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CN118460656B - A preparation method of Lumasiran - Google Patents

A preparation method of Lumasiran Download PDF

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CN118460656B
CN118460656B CN202410921759.7A CN202410921759A CN118460656B CN 118460656 B CN118460656 B CN 118460656B CN 202410921759 A CN202410921759 A CN 202410921759A CN 118460656 B CN118460656 B CN 118460656B
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substrate fragment
seq
sense strand
strand substrate
antisense strand
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CN118460656A (en
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洪浩
詹姆斯·盖吉
张娜
焦学成
王磊
冯骏晨
张冉冉
胡守俊
蒋相军
陈仁芳
贾旭
李少贺
王吉忠
严思堂
金星
刘永贤
王思源
傅绪飞
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Tianjin Kailaiying Biotechnology Co ltd
Asymchem Laboratories Tianjin Co Ltd
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Tianjin Kailaiying Biotechnology Co ltd
Asymchem Laboratories Tianjin Co Ltd
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Abstract

The invention provides a preparation method of Lumasiran. The Lumasiran is double-stranded RNA formed by complementary pairing of a sense strand and an antisense strand, the method comprises the steps of mixing a sense strand substrate fragment, an antisense strand substrate fragment and RNA ligase, wherein the sense strand substrate fragment can form the sense strand, the antisense strand substrate fragment can form the antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, the head base and the tail base of the sense strand substrate fragment and the tail base of the antisense strand substrate fragment are not connected with each other to form a double-stranded nucleotide structure containing nicks, and the bases at the two ends of the nicks are connected through phosphodiester bonds by using the RNA ligase to form Lumasiran. Can solve the problem of lower purity of Lumasiran prepared in the prior art, and is suitable for the technical field of medicine biosynthesis.

Description

Preparation method of Lumasiran
Technical Field
The invention relates to the field of medicine biosynthesis, in particular to a preparation method of Lumasiran.
Background
In the prior art, diseases caused by gene expression and mutation cannot be cured by traditional small molecule drugs, and the diseases can only be treated by intervention of gene expression. The siRNA is double-stranded RNA oligonucleotide with the length of 19-25nt, can specifically bind to mRNA of a target gene, and can achieve the effect of blocking the expression of the target gene by preventing the translation process, namely, causing gene silencing by mediating RNA interference. The research and development of siRNA drugs bring great development prospects to gene therapy, and are widely applied to the field of disease treatment. The siRNA acts on mRNA, and the patentable target point is obviously larger than the traditional small molecular medicine with the acting site being protein, and can act on a new target point by changing the sequence. Intervention in gene expression.
Lumasiran is a gene targeting therapeutic siRNA double-stranded RNA drug developed by Alnylam company for treating primary hyperoxalic acid urea type I (primary hyperoxaluria I, PH I) and approved by FDA in 2020. Lumasiran is the first drug approved for treatment of PH1, and phase 3 clinical data shows that Lumasiran treatment significantly reduces oxalic acid production in the liver, which can facilitate resolution of the underlying pathophysiological problems of PH 1.
The Lumasiran synthesis method mainly uses chemical solid phase synthesis method to prepare, in the synthesis method, solid phase carrier is used, cyclic synthesis is carried out by phosphoramidite triester method, after the synthesis cycle is finished, lumasiran chain is excised from the solid phase carrier by ammonolysis, and then the target product is obtained through purification. The yield of the product synthesis in the preparation method is reduced along with the increase of the synthetic chain length, and impurities generated in the synthesis process, such as impurities (N+1 impurities) with one nucleotide more than a target sequence or impurities (N-1 impurities) with one nucleotide less than the target sequence, are also increased along with the increase of the synthetic chain length, are not easy to remove, and lead to complex purification process and low efficiency. With Lumasiran increasingly being widely used in the treatment of primary hyperbilirubinemia, the synthesis scale is limited by the limitations of synthesis equipment, the cost is high, and the large-scale production is difficult, so that a more efficient Lumasiran synthesis method needs to be developed.
Disclosure of Invention
The invention mainly aims to provide a preparation method of 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, there is provided a method for preparing Lumasiran, which Lumasiran is a double-stranded siRNA consisting of a sense strand and an antisense strand complementarily paired, comprising:
mixing a sense strand substrate fragment, an antisense strand substrate fragment and RNA ligase, wherein the sense strand substrate fragment can form a sense strand, the antisense strand substrate fragment can form an antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are connected through hydrogen bonds formed by base complementation, NO end base of the sense strand substrate fragment and NO end base of the antisense strand substrate fragment are connected with each other to form a double-stranded nucleotide structure containing a notch, the bases at the two ends of the notch are connected through a phosphodiester bond by utilizing 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 a phosphate radical, the 3' end is a hydroxyl radical, the phosphate diester bond is formed by utilizing the RNA ligase to connect the phosphate radical at the 5 'end and the 3' end at the upstream and downstream of the notch, and the RNA ligase of Lumasiran is obtained, preferably, the RNA ligase of the RNA ligase family 1 comprises RNA ligase family 1 and RNA ligase or RNA ligase of the RNA ligase family 2, the RNA ligase of the RNA ligase family 1 comprises RNA ligase having the nucleotide sequence shown in SEQ ID3 and SEQ ID2 or the RNA ligase having the nucleotide sequence shown in SEQ ID2 or the sequence shown in SEQ ID 1-NO. 1 has the activity.
Further, the nucleotide sequence of the sense strand is the nucleotide sequence shown as SEQ ID NO. 23, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown as SEQ ID NO. 24.
Further, the sense strand substrate fragment comprises 2 or more, the antisense strand substrate fragment comprises 2 or more, preferably the sense strand substrate fragment has a length of 5 to 14nt, more preferably 8 to 12nt, and preferably the antisense strand substrate fragment has a length of 4 to 16nt, more preferably 7 to 12nt.
Further, 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, the antisense strand substrate fragment comprises a first antisense strand substrate fragment and a second antisense strand substrate fragment, preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 9, the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 10, the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 12, the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 11, preferably, the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 13, the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 14, the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 16, and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 15.
Further, the 3 'end of the first sense strand substrate fragment is connected with the 5' end of the second sense strand substrate fragment under the catalysis of RNA ligase to form a sense strand, the 3 'end of the first antisense strand substrate fragment is connected with the 5' end of the second antisense strand substrate fragment under the catalysis of RNA ligase to form an antisense strand, preferably, the 5 'end of the first sense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, the 5 'end of the second sense strand substrate fragment is a phosphate group, the 3' end is an L96 group, preferably, the 5 'end of the first antisense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, and the 5 'end of the second antisense strand substrate fragment is a phosphate group, and the 3' end is a hydroxyl group.
