Method for preparing QPI-1002

Abstract

Provided is a method for preparing QPI-1002. QPI-1002 is a double-stranded RNA composed of a complementarily paired sense strand and antisense strand. The preparation method therefor comprises mixing a sense strand substrate fragment, an antisense strand substrate fragment and an RNA ligase, wherein the sense strand substrate fragment can form the sense strand, the antisense strand substrate fragment can form the antisense strand, the sense strand substrate fragment and the antisense strand substrate fragment are linked via a hydrogen bond formed by means of base complementary pairing, and the terminal bases of the sense strand substrate fragment and antisense strand substrate fragment are not linked to each other, thereby forming a double-stranded nucleotide structure containing a nick. The RNA ligase is used to link the bases at both ends of the nick via a phosphodiester bond to form QPI-1002. The preparation method can solve the problem of the relatively low purity of QPI-1002 in the preparation in the prior art.

Description

A method for preparing QPI-1002

[0001] This application is based on and claims priority to Chinese application CN application number 2024109217281 filed on July 10, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of drug biosynthesis, and more specifically, to a method for preparing QPI-1002. Background Technology

[0003] Small interfering RNA (siRNA) is a double-stranded RNA of 19-25 nt in length. After entering the cell, siRNA dissociates into single strands. The sense strand specifically binds to the messenger RNA (mRNA) of the target gene through base matching, inducing a series of actions that ultimately degrade the target gene's mRNA, preventing mRNA translation and thus inhibiting target gene expression. In recent years, the development of siRNA drugs has received widespread attention. siRNA drugs act on mRNA, making their target sites significantly larger than those of traditional small molecule drugs that act on proteins. Furthermore, siRNA drugs can target new sites by changing their sequence, resulting in relatively short development times.

[0004] QPI-1002 is a double-stranded siRNA drug developed by Quark and is currently in Phase 3 clinical trials. QPI-1002 can be used for kidney-related diseases, such as inhibiting major adverse kidney events (MAKE).

[0005] Currently, QPI-1002 is prepared using a chemical method, employing a solid-phase support such as controlled-porous glass (CPG) or polystyrene resin. The oligonucleotide chain is extended along the 3' to 5' direction via a cyclic synthesis using a phosphoramidite method. After the synthesis cycle is complete, the QPI-1002 chain is removed from the solid-phase support through ammonolysis, followed by purification to obtain the pure product. However, this solid-phase synthesis method is cyclical, and the yield decreases with increasing chain length. Furthermore, impurities generated during synthesis, such as those with one more nucleotide than the target sequence (N+1 impurities) or one less nucleotide than the target sequence (N-1 impurities), also increase with increasing chain length. These impurities are difficult to remove, leading to a complex and inefficient purification process. Therefore, the cost of QPI-1002 preparation is high, making large-scale production difficult. Thus, a more efficient QPI-1002 synthesis method needs to be developed. Summary of the Invention

[0006] The main objective of this invention is to provide a method for preparing QPI-1002, so as to solve the problem of low purity of QPI-1002 prepared in the prior art.

[0007] To achieve the above objectives, according to one aspect of the present invention, a method for preparing QPI-1002 is provided, wherein QPI-1002 is a double-stranded RNA composed of complementary positive and negative strands; the preparation method includes: mixing a positive strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the positive strand substrate fragment can form a positive strand, and the antisense strand substrate fragment can form an antisense strand; the positive strand substrate fragment and the antisense strand substrate fragment are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive strand substrate fragment and the antisense strand substrate fragment are not interconnected, forming a double-stranded RNA containing... The product has a nicked double-stranded nucleotide structure; the bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form QPI-1002; the bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; the 5' end phosphate group and the 3' end hydroxyl group of the upstream and downstream of the nick are linked by RNA ligase to form phosphodiester bonds, thus obtaining QPI-1002; the RNA ligase includes RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase of RNA ligase family 2 is selected from one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or an enzyme that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has catalytic activity in forming phosphodiester bonds.

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

[0009] Furthermore, the positive chain substrate fragment includes two or more segments, and the negative chain substrate fragment includes two or more segments; preferably, the length of the positive chain substrate fragment is 5-12 nt, more preferably 6-11 nt; preferably, the length of the negative chain substrate fragment is 2-16 nt, more preferably 3-14 nt.

