CN118460656A - A preparation method of Lumasiran - Google Patents

Abstract

The present invention provides a method for preparing Lumasiran. The Lumasiran is a double-stranded RNA composed of a sense chain and an antisense chain through complementary pairing; the method comprises: mixing a sense chain substrate fragment, an antisense chain substrate fragment and an RNA ligase, wherein the sense chain substrate fragment can form a sense chain, and the antisense chain substrate fragment can form an antisense chain; the sense chain substrate fragment and the antisense chain substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense chain substrate fragment and the antisense chain substrate fragment 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 Lumasiran. The method can solve the problem of low purity of Lumasiran prepared in the prior art, and is applicable to the field of drug biosynthesis technology.

Description

A preparation method of Lumasiran

Technical Field

The present invention relates to the field of drug biosynthesis, and in particular to a method for preparing Lumasiran.

Background Art

In the prior art, diseases caused by gene expression and mutation cannot be cured by traditional small molecule drugs, and 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 by blocking the translation process, it can achieve the effect of hindering the expression of the target gene, that is, by mediating RNA interference to cause gene silencing. The research and development of siRNA drugs has brought huge 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.

Lumasiran is a gene-targeted siRNA-type double-stranded RNA drug developed by Alnylam for the treatment of primary hyperoxaluria I (PH I) and was approved by the FDA in 2020. Lumasiran is the first drug approved for the treatment of PH1. Phase 3 clinical data showed that Lumasiran treatment significantly reduced the production of oxalate in the liver and can promote the resolution of the intrinsic pathophysiological problems of PH1.

The synthesis method of Lumasiran mainly uses a chemical solid phase synthesis method for preparation. In the synthesis method, a solid phase carrier is used, and the synthesis is cyclically performed by the phosphoramidite triester method. After the synthesis cycle is completed, the Lumasiran chain is cut off from the solid phase carrier by aminolysis, and then purified to obtain the target product. The yield of the product synthesis in this preparation method decreases with the increase of the synthesis chain length. 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. It is not easy to remove, resulting in a complex purification process and low efficiency. As Lumasiran is increasingly widely used in the treatment of primary hyperbilirubinemia, 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 Lumasiran synthesis method.

Summary of the invention

The main purpose of the present invention is to provide a method for preparing Lumasiran to solve the problem of low purity of Lumasiran prepared in the prior art.

In order to achieve the above object, according to a first aspect of the present invention, a method for preparing Lumasiran is provided, wherein Lumasiran is a double-stranded siRNA consisting of a complementary sense strand and an antisense strand, and the preparation method comprises:

Mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment 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 a phosphodiester bond to form Lumasiran; the bases at both ends of the nick are the 5' end and the 3' end of different substrate fragments, 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 of the upstream and downstream of the nick to form a phosphodiester bond to obtain Lumasiran; the RNA ligase includes an RNA ligase of RNA ligase family 1 and\or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 includes: a RNA ligase having SEQ ID NO: 3 and\SEQ ID NO:5; an RNA ligase of RNA ligase family 2 selected from: one or more RNA ligases with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:4; or an enzyme that has more than 70% identity with any RNA ligase shown in SEQ ID NO:1 to SEQ ID NO:5 and has activity of catalyzing phosphodiester bond formation.

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.

Furthermore, the sense strand substrate fragment includes 2 or more strands, and the antisense strand substrate fragment includes 2 or more strands; preferably, the length of the sense strand substrate fragment is 5-14 nt, more preferably 8-12 nt; preferably, the length of the antisense strand substrate fragment is 4-16 nt, more preferably 7-12 nt.

Further, the sense chain substrate fragment and the antisense chain substrate fragment each include 2 fragments, the sense chain substrate fragment includes a first sense chain substrate fragment and a second sense chain substrate fragment, and the antisense chain substrate fragment includes a first antisense chain substrate fragment and a second antisense chain substrate fragment; preferably, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 10; the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 11; preferably, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14; the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15.