Further, when the sense strand substrate fragment and the antisense strand substrate fragment each comprise 2, the preparation method comprises mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a first antisense strand substrate fragment and a second antisense strand substrate fragment, and under the catalysis of RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are connected to form a sense strand, the first antisense strand substrate fragment and the second antisense strand substrate fragment are connected to form an antisense strand, and the sense strand and the antisense strand are formed Lumasiran through base complementary pairing, preferably annealing the sense strand substrate fragment and the antisense strand substrate fragment, and mixing with the RNA ligase to obtain Lumasiran.
Further, the sense strand substrate fragment and the antisense strand substrate fragment comprise 3 pieces, the sense strand substrate fragment comprises a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment, the antisense strand substrate fragment comprises a first antisense strand substrate fragment, a second antisense strand substrate fragment and a third sense strand substrate fragment, preferably, the nucleotide sequence of the first sense strand substrate fragment is a nucleotide sequence shown as SEQ ID NO. 17, the nucleotide sequence of the second sense strand substrate fragment is a nucleotide sequence shown as SEQ ID NO. 18, the nucleotide sequence of the third sense strand substrate fragment is a nucleotide sequence shown as SEQ ID NO. 19, preferably, the nucleotide sequence of the first antisense strand substrate fragment is a nucleotide sequence shown as SEQ ID NO. 22, the nucleotide sequence of the third sense strand substrate fragment is a nucleotide sequence shown as SEQ ID NO. 20, preferably, the preparation method comprises the steps of forming a base pair of the first sense strand substrate, the second sense strand substrate fragment, the third sense strand substrate fragment and the third sense strand substrate fragment through the reaction, and the third sense strand substrate fragment are formed by the coupling, and the antisense strand substrate fragments.
Further, the concentration of the sense strand substrate fragment and the antisense strand substrate fragment is independently selected from 0.1-4.5mM, preferably, 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, preferably, the reaction temperature of the preparation method is 10-40 ℃, more preferably 15-30 ℃, and the reaction time of the preparation method is 2-48h, more preferably 12-24h.
By applying the technical scheme of the application, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form Lumasiran sense strands, and the antisense strand substrate fragments are connected to form Lumasiran antisense strands, so that the siRNA medicine is prepared by utilizing a biosynthesis mode. Compared with the method for preparing Lumasiran by chemical synthesis, the preparation method provided by the application has the advantages of high purity of the obtained product, few generated impurities, simple preparation process, mild reaction conditions, low dosage of organic reagent, low production cost and convenience for realizing large-scale industrial production.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a schematic of an enzyme-catalyzed ligation reaction according to example 1 of the invention.
FIG. 2 shows a graph of the electrophoresis results of catalytic products of RNA Ligase 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 of HPLC detection of the RNA Ligase Ligase 25 catalytic product according to example 2 of the present invention.
FIG. 5 shows a graph of LC-MS detection results of the catalytic product of RNA Ligase 25 according to example 2 of the present invention.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The present application will be described in detail with reference to examples.
Term interpretation:
n+1 impurities nucleic acid impurities having a single nucleotide attached in addition to the target synthetic sequence.
N-1 impurities nucleic acid impurities having a single nucleotide deletion compared to the target synthetic sequence.
As mentioned in the background art, the preparation of Lumasiran in the prior art is carried out by adopting a chemical synthesis preparation method, so that the process is complex, the cost is high, and the generated N+1 and N-1 impurities are more, thereby influencing the subsequent purification of the product. In the present application, the inventors tried to develop a preparation method Lumasiran for preparing Lumasiran by enzyme-catalyzed synthesis, and thus proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the application, there is provided a method of preparing Lumasiran, lumasiran is double stranded RNA consisting of complementarily paired sense and antisense strands; the preparation method comprises mixing sense strand substrate fragment, antisense strand substrate fragment and RNA ligase, wherein the sense strand substrate fragment can form sense strand and the antisense strand substrate fragment can form 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 the nicks; the bases at two ends of the notch are connected by phosphodiester bonds to form Lumasiran, the bases at two ends of the notch are respectively the 5 'end and the 3' end of different substrate fragments, the 5 'end is phosphate, the 3' end is hydroxyl, the phosphate at the 5 'end and the hydroxyl at the 3' end at the upstream and downstream of the notch are connected by using RNA ligase to form phosphodiester bonds to form Lumasiran, the RNA ligase comprises RNA ligase family 1 and/or RNA ligase family 2, preferably the RNA ligase of the RNA ligase family 1 is selected from one or more of the RNA ligases with the amino acid sequences shown as SEQ ID NO:3 and/or SEQ ID NO:5, the RNA ligase of the RNA ligase family 2 is selected from the RNA ligases with the amino acid sequences shown as SEQ ID NO:1 and/or SEQ ID NO:2, or the RNA ligases with more than 70% identity with any one of the RNA ligases shown as SEQ ID NO: 1-5, including but not limited to, 80%, 89%, 99%, 85%, 90% or more than one another 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, even 99.9% or more) and has an enzyme that catalyzes the formation of a phosphodiester bond.
In the above preparation method, the sense strand substrate fragment is a nucleotide sequence of 2 or more that can constitute the sense strand, i.e., a plurality of nucleotide sequences of the sense strand substrate fragment can be spliced to constitute the same sequence as the sense strand sequence, and is different from the sense strand in that there is a nick between the sense strand substrate fragments, which are not linked by a phosphodiester bond. Similarly, the antisense strand substrate fragment and antisense strand have the features described above. The sense strand and the antisense strand of Lumasiran are obtained by connecting 2 or more sense strand substrate fragments or antisense strand substrate fragments with each other via phosphodiester bonds using RNA ligase.
In the above preparation method, lumasiran can be prepared by mixing the sense strand substrate fragment and the antisense strand substrate fragment with RNA ligase. In this preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementarily paired to form a nucleotide with a cohesive end, the nucleotide with a double-stranded structure with a cohesive end is further combined with other substrates to finally form a double-stranded nucleotide structure with an nick, the nick can be identified by RNA ligase, and the nick is connected by a phosphodiester bond, so that a target product Lumasiran is obtained, preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain Lumasiran.
In the above preparation method, it is preferable that the sense strand substrate fragment and the antisense strand substrate fragment are mixed and annealed, and the sense strand substrate fragment and the antisense strand substrate fragment can be formed into a double-stranded RNA structure by base complementary pairing, and a notch between different substrate fragments exists in the double-stranded RNA structure. And mixing the annealed reaction system with RNA ligase, connecting phosphate groups and hydroxyl groups at two sides of the notch by using the RNA ligase through a phosphodiester bond, and repairing the notch, thereby obtaining a target product Lumasiran with a complete double-chain structure.
In a preferred embodiment, the nucleotide sequence of the sense strand is the nucleotide sequence shown as SEQ ID NO. 23 and the nucleotide sequence of the antisense strand is the nucleotide sequence shown as SEQ ID NO. 24.
