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CN118460654A - Preparation method of Givosiran - Google Patents

Preparation method of Givosiran Download PDF

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Publication number
CN118460654A
CN118460654A CN202410921746.XA CN202410921746A CN118460654A CN 118460654 A CN118460654 A CN 118460654A CN 202410921746 A CN202410921746 A CN 202410921746A CN 118460654 A CN118460654 A CN 118460654A
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substrate fragment
seq
sense strand
antisense strand
strand substrate
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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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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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Abstract

The invention provides a preparation method of Givosiran. In the above method, givosiran is a double-stranded siRNA consisting of a sense strand and an antisense strand paired by complementation; the method comprises the following steps: mixing a sense strand substrate, an antisense strand substrate and RNA ligase, wherein the sense strand substrate can form a sense strand, the antisense strand substrate can form an antisense strand, the sense strand substrate and the antisense strand substrate are connected through hydrogen bonds formed by base complementation, and the head base and the tail base of the sense strand substrate are not connected with each other, so that a double-stranded nucleotide structure containing the nicks is formed; the bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form Givosiran. Can solve the problem of lower purity of Givosiran prepared in the prior art, and is suitable for the technical field of medicine biosynthesis.

Description

Preparation method of Givosiran
Technical Field
The invention relates to the field of medicine biosynthesis, in particular to a preparation method of Givosiran.
Background
The small interfering RNA (SMALL INTERFERING RNA, SIRNA) is double-stranded RNA with the length of 19-25nt, the siRNA can be dissociated into single strands after entering cells, wherein the sense strand can be specifically combined with messenger RNA (MESSENGER RNA, MRNA) of a target gene through base matching, a series of effects are induced, mRNA of the target gene is finally degraded, and translation of the mRNA is prevented so as to achieve the effect of obstructing target gene expression. In recent years, the development of siRNA drugs has achieved extensive attention, the siRNA drugs act on mRNA, and the target point of the ready-made medicine is obviously larger than that of the traditional small molecular medicine with the acting site being protein; in addition, siRNA drugs can act on new targets by transforming sequences, and development time is relatively short.
Givosiran is a siRNA double-stranded RNA drug developed by Alnylam company and used for treating adult acute hepatoporphyria (acute hepatic porphyria, AHP), and is approved by FDA in 2019 to be marketed under the trade name GIVLAARI.
Currently, the preparation method of Givosiran is a chemical method, solid phase carriers such as controllable microporous glass beads (Controlled Pore Glass, CPG) or polystyrene resin are used for cyclic synthesis by a phosphoramidite triester method, so that an oligonucleotide chain extends in the 3 'to 5' direction, after the synthesis cycle is finished, givosiran chains are excised from the solid phase carriers by ammonolysis, and then purified to obtain a pure product. Solid phase synthesis is a cyclic method, the yield of synthesis decreases with increasing synthetic chain length, and impurities generated in the synthesis process, such as impurities (n+1 impurities) one nucleotide more than the target sequence or impurities (N-1 impurities) one nucleotide less than the target sequence, also increase with increasing synthetic chain length, such impurities are not easily removed, resulting in complicated purification process and low efficiency. Therefore Givosiran is expensive to prepare and difficult to scale up, and therefore a more efficient Givosiran synthesis method needs to be developed.
Disclosure of Invention
The invention mainly aims to provide a preparation method of Givosiran to solve the problem of low purity of Givosiran 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 preparation method of Givosiran, which Givosiran is a double-stranded siRNA consisting of a sense strand and an antisense strand by complementary pairing; the preparation method comprises the following steps: mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase;
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 a sense strand and the antisense strand substrate fragment is capable of constituting an 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; connecting bases at the two ends of the notch by using RNA ligase through phosphodiester bonds to form Givosiran; 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 and nicking a phosphate radical at the 5 'end and a hydroxyl radical at the 3' end at the upstream and downstream by using RNA ligase to form a phosphodiester bond to obtain Givosiran; the RNA ligase is RNA ligase of RNA ligase family 1 and/or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is selected from: has the sequence shown in SEQ ID NO:3, a protein having an amino acid sequence shown in FIG. 3; the RNA ligase of RNA ligase family 2 is selected from: has the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no; or with a polypeptide having the sequence of SEQ ID NO: 1-SEQ ID NO:3, any one of the RNA ligases having the amino acid sequence shown in 3 has an identity of 70% or more, and has an enzyme that catalyzes the formation of a phosphodiester bond.