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

Furthermore, when the sense chain substrate fragment and the antisense chain substrate fragment each include 2 strands, the preparation method includes: mixing the first sense chain substrate fragment, the second sense chain substrate fragment, the first antisense chain substrate fragment and the second antisense chain substrate fragment, and under the catalytic action of RNA ligase, connecting the first sense chain substrate fragment and the second sense chain substrate fragment to form a sense chain, connecting the first antisense chain substrate fragment and the second antisense chain substrate fragment to form an antisense chain, and the sense chain and the antisense chain form Lumasiran through base complementary pairing; preferably, the sense chain substrate fragment and the antisense chain substrate fragment are annealed and then mixed with RNA ligase to obtain Lumasiran.

Further, the sense strand substrate fragment and the antisense strand substrate fragment each include 3 fragments, the sense strand substrate fragment includes a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment; the antisense strand substrate fragment includes a first antisense strand substrate fragment, a second antisense strand substrate fragment and a third antisense strand substrate fragment; preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; preferably, the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 22; the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: NO: The nucleotide sequence shown in 20; preferably, the preparation method comprises: mixing a first sense chain substrate fragment, a second sense chain substrate fragment, a third sense chain substrate fragment, a first antisense chain substrate fragment, a second antisense chain substrate fragment, a third antisense chain substrate fragment and RNA ligase; under the catalytic action of RNA ligase, the first sense chain substrate fragment, the second sense chain substrate fragment and the third sense chain substrate fragment are connected to form a sense chain, the first antisense chain substrate fragment, the second antisense chain substrate fragment and the third antisense chain substrate fragment are connected to form an antisense chain, and the sense chain and the antisense chain form Lumasiran through base complementary pairing.

Furthermore, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase also includes ATP, Tris-HCl, MgCl ₂ 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.

By applying the technical solution of the present invention, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form the Lumasiran sense strand, and the antisense strand substrate fragments are connected to form the Lumasiran antisense strand, thereby realizing the preparation of such siRNA drugs by biosynthesis. Compared with the method of preparing Lumasiran by chemical synthesis, the preparation method of the present application has a high purity of the product, generates less impurities, has a simple preparation process, mild reaction conditions, and a low amount of organic reagents, which reduces production costs and facilitates the realization of large-scale industrial production.

BRIEF DESCRIPTION OF THE DRAWINGS

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:

FIG. 1 shows a schematic diagram of an enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

FIG. 2 shows the electrophoresis results of the catalytic products of RNA ligases Ligase25, Ligase26, Ligase31 and Ligase41 according to Example 1 of the present invention.

FIG. 3 shows a schematic structural diagram of L96 according to an embodiment of the present invention.

FIG. 4 shows a graph showing the HPLC detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

FIG. 5 shows the LC-MS detection result of the product catalyzed by RNA ligase Ligase 25 according to Example 2 of the present invention.

DETAILED DESCRIPTION

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.

Terminology explanation:

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

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

As mentioned in the background technology, the prior art uses a chemical synthesis method to prepare Lumasiran, 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 Lumasiran, using enzyme-catalyzed synthesis to prepare Lumasiran, and thus proposes a series of protection schemes of this application.

In a first typical embodiment of the present application, a method for preparing Lumasiran is provided, wherein Lumasiran is a double-stranded RNA composed of a complementary sense strand and an antisense strand; the preparation method comprises: mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form a sense strand, and the antisense strand substrate fragment can form an antisense strand; the sense strand substrate fragment and the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity, and the head and tail bases of the sense strand substrate fragment and the antisense strand substrate fragment are not connected to each other, so as to form a double-stranded RNA containing a nick. Nucleotide structure; using RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Lumasiran; the bases at both ends of the nick are the 5' end and 3' end of different substrate fragments, respectively, the 5' end is a phosphate, and the 3' end is a hydroxyl; using RNA ligase 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 Lumasiran; the RNA ligase includes RNA ligase family 1 and\or RNA ligase family 2 RNA ligase; preferably, the RNA ligase of RNA ligase family 1 is selected from: having SEQ ID NO: 3 and/or SEQ ID NO: 5 as shown in one or more RNA ligases; RNA ligases of RNA ligase family 2 are selected from: RNA ligases having amino acid sequences shown in SEQ ID NO: 1 and/or SEQ ID NO: 2; or RNA ligases having more than 70% identity with any RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 5, 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.