[0010] Furthermore, both the sense and antisense substrate fragments consist of two components: the sense substrate fragment includes a first sense substrate fragment and a second sense substrate fragment, and the antisense substrate fragment includes a first antisense substrate fragment and a second antisense substrate fragment. The preparation method includes: S1) mixing the first sense substrate fragment, the second sense substrate fragment, the first antisense substrate fragment, the second antisense substrate fragment, and RNA ligase; S2) under the catalysis of RNA ligase, the first and second sense substrate fragments are ligated to form the sense strand, and the first and second antisense substrate fragments are ligated to form the antisense strand. The sense and antisense strands form QPI-1002 through complementary base pairing; preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, the first sense and antisense substrate fragments are annealed to form a first double-stranded RNA fragment, and the second sense and antisense substrate fragments are annealed to form a second double-stranded RNA fragment, and both the first and second double-stranded RNA fragments have ≥3nt complementary base pairing; preferably, the first and second double-stranded RNA fragments can form sticky ends with a length ≥2nt.

[0011] Further, in S1), the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate fragment is CmCUmUCmA; preferably, the nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate fragment is AUmAUmUCmUCm sequence.

[0012] Further, in S1), the nucleotide sequence of the first sense substrate fragment is GAmGAmAUmAUm, and the nucleotide sequence of the second sense substrate fragment is SEQ ID NO: 14; preferably, the first antisense substrate is SEQ ID NO: 16, and the second antisense substrate is UmUCmUCm.

[0013] Further, in S1, the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by 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 a hydroxyl 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.

[0014] Furthermore, both the sense and antisense substrate fragments comprise three components: a first sense substrate fragment, a second sense substrate fragment, and a third sense substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the preparation method includes: S1) mixing the first, second, and third sense substrate fragments, the first and second antisense substrate fragments, the third and third antisense substrate fragments, and an RNA ligase; S2) under the catalysis of the RNA ligase, the first and second sense substrate fragments are ligated to form a sense strand, and the ligation of the first and second antisense substrate fragments is catalyzed. The antisense strand is then formed, and the sense and antisense strands form QPI-1002 through base complementarity pairing; preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, in S1), the nucleotide sequence of the first sense substrate fragment is GAmGAmAUm; the nucleotide sequence of the second sense substrate fragment is AUmUUmCAmC; and the nucleotide sequence of the third sense substrate fragment is CmCUmUCmA; preferably, in S1), the nucleotide sequence of the first antisense substrate fragment is CmUCm; the nucleotide sequence of the second antisense substrate fragment is AAmAUmAUmU; and the nucleotide sequence of the third antisense substrate fragment is UmGAmAGmGGmUGm.

[0015] Furthermore, the concentrations of the sense and antisense substrate fragments are each independently selected from 0.1-4.5 mM, preferably 0.8-1.6 mM; preferably, the reaction system formed by mixing the sense and antisense substrate fragments and RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the reaction temperature of the preparation method is 0℃-60℃, more preferably 4℃-37℃; preferably, the reaction time of the preparation method is 0.5h-24h, more preferably 12-16h; preferably, the pH of the preparation method is 6-8.5.

[0016] By applying the technical solution of this invention and utilizing the above-described preparation method, under the catalysis of RNA ligase, the sense strand substrate fragments are ligated to form the QPI-1002 sense strand, and the antisense strand substrate fragments are ligated to form the QPI-1002 antisense strand, thereby realizing the preparation of this siRNA drug through biosynthesis. Compared with the chemical synthesis method for preparing QPI-1002, the preparation method of this application yields a product with high purity, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0018] Figure 1 shows a schematic diagram of the enzyme-catalyzed ligation reaction according to Example 1 of the present invention.

[0019] Figure 2 shows the electrophoretic results of the RNA ligase Ligase 31, Ligase 25 and Ligase 11 catalyzed products according to Example 1 of the present invention.

[0020] Figure 3 shows the HPLC detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention.

[0021] Figure 4 shows the LC-MS detection results of the RNA ligase Ligase 25 catalytic product according to Example 2 of the present invention. Detailed Implementation

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0023] Terminology Explanation:

[0024] N+1 impurities: Nucleic acid impurities that have an additional single nucleotide link compared to the target synthetic sequence.

[0025] N-1 impurities: Nucleic acid impurities that have a single nucleotide deletion compared to the target synthetic sequence.

[0026] As mentioned in the background section, existing technologies for preparing QPI-1002 employ chemical synthesis methods, which are not only complex and costly but also generate numerous N+1 and N-1 impurities, affecting subsequent product purification. In this application, the inventors attempt to develop a method for preparing QPI-1002 using enzyme-catalyzed synthesis, thus proposing a series of protective solutions.

