Double-stranded RNA molecule and modification method therefor

Abstract

Provided are a double-stranded RNA molecule and a modification method therefor, relating to the technical field of nucleic acids.

Description

Double-stranded RNA molecules and their modification methods

[0001] This application claims priority to Chinese Patent Application No. 202510177782.4, filed February 18, 2025; Chinese Patent Application No. 202510341076.9, filed March 21, 2025; Chinese Patent Application No. 202510402460.5, filed April 1, 2025; and Chinese Patent Application No. 202511124329.3, filed August 12, 2025. The entire contents of each application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of nucleic acid technology, specifically relating to the modification methods of double-stranded RNA molecules. Background Technology

[0003] Small interfering RNA (siRNA), as a gene silencing technology, has broad application prospects in the field of disease treatment. In recent years, research progress on siRNA modification methods has mainly focused on improving siRNA stability, specificity, reducing immunogenicity, and developing efficient delivery systems. siRNA molecules can be chemically modified to enhance their stability and reduce off-target effects. Common chemical modifications include phosphate thiocyanate backbone modification, methoxylation, fluorination, GNA modification, and 5'-(E)-VP modification. These modifications help improve the stability of siRNA in vivo, reduce the risk of degradation by nucleases, and reduce non-specific binding and immune responses. However, while reducing off-target effects, a decrease in activity is inevitable. There is still a need in this field for a new combination of modifications that can enhance activity while further reducing siRNA off-target effects and maintaining its activity in animals. Summary of the Invention

[0004] The technical problem this application aims to solve is: how to improve the effectiveness of siRNA, such as improving siRNA stability, specificity, reducing immunogenicity, and delivery efficiency.

[0005] To address the aforementioned technical problems, this application provides a double-stranded RNA molecule comprising a sense strand and an antisense strand that at least partially form a double-stranded region, wherein each nucleotide of the sense strand and the antisense strand is independently modified, wherein...

[0006] Following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluorinated nucleotides, the nucleotides at positions 2, 10, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotide at position 6 or 8 of the antisense strand is a 2'-fluorinated nucleotide.

[0007] In some embodiments, the 12th nucleotide of the antisense strand is a deoxyribonucleotide, oriented from the 5' end to the 3' end. In some embodiments, when the 12th ribonucleotide is uracil ribonucleotide, thymine deoxyribonucleotide is used instead of uracil ribonucleotide.

[0008] In some embodiments, the other nucleotides of the sense and / or antisense strands are 2'-methoxy modified.

[0009] In some embodiments, at least one phosphate group in the sense or antisense chain is a phosphate group with a modifying group.

[0010] Furthermore, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0011] Furthermore, the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

[0012] In some implementations, the double-stranded RNA molecule may also be linked to an inverted abase-free nucleotide.

[0013] Furthermore, the first nucleotide at the 5' end and / or the first nucleotide at the 3' end of the positive strand are both linked to a reverse abasic nucleotide via a thiophosphate group.

[0014] In some implementations, the first nucleotide of the antisense strand, starting from the 5' end, contains a VP modification.

[0015] In some embodiments, the RNA molecule includes:

[0016] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are 2' fluorinated nucleotides, and the nucleotides at position 1 and the penultimate position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group.

[0017] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and position 12 is a deoxyribonucleotide. When the ribonucleotide at this position is uracil ribonucleotide, it is replaced with thymine deoxyribonucleotide. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups.

[0018] Other nucleotides are nucleotides modified with a methoxy group at the 2' position; or

[0019] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are 2' fluorinated nucleotides, and the nucleotides at position 1 and the penultimate position are each connected to a reverse non-base nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group.

[0020] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and position 12 is a deoxyribonucleotide. When the ribonucleotide at this position is uracil ribonucleotide, it is replaced with thymine deoxyribonucleotide. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups.

[0021] The other nucleotides are nucleotides modified with a methoxy group at the 2' position.

[0022] Furthermore, the sense strand and antisense strand of the double-stranded RNA molecule are complementary to form the double-stranded region of siRNA, the 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region;

[0023] Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, the 5' end of the sense strand forms a blunt end, and the 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region.

[0024] Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, with the 3' end of the sense strand forming a blunt end and the 3' end of the antisense strand forming a blunt end;

[0025] Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, and the 3' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region, or the 5' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region.

[0026] Further, the antisense strand may be 15-30 nucleotides in length; further, the antisense strand may be 19-30 nucleotides in length; further, the antisense strand may be 19-25 nucleotides in length; further, the antisense strand may not exceed 23 nucleotides in length; further, the antisense strand may be 21 nucleotides in length; or, the antisense strand may be 23 nucleotides in length.

[0027] Further, in the double-stranded RNA molecule, the length of the double-stranded region can be 17-21 nucleotides; further, in the double-stranded RNA molecule, the length of the double-stranded region can be 19-21 nucleotides; further, in the double-stranded RNA molecule, the length of the double-stranded region can be 21 nucleotides; or, in the double-stranded RNA molecule, the length of the double-stranded region can be 19 nucleotides.

[0028] Further, in the double-stranded RNA molecule, the length of the sense strand may not exceed 30 nucleotides; further, the length of the sense strand may be 15-30 nucleotides; further, the length of the sense strand may be 19-30 nucleotides; further, the length of the sense strand may be 19-25 nucleotides; further, the length of the sense strand may be 23 nucleotides; or, the length of the sense strand may be 21 nucleotides; or, the length of the sense strand may be 19 nucleotides.