In the above preparation method, the sense strand substrate fragment is a nucleotide sequence that can form two or more sense strands, that is, multiple nucleotide sequences of the sense strand substrate fragment 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 substrate fragments and they are not connected by a phosphodiester bond. Similarly, the antisense strand substrate fragment and the antisense strand have the above characteristics. Two or more sense strand substrate fragments or antisense strand substrate fragments are connected by a phosphodiester bond using RNA ligase to obtain the sense strand and antisense strand of Lumasiran.

In the above-mentioned preparation method, the sense strand substrate fragment and the antisense strand substrate fragment are mixed with RNA ligase to prepare Lumasiran. In this preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementary paired to form a nucleotide with a sticky end, and the double-stranded structure nucleotide with a sticky end further continues to combine with other substrates to eventually form a double-stranded nucleotide structure containing a nick, and RNA ligase can recognize such a nick and connect the nick with a phosphodiester bond, thereby preparing the target product Lumasiran; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and mixed with RNA ligase to obtain Lumasiran.

In the above preparation method, it is preferred to first mix the sense strand substrate fragment and the antisense strand substrate fragment for annealing, so that the sense strand substrate fragment and the antisense strand substrate fragment can form a double-stranded RNA structure through base complementary pairing, and there are nicks between different substrate fragments in the double-stranded RNA structure. Then, the annealed reaction system is mixed with RNA ligase, and 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 Lumasiran with a complete double-stranded structure.

In a preferred embodiment, 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.

SEQ ID NO: 23:

GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm.

SEQ ID NO: 24:

UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm.

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 "sAm", "sCm" and the like indicates thio modification of the 5' phosphate of the ribonucleotide.

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

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

MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSE KRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.

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

MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVE EAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY.

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

MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAH ADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA.

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

MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATA HEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV.

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

MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDV KSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.

SEQ ID NO: 6 (Ligase 11, Thermococcus):

MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKV GELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.

SEQ ID NO: 7 (Ligase 20, Archaea):

MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGS GNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQV ASDYLRIMERAIRKYRIPWPDRELLVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYK NVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

SEQ ID NO: 8 (Ligase 32, bacteria):

MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDE LYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.

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.

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, their active sites, active pockets, active mechanisms, protein structures, etc. are likely to be the same as the proteins provided by the above sequences.

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).

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:

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

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

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

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

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.

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 Lumasiran. In the relevant experiments of this application, the inventors obtained the RNA ligase shown in SEQ ID NO: 1 to SEQ ID NO: 8 that can synthesize Lumasiran by screening from a large number of enzymes. The negative results, which accounted for a large proportion of the experiments, showed that most RNA ligases were difficult to catalyze the synthesis of Lumasiran, 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 Lumasiran synthesis.

In a preferred embodiment, the sense chain substrate fragment includes 2 or more substrate fragments, and the antisense chain substrate fragment includes 2 or more substrate fragments; preferably, the length of the sense chain substrate fragment is 5-14nt, more preferably 8-12nt; preferably, the length of the antisense chain substrate fragment is 4-16nt, more preferably 7-12nt.

In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each include 2 strands, the sense strand substrate fragment includes a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment includes a first antisense strand substrate fragment and a second antisense strand substrate fragment.

In a preferred embodiment, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment 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.

Preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 14. Preferably, the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 15.

Lumasiran can be prepared using the above preparation method and the substrates shown in SEQ ID NO: 9-SEQ ID NO: 12 or SEQ ID NO: 13-SEQ ID NO: 16. However, it should be noted that the selection of substrates is not limited to the above substrates. 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 Lumasiran 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 Lumasiran. The number of sense chain substrates or antisense chain substrates includes but is not limited to 2, 3, 4 or even more.

SEQ ID NO: 9: GmsAmsCmUmUmUmCfAmUfCfCfUmGmGm.

SEQ ID NO: 10: AmAmAmUmAmUmAm.

SEQ ID NO: 11: AfAmAmGmUmCmsCmsAm.

SEQ ID NO: 12: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGm.