SEQ ID NO:23:
GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm。
SEQ ID NO:24:
UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm。
In the present application, A, C, G or m after U represents 2' methoxy modification of the ribonucleotide, f represents modification of 2' fluoro of the ribonucleotide, s before the ribonucleotide in the writing methods of "sAm", "sCm" and the like represents thio modification of 5' phosphate of the ribonucleotide.
The RNA ligase can recognize a double-chain structure with a notch formed by complementary pairing of substrate fragments, so that the formation of a phosphodiester bond between a phosphate group and a hydroxyl group is catalyzed.
SEQ ID NO:1(Ligase-25,Vibrio phage NT-1):
MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.
SEQ ID NO:2(Ligase 26,Escherichia phage AR1):
MQELFNNLMELCKDSQRKFFYSDDVSASGRTYRIFSYNYASYSDWLLPDALECRGIMFEMDGEKPVRIASRPMEKFFNLNENPFTMNIDLNDVDYILTKEDGSLVSTYLDGDEILFKSKGSIKSEQALMANGILMNINHHQLRDRLKELAEDGFTANFEFVAPTNRIVLAYQEMKIILLNIRENETGEYISYDDIYKDAALRPYLVERYEIDSPKWVEEAKNAENIEGYVAVMKDGSHFKIKSDWYVSLHSTKSSLDNPEKLFKTIIDGASDDLKAMYADDEYSYRKIEAFETTYLKYLDRALFLVLDCHNKHCGKDRKTYAMEAQGVAKGAGMDHLFGIIMSLYQGYDSQEKVMCEIEQNFLKNYKKFIPEGY.
SEQ ID NO:3(Ligase 31,Vibrio phage VH12019):
MTTQELYNHLMTLTDDAEGKFFFADHISPLGEKLRVFSYHIASYSDWLLPGALEARGIMFQLDEQDKMVRIVSRPMEKFFNLNENPFTMDLDLTTTVQLMDKADGSLISTYLTGENFALKSKTSIFSEQAVAANRYIKLPENRDLWEFCDDLTQAGCTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPLPALMRVKKWLVDEYDPETAHADDFVEKLRATKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNNNHDDLYALFADDKPTIDRIREFDSHVSKTVSASFHAVSQFYVKNRHMSRKDYAIAGQKTLKPWEFGVAMIAYQNQTVEGVYEALVGAYLKRPELLIPEKYLNEA.
SEQ ID NO:4(Ligase 41,Vibrio phage VH7D):
MNVQELYKNLMSLADDAEGKFFFADHLSPLGEKFRVFSYHIASYSDWLLPGALEARGIMFQLDDNDEMIRIVSRPMEKFFNLNENPFTMELDLTTTVQLMDKADGSLISTYLSGENFALKSKTSIFSEQAVAANRYIKKPENRDLWEFCDDCTQAGLTVNMEWCAPNNRIVLEYPEAKLVILNIRDNETGDYVSFDDIPQSALMRVKQWLVDEYDPATAHEPDFVEKLRDTKGIEGMILRLANGQSVKIKTQWYVDLHSQKDSVNVPKKLVTTILNGNHDDLYALFADDKPTIERIREFDSHVTKTLTNSFNAVRQFYARNRHLARKDYAIAGQKVLKPWEFGVAMIAYQKQTVEGVYESLVTAYLKRPELAIPEKYLNGV.
SEQ ID NO:5(Ligase 42,Escherichia phage JN02):
MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.
SEQ ID NO:6(Ligase 11,Thermococcus):
MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.
SEQ ID NO:7(Ligase 20,Archaea):
MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.
SEQ ID NO:8(Ligase 32,bacteria):
MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.
Identity (Identity) in the present application refers to "Identity" between amino acid sequences or nucleotide sequences, i.e. the sum of the ratios of amino acid residues or nucleotides of the same kind in an amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using the alignment programs BLAST (Basic Local ALIGNMENT SEARCH Tool), FASTA, etc.
Proteins that are 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., 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) identical and have the same function have the same active site, active pocket, active mechanism, protein structure, etc. as those provided by the above sequences with a high probability.
As used herein, amino acid residues are abbreviated as 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).
The rules of substitution, replacement, etc., generally, which amino acids are similar in nature, and the effect after replacement is similar. For example, in the above homologous proteins, conservative amino acid substitutions may occur. "conservative amino acid substitutions" include, but are not limited to:
The hydrophobic amino acid (Ala, cys, gly, pro, met, val, ile, leu) is substituted with other hydrophobic amino acids;
the hydrophobic amino acid (Phe, tyr, trp) with a coarse side chain is replaced by other hydrophobic amino acids with a coarse side chain;
the positively charged amino acid (Arg, his, lys) of the side chain is replaced by other positively charged amino acids of the side chain;
The amino acid (Ser, thr, asn, gln) with a side chain having a polarity that is uncharged is substituted with other amino acids with a side chain having a polarity that is uncharged.
The amino acids may also be conservatively substituted by those skilled in the art according to amino acid substitution rules well known to those skilled in the art as the "blosum62 scoring matrix" in the art.
In the application, the phosphodiester bond is formed by the phosphate group and the hydroxyl group of the substrate only through the RNA ligase shown in SEQ ID NO. 1-SEQ ID NO. 5 or the enzyme which has more than 70% of the same degree with any RNA ligase shown in SEQ ID NO. 1-SEQ ID NO. 5, so that the product Lumasiran is obtained. In the related experiments of the application, the inventors obtained RNA ligase shown in SEQ ID NO. 1-8 capable of synthesizing Lumasiran by screening a large number of enzymes. The negative results with the extremely large ratio in the experiment show that most RNA ligases are difficult to catalyze Lumasiran to synthesize, including but not limited to RNA ligases shown in SEQ ID NO. 6-SEQ ID NO. 8, and the RNA ligases without activity of catalyzing Lumasiran synthesis are only shown by taking SEQ ID NO. 6-SEQ ID NO. 8 as examples in the specification.
In a preferred embodiment, the sense strand substrate fragment comprises 2 or more substrate fragments and the antisense strand substrate fragment comprises 2 or more substrate fragments, preferably the sense strand substrate fragment has a length of 5 to 14nt, more preferably 8 to 12nt, preferably the antisense strand substrate fragment has a length of 4 to 16nt, more preferably 7 to 12nt.
In a preferred embodiment, 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.
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 as SEQ ID NO. 12 and the nucleotide sequence of the second antisense strand substrate is the nucleotide sequence shown as 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 as SEQ ID NO. 16 and the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO. 15.
