CN118460652B - Preparation method of siRNA for treating non-arterial ischemic optic neuropathy - Google Patents

Abstract

The application provides a preparation method of siRNA for treating non-arterial ischemic optic neuropathy. siRNA Cosdosiran, cosdosiran is double-stranded RNA consisting of complementarily paired sense and antisense strands; the preparation method comprises the following steps: mixing a sense strand substrate, an antisense strand substrate and RNA ligase, catalyzing the connection between the sense strand substrate and the antisense strand substrate by using the RNA ligase and connecting by a phosphodiester bond to obtain a sense strand and an antisense strand, thereby obtaining Cosdosiran; sense strand substrates can constitute the sense strand and antisense strand substrates can constitute the antisense strand. Compared with Cosdosiran prepared by chemical synthesis, the preparation method provided by the application has the advantages that the purity of the obtained product is higher, the impurity is less, the reaction condition is mild, and the industrial scale-up production is convenient to realize.

Description

Preparation method of siRNA for treating non-arterial ischemic optic neuropathy

Technical Field

The invention relates to the field of medicine biosynthesis, in particular to a preparation method of siRNA for treating non-arterial ischemic optic neuropathy.

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.

Cosdosiran is siRNA double-stranded RNA medicine jointly developed by Quark corporation, and is currently in clinical phase 3 experiment. Cosdosiran can be used for treating Non-arterial ischemic optic neuropathy (Non-ARTERITIC ANTERIOR ISCHEMIC OPTIC NEUROPATHY, NAION).

Cosdosiran is a chemical method solid phase synthesis, using a solid phase carrier such as controllable microporous glass beads (Controlled Pore Glass, CPG) or polystyrene resin, circularly synthesizing by a phosphoramidite triester method, enabling an oligonucleotide chain to extend along the 3 'to 5' direction, cutting Cosdosiran chain from the solid phase carrier by ammonolysis after the synthesis cycle is finished, and purifying to obtain a pure product. The above synthesis process requires an expensive nucleic acid synthesizer, and the scale of synthesis is limited by the scale of the synthesizer, the scale of batch process is in the order of kg, increasing the synthesis throughput requires adding more instruments or more batches, and the cost of scale-up is very high. In addition, the solid phase synthesis method is a cyclic method, the yield of the synthesis decreases with increasing synthetic chain length, and impurities generated during the synthesis, 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, and the contents of the final n+1 and N-1 impurities are 1 to 3%.

Disclosure of Invention

The invention mainly aims to provide a preparation method of siRNA for treating non-arterial ischemic optic neuropathy, which aims to solve the problem of low purity of Cosdosiran prepared in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing siRNA for treating non-arterial ischemic optic neuropathy, wherein the siRNA is Cosdosiran, and Cosdosiran is double-stranded RNA consisting of a sense strand and an antisense strand which are complementarily paired; the method comprises the following steps: mixing a sense strand substrate, an antisense strand substrate, and an RNA ligase, wherein the sense strand substrate is capable of constituting a sense strand and the antisense strand substrate is capable of constituting 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 and the antisense strand substrate 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 Cosdosiran; the bases at the two ends of the notch are respectively the 5 'end and the 3' end of different substrates, 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 Cosdosiran; the RNA ligase is selected from RNA ligase family 2; RNA ligase comprises SEQ ID NO:1 or SEQ ID NO:2, and one or more of the RNA ligases shown in fig. 2; or with SEQ ID NO:1 or SEQ ID NO:2, and has an activity of catalyzing the formation of phosphodiester bonds.

Further, the sense strand substrate and the antisense strand substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method; preferably, the sense strand is SEQ ID NO:10, and the antisense strand is SEQ ID NO:11, a nucleic acid sequence as set forth in seq id no; preferably, the sense strand consists of 2 and more sense strand substrates, the length of which is 3-16nt, more preferably 5-12nt; preferably, the antisense strand consists of 2 and more antisense strand substrates, the length of which is 3-16nt, more preferably 7-14nt.

Further, the double-stranded RNA formed by annealing the sense strand substrate and the antisense strand substrate has more than 3 base combinations capable of complementary pairing; preferably, the double stranded RNA ends are cohesive ends; preferably, the adhesive tip length is 2-7nt.