Further, the nucleotide sequence of the sense strand is SEQ ID NO:21, and the nucleotide sequence of the antisense strand is SEQ ID NO:22, and a nucleotide sequence shown in seq id no.
Further, the sense strand substrate fragment comprises 2 or more, and the antisense strand substrate fragment comprises 2 or more; preferably, the sense strand substrate fragment is 2-19nt in length, more preferably 8-12nt; preferably, the antisense strand substrate fragment is 2-21nt in length, more preferably 4-15nt.
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, and the antisense strand substrate fragment comprises a first antisense strand substrate fragment and a second antisense strand substrate fragment; the preparation method comprises the following steps: 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, connecting the first sense strand substrate fragment and the second sense strand substrate fragment to form a sense strand, catalyzing the first antisense strand substrate fragment and the second antisense strand substrate fragment to connect to form an antisense strand, wherein the sense strand and the antisense strand are formed Givosiran through base complementation pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain Givosiran.
Further, 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 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 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; preferably, 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.
Further, the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:7, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:8, a nucleotide sequence shown in seq id no; preferably, the nucleotide sequence of the first antisense strand substrate fragment SEQ ID NO:10, the nucleotide sequence of the second antisense strand substrate fragment is set forth in SEQ ID NO:9, and a nucleotide sequence shown in the sequence No.
Further, the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:11, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:12, a nucleotide sequence shown in sequence no; preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:14, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 13.
Further, 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 fragments include 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 SEQ ID NO:15, a nucleotide sequence shown in seq id no; the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:16, a nucleotide sequence shown in seq id no; the nucleotide sequence of the third sense strand substrate fragment is SEQ ID NO:17, a nucleotide sequence shown in seq id no; preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:20, a nucleotide sequence shown in seq id no; the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:19, a nucleotide sequence shown in seq id no; the nucleotide sequence of the third antisense strand substrate fragment is SEQ ID NO:18, and a nucleotide sequence shown in seq id no.
Further, the concentration of the sense strand substrate fragment and the antisense strand substrate fragment are each independently selected from 0.1 to 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 process is 10-40 ℃, more preferably 15-30 ℃; preferably, the reaction time of the preparation process is 2 to 48 hours, more preferably 12 to 24 hours.
By applying the technical scheme of the application, the preparation method is utilized, under the catalysis of RNA ligase, the sense strand substrate fragments are connected to form Givosiran sense strands, and the antisense strand substrate fragments are connected to form Givosiran antisense strands, so that the siRNA medicine is prepared by utilizing a biosynthesis mode. Compared with the method for preparing Givosiran 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 structural diagram of an L96 group according to an embodiment of the present invention.
FIG. 2 shows a schematic of an enzyme-catalyzed ligation reaction according to example 1 of the invention.
FIG. 3 shows a graph of electrophoresis results of catalytic products of RNA Ligase Ligase25 and Ligase26 according to example 1 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 impurity: nucleic acid impurities having a single nucleotide attached in addition to the target synthetic sequence.
N-1 impurity: nucleic acid impurities having a single nucleotide deletion as compared to the target synthetic sequence.
As mentioned in the background art, the preparation of Givosiran 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 Givosiran for preparing Givosiran by enzyme-catalyzed synthesis, and thus proposed a series of protection schemes of the present application.
