: 13: GfsGmsUfUmAfAmCfAmCfCfAfUmUf.

[0078] SEQ ID NO: 14: UmAfCmUfUmCfAmAf.

[0079] SEQ ID NO: 15: UfGmUfUmAfAmCfCmsAmsGm.

[0080] SEQ ID NO: 16: UmsUfsGmAfAmGfUmAfAmAfUmGmGm.

[0081] In a preferred embodiment, 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 nucleotide sequence of the first sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 20; the nucleotide sequence of the second sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third sense chain substrate is the nucleotide sequence shown in SEQ ID NO: 22; preferably, the nucleotide sequence of the first antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 25; the nucleotide sequence of the second antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 24; the nucleotide sequence of the third antisense chain substrate is the nucleotide sequence shown in SEQ ID NO: 23.

[0082] SEQ ID NO: 17: GfsGmsUfUmAfAmCf.

[0083] SEQ ID NO: 18: AmCfCfAfUmUfUmAfCmUf.

[0084] SEQ ID NO: 19: UmCfAmAf.

[0085] SEQ ID NO: 20: AmCfCmsAmsGm.

[0086] SEQ ID NO: 21: AfAmAfUmGmGmUfGmUfUmAf.

[0087] SEQ ID NO: 22: UmsUfsGmAfAmGfUm.

[0088] In a preferred embodiment, the concentrations of the sense strand substrate and the antisense strand substrate are independently selected from 0.1-4.5 mM; 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 reaction temperature of the preparation method is 10-40°C, more preferably 15-30°C; preferably, the reaction time of the preparation method is 2-48h, more preferably 12-24h.

[0089] 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.

[0090] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0091] Example 1

[0092] Substrates 1 to 4 (length: nt) were added to a clean reagent bottle in an equimolar ratio and mixed to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrates 1 to 4 are shown in Table 1.

[0093] Table 1

[0094] .

[0095] Among them, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, s before the ribonucleotide in the sequence of "sGm", "sAm" etc. indicates thio modification of the 5' phosphate of the ribonucleotide, L96 indicates that the 3' end of the sense strand is modified with L96 group, and the structure is as follows Figure 1 As shown, the wavy line in the figure represents the base connected to the L96 group.

[0096] After the substrate mixture is annealed, the RNA fragment mixture of substrates 1 to 4 is obtained. The reaction system is set to 10 μL, and the reaction system includes 100 μM RNA fragment mixture, 50 mM Tris-HCl, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM); RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase41, Ligase 42, Ligase 11, Ligase 20 and Ligase32 are added at a final concentration of 0.2 mg / mL respectively. The schematic diagram of the enzyme ligation reaction is shown in the figure. Figure 2 The reaction system was reacted at 16°C for 16 h. The obtained reaction system was inactivated by heating at 80°C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm.

[0097] The products catalyzed by different RNA ligases were subjected to Urea-PAGE detection. The electrophoresis results of Ligase25, Ligase31 and Ligase11 are shown in Figure 3 As shown, Figure 3 Lane M represents the RNA molecule standard (marker), lane 1 represents the reaction system of Ligase 25, lane 2 represents the reaction system of Ligase 31, and lane 3 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis results of the target band in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0098] Substrate 1 has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 8, 12 and 14, and 2' fluorine modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11, 13 and 15. Ribonucleotides at positions 2 and 3 have thio modifications on their 5' phosphates.

[0099] The substrate 2 has 2' methoxy modifications on the 1st, 3rd and 5th ribonucleotides and 2' fluorine modifications on the 2nd, 4th and 6th ribonucleotides.

[0100] The substrate 3 has 2' methoxy modification on the 1st, 3rd, 5th, 7th, 9th, 10th and 11th ribonucleotides, 2' fluorine modification on the 2nd, 4th, 6th and 8th ribonucleotides, and thio modification on the 5' phosphate on the 10th and 11th ribonucleotides.

[0101] Substrate 4 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 11 and 12, 2' fluorine modifications on ribonucleotides at positions 2, 4, 6, 8 and 10, and thio modifications on the 5' phosphates at positions 2 and 3.

[0102] The sense strand of the prepared Fitusiran is GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23), and the antisense strand is UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0103] Table 2

[0104] .

[0105] “None” means no target product band was detected, “++” means the yield is 25-50% (excluding the endpoint value of 50%), “+++” means the yield is 50-90%, and “++++” means the yield is >90%.

[0106] 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 this embodiment is: yield = product grayscale data / (product grayscale data + substrate grayscale data).