Further, the sense strand substrate and the antisense strand substrate each comprise 2 substrates, the sense strand substrate comprises a first sense strand substrate and a second sense strand substrate, and the antisense strand substrate comprises a first antisense strand substrate and a second antisense strand substrate; the preparation method comprises the following steps: mixing a first sense strand substrate, a second sense strand substrate, a first antisense strand substrate and a second antisense strand substrate, catalyzing the connection of the first sense strand substrate and the second sense strand substrate by using RNA ligase to form a sense strand, catalyzing the connection of the first antisense strand substrate and the second antisense strand substrate to form an antisense strand, and complementarily pairing the sense strand and the antisense strand to form Cosdosiran.

Further, the 3 'end of the first sense strand substrate is linked to the 5' end of the second sense strand substrate under the catalysis of an RNA ligase to form a sense strand; the 3 'end of the first antisense strand substrate is connected with the 5' end of the second antisense strand substrate under the catalysis of RNA ligase to form an antisense strand; preferably, the 5 'end of the first sense strand substrate is an ia protecting group and the 3' end is a hydroxyl group; the 5 'end of the second sense strand substrate is a phosphate group, and the 3' end is a hydroxyl protecting group; preferably, the 5 'end of the first antisense strand substrate is a hydroxyl protecting group and the 3' end is a hydroxyl group; the 5 'end of the second antisense strand substrate is a phosphate group, and the 3' end is a hydroxyl protecting group.

Further, the first sense strand substrate is SEQ ID NO:6 or 12, and the second sense strand substrate is SEQ ID NO:7 or 13.

Further, the first antisense strand substrate is SEQ ID NO:9 or 15, and the second antisense strand substrate is SEQ ID NO:8 or 14.

Further, the sense strand substrate and the antisense strand substrate each comprise 3 substrates,

The sense strand substrates include a first sense strand substrate, a second sense strand substrate, and a third sense strand substrate;

Antisense strand substrates include a first antisense strand substrate, a second antisense strand substrate, and a third antisense strand substrate; preferably, the first sense strand substrate is SEQ ID NO:16, a nucleic acid sequence as set forth in seq id no; the second sense strand substrate is SEQ ID NO:17, a nucleic acid sequence as set forth in seq id no; the third sense strand substrate is SEQ ID NO:18, a nucleic acid sequence shown in seq id no; preferably, the first antisense strand substrate is SEQ ID NO:21, a nucleic acid sequence as set forth in seq id no; the second antisense strand substrate is SEQ ID NO:20, a nucleic acid sequence shown in seq id no; the third sense strand substrate is SEQ ID NO:19, and a nucleic acid sequence as set forth in seq id no.

Further, the concentration of the sense strand substrate and the antisense strand substrate is 0.1 to 4.5mM, more preferably 0.8 to 1.6 mM; preferably, the concentration of RNA ligase is 0.05-0.6mg/mL, more preferably 0.2mg/mL; preferably, in the reaction system formed by mixing the sense strand substrate, the antisense strand substrate and the RNA ligase, ATP, tris-HCl, mgCl ₂ and DTT are also included; preferably, the temperature of the enzyme-catalyzed reaction is from 0 to 60 ℃, more preferably from 4 to 37 ℃; preferably, the time of the enzyme-catalyzed reaction is from 0.5 to 24h, more preferably from 16 to 24 hours; preferably, the pH of the enzyme-catalyzed reaction is from 6.0 to 8.5; preferably, after the enzymatic reaction, purification is performed, and Cosdosiran is obtained after lyophilization.

By applying the technical scheme of the application, the preparation method is utilized, the sense strand of Cosdosiran is formed by connecting the sense strand substrates through RNA ligase catalysis, and the antisense strand of Cosdosiran is formed by connecting the antisense strand substrates, so that the siRNA with complex structure and multiple modifications is prepared by utilizing a biosynthesis mode. Compared with Cosdosiran prepared by chemical synthesis, the preparation method provided by the application has the advantages that the purity of the obtained product is higher, the impurity is less, the reaction condition is mild, and the industrial scale-up production is convenient to realize.

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 representation of the synthesis of Cosdosiran using RNA ligase according to the present invention;

FIG. 2 shows a chart of the Urea-PAGE detection of the catalytic products of ligase 25, ligase 48 and ligase 11 in example 1 of the present invention;

FIG. 3 shows the results of HPLC detection of ligase 48 in 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 deletion as compared to the target synthetic sequence.

N+1 impurity: nucleic acid impurities having a single nucleotide attached in addition to the target synthetic sequence.