In a first exemplary embodiment of the application, a method of preparation of Givosiran is provided, the Givosiran being a double stranded RNA consisting of complementarily paired sense and antisense strands; the preparation method comprises the following steps: mixing a sense strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase; 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 a sense strand and the antisense strand substrate fragment is capable of constituting an 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; connecting bases at the two ends of the notch by using RNA ligase through phosphodiester bonds to form Givosiran; 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 and nicking a phosphate radical at the 5 'end and a hydroxyl radical at the 3' end at the upstream and downstream by using RNA ligase to form a phosphodiester bond to obtain Givosiran;
The RNA ligase is RNA ligase of RNA ligase family 1 and/or RNA ligase family 2; the RNA ligase of RNA ligase family 1 is selected from the group consisting of RNA ligases having the sequence as set forth in SEQ ID NO:3, a protein having an amino acid sequence shown in FIG. 3; the RNA ligase of RNA ligase family 2 is selected from the group consisting of RNA ligases having the sequence as set forth in SEQ ID NO:1 and/or SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no; or with a polypeptide having the sequence of SEQ ID NO: 1-SEQ ID NO:3, including but not limited to, 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), and has an activity of catalyzing phosphodiester bond formation. 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.
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 Givosiran 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, givosiran can be prepared by mixing the sense strand substrate fragment and the antisense strand substrate fragment with RNA ligase. In the preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be complementarily paired to form a double-stranded structure nucleotide with a sticky end, and the double-stranded structure nucleotide with the sticky end is continuously combined with other substrates to form a double-stranded nucleotide structure with an nick; the RNA ligase can recognize the specific recognition site of the double-stranded nucleotide containing the nick, and the nick is connected, so that the target product Givosiran is prepared.
In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO:21, and the nucleotide sequence of the antisense strand is SEQ ID NO:22, and a nucleotide sequence shown in seq id no.
SEQ ID NO：21：CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm。
SEQ ID NO：22：UmsAfsAfGfAmUfGmAfGmAfCmAf CmUfCmUfUmUfCmUfGmsGmsUm。
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", "sGm" and the like represents thio modification of 5' phosphate of the ribonucleotide.
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 42，Escherichia phage JN02)：
MEKLYYNLLSLCKSSSDRKFFYSDDVSPIGKKYRIFSYNFASYSDWLLPDALECRGIMFEMDGETPVRIASRPMEKFFNLNENPFTLSINLDDVKYLMTKEDGSLVSTYLDGGTVRFKSKGSIKSDQAVSATSILLDIDHKNLADRLLELCNDGFTANFEYVAPTNKIVLTYPEKRLILLNIRDNNTGEYIEYDDIYLDPVFRKYLVDRFEVPEGDWTSDVKSSTNIEGYVAVMKDGSHFKLKTDWYVALHTTRDSISSPEKLFLAIVNGASDDLKAMYADDEFSFKKVELFEKAYLDFLDRSFYICLDTYDKHKGKDRKTYAIEAQAVCKGAQTPWLFGIIMNLYQGGSKEQMMTALESVFIKNHKNFIPEGY.
SEQ ID NO：4(Ligase 11，Thermococcus)：
MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.
SEQ ID NO：5(Ligase 20，Archaea)：
MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.
SEQ ID NO：6(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 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).
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, only the nucleotide sequence represented by SEQ ID NO: 1-SEQ ID NO:3, or an RNA ligase as set forth in SEQ ID NO: 1-SEQ ID NO:3, and the enzyme having an identity of 70% or more catalyzes the formation of a phosphodiester bond between a phosphate group and a hydroxyl group of a substrate of the present application to obtain a product Givosiran. In the experiments related to the present application, the inventors obtained the above-mentioned SEQ ID NO capable of synthesizing Givosiran by screening from a large number of enzymes: 1-SEQ ID NO: 3. Whereas the very large ratio of negative results in experiments showed that most RNA ligases are difficult to catalyze Givosiran synthesis, including but not limited to SEQ ID NO: 4-SEQ ID NO:6, in the present description only the RNA ligase shown in SEQ ID NO: 4-SEQ ID NO:6 shows the RNA ligase without catalytic Givosiran synthesis activity.