[0107] Example 2

[0108] Substrates 1 to 4 were added to a clean reagent bottle in an equimolar ratio, and mixed to obtain a substrate mixture, in which the concentration of each substrate was 2.5 mM. After the substrate mixture was annealed, a mixture of RNA fragments of substrates 1 to 4 was obtained. The reaction system was set to 50 μL, and the reaction system included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM); the RNA ligases Ligase 25 and Ligase 31 with higher catalytic activity in Example 1 were added at a final concentration of 0.2 mg / mL. The reaction system was reacted at 16 ° C for 16 h. After the reaction was completed, the protein was inactivated by heating at 80 ° C for 5 min, and the precipitate was removed by centrifugation.

[0109] The product catalyzed by Ligase25 was detected by HPLC and LC-MS. Figure 4 The yield was calculated based on the statistical results of the target peak area in the HPLC results, and the results are shown in Table 3.

[0110] Table 3

[0111] .

[0112] “++” indicates a yield of 25-50% (excluding the endpoint value of 50%), “+++” indicates a yield of 50-90%, and “++++” indicates a yield of >90%.

[0113] According to LC-MS detection, the molecular weight of the sense chain product was 8568.88, and the molecular weight of the antisense chain product was 7671.11. The theoretical value of the sense chain product was 8568.86±8, and the theoretical value of the antisense chain product was 7671.11±8, indicating that Ligase 25 and Ligase31 were able to connect to the substrate and generate Fitusiran.

[0114] Example 3

[0115] Add substrates 1 to 4 in an equimolar ratio to a clean reagent bottle and mix to obtain a substrate mixture. The concentration of each substrate in the substrate mixture is 2.5 mM. After the substrate mixture is annealed, a mixture of RNA fragments of substrates 1 to 4 is obtained. The reaction system is set to 10 mL, and the reaction system includes 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM); add RNA ligase Ligase 25 at a final concentration of 0.2 mg / mL. The reaction system is reacted at 16 ° C for 16 hours. The obtained reaction system was heated at 50°C for 10-20 min to inactivate the protein, and the precipitate was removed by centrifugation at 12000 rpm. The supernatant was purified using a Nano-Q column, and the obtained product was gradient eluted with NaCl and desalted by membrane packaging (molecular weight cutoff 1 kDa). The product was freeze-dried, and the yield was calculated to be 76.16%, and the analytical purity by HPLC was 97.53%.

[0116] Example 4

[0117] Substrates 5-8 (length: nt) were added to a clean reagent bottle in an equimolar ratio and mixed to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrates 5-8 are shown in Table 4. After the substrate mixture was annealed, a mixture of RNA fragments of substrates 5-8 was obtained. The reaction system was set to 50 μL, and the reaction system included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added at a final concentration of 0.2 mg / mL. The reaction system was reacted at 16 ° C for 16 h. The obtained reaction system was inactivated by 80 ° C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The results showed that the target peak accounted for 83.4% in the sample.

[0118] Table 4

[0119] .

[0120] Among them, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in the sequences such as "sGm" and "sAm" represents thio modification of the 5' phosphate of the ribonucleotide.

[0121] Substrate 5 has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 8 and 12, and 2' fluorine modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11 and 13. Ribonucleotides at positions 2 and 3 have thio modifications on their 5' phosphates.

[0122] The substrate 6 has 2' methoxy modification on the 1st, 3rd, 5th and 7th ribonucleotides and 2' fluorine modification on the 2nd, 4th, 6th and 8th ribonucleotides.

[0123] Substrate 7 has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 8, 9 and 10, 2' fluorine modifications on ribonucleotides at positions 1, 3, 5 and 7, and thio modifications on the 5' phosphates at positions 9 and 10.

[0124] Substrate 8 has 2' methoxy modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 11, 12 and 13, 2' fluorine modifications on ribonucleotides at positions 2, 4, 6, 8 and 10, and thio modifications on the 5' phosphates at positions 2 and 3.

[0125] The sense strand of the prepared Fitusiran is GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23), and the antisense strand is UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0126] Example 5

[0127] Substrates 9 to 14 (length: nt) were added to a clean reagent bottle in an equimolar ratio and mixed to obtain a substrate mixture. The concentration of each substrate in the substrate mixture was 2.5 mM. The sequences of substrates 9 to 14 are shown in Table 5. After the substrate mixture was annealed, a mixture of RNA fragments of substrates 9 to 14 was obtained. The reaction system was set to 50 μL, and the reaction system included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM); RNA ligase Ligase 25 was added at a final concentration of 0.2 mg / mL. The reaction system was reacted at 16 ° C for 16 h. The obtained reaction system was inactivated by 80 ° C for 5 min, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The results showed that the target peak accounted for 83.4% in the sample.