As mentioned in the background art, the preparation of Cosdosiran in the prior art is performed by adopting a chemical synthesis mode, so that a large amount of organic solvents are used, the development concept of green chemistry is not met, and the cost of scale-up of the synthesis is very high. In addition, there are N+1 impurities and N-1 impurities which have low purity, high impurity content, and particularly are difficult to remove in the prepared product, and the subsequent purification of the product is affected. In the present application, the inventors have tried to develop a method for preparing siRNA for treating non-arterial ischemic optic neuropathy using enzyme-catalyzed synthesis Cosdosiran, and thus proposed a series of protection schemes of the present application.

In a first exemplary embodiment of the present application, there is provided a method for preparing an siRNA for treating non-arterial ischemic optic neuropathy, wherein the siRNA is Cosdosiran and Cosdosiran is double-stranded RNA consisting of complementary paired sense and antisense strands; the method comprises the following steps: mixing a sense strand substrate, an antisense strand substrate, and an RNA ligase, wherein the sense strand substrate is capable of constituting a sense strand and the antisense strand substrate is capable of constituting 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 and the antisense strand substrate 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 Cosdosiran; the bases at the two ends of the notch are respectively the 5 'end and the 3' end of different substrates, 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 Cosdosiran; the RNA ligase is selected from RNA ligase family 2; RNA ligase comprises SEQ ID NO:1 or SEQ ID NO:2, and one or more of the RNA ligases shown in fig. 2; or with SEQ ID NO:1 or SEQ ID NO:2, and has an activity of catalyzing the formation of phosphodiester bonds. Wherein, the double-stranded nucleotide structure containing the nick formed by the sense strand substrate and the antisense strand substrate has an adhesive end.

In the above preparation method, the sense strand substrate is 2 or more nucleotide sequences capable of constituting the sense strand, i.e., a plurality of nucleotide sequences of the sense strand substrate can be spliced to form the same sequence as the sense strand sequence, and is different from the sense strand in that there is a break between the sense strand substrates and is not linked by a phosphodiester bond. Similarly, 2 or more antisense strand substrates were linked by phosphodiester bonds using RNA ligase to obtain Cosdosiran antisense strands.

In the preparation method, the sense strand substrate, the antisense strand substrate and the RNA ligase are mixed together, and Cosdosiran is directly prepared by a one-pot method. In one-pot ligation, base complementary pairing can be performed between the sense strand substrate and the antisense strand substrate to form a double-stranded structure, and the RNA ligase recognizes the double-stranded structure and then joins the nicks existing in the double-stranded structure, thereby preparing the target product Cosdosiran.

Any method capable of performing RNA synthesis is suitable for use in the present application, and in a preferred embodiment, the sense strand substrate and the antisense strand substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method. In a preferred embodiment, the sense strand is SEQ ID NO:10, and the antisense strand is SEQ ID NO:11, and a nucleic acid sequence as set forth in seq id no.

SEQ ID NO：10：

GCCAGAAUGUGGAACUCCU。

SEQ ID NO：11：

AGmGAmGUmUCmCACmAUmUCmUGmGCm。

In the application, A, C, G or m after U represents 2' methoxy modification of the ribonucleotide; SEQ ID NO:10, and C at position 18 is cytosine deoxyribonucleotide in L form.

Cosdosiran the sense strand length is 19nt, the antisense strand is 19nt, the sense strand/antisense strand is divided into sections of sense strand/antisense strand substrates with different lengths by the connection efficiency, and the substrates are connected by enzyme catalytic reaction, so that a product with higher purity can be obtained. In a preferred embodiment, the sense strand consists of 2 and more sense strand substrates, the sense strand substrates having a length of 3-16nt, more preferably 4-12nt; preferably, the antisense strand consists of 2 and more antisense strand substrates, the length of which is 3-16nt, more preferably 5-14nt.

When Cosdosiran is synthesized by a one-pot method, as shown in fig. 1, base complementary pairing can be performed between a sense strand substrate and an antisense strand substrate to form a double-stranded structure, an RNA ligase recognizes the double-stranded structure formed by the complementary pairing of the substrates and provided with a notch, a phosphodiester reaction is catalyzed between two single-stranded RNA fragments to generate a new phosphodiester bond, and a molecule of water is released, so that the sense strand substrate and the antisense strand substrate are respectively connected to obtain Cosdosiran. In a preferred embodiment, the double-stranded RNA formed by annealing the sense strand substrate and the antisense strand substrate has 3 or more base combinations capable of complementary pairing. In a preferred embodiment, the double stranded RNA ends are cohesive ends; the adhesive end length is 2-7nt.