[0027] In a first typical embodiment of this application, a method for preparing QPI-1002 is provided. QPI-1002 is a double-stranded RNA composed of complementary positive and negative strands. The preparation method includes: mixing a positive strand substrate fragment, an antisense strand substrate fragment, and an RNA ligase, wherein the positive strand substrate fragment can form a positive strand, and the antisense strand substrate fragment can form an antisense strand; the positive and antisense strand substrate fragments are linked by hydrogen bonds formed by complementary bases, and the head and tail bases of the positive and antisense strand substrate fragments are not interconnected, forming a double-stranded RNA containing a missing base. The nick is a double-stranded nucleotide structure; the bases at both ends of the nick are linked by phosphodiester bonds using RNA ligase to form QPI-1002; the bases at both ends of the nick are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; the phosphate group at the 5' end and the hydroxyl group at the 3' end of the nick are linked by RNA ligase to form phosphodiester bonds, thus obtaining QPI-1002; the RNA ligase includes RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase of RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase of RNA ligase family 2 is selected from one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; or an enzyme that has more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5 and has catalytic activity in forming phosphodiester bonds.

[0028] In the above preparation method, the positive strand substrate fragment is two or more nucleotide sequences that can form the positive strand. That is, multiple nucleotide sequences of the positive strand substrate fragment can be spliced ​​together to form a sequence identical to the positive strand sequence. The difference between the positive strand and the positive strand is that the positive strand substrate fragment has nicks and is not linked by phosphodiester bonds. Similarly, the antisense strand substrate fragment and the antisense strand have the above characteristics. Using RNA ligase, two or more positive strand substrate fragments or antisense strand substrate fragments are linked by phosphodiester bonds to obtain the positive and antisense strands of QPI-1002.

[0029] In the above preparation method, QPI-1002 can be prepared by mixing the sense and antisense substrate fragments with RNA ligase. In this preparation method, the sense and antisense substrate fragments can complement each other to form a double-stranded nucleotide with sticky ends. This double-stranded nucleotide with sticky ends can then bind to other substrate fragments to form a double-stranded nucleotide with nicks. RNA ligase can recognize the nicks in this double-stranded structure and link the nicks with phosphodiester bonds, thereby preparing the target product QPI-1002.

[0030] In a preferred embodiment, the nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO: 24.

[0031] SEQ ID NO: 23:

[0032] GAmGAmAUmAUmUUmCAmCCmCUmUCmA.

[0033] SEQ ID NO: 24:

[0034] UmGAmAGmGGmUGmAAmAUmAUmUCmUCm.

[0035] In this application, the m following A, C, G, or U indicates the 2' methoxy modification of the ribonucleotide.

[0036] In a preferred embodiment, the RNA ligase comprises RNA ligases from RNA ligase family 1 and / or RNA ligase family 2; preferably, the RNA ligase from RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; the RNA ligase from RNA ligase family 2 is selected from one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4; or in combination with SEQ ID NO: 1 to SEQ ID NO: 4. Any RNA ligase shown in NO:5 has more than 70% identity, including but not limited to 75%, 80%, 85%, 90%, 95%, 99% or more (e.g., more than 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 even more than 99.9%) and is an enzyme that catalyzes the formation of phosphodiester bonds.

[0037] All of the above RNA ligases can recognize the notched double-stranded structure formed by complementary pairing of substrate fragments, thereby catalyzing the formation of phosphodiester bonds between phosphate groups and hydroxyl groups.

[0038] SEQ ID NO: 1 (Ligase 25, Vibrio phage NT-1):

[0039] SEQ ID NO: 2 (Ligase 26, Escherichia phage AR1):

[0040] SEQ ID NO: 3 (Ligase 31, Vibrio phage VH12019):

[0041] SEQ ID NO: 4: (Ligase 41, Vibrio phage VH7D):

[0042] SEQ ID NO: 5 (Ligase 42, Escherichia phage JN02):

[0043] SEQ ID NO: 6: (Ligase 11, Thermococcus):

[0044] SEQ ID NO: 7: (Ligase 20, Archaea):

[0045] SEQ ID NO: 8: (Ligase 32, Bacteria):

[0046] In this application, "identity" refers to the "identity" between amino acid sequences or nucleotide sequences, that is, the total ratio of the same type of amino acid residues or nucleotides in the amino acid sequence or nucleotide sequence. The identity of amino acid sequences or nucleotide sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0047] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, or more than 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 even more than 99.9%) of identicality and function are highly likely to have the same active site, active pocket, active mechanism, and protein structure as the proteins provided by the above sequences.

[0048] As used herein, the amino acid residue abbreviations are 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).