SEQ ID NO: 13: GmsAmsCmUmUmUmCfAmUf.

SEQ ID NO: 14: CfCfUmGmGmAmAmAmUmAmUmAm.

SEQ ID NO: 15: AmAmGmUmCmsCmsAm.

SEQ ID NO: 16: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAf.

In a preferred embodiment, the 3' end of the first sense chain substrate fragment and the 5' end of the second sense chain substrate fragment are connected under the catalysis of RNA ligase to form a sense chain; the 3' end of the first antisense chain substrate fragment and the 5' end of the second antisense chain substrate fragment 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 fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense chain substrate fragment is a phosphate group, and the 3' end is an L96 group; the 5' end of the first antisense chain substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second antisense chain substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

In a preferred embodiment, the preparation method comprises: mixing a first sense chain substrate fragment, a second sense chain substrate fragment, a first antisense chain substrate fragment and a second antisense chain substrate fragment, using RNA ligase to catalyze the connection of the first sense chain substrate fragment and the second sense chain substrate fragment to form a sense chain, catalyzing the connection of the first antisense chain substrate fragment and the second antisense chain substrate fragment to form an antisense chain, and forming Lumasiran through base complementary pairing of the sense chain and the antisense chain.

In a preferred embodiment, the sense chain substrate fragments and the antisense chain substrate fragments each include 3 fragments, the sense chain substrate fragments include a first sense chain substrate fragment, a second sense chain substrate fragment and a third sense chain substrate fragment; the antisense chain substrate fragments include a first antisense chain substrate fragment, a second antisense chain substrate fragment and a third antisense chain substrate fragment; preferably, the nucleotide sequence of the first sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; the nucleotide sequence of the second sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; the nucleotide sequence of the third sense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; preferably, the nucleotide sequence of the first antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 22; the nucleotide sequence of the second antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of the third antisense chain substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20.

SEQ ID NO: 17: GmsAmsCmUmUm.

SEQ ID NO: 18: UmCfAmUfCfCfUmGm.

SEQ ID NO: 19: GmAmAmAmUmAmUmAm.

SEQ ID NO: 20: UmCmsCmsAm.

SEQ ID NO: 21: AmUfGmAfAmAmGm.

SEQ ID NO: 22: UmsAfsUmAmUmUfUmCfCfAmGmGm.

In a preferred embodiment, the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM; preferably, the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and RNA ligase also includes ATP, Tris-HCl, MgCl ₂ 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.

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.

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

Example 1

Substrate fragments 1 to 4 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 substrate fragments 1 to 4 are shown in Table 1, and the length unit is nt. After the substrate fragment mixture is annealed, an RNA fragment mixture is obtained. The reaction system was set to 10 μL, and the reaction system included 100 μM RNA fragment mixture, 50 mM Tris-HCl, 10 eq ATP, 100 eq MgCl ₂ , 10 eq DTT (1 eq = 100 μM), and RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase42, Ligase 11, Ligase20 and Ligase32 were 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 schematic diagram of the enzyme-catalyzed ligation reaction is shown in FIG1 .

The products catalyzed by RNA ligase Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase11, Ligase20 and Ligase32 were detected by Urea-PAGE. The electrophoresis results of the products catalyzed by RNA ligase Ligase25, Ligase26, Ligase31 and Ligase41 are shown in Figure 2. In Figure 2, 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 26, lane 3 represents the reaction system of Ligase 31, and lane 4 represents the reaction system of Ligase 41. 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.

Four single-stranded RNA fragments were prepared using a solid phase synthesis method.

Table 1

.

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 the sequences such as "sAm", "sGm" represents thio modification of the 5' phosphate of the ribonucleotide, L96 represents that the 3' end of the positive chain is modified with the L96 group, and the structure is shown in Figure 3, wherein the wavy line represents the base connected to the L96 group.

Substrate 1 has 2' methoxy modifications at ribonucleotides 1, 2, 3, 4, 5, 6, 8, 12, 13 and 14, and 2' fluorine modifications at ribonucleotides 7, 9, 10 and 11. The 5' phosphates on ribonucleotides 2 and 3 have thio modifications.