[0029] In some implementations, the antisense strand is 21 nucleotides long, and the sense strand is 21 nucleotides long.

[0030] The present invention also provides a conjugate comprising the double-stranded RNA molecule described above, and a ligand, conjugating group or targeting group conjugated thereto.

[0031] Furthermore, the ligand is one or more GalNAcs attached using divalent or trivalent branched bonds;

[0032] Furthermore, the structure of the coupling is shown in formula (34).

[0033] Optionally, the GalNAc is connected to the 3' end of the positive chain via a thiophosphate group or a phosphate group.

[0034] Optionally, the GalNAc is connected to the invAb linked to the 3' end of the positive chain via a thiophosphate group or a phosphate group.

[0035] The present invention also provides a composition comprising the double-stranded RNA molecule or conjugate of the present invention.

[0036] Furthermore, the composition is a pharmaceutical product and further comprises a pharmaceutically acceptable carrier.

[0037] Furthermore, the composition is a kit for diagnosing, preventing, or treating diseases.

[0038] The present invention also provides the application of the double-stranded RNA molecule, conjugate or composition of the present invention for the preparation of drugs or reagents for the diagnosis, prevention or treatment of diseases.

[0039] The present invention also provides methods for diagnosing, preventing or treating diseases, including providing a subject with the double-stranded RNA molecule, conjugate or composition of the present invention.

[0040] The present invention also provides a method for modifying a double-stranded RNA molecule, said double-stranded RNA molecule comprising a sense strand and an antisense strand that at least partially form a double-stranded region, wherein each nucleotide of said sense strand and antisense strand is independently modified, wherein said modification method comprises:

[0041] (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are fluorinated at position 2, and the nucleotides at position 1 and the last position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the nucleotides at the last position and the second-to-last position of the positive strand are connected through a thiophosphate group.

[0042] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated at position 2, and the ribonucleotide at position 12 is replaced by a deoxyribonucleotide; the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups;

[0043] Other nucleotides are modified with a methoxy group at the 2' position; or

[0044] (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are fluorinated at position 2, and the nucleotides at position 1 and the penultimate position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group.

[0045] Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and the 12th position is a deoxyribonucleotide replacing a ribonucleotide; the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate, and the penultimate and penultimate are linked by thiophosphate groups;

[0046] Other nucleotides are modified with a methoxy group at the 2' position.

[0047] The modification method of this invention can effectively enhance the activity of siRNA molecules. The double-stranded RNA molecules and their conjugates of this invention can effectively inhibit mRNA expression and have good stability, providing a new direction for siRNA drug screening. Attached Figure Description

[0048] Figure 1 shows the inhibition results of siRNA on hC3 mRNA in Example 2;

[0049] Figure 2 shows the off-target effects of compound GRD3408037AM7GVP in Hep3B cells;

[0050] Figure 3 shows the off-target effects of compound GRD3408037M3GVP in Hep3B cells;

[0051] Figure 4 shows the off-target effects of compound GRD3408037AM14GVP in Hep3B cells;

[0052] Figure 5 shows the inhibition results of siRNA on APOC3 mRNA in Example 9;

[0053] Figure 6 shows the off-target effects of the ARO-APOC3AM13GVP compound in Hep3B cells;

[0054] Figure 7 shows the off-target effects of the ARO-APOC3G compound in Hep3B cells. Detailed Implementation

[0055] In this article, the capital letters C, G, U, A, and T represent nucleotides and have meanings known in the art.

[0056] In this article, when referring to nucleotide modifications, the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the letter s are linked by a thiophosphate group; (d) indicates that the nucleotide adjacent to its left is a 2'-deoxyribonucleotide; (GNA) indicates that the nucleotide adjacent to its left is a GNA modified nucleotide; I represents inosine nucleotide, (Im) represents methoxy modified inosine nucleotide; VP indicates that the nucleotide adjacent to its right is a vinyl phosphate (VP) modified nucleotide; and (invAb) indicates that the adjacent nucleotide is linked to an invAb residue.

[0057] In this document, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired with the bases of the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the strand can be inferred from the sequence of its complementary strand.

[0058] In this document, when referring to methods for preparing siRNA, siRNA conjugates, or siRNA molecules, unless otherwise specified, a nucleoside monomer refers to a modified or unmodified nucleoside phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the desired siRNA, siRNA conjugate, or siRNA molecule. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this invention are commercially available.

[0059] In this document, "coupling" refers to the covalent connection between two or more chemical parts, each with a specific function; correspondingly, "coupling compound" refers to a compound formed by the covalent connection of these chemical parts. Further, "siRNA conjugate" refers to a compound formed by the covalent attachment of one or more chemical parts (ligands) with specific functions to siRNA. siRNA conjugates should be understood, depending on the context, as a collective term for multiple siRNA conjugates or a siRNA conjugate represented by a specific chemical formula. In the context of this invention, "coupling molecule" or "ligand" should be understood as a specific compound that can be reactively coupled to siRNA to ultimately form the siRNA conjugate of this invention.

[0060] In this document, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the possibility that the event or condition occurs and the possibility that it does not occur. For example, "alkyl" in "optionally substituted" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0061] In this article, the terms “treatment,” “relief,” or “improvement” are used interchangeably. These terms refer to methods of achieving beneficial or desired outcomes, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, even though the subject may still be suffering from the underlying disorder.