Lumasiran can be prepared by the above preparation method and the substrate shown in SEQ ID NO 9-SEQ ID NO 12 or SEQ ID NO 13-SEQ ID NO 16. However, the selection of the substrate is not limited to the above substrates, and the substrates capable of being combined to form the sense strand and the antisense strand can be used in the above preparation method, which is applicable to the preparation of Lumasiran but not limited to the difference in the substrate ligation positions, and the above preparation has a good ligation effect for the ligation of the sense strand sequence and the antisense strand sequence of Lumasiran. The number of sense strand substrates or antisense strand 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 strand substrate fragment is linked to the 5' end of the second sense strand substrate fragment under the catalysis of RNA ligase to form a sense strand, the 3 'end of the first antisense strand substrate fragment is linked to the 5' end of the second antisense strand substrate fragment under the catalysis of RNA ligase to form an antisense strand, preferably, in the preparation method, the 5 'end of the first sense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, the 5 'end of the second sense strand substrate fragment is a phosphate group, the 3' end is an L96 group, the 5 'end of the first antisense strand substrate fragment is a hydroxyl group, the 3' end is a hydroxyl group, and the 5 'end of the second antisense strand substrate fragment is a phosphate group, the 3' end is a hydroxyl group.
In a preferred embodiment, the above preparation method comprises mixing a first sense strand substrate fragment, a second sense strand substrate fragment, a first antisense strand substrate fragment, and a second antisense strand substrate fragment, catalyzing the ligation of the first sense strand substrate fragment and the second sense strand substrate fragment to form a sense strand using an RNA ligase, catalyzing the ligation of the first antisense strand substrate fragment and the second antisense strand substrate fragment to form an antisense strand, and forming Lumasiran by base complementary pairing of the sense strand and the antisense strand.
In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3, the sense strand substrate fragment comprises a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment, the antisense strand substrate fragment comprises 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 as SEQ ID NO:17, the nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO:18, the nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO:19, preferably the nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO:22, the nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown as SEQ ID NO:21, and the nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown as 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 concentration of the sense strand substrate fragment and the antisense strand substrate fragment is each independently selected from 0.1 to 4.5mM, preferably, ATP, tris-HCl, mgCl 2 and DTT are further included in the reaction system formed by mixing the sense strand substrate fragment, the antisense strand substrate fragment and the RNA ligase, preferably, the reaction temperature of the preparation method is 10 to 40 ℃, more preferably 15 to 30 ℃, and the reaction time of the preparation method is 2 to 48 hours, more preferably 12 to 24 hours.
The concentration of the sense strand substrate fragment and the antisense strand substrate fragment is selected from the group consisting of, but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, and 4.5mM, the reaction temperature of the preparation method is 10, 15, 16, 20, 25, 30, 35, and 40 ℃, and the reaction time of the preparation method is 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, and 48 hours.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1
Adding the substrate fragments 1-4 into a clean reagent bottle in an equimolar ratio, and uniformly mixing to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture is 2.5mM. The sequences of the substrate fragments 1 to 4 are shown in Table 1, and the length units are nt. After annealing the substrate fragment mixture or obtaining an RNA fragment mixture. The reaction system was set to 10. Mu.L, and the reaction system contained 100. Mu.M of an RNA fragment mixture, 50mM Tris-HCl,10eq of ATP,100eq of MgCl 2, 10eq of DTT (1 eq=100. Mu.M), and RNA Ligase Ligase 25, ligase 26, ligase 31, ligase 41, ligase 42, ligase 11, ligase20 and Ligase32 each having a final concentration of 0.2mg/mL were added. The reaction was reacted at 16 ℃ for 16h. The resulting reaction system was subjected to enzyme inactivation of ligase at 80℃for 5min and centrifugation at 12000rpm to remove precipitate. The enzyme-catalyzed ligation reaction is schematically depicted in FIG. 1.
The products obtained by catalyzing RNA ligases Ligase25, ligase26, ligase31, ligase41, ligase 42, ligase 11, ligase20 and Ligase32 were subjected to Urea-PAGE detection, and the electrophoresis results of the products obtained by catalyzing RNA ligases Ligase25, ligase26, ligase31 and Ligase41 are shown in FIG. 2, wherein lane M in FIG. 2 represents RNA molecular standard (marker), lane 1 represents the reaction system of Ligase25, lane 2 represents the reaction system of Ligase26, lane 3 represents the reaction system of Ligase31, and lane 4 represents the reaction system of Ligase 41. The yields were estimated from the gray scale analysis of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.
The solid phase synthesis method was used to prepare 4 single stranded RNA fragments.
TABLE 1
。
Wherein, m after A, C, G or U represents 2 'methoxy modification of the ribonucleotide, f represents modification of 2' fluoro of the ribonucleotide, s in the sequences of 'sAm', 'sGm' and the like represents thio modification of 5 'phosphoric acid of the ribonucleotide, L96 represents modification of L96 group at 3' end of sense strand, the structure is shown in figure 3, wherein wavy line represents base connected with L96 group.
Ribonucleotides at positions 1,2,3,4, 5, 6, 8, 12, 13 and 14 of substrate 1 have 2 'methoxy modifications and ribonucleotides at positions 7, 9, 10 and 11 have 2' fluoro modifications. Having thio modifications of the 5' phosphates on ribonucleotides 2 and 3.
The ribonucleotides at positions 1-7 of substrate 2 have 2' methoxy modifications.
Ribonucleotides at positions 1, 2,3, 4,5, 6 and 7 of substrate 3 have 2 'methoxy modifications and ribonucleotides at position 8 have 2' fluoro modifications. The 5' phosphates at ribonucleotides 7 and 8 have thio modifications.
Ribonucleotides at positions 1,3, 4, 5, 6, 9, 11, 12, 13 and 15 of substrate 4 have 2 'methoxy modifications and ribonucleotides at positions 2, 7, 8, 10 and 14 have 2' fluoro modifications. Having thio modifications of the 5' phosphates on ribonucleotides 2 and 3.
The sense strand of Lumasiran obtained was GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23) and the antisense strand was UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).
TABLE 2
。
In the representation of the product yield, n.d. indicates that no product formation was detected, "++" indicates a yield of 25-50% (excluding the end point values of 50%), "++" indicates a yield of 50-75%, and "++ + ++" indicates a yield of >75%.
The yield calculation formula in this example is yield = product gray data/(product gray data + substrate gray data).
Example 2
Adding the substrate fragments 1-4 into a clean reagent bottle in an equimolar ratio, and uniformly mixing to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture is 2.5mM. Annealing the substrate mixture or obtaining the RNA fragment mixture of the substrates 1-4. The reaction system was set at 50. Mu.L, and the reaction system comprised 800. Mu.M RNA fragment mixture, 50mM Tris-HCl,4eq ATP,100eq MgCl 2, 10eq DTT (1 eq=800. Mu.M) and the catalytically higher RNA ligases Ligase 25, ligase 26, ligase 31, ligase 41 and Ligase 42 of example 1 were added at a final concentration of 0.2mg/mL, respectively. The reaction was reacted at 16 ℃ for 16h. And heating at 80 ℃ for 5min after the reaction is finished to inactivate proteins, and centrifuging to remove precipitates.