[0049] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substitution are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0050] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0051] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0052] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0053] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0054] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0055] In this application, only RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, or enzymes with more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, can catalyze the formation of phosphodiester bonds between the phosphate group and the hydroxyl group of the substrate in this application to obtain the product QPI-1002. In the relevant experiments of this application, the inventors obtained the aforementioned RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5 that can synthesize QPI-1002 by screening from a large number of enzymes. However, the large proportion of negative results in the experiments showed that most RNA ligases are difficult to catalyze the synthesis of QPI-1002, including but not limited to the RNA ligases shown in SEQ ID NO: 6 to SEQ ID NO: 8. In this application specification, only SEQ ID NO: 6 to SEQ ID NO: 8 are used as examples to illustrate this type of RNA ligase that does not have the activity to catalyze the synthesis of QPI-1002.

[0056] In a preferred embodiment, the positive chain substrate fragment includes two or more segments, and the negative chain substrate fragment includes two or more segments; preferably, the length of the positive chain substrate fragment is 5-12 nt, more preferably 6-11 nt; preferably, the length of the negative chain substrate fragment is 2-16 nt, more preferably 3-14 nt.

[0057] In a preferred embodiment, both the sense and antisense substrate fragments comprise two fragments. The sense substrate fragment includes a first sense substrate fragment and a second sense substrate fragment, and the antisense substrate fragment includes a first antisense substrate fragment and a second antisense substrate fragment. The preparation method includes: S1) mixing the first sense substrate fragment, the second sense substrate fragment, the first antisense substrate fragment, and the second antisense substrate fragment; S2) under the catalysis of RNA ligase, the first sense substrate fragment and the second sense substrate fragment are ligated to form a sense strand, and the first antisense substrate fragment and the second antisense substrate fragment are ligated to form an antisense strand. The sense and antisense strands form QPI-1002 through base complementarity pairing; preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, the first sense and antisense substrate fragments are annealed to form a first double-stranded RNA fragment, and the second sense and antisense substrate fragments are annealed to form a second double-stranded RNA fragment, and both the first and second double-stranded RNA fragments have ≥3nt of base complementarity pairing; preferably, the first and second double-stranded RNA fragments can form sticky ends with a length ≥2nt.

[0058] In the above preparation method, the sense and antisense substrate fragments are first mixed and annealed. The sense and antisense substrate fragments can form a double-stranded RNA structure through base complementarity, and this double-stranded RNA structure contains nicks between the different substrate fragments. The annealed reaction system is then mixed with RNA ligase, which uses phosphodiester bonds to connect the phosphate and hydroxyl groups on both sides of the nick, repairing the nick and thus obtaining the target product QPI-1002 with a complete double-stranded structure.

[0059] In a preferred embodiment, in S1), the nucleotide sequence of the first sense substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second sense substrate fragment is CmCUmUCmA. Preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate is AUmAUmUCmUCm.

[0060] In a preferred embodiment, in S1), the nucleotide sequence of the first sense substrate is GAmGAmAUmAUm, and the nucleotide sequence of the second sense substrate is SEQ ID NO: 14. Preferably, the nucleotide sequence of the first antisense substrate is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate is UmUCmUCm.

[0061] In a preferred embodiment, in S2), the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by RNA ligase to form a sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by 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 a hydroxyl 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.

[0062] QPI-1002 can be prepared using the above-described preparation method and substrate fragments. However, it should be noted that the selection of substrate fragments is not limited to those described above; any substrate fragment capable of forming both the sense and antisense strands can be used in the above preparation method. The above preparation method is applicable to the preparation of QPI-1002 but is not limited to different substrate fragment connection positions. The above preparation method shows good ligation effects for both the sense and antisense strand sequences of QPI-1002. The number of sense or antisense substrate fragments includes, but is not limited to, 2, 3, 4, or even more.

[0063] SEQ ID NO: 9: GAmGAmAUmAUmUUmCAmC.

[0064] SEQ ID NO: 12: UmGAmAGmGGmUGmAAm.

[0065] SEQ ID NO: 14: UUmCAmCCmCUmUCmA.

[0066] SEQ ID NO: 15: UmUCmUCm.

[0067] SEQ ID NO: 16: UmGAmAGmGGmUGmAAmAUmA.

[0068] In a preferred embodiment, both the positive and negative substrate fragments comprise three segments: a first positive substrate fragment, a second positive substrate fragment, and a third positive substrate fragment; and a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment. Preferably, the preparation method includes: S1) mixing the first, second, and third positive substrate fragments, the first and second antisense substrate fragments, the third and first antisense substrate fragments, and an RNA ligase; S2) under the catalysis of the RNA ligase, the first and second positive substrate fragments are ligated to form a positive strand, catalyzing the ligation of the first and second antisense substrate fragments. Fragments are joined to form an antisense strand, and the sense and antisense strands form QPI-1002 through base complementarity pairing; preferably, the sense and antisense substrate fragments are annealed and then mixed with RNA ligase to obtain QPI-1002; preferably, in S1), the nucleotide sequence of the first sense substrate fragment is GAmGAmAUm; the nucleotide sequence of the second sense substrate fragment is AUmUUmCAmC; and the nucleotide sequence of the third sense substrate fragment is CmCUmUCmA; preferably, in S1), the nucleotide sequence of the first antisense substrate fragment is CmUCm; the nucleotide sequence of the second antisense substrate fragment is AAmAUmAUmU; and the nucleotide sequence of the third antisense substrate fragment is UmGAmAGmGGmUGm.