Any RNA ligase capable of recognizing a double-stranded structure with an absence of a complementary pairing of substrates that catalyzes the formation of a phosphodiester bond between a phosphate group and a hydroxyl group is suitable for use in the present application, and in a preferred embodiment, the RNA ligase is selected from the group consisting of RNA ligase family 2.RNA ligase family 2 is capable of catalyzing phosphodiester bond formation and of ligating multiple RNA molecules into longer strands. The ligase family 2 has high ligation activity on double-stranded RNA, can complete ligation reaction of single-stranded RNA, and can select proper enzymes for reaction according to the requirement of the reaction.

In a preferred embodiment, the RNA ligase comprises SEQ ID NO:1 or SEQ ID NO:2, and one or more of the RNA ligases shown in fig. 2; or with SEQ ID NO:1 or SEQ ID NO:2, including but not limited to, 75%, 80%, 85%, 90%, 95%, 99% (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, and even 99.9% or more), and has an activity of catalyzing phosphodiester bond formation.

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

MFKKYSSLENHYNSKFIEKLYTNGLTTGVWVAREKIHGTNFSLIIERDNVTCAKRTGPILPAEDFYGYEIVLKKYDKAIKAVQEVMESISTSVPVSYQVFGEFAGGGIQKGVDYGEKDFYVFDIIINTESDDTYYMSDYEMQDFCNTFGFKMAPMLGRGTFDSLIMIPNDLDSVLAAYNSTASEDLVEANNCVFDANVIGDNTAEGYVLKPCFPKWLSNGTRVAIKCKNSKFSEKKKSDKPVKTQVPLTEIDKNLLDVLACYVTLNRVNNVISKIGTVTPKDFGKVMGLTVQDILEETSREGIVLTSSDNPNLVKKELVRMVQDVLRPAWIELVS.

SEQ ID NO:2: (ligase 25,Vibrio phage NT-1)

MSFVKYTSLENSYRQAFVDKCDMLGVRDWVALEKIHGANFSFIVEFDGGYTVTPAKRTSIIGATATGDYDFYGCTSVVEAHKEKVELVANFLWLNEYINLYEPIIIYGELAGKGIQKEVNYGDKDFWAFDIFLPQREEFVDWDTCVAAFTNAEIKYTKELARGTLDELLRIDPLFKSLHTPAEHEGDNVAEGFVVKQLHSEKRLQSGSRAILKVKNEKFKEKKKKEGKTPTKLVLTPEQEKLHAEFSCYLTENRLKNVLSKLGTVNQKQFGMISGLFVKDAKDEFERDELNEVAIDRDDWNAIRRSLTNIANEILRKNWLNILDGNF.

SEQ ID NO:3: (ligase 11, thermococcus)

MVSSYFRNLLLKLGLPEERLEVLEGKGALAEDEFEGIRYVRFRDSARNFRRGTVVFETGEAVLGFPHIKRVVQLENGIRRVFKNKPFYVEEKVDGYNVRVVKVKDKILAITRGGFVCPFTTERIEDFVNFDFFKDYPNLVLVGEMAGPESPYLVEGPPYVKEDIEFFLFDIQEKGTGRSLPAEERYRLAEEYGIPQVERFGLYDSSKVGELKELIEWLSEEKREGIVMKSPDMRRIAKYVTPYANINDIKIGSHIFFDLPHGYFMGRIKRLAFYLAENHVRGEEFENYAKALGTALLRPFVESIHEVANGGEVDETFTVRVKNITTAHKMVTHFERLGVKIHIEDIEDLGNGYWRITFKRVYPDATREIRELWNGLAFVD.

SEQ ID NO:4: (ligase 20, archaea)

MVVPLKRIDKIRWEIPKFDKRMRVPGRVYADEVLLEKMKNDRTLEQATNVAMLPGIYKYSIVMPDGHQGYGFPIGGVAAFDVKEGVISPGGIGYDINCGVRLIRTNLTEKEVRPRIKQLVDTLFKNVPSGVGSQGRIKLHWTQIDDVLVDGAKWAVDNGYGWERDLERLEEGGRMEGADPEAVSQRAKQRGAPQLGSLGSGNHFLEVQVVDKIFDPEVAKAYGLFEGQVVVMVHTGSRGLGHQVASDYLRIMERAIRKYRIPWPDRELVSVPFQSEEGQRYFSAMKAAANFAWANRQMITHWVRESFQEVFKQDPEGDLGMDIVYDVAHNIGKVEEHEVDGKRVKVIVHRKGATRAFPPGHEAVPRLYRDVGQPVLIPGSMGTASYILAGTEGAMKETFGSTCHGAGRVLSRKAATRQYRGDRIRQELLNRGIYVRAASMRVVAEEAPGAYKNVDNVVKVVSEAGIAKLVARMRPIGVAKGAAALEH.