The ribonucleotides at positions 1-7 of substrate 2 have 2' methoxy modifications.

The ribonucleotides at positions 1, 2, 3, 4, 5, 6 and 7 of substrate 3 have 2' methoxy modifications, and the ribonucleotide at position 8 has a 2' fluorine modification. The 5' phosphates on the ribonucleotides at positions 7 and 8 have thio modifications.

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

The sense chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the antisense chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

Table 2

.

In expressing the product yield, N.D. means that no product formation was detected, “++” means the yield is 25-50% (excluding the endpoint value of 50%), “+++” means the yield is 50-75%, and “++++” means the yield is >75%.

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).

Example 2

Substrate fragments 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, 100 eq MgCl ₂ , 10 eq DTT (1 eq = 800 μM), and the RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41 and Ligase 42 with high 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 hours. After the reaction, the protein was inactivated by heating at 80°C for 5 minutes, and the precipitate was removed by centrifugation.

The product catalyzed by Ligase 25 was subjected to HPLC and LC-MS detection, as shown in FIG4 . 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 .

Table 3

.

In expressing the product yield, “++” indicates a yield of 50-70% (excluding the endpoint value of 70%), “+++” indicates a yield of 70-80%, and “++++” indicates >80%.

The molecular weight of the positive chain product identified by LC-MS was 8705.03, and the molecular weight of the antisense chain product was 7627.16. The theoretical values of the positive chain product were 8705.03±8, and the theoretical values of the antisense chain product were 7627.16±8, indicating that Lumasiran was generated by Ligase 25 ligation. The LC-MS detection results are shown in Figure 5.

Example 3

Substrates 1 to 4 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. After the substrate mixture was annealed, a mixture of RNA fragments of substrates 1 to 4 was obtained. The reaction system was set to 10 mL, and the reaction system included 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl ₂ , 1.25 eq DTT (1 eq = 800 μM), and 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 heated at 50 ° C for 15 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, desalted by membrane package (molecular weight cutoff 1 kDa), and the product was lyophilized. After calculation, the yield was 87.53% and the purity was 96.09%.

Example 4

Add substrates 5 to 8 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5 mM. The sequences of substrates 5 to 8 are shown in Table 4, and the length unit is nt. After the substrate mixture is annealed, the RNA fragment mixture of substrates 9 to 14 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 MgCl ₂ , 1.25 eq DTT (1 eq = 800 μM); add RNA ligase Ligase 8 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 inactivated by 80°C for 5 min to inactivate the ligase, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The enzyme activity was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the yield was "+++" and the target peak accounted for 80.6% in the sample.

Four single-stranded RNA fragments were prepared using a solid phase synthesis method.

Table 4

.

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 in sequences such as "sAm", "sGm" represents thio modification of 5' phosphate of the ribonucleotide.

The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 5 have 2' methoxy modifications, the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modifications, and the ribonucleotides at positions 7 and 9 have 2' fluoro modifications.

The ribonucleotides at positions 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of substrate 6 have 2' methoxy modifications, and the ribonucleotides at positions 1 and 2 have 2' fluorine modifications.

The ribonucleotides at positions 1, 2, 3, 4, 5, 6 and 7 of substrate 7 have 2' methoxy modifications, the ribonucleotides at positions 6 and 7 have 2' fluorine modifications, and the 5' phosphates of the ribonucleotides at positions 6 and 7 have 5' thio modifications.

The ribonucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13 and 15 of substrate 8 have 2' methoxy modifications, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 have 2' fluorine modifications, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modifications.

The sense chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the antisense chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

Example 5

Add substrates 9 to 14 in an equimolar ratio to a clean reagent bottle, mix well to obtain a substrate mixture, and the concentration of each substrate in the substrate mixture is 2.5 mM. The sequences of substrates 9 to 14 are shown in Table 5, and the length unit is nt. After the substrate mixture is annealed, a mixture of RNA fragments of substrates 9-14 is obtained. The reaction system is set to 10 mL. The reaction system includes 800 μM RNA fragment mixture, 50 mM Tris-HCl, 4 eq ATP, 12.5 eq MgCl ₂ , 1.25 eq DTT (1 eq = 800 μM), and RNA ligase Ligase 8 is added 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 inactivated by 80°C for 5 min to inactivate the ligase, and the precipitate was removed by centrifugation at 12000 rpm. The product catalyzed by Ligase 25 was detected by HPLC. The enzyme activity was measured by the rough estimated proportion of the product peak in the HPLC data of the reaction system sample. The results showed that the yield was +++, and the proportion of the target peak in the sample was 76.1%.