[0062] In this document, “prevention” and “protection” are used interchangeably. These terms refer to methods of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” the composition may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more pathological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0063] In this article, "modified nucleotide" or similar terms refer to nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosome with another group, or nucleotides in which the bases on the nucleotide are modified. "Fluorinated nucleotide" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and "non-fluorinated nucleotide" refers to nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosome with a non-fluorinated group. "Nucleotide analog" refers to a group that can replace a nucleotide in nucleic acids but has a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotide" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0064] In this article, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosyl group with fluorine, which has the structure shown in formula (1).

[0065] In this document, the non-fluorinated nucleotides are independently selected from nucleotides or nucleotide analogs formed by substituting the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group. The nucleotides formed by substituting the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxyribonucleotides.

[0066] In this article, in some specific cases, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe) modified nucleotide, as shown in formula (2), i.e., methoxy modified; the 2'-substituted alkoxy modified nucleotide can be, for example, a 2'-O-methoxyethyl (2'-MOE) modified nucleotide, as shown in formula (3), a 2'-amino (2'-NH2) modified nucleotide, as shown in formula (4), and a 2'-deoxyribonucleotide (DNA), as shown in formula (5), where Base refers to the modified or unmodified nucleotide base A, U, G, C, T or other nucleotide bases:

[0067] In this document, nucleotide analogues refer to groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. These nucleotide analogues can be isonucleotides, bridged nucleotides, or acyclic nucleotides.

[0068] In some embodiments described herein, the bridged nucleic acid (BNA) refers to a restricted or inaccessible nucleotide. The BNA may contain a five-, six-, or seven-membered ring with a "fixed" C3'-endoglucan condensation. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide. The BNA may be LNA, ENA, cET BNA, etc., where LNA is shown in formula (6), ENA in formula (7), and cET BNA in formula (8), where Base refers to the modified or unmodified nucleotide bases A, U, G, C, T, or other nucleotide bases.

[0069] In this article, non-fluorinated nucleotides may be thermally unstable nucleotides, and the thermally unstable modifications may include, but are not limited to, debasement modification, mismatch with relative nucleotides in the opposite chain, and sugar modification.

[0070] In this document, the debasement modifications mentioned are such as 2'-deoxy modifications or acyclic nucleotides, for example, unlocked nucleic acids (UNA) or glycol nucleic acids (GNA). Some exemplary debasement modifications include, but are not limited to, the following structural formulas (Formulas (9) to (15)):

[0071] Where R = H, Me, Et or OMe; R' = H, Me, Et or OMe; R” = H, Me, Et or OMe.

[0072] In some embodiments described herein, sugar modifications include, but are not limited to, the following: 2'-deoxyribonucleotides, unlocked nucleic acids, and glycol-based nucleic acids.

[0073] In some embodiments herein, an acyclic nucleotide refers to any nucleotide having a noncyclic ribose, for example, wherein any bond between ribose carbons in the nucleotide (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent and / or at least one of the ribose carbons or oxygen (e.g., C1', C2', C3', C4', or O4') is absent independently or in combination.

[0074] In some embodiments, GNA represents ethylene glycol nucleic acid, which is a polymer similar to DNA or RNA, but with a different "backbone" composed of repeating glycerol units linked by phosphodiester bonds. The structure of A (GNA) is shown in Formula (16), the structure of G (GNA) is shown in Formula (17), the structure of C (GNA) is shown in Formula (18), the structure of U (GNA) is shown in Formula (19), and the structure of T (GNA) is shown in Formula (20).

[0075] In some embodiments described herein, inosine nucleotides are also thermally unstable modifications. Inosine nucleotides are represented by the following structures: I(d) structure as shown in formula (21), I structure as shown in formula (22), (If) structure as shown in formula (23), and (Im) structure as shown in formula (24).

[0076] In this invention, the phosphate group with the modifying group is a thiophosphate group having the structure shown in formula (25). In one embodiment of the invention, the nucleotide linked to the thiophosphate group is shown in formula (26), and in another embodiment of the invention, the nucleotide is modified with vinyl phosphate (VP). In one embodiment of the invention, the nucleotide modified with VP and methoxy, i.e., the nucleotide modified with 5'-(E)-vinyl-2'-methoxy phosphate group (5'-(E)-VP-2'-OMe), is shown in formula (27); in another embodiment of the invention, the nucleotide modified with VP, methoxy, and thiophosphate group, i.e., the nucleotide modified with 5'-PS (i.e., the nucleotide modified with 5'-(E)-vinyl-2'-methoxy thiophosphate group), is shown in formula (28).

[0077] In this article, in one embodiment of the invention, invAb represents a reverse abase-free nucleotide, and the structural formula of invAb is shown in formula (33).

[0078] Specifically, the structural formulas of the reverse nucleotide modified nucleotides are shown in formulas (29) to (32).

[0079] In this article, GalNAc is a type of N-acetylgalactosamine, which forms an siRNA conjugate with siRNA, and its structure is shown in formula (34) below:

[0080] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0081] For any experimental steps or conditions not specifically specified in the following examples, the procedures and conditions described in the literature in this field should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Nucleic acid electrophoresis, real-time quantitative PCR (RT-qPCR), and other operations used were performed according to the methods described in *Molecular Biology (4th Edition)* (Alexander McLennan et al., 2019).

[0082] The siRNAs involved in the following examples are siRNAs synthesized via phosphoramide solid-phase synthesis.

[0083] Unless otherwise specified, all reagent ratios provided below are calculated on a volume ratio (v / v).

[0084] The siRNA designed based on gene transcripts was synthesized by Suzhou GeneGene Co., Ltd.