[0128] Table 5

[0129] .

[0130] Among them, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents 2' fluorine modification of the ribonucleotide, and s before the ribonucleotide in the sequences such as "sGm" and "sAm" represents thio modification of the 5' phosphate of the ribonucleotide.

[0131] Substrate 9 has 2' methoxy modifications on ribonucleotides at positions 2, 4 and 6, and 2' fluorine modifications on ribonucleotides at positions 1, 4, 5 and 7. Ribonucleotides at positions 2 and 3 have thio modifications on their 5' phosphates.

[0132] The substrate 10 has 2' methoxy modifications on the 1st, 5th, 7th and 9th ribonucleotides, and 2' fluorine modifications on the 2nd, 3rd, 4th, 6th, 8th and 10th ribonucleotides.

[0133] The substrate 11 has 2' methoxy modification on the 1st and 3rd ribonucleotides and 2' fluorine modification on the 2nd and 4th ribonucleotides.

[0134] The substrate 12 has 2' methoxy modifications on the 1st, 3rd, 4th and 5th ribonucleotides, 2' fluorine modifications on the 2nd ribonucleotide, and thio modifications on the 5' phosphates on the 4th and 5th ribonucleotides.

[0135] The substrate 13 has 2' methoxy modification on the 2nd, 4th, 5th, 6th, 8th and 10th ribonucleotides, and 2' fluorine modification on the 1st, 3rd, 7th, 9th and 11th ribonucleotides.

[0136] The substrate 14 has 2' methoxy modification on the 1st, 3rd, 5th and 7th ribonucleotides, 2' fluorine modification on the 2nd and 3rd ribonucleotides, and thio modification on the 5' phosphate on the 2nd and 3rd ribonucleotides.

[0137] The sense strand of the prepared Fitusiran is GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAfCmUfUmCfAmAf-L96 (SEQ ID NO: 23), and the antisense strand is UmsUfsGmAfAmGfUmAfAmAfUmGmGmUfGmUfUmAfAmCfCmsAmsGm (SEQ ID NO: 24).

[0138] Comparative Example 1

[0139] The yield of the Fitusiran product prepared by the enzyme-linked method of the present invention is 30.9%, which is comparable to the average yield (31.2%) of the full-length Fitusiran product synthesized by solid phase synthesis. The total proportion of N+1 and N-1 impurities in the Fitusiran product synthesized by the preparation method of the present application is 0.4%, which is lower than the total proportion of N+1 and N-1 impurities (1.42%) of the full-length Fitusiran product synthesized by solid phase synthesis.

[0140] 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, single-stranded RNA fragments designed based on the sequence of Fitusiran are catalyzed by RNA ligase to form Fitusiran, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the chemical synthesis preparation method, the product obtained by the preparation method of the present application has high purity, less impurities, a simple preparation process, mild reaction conditions, low organic reagent dosage, reduced production costs, and is easy to achieve scale-up industrial production.

[0141] 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 siRNA for inhibiting FXI gene expression, characterized in that: The siRNA is Fitusiran, which 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, wherein the sense strand substrate can form the sense strand, and the antisense strand substrate can form 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 the sticky end continues to connect with the remaining portion of the sense strand substrate fragment and the remaining portion of the antisense strand substrate fragment through hydrogen bonds formed by base complementarity; 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 the Fitusiran; 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 the Fitusiran; The RNA ligase is one or more of the RNA ligases having the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5; The sense strand substrate comprises 2 or 3 strands, and the antisense strand substrate comprises 2 or 3 strands; The length of the sense strand substrate is 2-19 nt; The length of the antisense strand substrate is 2-21 nt.

2. The preparation method according to claim 1, 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 the 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 form the Fitusiran through base complementary pairing.

3. The preparation method according to claim 2, 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.

4. The preparation method according to claim 3, 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.

5. The preparation method according to claim 3, characterized in that: The nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 10; The nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 11; SEQ ID NO: 9: GfsGmsUfUmAfAmCfAmCfCfAfUmUfUmAf; SEQ ID NO: 10: CmUfUmCfAmAf; SEQ ID NO: 11: GmUfGmUfUmAfAmCfCmsAmsGm; SEQ ID NO: 12: UmsUfsGmAfAmGfUmAfAmAfUmGm; The first sense strand substrate has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 8, 12 and 14, 2' fluorine modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11, 13 and 15, and thio modifications on the 5' phosphates at positions 2 and 3; The second sense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 3 and 5, and 2' fluorine modification on ribonucleotides at positions 2, 4 and 6; The first antisense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 3, 5, 7, 9, 10, and 11, 2' fluorine modification on ribonucleotides at positions 2, 4, 6, and 8, and thio modification on 5' phosphate on ribonucleotides at positions 10 and 11; The second antisense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 3, 5, 7, 9, 11 and 12, 2' fluorine modification on ribonucleotides at positions 2, 4, 6, 8 and 10, and thio modification on 5' phosphate on ribonucleotides at positions 2 and 3.