HPLC and LC-MS detection are carried out on the product obtained by catalyzing Ligase 25, the yield is calculated according to the statistical result of the target peak area in the HPLC result, and the result is shown in Table 3.
TABLE 3 Table 3
。
In the representation of the product yield, "++" means that the yield is 50 to 70% (excluding 70% of the end point value), "+++" means the yield is 70-80%, the yield is 70-80%.
LC-MS is used for identifying that the molecular weight of the sense strand product is 8705.03, the molecular weight of the antisense strand product is 7627.16, the theoretical value of the sense strand product is 8705.03 +/-8, and the theoretical value of the antisense strand product is 7627.16 +/-8, which shows that Lumasiran is generated by ligation of Ligase 25, and the detection result of the LC-MS is shown in FIG. 5.
Example 3
Adding the substrates 1-4 into a clean reagent bottle in an equimolar ratio, and uniformly mixing to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture is 2.5mM. Annealing the substrate mixture or obtaining the RNA fragment mixture of the substrates 1-4. The reaction system was set to 10mL, and the reaction system included 800. Mu.M RNA fragment mixture, 50mM Tris-HCl,4eq ATP,12.5eq MgCl 2, 1.25eq DTT (1 eq=800. Mu.M) and RNA Ligase Ligase 25 was added at a final concentration of 0.2 mg/mL. The reaction was reacted at 16 ℃ for 16h. The obtained reaction system is heated for 15min at 50 ℃ to inactivate proteins, the precipitate is removed by centrifugation at 12000rpm, a supernatant is purified by a Nano-Q column, the obtained product is subjected to gradient elution by NaCl, membrane-packed desalting treatment (molecular weight cut-off of 1 kDa) is carried out, the product is subjected to freeze-drying treatment, and the yield is 87.53% and the purity is 96.09% after calculation.
Example 4
Adding 5-8 substrates into a clean reagent bottle in an equimolar ratio, and uniformly mixing to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture is 2.5mM. The sequences of substrates 5 to 8 are shown in Table 4, and the length unit is nt. Annealing the substrate mixture or obtaining the RNA fragment mixture of the substrates 9-14. The reaction system was set at 10mL and consisted of 800. Mu.M RNA fragment mixture, 50mM Tris-HCl,4eq ATP,12.5eq MgCl 2, 1.25eq DTT (1 eq=800. Mu.M) and RNA Ligase Ligase 8 was added at a final concentration of 0.2 mg/mL. The reaction was reacted at 16 ℃ for 16h. The obtained reaction system is deactivated by Ligase at 80 ℃ for 5min, precipitates are removed by centrifugation at 12000rpm, HPLC detection is carried out on a product obtained by Ligase 25 catalysis, the enzyme activity is measured by roughly estimated proportion of a product peak in HPLC data of a reaction system sample, the result shows that the yield is "++", and the proportion of a target peak in the sample is 80.6%.
The solid phase synthesis method was used to prepare 4 single stranded RNA fragments.
TABLE 4 Table 4
。
Wherein, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents modification of 2' fluoro of the ribonucleotide, s in the sequences of ' sAm ', ' sGm ' and the like represents thio modification of 5' phosphate of the ribonucleotide.
Ribonucleotides at positions 1,2,3,4,5 and 6 of substrate 5 have 2 'methoxy modifications, 5' phosphate of ribonucleotides at positions 2 and 3 have 5 'thio modifications, and ribonucleotides at positions 7 and 9 have 2' fluoro modifications.
Ribonucleotides at positions 3,4, 5, 6, 7, 8, 9, 10, 11 and 12 of substrate 6 have 2 'methoxy modifications and ribonucleotides at positions 1 and 2 have 2' fluoro modifications.
Ribonucleotides at positions 1,2, 3, 4, 5, 6 and 7 of substrate 7 have 2 'methoxy modifications, ribonucleotides at positions 6 and 7 have 2' fluoro modifications, and the 5 'phosphate of ribonucleotides at positions 6 and 7 has 5' thio modifications.
Ribonucleotides at positions 1,3, 4, 5, 7, 10, 11, 12, 13 and 15 of substrate 8 have 2 'methoxy modifications, ribonucleotides at positions 2, 6, 8, 9, 14 and 16 have 2' fluoro modifications, and the 5 'phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.
The sense strand of Lumasiran obtained was GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23) and the antisense strand was UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).
Example 5
Adding substrates 9-14 into a clean reagent bottle in an equimolar ratio, and uniformly mixing to obtain a substrate mixture, wherein the concentration of each substrate in the substrate mixture is 2.5mM. The sequences of substrates 9 to 14 are shown in Table 5, and the length unit is nt. After annealing the substrate mixture or obtaining RNA fragment mixture of substrates 9-14. The reaction system was set to 10mL. The reaction system consisted of 800. Mu.M RNA fragment mixture, 50mM Tris-HCl,4eq ATP,12.5eq MgCl 2, 1.25eq DTT (1 eq=800. Mu.M) and RNA Ligase Ligase 8 was added at a final concentration of 0.2 mg/mL. The reaction was reacted at 16 ℃ for 16h. The obtained reaction system is deactivated by Ligase at 80 ℃ for 5min, precipitates are removed by centrifugation at 12000rpm, HPLC detection is carried out on a product obtained by Ligase 25 catalysis, the enzyme activity is measured by roughly estimated proportion of a product peak in HPLC data of a reaction system sample, the result shows that the yield is++, and the target peak proportion in the sample is 76.1%.
The solid phase synthesis method was used to prepare 6 single stranded RNA fragments.
TABLE 5
。
Wherein, m after A, C, G or U represents 2' methoxy modification of the ribonucleotide, f represents modification of 2' fluoro of the ribonucleotide, s in the sequences of ' sAm ', ' sGm ' and the like represents thio modification of 5' phosphate of the ribonucleotide.
Ribonucleotides at positions 1,2, 3, 4 and 5 of substrate 9 have 2' methoxy modifications and the 5' phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.
Ribonucleotides at positions 1,3, 7 and 8 of the substrate 10 have 2 'methoxy modifications and ribonucleotides at positions 2, 4,5 and6 have 2' fluoro modifications.
Ribonucleotides at positions 1-8 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' phosphate of the ribonucleotides at positions 3 and 4 have 5' thio modifications.
Ribonucleotides at positions 1, 3, 5, 6 and 7 of substrate 13 have 2 'methoxy modifications and ribonucleotides at positions 2 and 4 have 2' fluoro modifications.