[0085] Example:

[0086] Example 1: Testing the activity of siRNA sequences in in vitro cell lines

[0087] The sequences used in this embodiment are shown in Table 1. The siRNA molecules with the following sequences were synthesized by Suzhou Gemma Gene Co., Ltd., and have been verified to be correct.

[0088] Table 1. siRNAs that inhibit C3

[0089] The activity of the synthesized siRNAs in Table 1 was tested in in vitro cell lines.

[0090] This experimental example provides a determination of the relative inhibitory level of the siRNA compound of the present invention on C3 mRNA in HepG2 cells using quantitative real-time PCR (qPCR).

[0091] The activity of siRNA inhibiting C3 was detected in HepG2 cells, and the experimental procedure is as follows:

[0092] 1. HepG2 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into MEM medium (purchased from Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 1% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h. After culture, the HepG2 cells were digested with trypsin (purchased from GIBCO, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM medium to obtain a cell concentration of 3 × 10⁶ cells / year. 5 Cell suspension of cells / mL;

[0093] 2. Dilute each siRNA in Table 1 using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilution buffer; mix 25 μL opti-MEM with 0.25 μL Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilution buffer; mix 25 μL of siRNA dilution buffer with the transfection reagent dilution buffer and incubate at room temperature (25℃) for 15 min to obtain siRNA transfection solution;

[0094] 3. After seeding the cell suspension into 96-well plates at a seeding rate of 50 μL / well, set up MOCK group (MOCK group is the transfection reagent control group), hC3_2702AM7 to hC3_913L23M2 groups in 96-well plates, with 3 replicates in each group;

[0095] 4. After setting up, add 50 μL of siRNA transfection buffer to each well of the experimental group (the hC3_2702AM7 experimental group was given transfection buffer containing hC3_2702AM7, the hC3_2702AM8 experimental group was given transfection buffer containing hC3_2702AM8, and so on, with the final concentration of siRNA in the wells being 1 nM and 0.1 nM, respectively). Incubate the cells in a 5% (v / v) CO2, 37℃ cell culture incubator for 48 h for transfection. After transfection, discard the liquid in the wells and collect the cells.

[0096] 5. After transfection, discard the liquid in the wells, collect the cells, and extract total RNA from the cells in each well using the magnetic bead method total RNA extraction kit (purchased from Suzhou Genegene Co., Ltd., catalog number E31008) according to the method described in the kit instructions.

[0097] 6. Using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01), the experimental procedures were performed according to the product instructions. A 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe the total RNA from the cells. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 37°C for 15 min, then at 85°C for 5 s. 80 μL of DEPC water was added to each reverse transcription reaction system to obtain a solution containing cDNA.

[0098] 7. For each reverse transcription reaction system, take 4 μL of the above-mentioned cDNA-containing solution as a template. Using the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02), prepare a 20 μL qPCR reaction system on an ice box according to Table 2. Primers 1 and 2 are the PCR primer sequences for amplifying the target gene C3 and the internal reference gene GAPDH, respectively (as shown in Table 3). Place each qPCR reaction system in an ABIStepOnePlus Real-Time... Amplification was performed using a three-step method on a PCR instrument. The amplification program was as follows: pre-denaturation at 95℃ for 10 min, followed by denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene C3 and internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of target gene C3 and internal reference gene GAPDH in product W were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of target gene C3 and internal reference gene GAPDH.

[0099] Table 2. DNA amplification reaction system

[0100] Table 3. Primer Information

[0101] The relative quantification of the target gene C3 in each test group was performed using the comparative Ct (ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group) ΔΔCt(test group) = ΔCt(test group) - ΔCt(average in control group) ΔΔCt(control group) = ΔCt(control group) - ΔCt(average in control group)

[0102] Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) for each sample in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value.

[0103] Using the control group as a baseline, the expression level of C3 mRNA in the test group was normalized, and the expression level of C3 mRNA in the control group was defined as 100%.

[0104] The relative expression level of C3 mRNA in the test group was 2- ΔΔCt(测试组) ×100%

[0105] The inhibition rate of C3 mRNA in the test group = 1 - the relative expression level of C3 mRNA in the test group.

[0106] The C3 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the control group. The data were expressed as a percentage relative to the control group and presented as the mean plus the standard deviation.

[0107] The experimental results are shown in Table 4.

[0108] Table 4. Inhibition levels of different modified siRNAs on C3 mRNA in HepG2 cells.

[0109] Example 2: Activity of siRNA sequences in animals

[0110] The activity of siRNA in Table 1 was tested in animals.

[0111] GalNAc-conjugated modified siRNAs were used in the hC3_2702AM13G and hC3_913AM13G groups. Three commercially available male hC3 humanized mice (4-6 weeks old) in each group were given a single subcutaneous dose of 3 mg / kg of GalNAc-conjugated siRNA or a saline control.

[0112] On the 14th day after administration, mice were sacrificed, liver samples were collected, liver mRNA was extracted, and the siRNA conjugates were analyzed by RT-qPCR according to the RT-qPCR method in Example 1. The detection results are shown in Figure 1.

[0113] The steps for extracting liver mRNA are as follows:

[0114] Step 1: Take 20 mg of mouse liver tissue, add 1 mL of Trizol Lysis Buffer (purchased from Life Technology, catalog number 410701), grind and lyse the tissue to obtain the grinding product; transfer the grinding product to an RNase-free 1.5 mL centrifuge tube, shake for 15 s to fully lyse the tissue cells, and then let it stand at room temperature (25℃) for 5 min to obtain the lysate;

[0115] Step 2: Open the cap of the centrifuge tube, add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925) to the centrifuge tube, shake for 20 seconds, then let stand at room temperature (25℃) for 3 minutes, and then centrifuge at 4℃ and 12000×g for 20 minutes. After centrifugation, transfer the supernatant to a new 2.0 mL centrifuge tube, and add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., catalog number 20210802) to the centrifuge tube. Invert and mix well to obtain the mixture.