Six single-stranded RNA fragments were prepared using solid phase synthesis method.

Table 5

.

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 in sequences such as "sAm", "sGm" represents thio modification of 5' phosphate of the ribonucleotide.

The ribonucleotides at positions 1, 2, 3, 4 and 5 of substrate 9 have 2' methoxy modifications, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modifications.

The ribonucleotides at positions 1, 3, 7 and 8 of substrate 10 have 2' methoxy modifications, and the ribonucleotides at positions 2, 4, 5 and 6 have 2' fluorine modifications.

The ribonucleotides at positions 1 to 8 of substrate 11 have 2’ methoxy modifications.

The ribonucleotides at positions 1, 2, 3 and 4 of substrate 12 have 2' methoxy modifications, and the 5' phosphates of the ribonucleotides at positions 3 and 4 have 5' thio modifications.

The ribonucleotides at positions 1, 3, 5, 6 and 7 of substrate 13 have 2' methoxy modifications, and the ribonucleotides at positions 2 and 4 have 2' fluorine modifications.

The ribonucleotides at positions 1, 3, 4, 5, 7, 8, 10, 11 and 12 of substrate 14 have 2' methoxy modification, the ribonucleotides at positions 2, 6, 8 and 9 have 2' fluorine modification, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modification.

The sense chain of the prepared Lumasiran is GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23), and the antisense chain is UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).

Comparative Example 1

The average yield of full-length Lumasiran product synthesized using solid phase synthesis was 29.6%, and the total proportion of N+1 and N-1 impurities was 1.45%.

The yield of Lumasiran product prepared by the enzyme-linked method of the present invention is 71.91%, and the yield of the solid phase synthesis substrate is 42.2% of the yield of the whole process. The overall yield is 30.35%, which is higher than the average yield of the product obtained by the solid phase synthesis method. The total proportion of N+1 and N-1 impurities is 0.41%, which is lower than the proportion of such impurities in the process of synthesizing Lumasiran by solid phase synthesis.

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 Lumasiran are catalyzed by RNA ligase to form Lumasiran, 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, simple preparation process, mild reaction conditions, low amount of organic reagents, reduced production costs, and is easy to achieve scale-up industrial production.

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 (14)

1. A method of preparing Lumasiran, wherein Lumasiran is a double-stranded siRNA consisting of complementarily paired sense and antisense strands, comprising:

Mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment is capable of constituting the sense strand and the antisense strand substrate fragment is capable of constituting the antisense strand;

The sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, and the head base and the tail base of the sense strand substrate fragment and the antisense strand substrate fragment are not connected with each other to form a double-stranded nucleotide structure containing a nick;

Connecting bases at two ends of the nick with a phosphodiester bond by using the RNA ligase to form Lumasiran;

the bases at the two ends of the notch are respectively the 5 'end and the 3' end of different substrate fragments, the 5 'end is phosphate radical, and the 3' end is hydroxyl radical;

connecting the phosphate at the 5 'end and the hydroxyl at the 3' end at the upstream and downstream of the nick by using the RNA ligase to form the phosphodiester bond, thereby obtaining Lumasiran;

the RNA ligase comprises RNA ligase of RNA ligase family 1 or RNA ligase family 2;

the RNA ligase of RNA ligase family 1 is selected from: has the sequence of SEQ ID NO:3 or SEQ ID NO:5, an RNA ligase having an amino acid sequence shown in SEQ ID NO. 5;

the RNA ligase of RNA ligase family 2 comprises: selected from SEQ ID NOs: 1. SEQ ID NO:2 or SEQ ID NO:4, and one or more of the RNA ligases of the amino acid sequence shown in fig. 4;

Or the RNA ligase is a sequence that hybridizes to SEQ ID NO: 1-SEQ ID NO:5, and has an enzyme that catalyzes the formation of said phosphodiester bond.