Ribonucleotides at positions 1, 3, 4, 5, 7, 8, 10, 11 and 12 of substrate 14 have 2 'methoxy modifications, ribonucleotides at positions 2, 6, 8 and 9 have 2' fluoro modifications, and the 5 'phosphate of the ribonucleotides at positions 2 and 3 has 5' thio modifications.
The sense strand of Lumasiran obtained was GmsAmsCmUmUmUmCfAmUfCfCfUmGmGmAmAmAmUmAmUmAm (SEQ ID NO: 23) and the antisense strand was UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAfAmAmGmUmCmsCmsAm (SEQ ID NO: 24).
Comparative example 1
The average yield of the full-length Lumasiran product by solid phase synthesis is 29.6%, and the total content of N+1 and N-1 impurities is 1.45%.
The yield of Lumasiran products obtained by the enzyme-linked method in the invention is 71.91%, the yield of substrates synthesized by the solid phase is 42.2% of the yield of the whole process, the total yield is 30.35% which is higher than the average yield of products obtained by the solid phase synthesis method, and the total impurity ratio of N+1 and N-1 is 0.41% which is lower than the impurity ratio in the process of synthesizing Lumasiran by the solid phase synthesis method.
From the above description, it can be seen that the above-described embodiments of the present application achieve the technical effect that in the preparation method of the present application, the formation of Lumasiran of single-stranded RNA fragments designed based on Lumasiran sequences is catalyzed by RNA ligase, thereby achieving the preparation of such siRNA drugs by means of biosynthesis. Compared with a chemical synthesis preparation method, the preparation method provided by the application has the advantages that the purity of the obtained product is high, the generated impurities are less, the preparation process is simple, the reaction condition is mild, the dosage of organic reagent is low, the production cost is reduced, and the large-scale industrial production is convenient to realize.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (12)

1.一种Lumasiran的制备方法，其特征在于，所述Lumasiran为由互补配对的正义链和反义链组成的双链siRNA，所述制备方法包括：1. A method for preparing Lumasiran, characterized in that the Lumasiran is a double-stranded siRNA consisting of a complementary sense strand and an antisense strand, and the preparation method comprises: 将正义链底物片段、反义链底物片段和RNA连接酶混合，其中，所述正义链底物片段能够组成所述正义链，所述反义链底物片段能够组成所述反义链；Mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the sense strand substrate fragment can constitute the sense strand, and the antisense strand substrate fragment can constitute the antisense strand; 部分所述正义链底物片段和部分所述反义链底物片段以碱基互补形成的氢键连接，形成具有粘性末端的双链核苷酸结构；Part of the sense strand substrate fragment and part of the antisense strand substrate fragment are connected by hydrogen bonds formed by base complementarity to form a double-stranded nucleotide structure with sticky ends; 所述含有粘性末端的双链核苷酸结构继续与剩余部分的所述正义链底物片段和剩余部分的所述反义链底物片段以碱基互补形成的氢键连接；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; 所述正义链底物片段和所述反义链底物片段的头尾碱基之间均未互相连接，形成含有缺刻的双链核苷酸结构；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; 利用所述RNA连接酶将所述缺刻两端的碱基以磷酸二酯键连接，形成所述Lumasiran；Using the RNA ligase to connect the bases at both ends of the nick with a phosphodiester bond to form Lumasiran; 所述缺刻两端的所述碱基分别为不同底物片段的5’端和3’端，所述5’端为磷酸根，所述3’端为羟基；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 group, and the 3' end is a hydroxyl group; 利用所述RNA连接酶连接所述缺刻上下游的所述5’端的磷酸根和所述3’端的羟基，形成所述磷酸二酯键，获得所述Lumasiran；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 Lumasiran; 所述RNA连接酶包括RNA连接酶家族1或RNA连接酶家族2的RNA连接酶；The RNA ligase includes an RNA ligase of RNA ligase family 1 or RNA ligase family 2; 所述RNA连接酶家族1的RNA连接酶选自：如SEQ ID NO：3或SEQ ID NO：5所示的氨基酸序列的RNA连接酶；The RNA ligase of RNA ligase family 1 is selected from: an RNA ligase having an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 5; 所述RNA连接酶家族2的RNA连接酶选自：如SEQ ID NO：1、SEQ ID NO：2或SEQ ID NO：4所示的氨基酸序列的RNA连接酶；The RNA ligase of RNA ligase family 2 is selected from: an RNA ligase having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4; 所述正义链底物片段包括2条或3条，反义链底物片段包括2条或3条；The sense strand substrate fragments include 2 or 3 fragments, and the antisense strand substrate fragments include 2 or 3 fragments; 所述正义链底物片段的长度为5-14nt；The length of the positive strand substrate fragment is 5-14 nt; 所述反义链底物片段的长度为4-16nt。The length of the antisense strand substrate fragment is 4-16 nt. 2.根据权利要求1所述的制备方法，其特征在于，所述正义链底物片段和所述反义链底物片段均包括2条，所述正义链底物片段包括第一正义链底物片段和第二正义链底物片段，所述反义链底物片段包括第一反义链底物片段和第二反义链底物片段。2. The preparation method according to claim 1, characterized in that 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. 3.