[0069] In a preferred embodiment, the concentrations of the sense and antisense substrate fragments are each selected from 0.1-4.5 mM, preferably 0.8-1.6 mM; preferably, the reaction system formed by mixing the sense and antisense substrate fragments and RNA ligase further includes ATP, Tris-HCl, MgCl2, and DTT; preferably, the reaction temperature of the preparation method is 0℃-60℃, more preferably 4℃-37℃; preferably, the reaction time of the preparation method is 0.5h-24h, more preferably 12-16h; preferably, the pH of the preparation method is 6-8.5.

[0070] The concentrations of the aforementioned sense and antisense substrate fragments are each selected from, but not limited to, 0.1, 0.5, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 mM; the reaction temperatures of the aforementioned preparation methods are, but not limited to, 10, 15, 16, 20, 25, 30, 35, or 40 °C; the reaction times of the aforementioned preparation methods are, but not limited to, 2, 5, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, or 48 h; and the pH of the aforementioned preparation methods is, but not limited to, 6, 6.5, 7, 7.5, 8, or 8.5.

[0071] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0072] Example 1

[0073] The sequence based on QPI-1002 is designed into four single-stranded RNA fragments as shown in Table 1, with the length unit being nt.

[0074] Table 1

[0075] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide.

[0076] The ribonucleotides at positions 2, 4, 6, 8, 10, and 12 of substrate 1 have a 2' methoxy group modification.

[0077] The ribonucleotides at positions 1, 3, and 5 of substrate 2 have 2' methoxy groups modified.

[0078] The ribonucleotides at positions 2, 4, 6, and 8 of substrate 3 have 2' methoxy groups modified.

[0079] The ribonucleotides at positions 1, 3, 5, 7, 9, and 11 of substrate 4 have 2' methoxy groups modified.

[0080] The above four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0081] Four single-stranded RNA fragments were mixed in equimolar proportions to obtain a substrate mixture with a final concentration of 2.5 mM (2.5 mM for each substrate). The mixture was then annealed to obtain an annealed RNA fragment mixture. The annealed RNA fragment mixture was subjected to an enzymatic ligation reaction in a 10 μL volume. The reaction system included reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, and dithiothreitol (DTT). RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase 11, Ligase 20, and Ligase 32 were added, respectively. The reaction system was incubated at 16 °C for 16 h. The resulting reaction system was then subjected to 80 °C for 5 min to inactivate the ligases, and the precipitate was removed by centrifugation at 12000 rpm. A schematic diagram of the enzyme-catalyzed ligation reaction is shown in Figure 1.

[0082] The products catalyzed by RNA ligases Ligase 25, Ligase 26, Ligase 31, Ligase 41, Ligase 42, Ligase 11, Ligase 20, and Ligase 32 were analyzed by SDS-PAGE. The electrophoresis results of the products catalyzed by RNA ligases Ligase 31, Ligase 25, and Ligase 11 are shown in Figure 2. In Figure 2, lane M represents the RNA molecule marker, lane 1 represents the reaction system of Ligase 31, lane 2 represents the reaction system of Ligase 25, and lane 3 represents the reaction system of Ligase 11. The yield was estimated based on the grayscale analysis of the target bands in the Urea-PAGE results, and the final yield results are shown in Table 2.

[0083] Activity screening of 10 RNA ligases revealed that Ligase25 exhibited good ligation performance, converting most of the substrate into QPI-1002.

[0084] The positive chain of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the negative chain is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

[0085] The reaction results are shown in Table 2.

[0086] Table 2

[0087] Remark:

[0088] 1) Reaction conditions: 100 μM substrate fragment, 10 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 100 μM), final concentration of 0.2 mg / mL enzyme, 2385 V, 50 mM Tris-HCl, pH 7.5, 16℃, 16 h;

[0089] 2) ND indicates no product generation detected, ++ indicates 25-50% (excluding the 50% endpoint value), +++ indicates 50-75%, and ++++ indicates >75%.

[0090] The grayscale data of the product and substrate were obtained by grayscale analysis of the Urea-PAGE gel electrophoresis results image. In this example, the yield calculation formula is: Yield = Product grayscale data / (Product grayscale data + Substrate grayscale data).