6. The preparation method according to claim 3, characterized in that: The nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 14; The nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 15; SEQ ID NO: 13: GfsGmsUfUmAfAmCfAmCfCfAfUmUf; SEQ ID NO: 14: UmAfCmUfUmCfAmAf; SEQ ID NO: 15: UfGmUfUmAfAmCfCmsAmsGm; SEQ ID NO: 16: UmsUfsGmAfAmGfUmAfAmAfUmGmGm; The first sense strand substrate has 2' methoxy modifications on ribonucleotides at positions 2, 4, 6, 8 and 12, 2' fluorine modifications on ribonucleotides at positions 1, 3, 5, 7, 9, 10, 11 and 13, and thio modifications on the 5' phosphates at positions 2 and 3; The second sense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 3, 5 and 7, and 2' fluorine modification on ribonucleotides at positions 2, 4, 6 and 8; The first antisense strand substrate has 2' methoxy modification on ribonucleotides at positions 2, 4, 6, 8, 9 and 10, 2' fluorine modification on ribonucleotides at positions 1, 3, 5 and 7, and thio modification on 5' phosphate on ribonucleotides at positions 9 and 10; The second antisense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 3, 5, 7, 9, 11, 12 and 13, 2' fluorine modification on ribonucleotides at positions 2, 4, 6, 8 and 10, and thio modification on 5' phosphate on ribonucleotides at positions 2 and 3.

7. The preparation method according to claim 1, 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.

8. The preparation method according to claim 7, characterized in that: The nucleotide sequence of the first sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 17; The nucleotide sequence of the second sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 18; The nucleotide sequence of the third sense strand substrate is the nucleotide sequence shown in SEQ ID NO: 19; The nucleotide sequence of the first antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 22; The nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 21; The nucleotide sequence of the third antisense strand substrate is the nucleotide sequence shown in SEQ ID NO: 20; SEQ ID NO: 17: GfsGmsUfUmAfAmCf; SEQ ID NO: 18: AmCfCfAfUmUfUmAfCmUf; SEQ ID NO: 19: UmCfAmAf; SEQ ID NO: 20: AmCfCmsAmsGm; SEQ ID NO: 21: AfAmAfUmGmGmUfGmUfUmAf; SEQ ID NO: 22: UmsUfsGmAfAmGfUm; The first sense strand substrate has 2' methoxy modification on ribonucleotides at positions 2, 4 and 6, 2' fluorine modification on ribonucleotides at positions 1, 4, 5 and 7, and thio modification on 5' phosphate on ribonucleotides at positions 2 and 3; The second sense strand substrate has 2' methoxy modification on ribonucleotides at positions 1, 5, 7 and 9, and 2' fluorine modification on ribonucleotides at positions 2, 3, 4, 6, 8 and 10; The third sense strand substrate has 2' methoxy modification on the 1st and 3rd ribonucleotides, and 2' fluorine modification on the 2nd and 4th ribonucleotides; The first antisense strand substrate has 2' methoxy modification on the 1st, 3rd, 4th and 5th ribonucleotides, 2' fluorine modification on the 2nd ribonucleotide, and thio modification on the 5' phosphate on the 4th and 5th ribonucleotides; The second antisense strand substrate has 2' methoxy modification on ribonucleotides at positions 2, 4, 5, 6, 8 and 10, and 2' fluorine modification on ribonucleotides at positions 1, 3, 7, 9 and 11; The third antisense strand substrate has 2' methoxy modification on the 1st, 3rd, 5th and 7th ribonucleotides, 2' fluorine modification on the 2nd and 3rd ribonucleotides, and thio modification on the 5' phosphate on the 2nd and 3rd ribonucleotides.

9. The preparation method according to claim 1, characterized in that: The concentrations of the sense strand substrate and the antisense strand substrate are each independently selected from 0.1-4.5 mM; 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.

10. The preparation method according to claim 1, characterized in that: The reaction temperature of the preparation method is 10-40°C; The reaction time of the preparation method is 2-48h.

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