In a preferred embodiment, the sense strand substrate fragment comprises 2 or more, and the antisense strand substrate fragment comprises 2 or more; preferably, the sense strand substrate fragment is 2-19nt in length, more preferably 8-12nt; preferably, the antisense strand substrate fragment is 2-21nt in length, more preferably 4-15nt.
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; the preparation method comprises the following steps: 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, connecting the first sense strand substrate fragment and the second sense strand substrate fragment to form a sense strand, catalyzing the first antisense strand substrate fragment and the second antisense strand substrate fragment to connect to form an antisense strand, wherein the sense strand and the antisense strand are formed Givosiran through base complementation pairing; preferably, the sense strand substrate fragment and the antisense strand substrate fragment are annealed and then mixed with RNA ligase to obtain Givosiran.
In the preparation method, the sense strand substrate fragment and the antisense strand substrate fragment can be mixed for annealing, and a double-stranded RNA structure can be formed by base complementary pairing between the sense strand substrate fragment and the antisense strand substrate fragment, and the double-stranded RNA structure has a notch between different substrate fragments. 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 Givosiran with a complete double-chain structure.
In a preferred embodiment, 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 an RNA ligase to form the sense strand; the 3 'end of the first antisense strand substrate fragment is linked 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 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; preferably, 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.
In a preferred embodiment, the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:7, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO. Preferably, the nucleotide sequence of the first antisense strand substrate is SEQ ID NO:10, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 9.
In a preferred embodiment, the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:11, and the second sense strand substrate fragment is SEQ ID NO:12, and a nucleotide sequence shown in seq id no. Preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:14, and the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO: 13.
Using the above preparation method and SEQ ID NO:7-SEQ ID NO:10 or SEQ ID NO:11-SEQ ID NO:14, can be prepared Givosiran. However, it should be noted that the selection of the substrate fragment is not limited to the above-mentioned SEQ ID NO:7-10 or SEQ ID NO:11-14, and the substrate fragments capable of being combined to form the sense strand and the antisense strand can be applied to the preparation method, wherein the preparation method is applicable to preparation of Givosiran but is not limited to the difference of the connection positions of the substrate fragments, and the preparation has a good connection effect on connection of the sense strand sequence and the antisense strand sequence of Givosiran. The number of sense strand substrate fragments or antisense strand substrate fragments includes, but is not limited to, 2, 3, 4, or even more.
SEQ ID NO：7：CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCf。
SEQ ID NO：8：AmUmCmUmUmAm。
SEQ ID NO：9：CmUfCmUfUmUfCmUfGmsGmsUm。
SEQ ID NO：10：UmsAfsAfGfAmUfGmAfGmAfCmAf。
SEQ ID NO：11：CmsAmsGmAmAmAmGfAmGfUm。
SEQ ID NO：12：GfUmCfUmCfAmUmCmUmUmAm。
SEQ ID NO：13：UfUmUfCmUfGmsGmsUm。
SEQ ID NO：14：UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCm。
In a preferred embodiment, the sense strand substrate fragment and the antisense strand substrate fragment each comprise 3, the sense strand substrate fragment comprising 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; preferably, the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:15, a nucleotide sequence shown in seq id no; the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:16, a nucleotide sequence shown in seq id no; the nucleotide sequence of the third sense strand substrate fragment is SEQ ID NO:17, a nucleotide sequence shown in seq id no; preferably, the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:20, a nucleotide sequence shown in seq id no; the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:19, a nucleotide sequence shown in seq id no; the nucleotide sequence of the third antisense strand substrate fragment is SEQ ID NO:18, and a nucleotide sequence shown in seq id no.