[0091] Example 2:

[0092] Ligase 25 with good reactivity was selected, and the annealed substrate fragment was used for enzyme-catalyzed ligation reaction. The reaction conditions were as follows: reaction buffer (50mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT) and RNA ligase were added sequentially to a 50μL reactor, and the reaction was carried out at 16℃ for 16h.

[0093] After the reaction, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The samples were analyzed by HPLC and LC-MS. The HPLC results of the Ligase 25 catalyzed product are shown in Figure 3. The yield was measured by the rough estimate of the proportion of the product peak in the HPLC data of the reaction system sample, and the results are shown in Table 3.

[0094] Table 3

[0095] Remark:

[0096] 1) Reaction conditions: 800 μM substrate fragment, 4 eq ATP, 100 eq MgCl2, 10 eq DTT (1 eq = 800 μM), final concentration of 0.2 mg / mL enzyme, 298 V, 50 mM Tris-HCl, pH 7.5, 16℃, 16 h;

[0097] 2) + indicates <50%.

[0098] The molecular weight of the positive chain product was determined by LC-MS to be 6089.9 and the molecular weight of the antisense chain product was 6223.9. The theoretical value of the positive chain product was 6089.96±8 and the theoretical value of the antisense chain product was 6224.0±8, indicating that Ligase 25 was linked to form QPI-1002. The detection results are shown in Figure 4.

[0099] Example 3

[0100] The annealed substrate fragment and Ligase 25 were used for enzymatic ligation under the following conditions: Reaction buffer (50 mM Tris-HCl, pH 7.5), adenosine triphosphate (ATP), MgCl2, dithiothreitol (DTT), and RNA ligase were added sequentially to a 10 mL reactor, and the reaction was carried out at 16 °C for 16 h. After the overnight reaction, the protein was inactivated by heating at 50 °C for 15 min. The supernatant was collected by centrifugation, purified using a Nano-Q column, eluted with a NaCl gradient, desalted by membrane coating (molecular weight cutoff 1 kDa), and lyophilized to obtain a dry powder. The calculated yield was 68.73%, and the purity (HPLC detection) was 95.44%.

[0101] Example 4

[0102] The sequence based on QPI-1002 is designed into four single-stranded RNA fragments as shown in Table 4, with the length unit being nt.

[0103] Table 4

[0104] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide.

[0105] The ribonucleotides at positions 2, 4, 6, and 8 of substrate 5 have 2' methoxy groups modified.

[0106] The ribonucleotides at positions 2, 4, 6, 8, and 10 of substrate 6 have 2' methoxy groups modified.

[0107] The ribonucleotides at positions 1, 3, and 5 of substrate 7 have a 2' methoxy group modification.

[0108] The ribonucleotides at positions 1, 3, 5, 7, 9, 11, and 13 of substrate 8 have 2' methoxy groups modified.

[0109] The above four single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0110] The positive chain of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the negative chain is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

[0111] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 50 μL. 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), and Ligase 25 at a final concentration of 0.2 mg / mL were added sequentially to the reactor. 50 mM Tris-HCl was added at 239V, pH 7.5, and the reaction was carried out at 16℃ for 16 h. After the reaction, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The results were analyzed by HPLC. The yield was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 72.3% of the sample, indicating a yield of +++.

[0112] Example 5

[0113] The six single-stranded RNA fragments based on the QPI-1002 sequence are shown in Table 5, with length units of nt.

[0114] Table 5

[0115] In this context, the 'm' following A, C, G, or U indicates a 2' methoxy group modification of the ribonucleotide.

[0116] The ribonucleotides at positions 2, 4, and 6 of substrate 9 have 2' methoxy groups modified.

[0117] The ribonucleotides at positions 2, 4, and 6 of substrate 10 have 2' methoxy groups modified.

[0118] The ribonucleotides at positions 1, 3, and 5 of substrate 11 have a 2' methoxy group modification.

[0119] The ribonucleotides at positions 1 and 3 of substrate 12 have 2' methoxy groups modified.

[0120] The ribonucleotides at positions 2, 4, and 6 of substrate 13 have 2' methoxy groups modified.

[0121] The ribonucleotides at positions 1, 3, 5, 7, and 9 of substrate 14 have 2' methoxy groups modified.

[0122] The above six single-stranded RNA fragments were prepared using a solid-phase synthesis method.

[0123] The positive chain of the prepared QPI-1002 is GAmGAmAUmAUmUUmCAmCCmCUmUCmA (SEQ ID NO: 23); the negative chain is UmGAmAGmGGmUGmAAmAUmAUmUCmUCm (SEQ ID NO: 24).