2. The method of claim 1, wherein the sense strand has a nucleotide sequence of SEQ ID NO:23, and the nucleotide sequence of the antisense strand is set forth in SEQ ID NO:24, and a nucleotide sequence shown in seq id no.

3. The method of claim 1, wherein the sense strand substrate fragment comprises 2 or more and the antisense strand substrate fragment comprises 2 or more;

the length of the sense strand substrate fragment is 5-14nt;

the antisense strand substrate fragment is 4-16nt in length.

4. The method of preparing according to claim 3, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 2, the sense strand substrate fragment comprises a first sense strand substrate fragment and a second sense strand substrate fragment, and the antisense strand substrate fragment comprises a first antisense strand substrate fragment and a second antisense strand substrate fragment.

5. The method of claim 4, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:9, the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:10 is shown in the figure;

The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:12, the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:11, are shown.

6. The method of claim 4, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:13, the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:14 sequence;

The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:16, the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 15.

7. The method of claim 4, wherein the 3 'end of the first sense strand substrate fragment is ligated to the 5' end of the second sense strand substrate fragment under the catalysis of the RNA ligase to form the sense strand; the 3 'end of the first antisense strand substrate fragment is ligated to the 5' end of the second antisense strand substrate fragment under the catalysis of the RNA ligase to form the antisense strand.

8. The method of claim 4, wherein the first sense strand substrate fragment has a hydroxyl group at the 5 'end and a hydroxyl group at the 3' end; the 5 'end of the second sense strand substrate fragment is a phosphate group, and the 3' end of the second sense strand substrate fragment is an L96 group;

The 5 'end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end of the first antisense strand substrate fragment is a hydroxyl group; the 5 'end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.

9. The method according to claim 4, wherein when the sense strand substrate fragment and the antisense strand substrate fragment each comprise 2, the method comprises:

Mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment and the second antisense strand substrate fragment, wherein the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form the sense strand under the catalysis of the RNA ligase, the first antisense strand substrate fragment and the second antisense strand substrate fragment are connected to form the antisense strand, and the sense strand and the antisense strand are formed into the Lumasiran through base complementary pairing.

10. The method according to claim 3, wherein the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3,

The sense strand substrate fragments comprise a first sense strand substrate fragment, a second sense strand substrate fragment, and a third sense strand substrate fragment;

The antisense strand substrate fragments include a first antisense strand substrate fragment, a second antisense strand substrate fragment, and a third antisense strand substrate fragment.

11. The method of claim 10, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:17, a nucleotide sequence shown in seq id no;

The nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:18, a nucleotide sequence shown in seq id no;

the nucleotide sequence of the third sense strand substrate fragment is SEQ ID NO:19, a nucleotide sequence shown in seq id no;

the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:22, a nucleotide sequence shown in seq id no;

The nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:21, a nucleotide sequence shown in seq id no;

The nucleotide sequence of the third antisense strand substrate fragment is SEQ ID NO:20, and a nucleotide sequence shown in seq id no.

12. The method of manufacturing according to claim 11, characterized in that the method of manufacturing comprises: mixing the first sense strand substrate fragment, the second sense strand substrate fragment, the third sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, the third antisense strand substrate fragment, and the RNA ligase;

Under the catalytic action of RNA ligase, the first sense strand substrate fragment, the second sense strand substrate fragment and the third sense strand substrate fragment are connected to form the sense strand, the first antisense strand substrate fragment, the second antisense strand substrate fragment and the third antisense strand substrate fragment are connected to form the antisense strand, and the sense strand and the antisense strand are formed into the Lumasiran through base complementary pairing.

13. The method of claim 1, wherein the sense strand substrate fragment and the antisense strand substrate fragment are each independently selected from 0.1 to 4.5mM;

The reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase further comprises ATP, tris-HCl, mgCl ₂ and DTT.

14. The preparation method according to claim 1, wherein the reaction temperature of the preparation method is 10 to 40 ℃;

the reaction time of the preparation method is 2-48h.