根据权利要求2所述的制备方法，其特征在于，所述第一正义链底物片段的核苷酸序列为SEQ ID NO：9所示，所述第二正义链底物片段的核苷酸序列为SEQ ID NO：10所示；3. The preparation method according to claim 2, characterized in that the nucleotide sequence of the first sense strand substrate fragment is shown in SEQ ID NO: 9, and the nucleotide sequence of the second sense strand substrate fragment is shown in SEQ ID NO: 10; 所述第一反义链底物片段的核苷酸序列为SEQ ID NO：12所示，所述第二反义链底物片段的核苷酸序列为SEQ ID NO：11序列所示；The nucleotide sequence of the first antisense strand substrate fragment is shown in SEQ ID NO: 12, and the nucleotide sequence of the second antisense strand substrate fragment is shown in SEQ ID NO: 11; SEQ ID NO：9：GmsAmsCmUmUmUmCfAmUfCfCfUmGmGm；SEQ ID NO: 9: GmsAmsCmUmUmUmCfAmUfCfCfUmGmGm; SEQ ID NO：10：AmAmAmUmAmUmAm；SEQ ID NO: 10: AmAmAmUmAmUmAm; SEQ ID NO：11：AfAmAmGmUmCmsCmsAm；SEQ ID NO: 11: AfAmAmGmUmCmsCmsAm; SEQ ID NO：12：UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGm；SEQ ID NO: 12: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGm; 所述第一正义链底物片段的第1、2、3、4、5、6、8、12、13和14位核糖核苷酸具有2’甲氧基修饰，第7、9、10和11位核糖核苷酸具有2’氟修饰，第2和第3位核糖核苷酸上的5’磷酸的具有硫代修饰；The first sense strand substrate fragment has 2' methoxy modification at ribonucleotides 1, 2, 3, 4, 5, 6, 8, 12, 13 and 14, 2' fluorine modification at ribonucleotides 7, 9, 10 and 11, and thio modification at 5' phosphate at ribonucleotides 2 and 3; 所述第二正义链底物片段的第1-7位核糖核苷酸具有2’甲氧基修饰；The ribonucleotides at positions 1 to 7 of the second sense strand substrate fragment have 2' methoxy modifications; 所述第一反义链底物片段的第1、2、3、4、5、6和7位核糖核苷酸具有2’甲氧基修饰，第8位核糖核苷酸具有2’氟修饰，第7和第8位核糖核苷酸上的5’磷酸的具有硫代修饰；The first antisense strand substrate fragment has 2' methoxy modifications at positions 1, 2, 3, 4, 5, 6 and 7, 2' fluorine modifications at position 8, and thio modifications at 5' phosphates at positions 7 and 8; 所述第二反义链底物片段的第1、3、4、5、6、9、11、12、13和15位核糖核苷酸具有2’甲氧基修饰，第2、7、8、10和14位核糖核苷酸具有2’氟修饰，第2和第3位核糖核苷酸上的5’磷酸的具有硫代修饰。The ribonucleotides at positions 1, 3, 4, 5, 6, 9, 11, 12, 13 and 15 of the second antisense strand substrate fragment have 2' methoxy modifications, the ribonucleotides at positions 2, 7, 8, 10 and 14 have 2' fluorine modifications, and the 5' phosphates on the ribonucleotides at positions 2 and 3 have thio modifications. 4.根据权利要求2所述的制备方法，其特征在于，所述第一正义链底物片段的核苷酸序列为SEQ ID NO：13所示，所述第二正义链底物片段的核苷酸序列为SEQ ID NO：14序列所示；4. The preparation method according to claim 2, characterized in that the nucleotide sequence of the first sense strand substrate fragment is shown in SEQ ID NO: 13, and the nucleotide sequence of the second sense strand substrate fragment is shown in SEQ ID NO: 14; 所述第一反义链底物片段的核苷酸序列为SEQ ID NO：16所示，所述第二反义链底物片段的核苷酸序列为SEQ ID NO：15所示；The nucleotide sequence of the first antisense strand substrate fragment is shown in SEQ ID NO: 16, and the nucleotide sequence of the second antisense strand substrate fragment is shown in SEQ ID NO: 15; SEQ ID NO：13：GmsAmsCmUmUmUmCfAmUf；SEQ ID NO: 13: GmsAmsCmUmUmUmCfAmUf; SEQ ID NO：14：CfCfUmGmGmAmAmAmUmAmUmAm；SEQ ID NO: 14: CfCfUmGmGmAmAmAmUmAmUmAm; SEQ ID NO：15：AmAmGmUmCmsCmsAm；SEQ ID NO: 15: AmAmGmUmCmsCmsAm; SEQ ID NO：16：UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAf；SEQ ID NO: 16: UmsAfsUmAmUmUfUmCfCfAmGmGmAmUfGmAf; 所述第一正义链底物片段的第1、2、3、4、5和6位上的核糖核苷酸具有2’甲氧修饰，第2和3位上的核糖核苷酸的5’磷酸具有5’硫代修饰，第7和9位上的核糖核苷酸具有2’氟代修饰；The ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of the first sense strand substrate fragment 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; 所述第二正义链底物片段的第3、4、5、6、7、8、9、10、11和12位上的核糖核苷酸具有2’甲氧修饰，第1和2位上的核糖核苷酸具有2’氟修饰；The ribonucleotides at positions 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 of the second sense strand substrate fragment have 2' methoxy modifications, and the ribonucleotides at positions 1 and 2 have 2' fluorine modifications; 所述第一反义链底物片段的第1、2、3、4、5、6和7位上的核糖核苷酸具有2’甲氧修饰，第6和7位上的核糖核苷酸具有2’氟修饰，第6和7位上的核糖核苷酸的5’磷酸具有5’硫代修饰；The ribonucleotides at positions 1, 2, 3, 4, 5, 6 and 7 of the first antisense strand substrate fragment 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; 所述第二反义链底物片段的第1、3、4、5、7、10、11、12、13和15位上的核糖核苷酸具有2’甲氧修饰，第2、6、8、9、14和16位上的核糖核苷酸具有2’氟修饰，第2和3位上的核糖核苷酸的5’磷酸具有5’硫代修饰。The ribonucleotides at positions 1, 3, 4, 5, 7, 10, 11, 12, 13 and 15 of the second antisense strand substrate fragment have 2' methoxy modification, the ribonucleotides at positions 2, 6, 8, 9, 14 and 16 have 2' fluorine modification, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modification. 5.根据权利要求2所述的制备方法，其特征在于，所述第一正义链底物片段的3’端与所述第二正义链底物片段的5’端在所述RNA连接酶的催化下连接，形成所述正义链；所述第一反义链底物片段的3’端与所述第二反义链底物片段的5’端在所述RNA连接酶的催化下连接，形成所述反义链。5. The preparation method according to claim 2 is characterized in that 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 the RNA ligase to form the 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 the RNA ligase to form the antisense chain. 6.根据权利要求2所述的制备方法，其特征在于，所述第一正义链底物片段的5’端为羟基基团，3’端为羟基基团；所述第二正义链底物片段的5’端为磷酸基团，3’端为L96基团；6. The preparation method according to claim 2, characterized in that the 5' end of the first sense strand substrate fragment is a hydroxyl group, and the 3' end is a hydroxyl group; the 5' end of the second sense strand substrate fragment is a phosphate group, and the 3' end is an L96 group; 所述第一反义链底物片段的5’端为羟基基团，3’端为羟基基团；所述第二反义链底物片段的5’端为磷酸基团，3’端为羟基。The 5' end of the first antisense strand substrate fragment is a hydroxyl group, and the 3' end 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. 7.根据权利要求2所述的制备方法，其特征在于，所述正义链底物片段和所述反义链底物片段均包括2条时，所述制备方法包括：7. The preparation method according to claim 2, characterized in that when the sense strand substrate fragment and the antisense strand substrate fragment each include 2 strands, the preparation method comprises: 将所述第一正义链底物片段、所述第二正义链底物片段、所述第一反义链底物片段和所述第二反义链底物片段混合，在所述RNA连接酶的催化作用下，所述第一正义链底物片段和所述第二正义链底物片段连接形成所述正义链，所述第一反义链底物片段和所述第二反义链底物片段连接形成所述反义链，所述正义链和所述反义链通过碱基互补配对形成所述Lumasiran。