SEQ ID NO:5: (ligase 32, bacterium)

MVSLHFKHILLKLGLDKERIEILEMKGGIVEDEFEGLRYLRFKDSAKGLRRGTVVFNESDIILGFPHIKRVVHLRNGVKRIFKSKPFYVEEKVDGYNVRVAKVGEKILALTRGGFVCPFTTERIGDFINEQFFKDHPNLILCGEMAGPESPYLVEGPPYVEEDIQFFLFDIQEKRTGRSIPVEERIKLAEEYGIQSVEIFGLYSYEKIDELYELIERLSKEGREGVVMKSPDMKKIVKYVTPYANVNDIKIGSRIFFDLPHGYFMQRIKRLAFYIAEKRIRREDFDEYAKALGKALLQPFVESIWDVAAGEMIAEIFTVRVKKIETAYKMVSHFERMGLNIHIDDIEELGNGYWKITFKRVYDDATKEIRELWNGHAFVD.

Identity (Identity) in the present application refers to "Identity" between amino acid sequences or nucleic acid sequences, i.e. the sum of the ratios of amino acid residues or nucleotides of the same kind in the amino acid sequences or nucleic acid sequences. The identity of amino acid sequences or nucleic acid 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, and have the same active site, active pocket, active mechanism, protein structure, etc. as those provided by the a) sequence 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.

The application passes through SEQ ID NO:1 or SEQ ID NO:2, or an RNA ligase as set forth in SEQ ID NO:1 or SEQ ID NO:2, and the enzyme having an identity of 70% or more catalyzes the formation of a phosphodiester bond between substrates in the present application to give a product Cosdosiran. In the experiments related to the present application, the inventors obtained the above-mentioned SEQ ID NO capable of synthesizing Cosdosiran by screening from a large number of enzymes (50): 1 or SEQ ID NO: 2. While negative results with a very large ratio (up to 70%) in experiments show that most RNA ligases are difficult to catalyze Cosdosiran synthesis, including but not limited to SEQ ID NO: 3-SEQ ID NO:5, in the present description only the RNA ligase shown in SEQ ID NO: 3-SEQ ID NO:5 shows, for example, the RNA ligase which has no catalytic Cosdosiran synthesis activity.

In a preferred embodiment, the sense strand substrate and the antisense strand substrate each comprise 2 substrates, the sense strand substrate comprises a first sense strand substrate and a second sense strand substrate, and the antisense strand substrate comprises a first antisense strand substrate and a second antisense strand substrate; the preparation method comprises the following steps: mixing a first sense strand substrate, a second sense strand substrate, a first antisense strand substrate and a second antisense strand substrate, catalyzing the connection of the first sense strand substrate and the second sense strand substrate by using RNA ligase to form a sense strand, catalyzing the connection of the first antisense strand substrate and the second antisense strand substrate to form an antisense strand, and complementarily pairing the sense strand and the antisense strand to form Cosdosiran.

In a preferred embodiment, the 3 'end of the first sense strand substrate is linked to the 5' end of the second sense strand substrate under the catalysis of an RNA ligase to form a sense strand; the 3 'end of the first antisense strand substrate is connected with the 5' end of the second antisense strand substrate under the catalysis of RNA ligase to form an antisense strand; preferably, the 5 'end of the first sense strand substrate is an ia protecting group and the 3' end is a hydroxyl group; the 5 'end of the second sense strand substrate is a phosphate group, and the 3' end is a hydroxyl protecting group; preferably, the 5 'end of the first antisense strand substrate is a hydroxyl protecting group and the 3' end is a hydroxyl group; the 5 'end of the second antisense strand substrate is a phosphate group, and the 3' end is a hydroxyl protecting group.

In a preferred embodiment, the first sense strand substrate is SEQ ID NO:6 or 12, and the second sense strand substrate is SEQ ID NO:7 or 13.