SEQ ID NO：15：CmsAmsGmAmAm。
SEQ ID NO：16：AmGfAmGfUmGfUm。
SEQ ID NO：17：CfUmCfAmUmCmUmUmAm。
SEQ ID NO：18：UfGmsGmsUm。
SEQ ID NO：19：CmUfCmUfUmUfCm。
SEQ ID NO：20：UmsAfsAfGfAmUfGmAfGmAfCmAf。
In a preferred embodiment, the concentration of the sense strand substrate fragment and the antisense strand substrate fragment are each independently selected from 0.1 to 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 process is 10-40 ℃, more preferably 15-30 ℃; preferably, the reaction time of the preparation process is 2 to 48 hours, more preferably 12 to 24 hours.
The concentration of the above sense strand substrate fragment and antisense strand substrate fragment are each selected from the group including, but not limited to, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5mM; the reaction temperature of the above preparation method includes, but is not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 ℃; the reaction time of the above preparation method includes, but is not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45 or 48 hours.
The advantageous effects of the present application will be explained in further detail below in connection with specific examples.
Example 1:
4 single-stranded RNA fragments (length: nt) as shown in Table 1 were designed based on the sense strand and the antisense strand of Givosiran sequences (SEQ ID NO:21 and SEQ ID NO: 22):
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 positioned before the ribonucleotide 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 the 3' end of the sense strand, the structure is shown as figure 1, and wavy lines represent bases connected with the L96 group.
Ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8, 10, 12 and 14 of substrate 1 have 2' methoxy modifications, nucleotides at positions 7, 9, 11, 13 and 15 have 2' fluoro modifications, and 5' phosphate at positions 2 and 3 have thio modifications.
Ribonucleotides at positions 1, 2, 3, 4, 5 and 6 of substrate 2 have 2' methoxy modifications.
Ribonucleotides at positions 1, 3, 5, 7, 9, 10 and 11 of substrate 3 have 2' methoxy modifications, nucleotides at positions 2, 4, 6 and 8 have 2' fluoro modifications, and nucleotides at positions 10 and 11 have thio modifications at the 5' phosphate.
Ribonucleotides at positions 1,5, 7, 9 and 11 of substrate 4 have 2' methoxy modifications, nucleotides at positions 2,3,4, 6, 8, 10 and 12 have 2' fluoro modifications, and 5' phosphate at positions 2 and 3 has thio modifications.
The above 4 single-stranded RNA fragments were prepared using a solid phase synthesis method.
The 4 single-stranded RNA fragments were mixed in an equimolar ratio to obtain a substrate mixture having a final concentration of 2.5mM (2.5 mM for each substrate), and annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to enzyme-catalyzed ligation reaction, the reaction system was set to 10. Mu.L, and the reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT), and RNA Ligase Ligase 25, ligase 26, ligase 11, ligase 20 and Ligase 32 were added, respectively. The reaction system 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. 2.
Products obtained by catalyzing RNA ligases Ligase25, ligase26, ligase 11, ligase 20 and Ligase 32 were subjected to SDS-PAGE detection. The results of electrophoresis of the products obtained by catalyzing the RNA Ligase25 and the Ligase26 are shown in FIG. 3, wherein the lane M in FIG. 3 represents the RNA molecular standard (marker), the lane 1 represents the reaction system of the Ligase25, and the lane 2 represents the reaction system of the Ligase 26. 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 activity screening of 6 RNA ligases shows that the Ligase25 has better connection effect and can convert most substrates into Givosiran.
The sense strand of Givosiran obtained was CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21) and the antisense strand was UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22). The reaction results are shown in Table 2.
TABLE 2
。
Remarks:
1) Reaction conditions: substrate fragment 100. Mu.M, ATP 10eq, mgCl 2 100eq, DTT 10eq (1 eq=100. Mu.M), 0.2mg/mL enzyme, 1891V, 50mM Tris-HCl pH 7.5, 16℃for 16h;
2) ++ means 25 to 50% (excluding 50% of the end points), ++ represents 50-75%.
The gray scale data of the product and substrate obtained by gray scale analysis of the pattern of the Urea-PAGE gel electrophoresis result is calculated as: yield = product gray data/(product gray data + substrate gray data).