[0116] Step 3: Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture obtained in Step 2 to the purification column, let it stand for 2 min, and then centrifuge the purification column at 4℃ and 10000×g for 1 min. After centrifugation, discard the filtrate; repeat the above steps for the remaining mixture.

[0117] Step 4: Add 700 μL of 80% (v / v) ethanol to the purification column, then centrifuge the column at 4°C and 10000×g for 1 min. After centrifugation, discard the filtrate.

[0118] Step 5: Add 700 μL of 80% (v / v) ethanol to the purification column, then centrifuge the purification column at 4℃ and 10000×g for 1 min. After centrifugation, discard the filtrate.

[0119] Step 6: Centrifuge the purification column at 10000×g for 2 min at 4℃. After centrifugation, remove the purification column with the collection tube (if there is liquid in the collection tube, please be careful not to splash the liquid onto the purification column), discard the collection tube, and put the purification column into a new 1.5 mL centrifuge tube; add 100 μL of DEPC water to the purification column, let it stand at room temperature (25℃) for 2 min, and then centrifuge at 10000×g for 1 min at 4℃. After centrifugation, collect the RNA solution for subsequent experiments.

[0120] The qPCR detection method is the same as described in Example 1, except that the above-mentioned RNA is used as the template and the primer and probe sequences are different.

[0121] Primer sequences are shown in Table 5, and experimental results are shown in Figure 1. The hC3_2702AM13G and hC3_913AM13G of the present invention can efficiently inhibit hC3 mRNA expression.

[0122] Table 5. Primer Information

[0123] Example 3: Detection of siRNA activity - IC50 in HepG2 cells

[0124] This study detected the activities (IC50, 120, 130, 130, 130, 130, 130, 130, 16 ...30, 130, 130, 130, 130, 130, 130, 130, 130, 140, 130, 130, 130, 130, 130, 140, 130, 50 ).

[0125] IC 50 The calculation method is as follows: siRNA concentration gradients are set: 10nM, 1nM, 0.33nM, 0.11nM, 0.037nM, 0.0123nM, and 0.0041nM; after detecting the effect of gradient concentration siRNA on the relative expression level of C3 mRNA according to the above detection method, the IC50 curves corresponding to different siRNAs are fitted using "nonlinear regression".

[0126] The experimental procedure is the same as in Example 1. The siRNA concentration gradient was set as follows: 10 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, 0.0123 nM, and 0.0041 nM for transfection, and the transfection time was 48 h. The experimental results are shown in Table 6 below.

[0127] Table 6 IC50

[0128] Example 4: Detection of siRNA activity in HepG2 cells

[0129] The siRNA used in this embodiment is shown in Table 7. The siRNA molecule with the following sequence was synthesized by Suzhou Genegene Co., Ltd., and the sequence was verified to be correct.

[0130] Table 7. siRNAs that inhibit SGLT2

[0131] The activity of the siRNAs in Table 7 was tested in vitro in cell lines, and the experimental procedure is as follows:

[0132] 1. Construction of stable cell line LV-SGLT2

[0133]

[0134] 2. Dilute each siRNA in Table 7 using opti-MEM (Gibco, catalog number 31985-070) to obtain siRNA dilution solutions; mix 25 μL opti-MEM with 0.25 μL Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to obtain transfection reagent dilution solutions; mix 25 μL of siRNA dilution solution with the transfection reagent dilution solutions and incubate at room temperature (25℃) for 15 min to obtain siRNA transfection solutions;

[0135] 3. (The last part appears to be incomplete and possibly contains errors. It doesn't translate directly.) 5 After seeding 50 μL of MEM complete medium containing LV-SGLT2 cells / mL into 96-well plates, the 96-well plates were divided into NC group, BLACK group (BLACK group is the cell group) as control, and hSLC5A2-185AM13 group to hSLC5A2-202L23M31 group, with 3 replicates in each group.

[0136] 4. After setting up, add 50 μL of siRNA transfection solution to each well of the experimental groups (hSLC5A2-185AM13 experimental group was given transfection solution containing hSLC5A2-185AM13, hSLC5A2-185AM14 experimental group was given transfection solution containing hSLC5A2-185AM14, and so on, with a final concentration of siRNA in the wells of 1 nM). Incubate for 24 h in a 5% (v / v) CO2, 37°C cell culture incubator for transfection. Approximately 6 h after transfection, discard the transfection complex, add 100 μL of complete culture medium to each well, and continue incubation. 24 h after transfection, discard the cell supernatant. Slowly add an equal volume of pre-chilled 1x PBS to the adherent cells for washing, slowly remove the PBS, and place on ice for later use.

[0137] 5. Cell sample processing:

[0138] Prepare the lysis buffer according to Table 8:

[0139] Table 8. Cell lysis reaction system Note: Prepare the lysis working solution according to the required volume of the well plate. The ratio of each component is FlysisAmp cell lysis buffer:DNase I:enhancing solution = 23:1:1. After preparation, invert and mix 10-15 times, avoiding vigorous vortexing; place on ice after mixing and use within 1 hour.

[0140] Add 50 μL of the prepared lysis buffer to each well of the cell culture plate, gently pipette 8-10 times to mix thoroughly, and let stand at room temperature for 5 minutes to lyse the cells.