In a preferred embodiment, the first antisense strand substrate is SEQ ID NO:9 or 15, and the second antisense strand substrate is SEQ ID NO:8 or 14.

Using the preparation method described above and SEQ ID NOs:6-9, SEQ ID NOs:12-15, can be prepared Cosdosiran. However, it should be noted that the choice of substrate is not limited to the above-mentioned SEQ ID NOs:6-9, SEQ ID NOs:12-15, the substrates capable of being combined to form the sense strand and the antisense strand can be applied to the preparation method, the preparation method is suitable for Cosdosiran preparation but not limited to the difference of substrate connection positions, and the preparation has good connection effect on the connection of the sense strand sequence and the antisense strand sequence of Cosdosiran. The number of sense strand substrates or antisense strand substrates includes, but is not limited to, 2, 3,4, or even more. Wherein, the C at the 12 th position in SEQ ID NO. 7 and the C at the 6 th position in SEQ ID NO. 13 are L-type cytosine deoxyribonucleotides.

SEQ ID NO：6：

GCCAGA。

SEQ ID NO：7：

AUGUGGAACUCCU。

SEQ ID NO：8：

CACmAUmUCmUGmGCm。

SEQ ID NO：9：

AGmGAmGUmUCm。

SEQ ID NO：12：

GCCAGAAUGUGG。

SEQ ID NO：13：

AACUCCU。

SEQ ID NO：14：

CmUGmGCm。

SEQ ID NO：15：

AGmGAmGUmUCmCACmAUmU。

In a preferred embodiment, the sense strand substrate and the antisense strand substrate each comprise 3 substrates, the sense strand substrate comprising a first sense strand substrate, a second sense strand substrate, and a third sense strand substrate; antisense strand substrates include a first antisense strand substrate, a second antisense strand substrate, and a third antisense strand substrate; preferably, the first sense strand substrate is SEQ ID NO:16, a nucleic acid sequence as set forth in seq id no; the second sense strand substrate is SEQ ID NO:17, a nucleic acid sequence as set forth in seq id no; the third sense strand substrate is SEQ ID NO:18, a nucleic acid sequence shown in seq id no; preferably, the first antisense strand substrate is SEQ ID NO:21, a nucleic acid sequence as set forth in seq id no; the second antisense strand substrate is SEQ ID NO:20, a nucleic acid sequence shown in seq id no; the third sense strand substrate is SEQ ID NO:19, and a nucleic acid sequence as set forth in seq id no. Wherein, the C at the 7 th position in SEQ ID NO. 18 is L-type cytosine deoxyribonucleotide.

SEQ ID NO：16：

GCCA。

SEQ ID NO：17：

GAAUGUG。

SEQ ID NO：18：

GAACUCCU。

SEQ ID NO：19：

AUmUCmUGmGCm。

SEQ ID NO：20：

UmUCmCACm。

SEQ ID NO：21：

AGmGAmG。

In a preferred embodiment, the concentration of sense and antisense strand substrates is 0.1-4.5mM, more preferably 0.8-1.6mM; preferably, the concentration of RNA ligase is 0.05-0.6mg/mL, more preferably 0.2mg/L; preferably, in the reaction system formed by mixing the sense strand substrate, the antisense strand substrate and the RNA ligase, ATP, tris-HCl, mgCl ₂ and DTT are also included; preferably, the temperature of the enzyme-catalyzed reaction is from 0 to 60 ℃, more preferably from 4 to 37 ℃; preferably, the time of the enzyme-catalyzed reaction is from 0.5 to 24h, more preferably from 16 to 24 hours; preferably, the pH of the enzyme-catalyzed reaction is from 6.0 to 8.5; preferably, after the enzymatic reaction, purification is performed, and Cosdosiran is obtained after lyophilization.

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 application is described in further detail below in connection with specific examples which are not to be construed as limiting the scope of the application as claimed.

Example 1

Based on Cosdosiran sequences, 4 single-stranded RNA fragments were designed as follows:

The above 4 single-stranded RNA fragments were prepared using a solid phase synthesis method. Wherein, A, C, G or m after U represents 2' methoxy modification of the ribonucleotide, and C at 12 th position in SEQ ID NO. 7 is L-type cytosine deoxyribonucleotide.