Example 2:
The substrates fragments annealed in example 1, which were able to catalyze the synthesis Givosiran, of Ligase 25, ligase 26 and Ligase 42, were used to carry out the respective enzyme-catalyzed ligation reactions under the following conditions: the reaction system was set at 50. Mu.L, and a reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT) and RNA ligase were sequentially added to the reactor to react at 16℃for 16 hours.
Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The HPLC and LC-MS measurements were performed, wherein the HPLC results of the Ligase 25 catalytic product are shown in FIG. 4, and the yield was measured as the roughly estimated ratio of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.
TABLE 3 Table 3
。
Remarks:
1) Reaction conditions: substrate fragment 800. Mu.M, ATP 4eq, mgCl 2 100eq, DTT 10eq (1 eq=800. Mu.M), 0.2mg/mL enzyme, 236V, 50mM Tris-HCl pH 7.5, 16℃for 16h;
2) N.d. indicates that no product formation was detected, ++ means 50 to 70% (excluding 70% of the end points), ++ represents 70-80%.
LC-MS was used to identify the sense strand product as having a molecular weight 8732.04, the antisense strand product as having a molecular weight 7560.03, the sense strand product as theoretical 8732.04.+ -.8, and the antisense strand product as theoretical 7560.03.+ -.8, indicating that ligation of Ligase 25, ligase 26, and Ligase 42 produced Givosiran. The LC-MS detection result diagram of Ligase 25 is shown in FIG. 5.
Example 3
The annealed substrate fragment and the Ligase 25 are used for carrying out enzyme catalytic ligation reaction, and the reaction conditions are as follows: the reaction system was set to 10 mL, and a reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (adenosine triphosphate, ATP), mgCl 2, dithiothreitol (DTT) and RNA ligase were sequentially added to the reactor to react at 16℃for 16 hours. After the overnight reaction, heating at 50 ℃ for 15min to inactivate proteins, centrifuging to obtain a supernatant, purifying the supernatant by using a Nano-Q column, eluting by using a NaCl gradient, desalting by using a membrane package (with a molecular weight cut-off of 1 kDa), and freeze-drying in a freeze dryer to obtain dry powder, wherein the calculated yield is 66.18%, and the purity (HPLC detection) is as follows: 91.88%.
Example 4
Based on Givosiran sequences, 4 single-stranded RNA fragments (length: nt) as shown in Table 4 were designed.
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 sequence of ' sAm ', ' sGm ', etc. before the ribonucleotide represents thio modification of 5' phosphate of the ribonucleotide.
Ribonucleotides at positions 1, 2, 3, 4, 5, 6, 8 and 10 of substrate 5 have 2' methoxy modifications, nucleotides at positions 7 and 9 have 2' fluoro modifications, and nucleotides at positions 2 and 3 have thio modifications at the 5' phosphate.
Ribonucleotides at positions 2,4, 6, 7, 8, 9, 10 and 11 of substrate 6 have 2 'methoxy modifications and nucleotides at positions 1,3 and 5 have 2' fluoro modifications. The 5' end is modified by an L96 group.
The ribonucleotides at positions 2, 4, 6, 7 and 8 of substrate 7 have 2' methoxy modifications, the nucleotides at positions 1, 3 and 5 have 2' fluoro modifications, and the ribonucleotides at positions 7 and 8 have thio modifications at the 5' phosphate.
Ribonucleotides at positions 1, 5, 7, 9, 11, 13 and 15 of substrate 8 have 2' methoxy modifications, nucleotides at positions 2, 3, 4, 6, 8, 10, 12 and 14 have 2' fluoro modifications, and 5' phosphate at positions 2 and 3 ribonucleotides have thio modifications.
The annealed substrate fragment and the Ligase 25 are used for carrying out enzyme catalytic ligation reaction, and the reaction conditions are as follows: the reaction system was set at 10mL, and 800. Mu.M of the substrate fragment, 4eq of ATP, 2 12.5eq,DTT 1.25eq of MgCl (1 eq=800. Mu.M), 0.2mg/mL of Ligase 25, 50mM of Tris-HCl pH7.5 were sequentially added to the reactor, and reacted at 16℃for 16 h. Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The assay HPLC was run and the yield was measured as the coarsely estimated occupancy of the product peak in the HPLC data of the reaction system samples. The result showed a yield of++ (75.2% product peak).