[0124] An annealed substrate fragment and ligase Ligase 25 were used for enzymatic ligation. The reaction conditions were as follows: the reaction system was set to 50 μL. 800 μM substrate fragment, 4 eq ATP, 12.5 eq MgCl2, 1.25 eq DTT (1 eq = 800 μM), and Ligase 25 at a final concentration of 0.2 mg / mL were added sequentially to the reactor. 50 mM Tris-HCl was added at 239V, pH 7.5, and the reaction was carried out at 16℃ for 16 h. After the reaction, the protein was inactivated by heating at 80℃ for 5 min, and the supernatant was collected by centrifugation. The results were analyzed by HPLC. The yield was measured by the approximate percentage of the product peak in the HPLC data of the reaction system sample. The results showed that the target peak accounted for 79.6% of the sample, indicating a yield of +++.

[0125] Comparative Example 1

[0126] The average yield of the full-length QPI-1002 product synthesized by solid-phase synthesis was 29.5%, with N+1 and N-1 impurities accounting for a total of 1.50%.

[0127] The yield of QPI-1002 product prepared by the enzyme-linked method of this invention was 66.18%, while the average yield of the substrates used in solid-phase synthesis was 45.9%. Multiplying these yields, the overall yield of the process was 30.3%, which is higher than the average yield of the product obtained by solid-phase synthesis. Furthermore, the total proportion of N+1 and N-1 impurities was 0.38%, which is lower than the proportion of such impurities in the solid-phase synthesis of QPI-1002.

[0128] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the preparation method of this application, four short substrate fragments (less than 19 nt in length) are first synthesized, and then the full-length QPI-1002 is synthesized by enzymatic ligation, thereby realizing the preparation of this siRNA drug by biosynthesis. Due to the shortening of the length of the synthesized fragments, the N+1 and N-1 impurities generated during the synthesis process are correspondingly reduced; the ligation efficiency of N+1 and N-1 impurities in the substrate fragments is reduced, further reducing the N+1 and N-1 impurities in the full-length product; in addition, the chain lengths of N+1 and N-1 impurities in the substrate fragments are significantly different from those of the full-length product, making them easy to remove during purification, and the final content of N+1 and N-1 impurities is <0.5%. Compared with the chemical synthesis preparation method, the preparation method of this application yields a product with high purity, generates fewer impurities, has a simple preparation process, mild reaction conditions, low organic reagent consumption, reduces production costs, and facilitates large-scale industrial production.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing QPI-1002, characterized in that, The QPI-1002 is a double-stranded RNA composed of complementary sense and antisense strands; The preparation method includes: A positive sense substrate fragment, an antisense substrate fragment, and an RNA ligase are mixed, wherein the positive sense substrate fragment is capable of forming the positive sense strand, and the antisense substrate fragment is capable of forming the antisense strand; The positive and negative substrate fragments are connected by hydrogen bonds formed by complementary bases. The head and tail bases of the positive and negative substrate fragments are not connected to each other, forming a double-stranded nucleotide structure with notches. The bases at both ends of the notch are linked by phosphodiester bonds using the RNA ligase to form QPI-1002; The bases at both ends of the notch are the 5' and 3' ends of different substrate fragments, respectively, with the 5' end being a phosphate group and the 3' end being a hydroxyl group; The phosphate group at the 5' end and the hydroxyl group at the 3' end of the notch are linked together using the RNA ligase to form the phosphodiester bond, thereby obtaining QPI-1002.

2. The preparation method according to claim 1, characterized in that, The RNA ligase includes RNA ligases from RNA ligase family 1 and / or RNA ligase family 2. The RNA ligase of the RNA ligase family 1 is the RNA ligase shown in SEQ ID NO: 5; The RNA ligase of the RNA ligase family 2 is selected from one or more of the RNA ligases shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4; It may have more than 70% identity with any of the RNA ligases shown in SEQ ID NO: 1 to SEQ ID NO: 5, and have the activity of catalyzing the formation of the phosphodiester bond.

3. The preparation method according to claim 1, characterized in that, The nucleotide sequence of the sense strand is SEQ ID NO: 23, and the nucleotide sequence of the antisense strand is SEQ ID NO:

24.

4. The preparation method according to any one of claims 1-3, characterized in that, The justice chain substrate fragment includes two or more fragments, and the antisense chain substrate fragment includes two or more fragments; The length of the substrate segment of the justice chain is 5-12 nt; The antisense substrate fragment is 2-16 nt in length.