Cosdosiran-1 to the 5' end of which is added an ia protecting group; cosdosiran-2 is deoxyribonucleotide at position 12 and cytosine (C); cosdosiran-3 having 2' methoxy modifications at ribonucleotides at positions 3,5, 7,9 and 11; cosdosiran-4 have 2' methoxy modifications on ribonucleotides at positions 2, 4,6 and 8.

The sense strand of Cosdosiran obtained was 5'-R-GCCAGAAUGUGGAACUCCU-3' (SEQ ID NO: 10) and the antisense strand was 5'-AGmGAmGUmUCmCACmAUmUCmUGmGCm-3' (SEQ ID NO: 11). Wherein the 5' end of the sense strand is protected by an ia group (3 '5' reverse-linked deoxyribose).

Uniformly mixing 4 single-stranded RNA fragments in an equimolar ratio to obtain a substrate mixture, and annealing to obtain an annealed RNA fragment mixed solution. And carrying out enzyme catalytic ligation reaction on the annealed RNA fragment mixed solution, wherein the reaction conditions are as follows: the substrate fragment 100. Mu.M, ATP 10eq, mgCl ₂ 100eq, DTT 10eq, RNA ligase at a final concentration of 0.2 mg/mL, 4814V 50mM Tris-HCl pH 7.5 were added sequentially to a 10 uL reactor and reacted at 16℃for 16h. After the reaction, the protein was inactivated by heating at 80℃to 5min, and the supernatant was collected by centrifugation, and the result of the Urea-PAGE detection is shown in FIG. 2. In FIG. 2, lane M represents RNA molecular standard (marker), lane 1 represents the reaction system of ligase 25, lanes 2 and 3 represent the reaction system of ligase 48, and lane 4 represents the reaction system of ligase 11. The yields were estimated as grey scale analysis of product bands in the Urea-PAGE results, and the final yield results are shown in the following table:

In example 1, the yield was estimated as the result of gray scale analysis of the target band in the Urea-PAGE result, and "none" means that the target band was not detected, "++" indicates a yield of 25 to 50% (excluding 50% of the end point values), "+++" indicates a yield of 50 to 90%, ++++ indicates a yield >90%.

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). The ligation of ligases 25 and 48 was found to be good and was able to convert most of the substrate to Cosdosiran.

Example 2:

The ligase 48 with better reactivity is selected, and the annealed substrate fragments are used for enzyme catalytic ligation reaction under the following reaction conditions: the substrate fragment 800. Mu.M, ATP 4eq, mgCl ₂ 12.5eq,DTT 1.25 eq, RNA ligase at a final concentration of 0.2 mg/mL, 239V 50 mM Tris-HCl pH 7.5 were sequentially added to a 50 uL reactor and reacted at 16℃for 16 h. After the reaction, the protein was inactivated by heating at 80℃to 5min, and the supernatant was collected by centrifugation. The HPLC results of the assay for ligase 48 are shown in FIG. 3.

The yield was measured as the roughly estimated ratio of product peaks in the HPLC data of the reaction system samples, and the results were as follows:

In example 2, the yield was calculated as a statistic of the target peak in the HPLC result, ++ represents 70 to 80%, +++ represents 80 to 90%, ++ represents 80-90%.

LC-MS was used to identify the sense strand product as having a molecular weight 6221.90, the antisense strand product as having a molecular weight 6160.99, the sense strand product as theoretical 6221.89.+ -.8, and the antisense strand product as theoretical 6160.99.+ -.8, indicating that ligase 48 ligation produced Cosdosiran.

Comparative example 1

The average yield of the full-length Cosdosiran product by solid phase synthesis was 25.2% and the total n+1 and N-1 impurities was 1.51%.

The yield of Cosdosiran products produced by the enzymatic connection related by the invention in the enzymatic synthesis step is 68.73%, the average value of the solid phase synthesis yield of the used substrate is 41.3%, the yield of the whole process obtained by multiplication is 28.39%, the average yield of the total solid phase synthesis full-length Cosdosiran products is higher than that of the total N+1 and N-1 impurities in the Cosdosiran products synthesized by the enzymatic connection is 0.39%, and the impurity ratio of the total N+1 and N-1 impurities in the total solid phase synthesis full-length Cosdosiran products is lower than that of the total solid phase synthesis full-length Cosdosiran products.