[0141] After lysis, add 5 μL of stop solution to each well of the cell culture plate, gently pipette 8-10 times to mix, and let stand at room temperature for 2 minutes to terminate the reaction. The lysis product can be stored on ice for 2 hours; for long-term storage, it should be stored at -80°C.

[0142] 6. Preparation of qPCR reaction system

[0143] For each reverse transcription reaction system, 50 μL of the cell lysis buffer was diluted with 150 μL of DEPC H2O to prepare RNA template. A 13 μL qPCR reaction system was prepared on an ice box according to Table 9, with primer sequences shown in Table 10. 9.75 μL of the prepared PCR reaction solution was aliquoted into each well of a 384-well plate, and 3.25 μL of RNA template was added to each well. Finally, 3 μL of paraffin oil was added, and the plates were sealed with a sealing film and centrifuged at 3000 rpm for 1 min. Detection: Real-time quantitative PCR was performed on an LC480, with the program shown in Table 11. The melting curves of the target gene SGLT2 and the internal reference gene GAPDH in product W were collected using a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene SGLT2 and the internal reference gene GAPDH.

[0144] Table 9. One-step RT-qPCR probe method reaction system

[0145] Table 10. Primer Information

[0146] Table 11. RT-qPCR reaction procedure

[0147] The relative quantification of the target gene SGLT2 in each test group was performed using the comparative Ct (ΔΔCt) method, as described in Example 1. Experimental results are shown in Table 12. The results indicate that, under the same naked siRNA sequence conditions, siRNA molecules modified with AM13 and AM14 modulo sequences exhibited better inhibitory effects.

[0148] Table 12. mRNA inhibition level of siRNA at 1 nM in the stable cell line LV-SGLT2

[0149] Example 5: Activity detection of siRNA sequence in A549 cells

[0150] The siRNAs used in this embodiment are shown in Table 13. These siRNA molecules with the following sequences were synthesized by Suzhou Gemma Gene Co., Ltd., and the sequences were verified to be correct. All sequences in Table 13 were modified using the AM13 pattern.

[0151] Table 13 siRNAs that inhibit STAT6

[0152] The relative inhibitory levels of siRNAs on STAT6 mRNA in Table 13 were detected in A549 cells (purchased from the Cell Bank of the Chinese Academy of Sciences). The experimental procedures were the same as in Example 1, except that the final concentrations of siRNAs were 10 nM, 1 nM, and 0.1 nM.

[0153] Primer sequences are shown in Table 14, and experimental results are shown in Table 15.

[0154] Table 14. Primer Information

[0155] Table 15. mRNA inhibition levels of siRNA in A549 cells at 10 nM / 1 nM / 0.1 nM.

[0156] Example 6: In vitro activity detection of siRNA sequences

[0157] The siRNA used in this embodiment is shown in Table 16. The siRNA molecule with the following sequence was synthesized by Suzhou Gemma Gene Co., Ltd., and the sequence was verified to be correct.

[0158] Table 16 siRNAs that inhibit GPR75

[0159] To verify the inhibitory activity of the siRNA on GPR75 gene expression, a plasmid vector, psiCHECK2, was constructed for detection in the experiment. The psiCHECK2 vector is a plasmid vector that can monitor changes in the expression of a target gene fused with a reporter gene. This vector uses Renal luciferase as the primary reporter gene. The target fragment is cloned into the multiple cloning site downstream of the translation stop codon of Renal luciferase. The RNAi process, initiated by the synthesized siRNA, targets the target gene, leading to the cleavage and subsequent degradation of the fusion mRNA. By detecting changes in Renal luciferase activity, it is possible to determine whether a targeting relationship exists between the siRNA and the target gene fragment.

[0160] The experimental procedure is as follows:

[0161] Step 1: Plasmid Construction Detection

[0162]

[0163] Step 2: Cell Culture and Transfection

[0164] The siRNA to be tested was diluted with DEPC water to obtain the siRNA dilution solution. Then, the siRNA dilution solution was added to the 96-well plate at a rate of 5 μL / well.

[0165] Add 12.5 μL of Opti-MEM (Gibco, catalog number 31985-070) containing 20 ng of detection plasmid to a 96-well plate, add 32.5 μL of Opti-MEM to a 96-well plate, add 0.3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to a 96-well plate, and let stand at room temperature (25°C) for 15 min to obtain the mixed system.

[0166] After settling, it will contain 2×10 5 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were added to 96-well plates at a rate of 50 μL / well using DMEM complete medium (purchased from Transgen Biotech, catalog number FI101-01) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 24 h for subsequent dual-luciferase assay.

[0167] The final concentrations of siRNA in the culture system were 10 nM, 1 nM, 0.3 nM, 0.1 nM, 0.01 nM, 0.01 nM, and 0.003 nM. A blank control group, MOCK, was set up in the experiment. MOCK was a group that only added interference reagents and no sequence.