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 are mixed, and under the catalytic action of the RNA ligase, the first sense chain substrate fragment and the second sense chain substrate fragment are connected to form the sense chain, the first antisense chain substrate fragment and the second antisense chain substrate fragment are connected to form the antisense chain, and the sense chain and the antisense chain form Lumasiran through base complementary pairing. 8.根据权利要求1所述的制备方法，其特征在于，所述正义链底物片段和所述反义链底物片段均包括3条，8. The preparation method according to claim 1, characterized in that the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3 strands, 所述正义链底物片段包括第一正义链底物片段、第二正义链底物片段和第三正义链底物片段；The sense strand substrate fragments include a first sense strand substrate fragment, a second sense strand substrate fragment and a third sense strand substrate fragment; 所述反义链底物片段包括第一反义链底物片段、第二反义链底物片段和第三反义链底物片段。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. 9.根据权利要求8所述的制备方法，其特征在于，所述第一正义链底物片段的核苷酸序列为SEQ ID NO：17所示的核苷酸序列；9. The preparation method according to claim 8, characterized in that the nucleotide sequence of the first sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 17; 所述第二正义链底物片段的核苷酸序列为SEQ ID NO：18所示的核苷酸序列；The nucleotide sequence of the second sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 18; 所述第三正义链底物片段的核苷酸序列为SEQ ID NO：19所示的核苷酸序列；The nucleotide sequence of the third sense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 19; 所述第一反义链底物片段的核苷酸序列为SEQ ID NO：22所示的核苷酸序列；The nucleotide sequence of the first antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 22; 所述第二反义链底物片段的核苷酸序列为SEQ ID NO：21所示的核苷酸序列；The nucleotide sequence of the second antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 21; 所述第三反义链底物片段的核苷酸序列为SEQ ID NO：20所示的核苷酸序列；The nucleotide sequence of the third antisense strand substrate fragment is the nucleotide sequence shown in SEQ ID NO: 20; SEQ ID NO：17：GmsAmsCmUmUm；SEQ ID NO: 17: GmsAmsCmUmUm; SEQ ID NO：18：UmCfAmUfCfCfUmGm；SEQ ID NO: 18: UmCfAmUfCfCfUmGm; SEQ ID NO：19：GmAmAmAmUmAmUmAm；SEQ ID NO: 19: GmAmAmAmUmAmUmAm; SEQ ID NO：20：UmCmsCmsAm；SEQ ID NO: 20: UmCmsCmsAm; SEQ ID NO：21：AmUfGmAfAmAmGm；SEQ ID NO: 21: AmUfGmAfAmAmGm; SEQ ID NO：22：UmsAfsUmAmUmUfUmCfCfAmGmGm；SEQ ID NO: 22: UmsAfsUmAmUmUfUmCfCfAmGmGm; 所述第一正义链底物片段的第1、2、3、4和5位上的核糖核苷酸具有2’甲氧修饰，第2和3位上的核糖核苷酸的5’磷酸具有5’硫代修饰；The ribonucleotides at positions 1, 2, 3, 4 and 5 of the first sense strand substrate fragment have 2' methoxy modifications, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modifications; 所述第二正义链底物片段的第1、3、7和8位上的核糖核苷酸具有2’甲氧修饰，第2、4、5和6位上的核糖核苷酸具有2’氟修饰；The ribonucleotides at positions 1, 3, 7 and 8 of the second sense strand substrate fragment have 2' methoxy modification, and the ribonucleotides at positions 2, 4, 5 and 6 have 2' fluorine modification; 所述第三正义链底物片段的第1~8位上的核糖核苷酸具有2’甲氧修饰；The ribonucleotides at positions 1 to 8 of the third sense strand substrate fragment have 2' methoxy modifications; 所述第一反义链底物片段的第1、2、3和4位上的核糖核苷酸具有2’甲氧修饰，第3和4位上的核糖核苷酸的5’磷酸具有5’硫代修饰；The ribonucleotides at positions 1, 2, 3 and 4 of the first antisense strand substrate fragment have 2' methoxy modifications, and the 5' phosphates of the ribonucleotides at positions 3 and 4 have 5' thio modifications; 所述第二反义链底物片段的第1、3、5、6和7位上的核糖核苷酸具有2’甲氧修饰，第2和4位上的核糖核苷酸具有2’氟修饰；The ribonucleotides at positions 1, 3, 5, 6 and 7 of the second antisense strand substrate fragment have 2' methoxy modifications, and the ribonucleotides at positions 2 and 4 have 2' fluorine modifications; 所述第三反义链底物片段的第1、3、4、5、7、8、10、11和12位上的核糖核苷酸具有2’甲氧修饰，第2、6、8和9位上的核糖核苷酸具有2’氟修饰，第2和3位上的核糖核苷酸的5’磷酸具有5’硫代修饰。The ribonucleotides at positions 1, 3, 4, 5, 7, 8, 10, 11 and 12 of the third antisense strand substrate fragment have 2' methoxy modifications, the ribonucleotides at positions 2, 6, 8 and 9 have 2' fluorine modifications, and the 5' phosphates of the ribonucleotides at positions 2 and 3 have 5' thio modifications. 10.根据权利要求9所述的制备方法，其特征在于，所述制备方法包括：将所述第一正义链底物片段、所述第二正义链底物片段、所述第三正义链底物片段、所述第一反义链底物片段、所述第二反义链底物片段、所述第三反义链底物片段和所述RNA连接酶混合；10. The preparation method according to claim 9, characterized in that the preparation method 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; 在RNA连接酶的催化作用下，所述第一正义链底物片段、所述第二正义链底物片段和所述第三正义链底物片段连接形成所述正义链，所述第一反义链底物片段、所述第二反义链底物片段和所述第三反义链底物片段连接形成所述反义链，所述正义链和所述反义链通过碱基互补配对形成所述Lumasiran。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 the 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 the antisense chain, and the sense chain and the antisense chain form Lumasiran through base complementary pairing. 11.根据权利要求1所述的制备方法，其特征在于，所述正义链底物片段及所述反义链底物片段浓度各自独立选自0.1-4.5mM；11. The preparation method according to claim 1, characterized in that the concentrations of the sense strand substrate fragment and the antisense strand substrate fragment are independently selected from 0.1-4.5 mM; 所述正义链底物片段、所述反义链底物片段和所述RNA连接酶混合形成的反应体系中，还包括ATP、Tris-HCl、MgCl2和DTT。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. 12.根据权利要求1所述的制备方法，其特征在于，所述制备方法的反应温度为10-40℃；12. The preparation method according to claim 1, characterized in that the reaction temperature of the preparation method is 10-40°C; 所述制备方法的反应时间为2-48h。The reaction time of the preparation method is 2-48h.
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