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, RNA ligase catalyzes the sense strand of Cosdosiran to form the sense strand of the sense strand substrate and catalyzes the antisense strand of Cosdosiran to form the antisense strand of the antisense strand substrate, so that the siRNA with complex structure and various modifications is prepared by utilizing a biosynthesis mode. Compared with Cosdosiran prepared by chemical synthesis, the preparation method provided by the application has the advantages that the purity of the product is higher, the impurities are few (the content of N+1 and N-1 impurities is less than 0.5%), the subsequent final product purification pressure is small, the reaction condition is mild, a large amount of organic reagents are not used, the production cost can be reduced, and the industrialized amplified production is conveniently realized.

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 siRNA for treating non-arterial ischemic optic neuropathy, wherein the siRNA is Cosdosiran and the Cosdosiran is double-stranded RNA consisting of a sense strand and an antisense strand in complementary pair;

the method comprises the following steps:

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

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

The bases at the two ends of the notch are respectively the 5 'end and the 3' end of different substrates, 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 Cosdosiran;

The RNA ligase is selected from RNA ligase family 2;

The RNA ligase is SEQ ID NO:1 or SEQ ID NO:2, and one or more of the RNA ligases shown in fig. 2;

The sense strand is SEQ ID NO:10, and the antisense strand is a nucleic acid sequence shown in SEQ ID NO:11, a nucleic acid sequence as set forth in seq id no;

The sense strand consists of 2 or more sense strand substrates, and the length of each sense strand substrate is 3-16nt;

the antisense strand consists of 2 or more antisense strand substrates, and the length of the antisense strand substrates is 3-16nt.

2. The method of claim 1, wherein the sense strand substrate and the antisense strand substrate are obtained by a solid phase synthesis method or a liquid phase synthesis method.

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

4. The method of making according to claim 2, wherein the sense strand substrate and the antisense strand substrate each comprise 2 substrates, the sense strand substrate comprises a first sense strand substrate and a second sense strand substrate, and the antisense strand substrate comprises a first antisense strand substrate and a second antisense strand substrate;

The preparation method comprises the following steps: mixing the first sense strand substrate, the second sense strand substrate, the first antisense strand substrate and the second antisense strand substrate, catalyzing the ligation of the first sense strand substrate and the second sense strand substrate to form the sense strand using an RNA ligase, catalyzing the ligation of the first antisense strand substrate and the second antisense strand substrate to form the antisense strand, and complementarily pairing the sense strand and the antisense strand to form the Cosdosiran.

5. The method of claim 4, wherein the 3 'end of the first sense strand substrate is linked to the 5' end of the second sense strand substrate under the catalysis of the RNA ligase to form the sense strand; the 3 'end of the first antisense strand substrate and the 5' end of the second antisense strand substrate are connected under the catalysis of the RNA ligase to form the antisense strand;

The 5 'end of the first sense strand substrate is an ia protecting group, and the 3' end of the first sense strand substrate is a hydroxyl group; the 5 'end of the second sense strand substrate is a phosphate group, and the 3' end of the second sense strand substrate is a hydroxyl protecting group;

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

6. The method of claim 5, wherein the first sense strand substrate is SEQ ID NO:6 or 12, and the second sense strand substrate is the nucleic acid sequence set forth in SEQ ID NO:7 or 13.

7. The method of claim 5, wherein the first antisense strand substrate is SEQ ID NO:9 or 15, and the second antisense strand substrate is the nucleic acid sequence set forth in SEQ ID NO:8 or 14.

8. The method of claim 2, wherein the sense strand substrate and the antisense strand substrate each comprise 3 substrates,

The sense strand substrates include a first sense strand substrate, a second sense strand substrate, and a third sense strand substrate;

the antisense strand substrates include a first antisense strand substrate, a second antisense strand substrate, and a third antisense strand substrate.

9. The method of claim 8, wherein the first sense strand substrate is SEQ ID NO:16, a nucleic acid sequence as set forth in seq id no;

the second sense strand substrate is SEQ ID NO:17, a nucleic acid sequence as set forth in seq id no;

the third sense strand substrate is SEQ ID NO:18, and a nucleic acid sequence shown in seq id no.

10. The method of claim 8, wherein the first antisense strand substrate is SEQ ID NO:21, a nucleic acid sequence as set forth in seq id no;

The second antisense strand substrate is SEQ ID NO:20, a nucleic acid sequence shown in seq id no;

the third sense strand substrate is SEQ ID NO:19, and a nucleic acid sequence as set forth in seq id no.

11. A method of preparation according to any one of claims 1 to 3, wherein the concentration of the sense strand substrate and the antisense strand substrate is 0.1 to 4.5mM.

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