[0168] Step 3: Dual-luciferase assay

[0169] After culture, the cells in the 96-well plate were subjected to dual-luciferase assay. Three replicates were used for each siRNA assay. The specific assay method is as follows:

[0170] Take the dual-luciferase assay kit (purchased from Promega, catalog number E2940), dilute the 5× lysis buffer in the kit with water to make 1× lysis buffer, and prepare substrate 1 and substrate 2 according to the kit instructions;

[0171] Take a 96-well plate, discard the supernatant, dilute each well with PBS buffer (purchased from Hyclone, catalog number SH30256.01) and wash twice, then add 1× lysis buffer at a rate of 50 μL / well to the 96-well plate and incubate at room temperature (25℃) for 20 min to lyse the cells in the 96-well plate to obtain the lysis buffer;

[0172] The lysis buffer was aspirated from the original 96-well plate and added to a new opaque 96-well plate at a rate of 30 μL / well. Substrate 1 and Substrate 2 were then added to the 96-well plate in portions of 30 μL / well / time. After each addition of substrate, the 96-well plate was analyzed using a multi-mode microplate reader to obtain the numerical results of firefly luciferase and Renilla luciferase, respectively.

[0173] The emission ratio of each well in a 96-well plate is calculated using the following formula:

[0174] Luminescence ratio = Renilla luciferase value / Firefly luciferase value (Renilla / Firefly), calculate the luminescence ratio of each well in the 96-well plate;

[0175] The luminescence ratio of each test group (i.e., the group with added siRNA), blank control group, and negative control group is the average of the luminescence ratios of the three replicates;

[0176] Based on the luminescence ratio of the blank control group, the luminescence ratios of each test group and the negative control group were normalized to obtain the ratio R of luminescence ratio (test) / luminescence ratio (control), which represents the expression level (i.e., relative residual activity) of the Renaissance luciferase reporter gene.

[0177] After obtaining the relative residual activity of the Renal luciferase reporter gene, the inhibition rate of siRNA on the GPR75 gene was calculated according to the formula: inhibition rate = (1-R)×100%.

[0178] After calculating the inhibition rate of GPR75 gene expression at different final concentrations, the IC50 of each siRNA group was calculated using GraphPad Prism software, resulting in the data in Table 17 below. The results show that the siRNAs in Table 16 can effectively inhibit GPR75 mRNA.

[0179] Table 17: IC50

[0180] Example 7: Activity detection of siRNA sequence in Hep3B cells

[0181] The siRNAs provided in Table 18 of this embodiment are siRNA molecules with the following sequences synthesized by Suzhou Gemma Gene Co., Ltd., and the sequences have been verified to be correct.

[0182] Table 18 siRNAs that inhibit C5

[0183] The relative inhibitory level of the siRNA of this invention on C5 mRNA was detected in Hep3B cells. The experimental procedure was the same as in Example 1, except that the final concentration of siRNA was 1 nM.

[0184] Primer sequences are shown in Table 19, and experimental results are shown in Table 20.

[0185] Table 19. Primer Information

[0186] Table 20. mRNA inhibition level of siRNA at 1 nM in Hep3B cells

[0187] Example 8: Safety evaluation of siRNA sequences

[0188] This embodiment used RNA sequencing (RNA-seq) to determine the off-target effects of GRD3408037M3GVP, GRD3408037AM7GVP, and GRD3408037AM14GVP compounds in Hep3B cells. 1 nM of the compounds were transfected into Hep3B cell lines, and RNA-seq was performed at Suzhou Genewiz Biotechnology Co., Ltd. The results are shown in Figures 2 to 4. The results show that the gene perturbation caused by the AM14 modulator was significantly reduced, and the off-target effects were significantly improved, further demonstrating that the modification method used in this invention can effectively improve the safety of siRNA molecules.

[0189] Example 9: Activity detection of siRNA sequence in Hep3B cells

[0190] The siRNA used in this embodiment is shown in Table 21. The siRNA molecule with the following sequence was synthesized by Suzhou Gemma Gene Co., Ltd., and the sequence was verified to be correct.

[0191] Table 21 siRNAs that inhibit APOC3

[0192] GalNAc-conjugated modified siRNA was used in the ARO-APOC3G and ARO-APOC3AM13GVP groups. Three APOC3 humanized mice (commercially available, female, 4-6 weeks old) in each group were given a single subcutaneous dose of 2 mg / kg of GalNAc-conjugated siRNA or a saline control.

[0193] On day 21 after administration, mice were sacrificed, liver samples were collected, liver mRNA was extracted, and the siRNA conjugates were analyzed by RT-qPCR according to the method in Example 1. The experimental steps are as described in Example 2, the primers are as described in Table 22, and the detection results are shown in Figure 5.

[0194] Table 22. Primer Information

[0195] Example 10: Safety evaluation of siRNA sequences

[0196] This experiment investigated the off-target effects of ARO-APOC3G and ARO-APOC3AM13GVP compounds in cells using RNA sequencing (RNA-seq). 1 nM of the compound was transfected into Hep3B cell lines, and the cells were sent to Suzhou Genewiz Biotechnology Co., Ltd. for RNA-seq 24 hours later. The results are shown in Figures 6 and 7. The results indicate that the gene perturbation caused by the compound in ARO-APOC3AM13GVP was significantly reduced, and the off-target effects were significantly improved, further demonstrating that the modification method used in this invention can effectively improve the safety of siRNA molecules.

Claims

1. A double-stranded RNA molecule comprising a sense strand and an antisense strand that at least partially form a double-stranded region, wherein each nucleotide of the sense strand and the antisense strand is independently modified, wherein, Following the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluorinated nucleotides, the nucleotides at positions 2, 10, 14, and 16 of the antisense strand are 2'-fluorinated nucleotides, and the nucleotide at position 6 or 8 of the antisense strand is a 2'-fluorinated nucleotide. Preferably, the 12th nucleotide of the antisense strand is a deoxyribonucleotide, following the direction from the 5' end to the 3' end; Preferably, the other nucleotides of the sense and / or antisense strands are 2'-methoxy modified.