The sense strand of Givosiran obtained was CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21) and the antisense strand was UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).
Example 5
The 6 single-stranded RNA fragments (length: nt) shown in Table 5 were designed based on the sequence of Givosiran.
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 sequence of ' sAm ', ' sGm ', etc. before the ribonucleotide 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' phosphate at the ribonucleotides at positions 2 and 3 have thio modifications.
Ribonucleotides at positions 1,3, 5 and 7 of substrate 10 have 2 'methoxy modifications and nucleotides at positions 2, 4 and 6 have 2' fluoro modifications.
Ribonucleotides at positions 2, 4, 5, 6, 7, 8 and 9 of substrate 11 have 2 'methoxy modifications and nucleotides at positions 1 and 3 have 2' fluoro modifications.
The ribonucleotides at positions 2, 3 and 4 of substrate 12 have 2' methoxy modifications, the nucleotide at position 1 has 2' fluoro modifications, and the 5' phosphate on the ribonucleotides at positions 3 and 4 has thio modifications.
Ribonucleotides at positions 1,3, 5 and 7 of substrate 13 have 2 'methoxy modifications and nucleotides at positions 2, 4 and 6 have 2' fluoro modifications.
Ribonucleotides at positions 1, 5, 7, 9 and 11 of substrate 14 have 2' methoxy modifications, nucleotides at positions 2, 3, 4, 6, 8, 10 and 12 have 2' fluoro modifications, and 5' phosphate at positions 2 and 3 has thio modifications.
The sense strand of Givosiran obtained was CmsAmsGmAmAmAmGfAmGfUmGfUmCfUmCfAmUmCmUmUmAm (SEQ ID NO: 21) and the antisense strand was UmsAfsAfGfAmUfGmAfGmAfCmAfCmUfCmUfUmUfCmUfGmsGmsUm (SEQ ID NO: 22).
The annealed substrate fragment and the Ligase 25 are used for carrying out enzyme catalytic ligation reaction, and the reaction conditions are as follows: the reaction system was set at 10mL, and 800. Mu.M of the substrate fragment, 4eq of ATP, 2 12.5eq,DTT 1.25eq of MgCl (1 eq=800. Mu.M) and 0.2mg/mL of Ligase 25, 50mM of Tris-HCl pH7.5 were sequentially added to the reactor and reacted at 16℃for 16 hours. Heating at 80deg.C for 5min to deactivate protein, and centrifuging to obtain supernatant. The assay HPLC was performed and the enzyme activity was measured as the coarsely estimated ratio of product peaks in the HPLC data of the reaction system samples. The result showed a yield of++ (70.3% product peak).
Comparative example 1
The average yield of the full-length Givosiran product by solid phase synthesis is 32.9%, and the total content of N+1 and N-1 impurities is 1.52%.
The yield of Givosiran products obtained by the enzyme-linked method in the invention is 87.53%, the average value of the solid phase synthesis yield of the used substrate is 39.1%, the yield of the multiplied integral process is 34.2%, the product average yield is higher than that 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 that of the impurity ratio in the process of synthesizing Givosiran by the solid phase synthesis method.