5. The preparation method according to claim 4, characterized in that, Both the justice chain substrate segment and the antisense chain substrate segment include two segments. The justice chain substrate segment includes a first justice chain substrate segment and a second justice chain substrate segment. The antisense chain substrate segment includes a first antisense chain substrate segment and a second antisense chain substrate segment. The preparation method includes: S1) Mix the first sense strand substrate fragment, the second sense strand substrate fragment, the first antisense strand substrate fragment, the second antisense strand substrate fragment, and the RNA ligase; S2) Under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are joined to form the sense strand, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are joined to form the antisense strand. The sense strand and the antisense strand form the QPI-1002 through complementary base pairing. The first sense strand substrate fragment and the first antisense strand substrate fragment anneal to form a first double-stranded RNA fragment, and the second sense strand substrate fragment and the second antisense strand substrate fragment anneal to form a second double-stranded RNA fragment. Both the first double-stranded RNA fragment and the second double-stranded RNA fragment have ≥3nt complementary base pairings. The first double-stranded RNA fragment and the second double-stranded RNA fragment can form a sticky end, the length of which is ≥2nt.

6. The preparation method according to claim 5, characterized in that, In S1), the nucleotide sequence of the first positive strand substrate fragment is SEQ ID NO: 9, and the nucleotide sequence of the second positive strand substrate fragment is CmCUmUCmA; The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 12, and the nucleotide sequence of the second antisense substrate fragment is AUmAUmUCmUCm.

7. The preparation method according to claim 5, characterized in that, In S1), the nucleotide sequence of the first positive-strand substrate fragment is GAmGAmAUmAUm, and the nucleotide sequence of the second positive-strand substrate fragment is SEQ ID NO:

14. The nucleotide sequence of the first antisense substrate fragment is SEQ ID NO: 16, and the nucleotide sequence of the second antisense substrate fragment is UmUCmUCm.

8. The preparation method according to claim 5, characterized in that, In step S1), the 3' end of the first sense strand substrate fragment and the 5' end of the second sense strand substrate fragment are ligated by the RNA ligase to form the sense strand; the 3' end of the first antisense strand substrate fragment and the 5' end of the second antisense strand substrate fragment are ligated by the RNA ligase to form the antisense strand.

9. The preparation method according to claim 8, characterized in that, The first positive-chain substrate fragment has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second positive-chain substrate fragment has a phosphate group at its 5' end and a hydroxyl group at its 3' end. The first antisense substrate fragment has a hydroxyl group at its 5' end and a hydroxyl group at its 3' end; the second antisense substrate fragment has a phosphate group at its 5' end and a hydroxyl group at its 3' end.

10. The preparation method according to claim 4, characterized in that, Both the justice chain substrate segment and the antisense chain substrate segment include three segments. The substrate segment of the justice chain includes a first justice chain substrate segment, a second justice chain substrate segment, and a third justice chain substrate segment; The antisense substrate fragment includes a first antisense substrate fragment, a second antisense substrate fragment, and a third antisense substrate fragment.

11. The preparation method according to claim 10, characterized in that, The positive and negative substrate fragments each comprise three fragments, and the preparation method includes: S1) mixing the first positive substrate fragment, the second positive substrate fragment, the third positive substrate fragment, the first negative substrate fragment, the second negative substrate fragment, the third negative substrate fragment, and the RNA ligase; S2) Under the catalysis of the RNA ligase, the first sense strand substrate fragment and the second sense strand substrate fragment are joined to form the sense strand, and the first antisense strand substrate fragment and the second antisense strand substrate fragment are joined to form the antisense strand. The sense strand and the antisense strand form the QPI-1002 through complementary base pairing.

12. The preparation method according to claim 11, characterized in that, In S1), the nucleotide sequence of the first positive-strand substrate fragment is GAmGAmAUm; The nucleotide sequence of the second positive-strand substrate fragment is AUmUUmCAmC; The nucleotide sequence of the third positive-strand substrate fragment is CmCUmUCmA; The first antisense chain substrate fragment is CmUCm; The nucleotide sequence of the second antisense substrate fragment is AAmAUmAUmU; The nucleotide sequence of the third antisense substrate fragment is UmGAmAGmGGmUGm.

13. The preparation method according to any one of claims 1-3, characterized in that, The concentrations of the sense substrate fragment and the antisense substrate fragment are each independently selected from 0.1-4.5 mM; The reaction system formed by mixing the sense substrate fragment, the antisense substrate fragment, and the RNA ligase also includes ATP, Tris-HCl, MgCl2, and DTT.

14. The preparation method according to claim 1, characterized in that, The reaction temperature of the preparation method is 0℃~60℃; The reaction time for the preparation method is 0.5 h to 24 h; The pH of the preparation method is 6 to 8.5.

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