2. The double-stranded RNA molecule according to claim 1, wherein, At least one phosphate group in the sense chain or antisense chain is a phosphate group with a modifying group; Preferably, the phosphate ester group having the modifying group is a thiophosphate ester group; Preferably, the thiophosphate group linkage is present at least at one of the following positions: between the first and second nucleotides at either end of the sense or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination thereof.

3. The double-stranded RNA molecule according to claim 1 or 2, wherein, The double-stranded RNA molecule is also linked to an inverted abase-free nucleotide. Preferably, the first nucleotide at the 5' end and / or the first nucleotide at the 3' end of the positive strand are both linked to a reverse abasic nucleotide via a thiophosphate group.

4. The double-stranded RNA molecule according to any one of claims 1 to 3, wherein, Starting from the 5' end of the antisense strand, the first nucleotide of the antisense strand contains a VP modification.

5. The double-stranded RNA molecule according to any one of claims 1 to 4, wherein, The RNA molecule includes: (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are 2' fluorinated nucleotides, and the nucleotides at position 1 and the penultimate position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group. Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and position 12 is a deoxyribonucleotide. When the ribonucleotide at this position is uracil ribonucleotide, it is replaced with thymine deoxyribonucleotide. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups. Other nucleotides are nucleotides modified with a methoxy group at the 2' position; or (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are 2' fluorinated nucleotides, and the nucleotides at position 1 and the penultimate position are each connected to a reverse non-base nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group. Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and position 12 is a deoxyribonucleotide. When the ribonucleotide at this position is uracil ribonucleotide, it is replaced with thymine deoxyribonucleotide. The nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups. The other nucleotides are nucleotides modified with a methoxy group at the 2' position.

6. The double-stranded RNA molecule according to any one of claims 1 to 5, wherein, The sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of siRNA. The 3' end of the sense strand forms a blunt end, and the 3' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region. Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, the 5' end of the sense strand forms a blunt end, and the 5' end of the antisense strand has 1-3 protruding nucleotides extending out of the double-stranded region. Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, with the 3' end of the sense strand forming a blunt end and the 3' end of the antisense strand forming a blunt end; Alternatively, the sense and antisense strands of the double-stranded RNA molecule are complementary to form the double-stranded region of the siRNA, and the 3' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region, or the 5' ends of the sense and antisense strands have 1-3 protruding nucleotides extending out of the double-stranded region.

7. The double-stranded RNA molecule according to any one of claims 1 to 6, wherein, The antisense strand is 19-30 nucleotides in length; the sense strand is 15-30 nucleotides in length. Preferably, the antisense strand is 21 nucleotides long and the sense strand is 21 nucleotides long.

8. A conjugate comprising a double-stranded RNA molecule according to any one of claims 1 to 7, and a ligand, conjugating group or targeting group conjugated thereto; Preferably, the ligand is one or more GalNAcs attached using divalent or trivalent branched bonds; More preferably, the structure of the coupling is as shown in formula (34); Optionally, the GalNAc is connected to the 3' end of the positive chain via a thiophosphate group or a phosphate group. Optionally, the GalNAc is connected to the invAb linked to the 3' end of the positive chain via a thiophosphate group or a phosphate group.

9. A composition comprising the double-stranded RNA molecule according to any one of claims 1 to 7 or the conjugate according to claim 8; Preferably, the composition is a drug, further comprising a pharmaceutically acceptable carrier; or The composition is a kit for the diagnosis, prevention or treatment of diseases.

10. The use of the double-stranded RNA molecule according to any one of claims 1 to 7, the conjugate according to claim 8, or the composition according to claim 9 for the preparation of medicaments or reagents for the diagnosis, prevention, or treatment of diseases.

11. A method for diagnosing, preventing, or treating a disease, comprising providing a subject with a double-stranded RNA molecule as described in any one of claims 1 to 7, a conjugate as described in claim 8, or a composition as described in claim 9.

12. A method for modifying a double-stranded RNA molecule, said double-stranded RNA molecule comprising a sense strand and an antisense strand that at least partially form a double-stranded region, wherein each nucleotide of the sense strand and the antisense strand is independently modified, wherein, The modification method includes: (1) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are fluorinated at position 2, and the nucleotides at position 1 and the last position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the nucleotides at the last position and the second-to-last position of the positive strand are connected through a thiophosphate group. Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 10, 14, and 16 of the antisense strand are fluorinated at position 2, and the ribonucleotide at position 12 is replaced by a deoxyribonucleotide; the nucleotides at positions 1 and 2, 2 and 3, penultimate and penultimate, and penultimate and penultimate are linked by phosphate thioester groups; Other nucleotides are modified with a methoxy group at the 2' position; or (2) In the direction from the 5' end to the 3' end, the nucleotides at positions 7, 9, 10, and 11 of the positive strand are fluorinated at position 2, and the nucleotides at position 1 and the penultimate position are each connected to a reverse abase-free nucleotide through a thiophosphate group; optionally, the penultimate and penultimate nucleotides of the positive strand are connected through a thiophosphate group. Following the direction from the 5' end to the 3' end, the nucleotides at positions 2, 8, 10, 14, and 16 of the antisense strand are 2' fluorinated nucleotides, and the 12th position is a deoxyribonucleotide replacing a ribonucleotide; the nucleotides at positions 1 and 2, 2 and 3, the penultimate and penultimate, and the penultimate and penultimate are linked by thiophosphate groups; Other nucleotides are modified with a methoxy group at the 2' position.

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