From the above description, it can be seen that the above embodiments of the present application achieve the following technical effects: in the preparation method of the application, the single-stranded RNA fragment designed based on Givosiran sequences is catalyzed by RNA ligase to form Givosiran, so that the siRNA medicine is prepared by a biosynthesis mode. 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. A method for preparing Givosiran, wherein Givosiran is a double-stranded siRNA, and consists of a sense strand and an antisense strand which are complementary to each other by base;
The preparation method comprises the following steps:
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 Givosiran;
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 Givosiran;
The RNA ligase is one or more of RNA ligase family 1 and/or RNA ligase family 2;
the RNA ligase of RNA ligase family 1 is selected from: has the sequence shown in SEQ ID NO:3, a protein having an amino acid sequence shown in FIG. 3;
the RNA ligase of RNA ligase family 2 is selected from: has the sequence shown in SEQ ID NO:1 and/or SEQ ID NO:2, a protein having an amino acid sequence shown in seq id no; or (b)
The RNA ligase is a nucleic acid sequence having SEQ ID NO: 1-SEQ ID NO:3, any one of the RNA ligases having the amino acid sequence shown in 3 has an identity of 70% or more, and has an enzyme that catalyzes the formation of the phosphodiester bond.
2. The method of claim 1, wherein the sense strand has a nucleotide sequence of SEQ ID NO:21, and the nucleotide sequence of the antisense strand is the nucleotide sequence shown in SEQ ID NO:22, and a nucleotide sequence shown in seq id no.
3. The method of any one of claims 1-2, 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 2-19nt;
The antisense strand substrate fragment is 2-21nt 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;
The preparation method comprises the following steps: 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, and ligating the first sense strand substrate fragment and the second sense strand substrate fragment to form the sense strand under the catalytic action of the RNA ligase;
catalyzing ligation of the first antisense strand substrate fragment and the second antisense strand substrate fragment to form the antisense strand under the catalysis of the RNA ligase;
the sense strand and the antisense strand form the Givosiran by base complementary pairing.
5. 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.
6. 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.
7. The method of claim 4, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:7, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:8, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:10, and the nucleotide sequence of the second antisense strand substrate fragment is shown in SEQ ID NO:9, and a nucleotide sequence shown in the sequence No.
8. The method of claim 4, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:11, and the nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:12, a nucleotide sequence shown in sequence no;
the nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:14, and the nucleotide sequence of the second antisense strand substrate fragment is set forth in SEQ ID NO: 13.
9. 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.
10. The method of claim 9, wherein the nucleotide sequence of the first sense strand substrate fragment is SEQ ID NO:15, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the second sense strand substrate fragment is SEQ ID NO:16, a nucleotide sequence shown in seq id no;
the nucleotide sequence of the third sense strand substrate fragment is SEQ ID NO:17, a nucleotide sequence shown in seq id no;
The nucleotide sequence of the first antisense strand substrate fragment is SEQ ID NO:20, a nucleotide sequence shown in seq id no;
the nucleotide sequence of the second antisense strand substrate fragment is SEQ ID NO:19, a nucleotide sequence shown in seq id no;
the nucleotide sequence of the third antisense strand substrate fragment is SEQ ID NO:18, and a nucleotide sequence shown in seq id no.
11. The method according to any one of claim 1 to 2, wherein,
The concentration of the sense strand substrate fragment and the antisense strand substrate fragment are each independently selected from 0.1-4.5mM;
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.
12. The preparation method according to claim 1, wherein the reaction temperature of the preparation method is 10-40 ℃;
the reaction time of the preparation method is 2-48h.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160244766A1 (en) * 2013-10-04 2016-08-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the alas1 gene
WO2023194537A1 (en) * 2022-04-08 2023-10-12 Glaxosmithkline Intellectual Property Development Limited Novel processes for the production of polynucleotides including oligonucleotides
CN117070583A (en) * 2023-10-16 2023-11-17 吉林凯莱英制药有限公司 Preparation method of siRNA for inhibiting PCSK9 gene expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160244766A1 (en) * 2013-10-04 2016-08-25 Alnylam Pharmaceuticals, Inc. Compositions and methods for inhibiting expression of the alas1 gene
WO2023194537A1 (en) * 2022-04-08 2023-10-12 Glaxosmithkline Intellectual Property Development Limited Novel processes for the production of polynucleotides including oligonucleotides
CN117070583A (en) * 2023-10-16 2023-11-17 吉林凯莱英制药有限公司 Preparation method of siRNA for inhibiting PCSK9 gene expression

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