Double-stranded RNA containing nucleotide analogs capable of reducing off-target toxicity

Abstract

The present invention provides a double-stranded RNA having a nucleotide analogue. The double stranded RNAs of the present invention exhibit one or more of enhanced stability, reduced off-target toxicity, and enhanced effectiveness.

Description

Double-stranded RNA containing nucleotide analogs to reduce off-target toxicity

[0001] The present invention claims priority to Chinese application CN202210744263.8 filed on June 27, 2022, Chinese application CN202210948347.3 filed on August 8, 2022, and Chinese application CN202211483699.2 filed on November 24, 2022, which are incorporated herein by reference in their entirety. Field of the Invention

[0002] The present invention belongs to the field of medicine, and in particular relates to double-stranded RNA with nucleotide analogs. Background Art

[0003] RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) induces the efficient and specific degradation of target mRNA. Incorporating heat-labile nucleotides (e.g., glycerol nucleic acid (GNA)) into the antisense strand of dsRNA can help improve interference efficiency and reduce off-target toxicity (see, for example, PCT Publication No. WO2018098328A1).

[0004] Therefore, there is a need in the art to develop a new nucleotide analog that can help reduce off-target toxicity after being incorporated into double-stranded RNA.

[0005] Summary of the Invention

[0006] The present invention solves the above problems by providing a novel nucleotide analog.

[0007] In one aspect, the present invention provides a nucleotide duplex represented by formula (A):

[0008] wherein each group is as defined below.

[0009] In another aspect, the present invention provides a double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand comprises one or more nucleotide monomers represented by formula (III) or (IV):

[0010] in,

[0011] The nucleotide monomers are shown in the order 5'=>3' from to connect;

[0012] Each group is as defined below.

[0013] In another aspect, the present invention provides a nucleic acid molecule comprising one or more nucleotide duplexes and / or nucleotide monomers as described herein in its nucleotide sequence.

[0014] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned double-stranded RNA.

[0015] In another aspect, the present invention provides a cell containing the aforementioned double-stranded RNA or the aforementioned vector.

[0016] In another aspect, the present invention relates to a pharmaceutical composition comprising a double-stranded RNA molecule as described herein, and a pharmaceutically acceptable carrier or excipient.

[0017] In another aspect, the present invention relates to a kit comprising a double-stranded RNA molecule as described herein.

[0018] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising the step of introducing a double-stranded RNA molecule as described herein into the cell.

[0019] In another aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell, comprising expressing a double-stranded RNA molecule as described herein in said cell.

[0020] In another aspect, the present invention relates to a method for reducing off-target toxicity in a cell, comprising the step of introducing the double-stranded RNA molecule described herein into the cell.

[0021] In another aspect, the present invention relates to a method for reducing off-target toxicity in a cell, comprising expressing a double-stranded RNA molecule described herein in said cell.

[0022] Upon incorporation of the nucleotides of the present invention into the antisense strand of dsRNA, the resulting double-stranded RNA exhibits one or more of enhanced stability, reduced off-target toxicity, and enhanced efficacy.

[0023] Detailed Description of the Invention

[0024] definition

[0025] Chemical definition

[0026] Definitions of specific functional groups and chemical terms are described in more detail below.

[0027] When a numerical range is listed, it is intended to include every value and sub-range within the stated range. For example, "C 1-6 "Alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C 3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0028] “C 1-6 "Alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl groups are preferred. 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term “C 1-6 "Alkyl" also includes heteroalkyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3), or i-Bu(-CH2CH(CH3)2).

[0029] “C 2-6 "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-4 Alkenyl is preferred. 2-6 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6"Alkenyl" also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). An alkenyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0030] “C 2-6 "Alkynyl" refers to a straight or branched chain hydrocarbon group having 2 to 6 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-4 Alkynyl is preferred. 2-6 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 "Alkynyl" also includes heteroalkynyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0031] "Halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

[0032] Therefore, “C 1-6 "Haloalkyl" refers to the above-mentioned "C 1-6 Alkyl", which is substituted by one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl is particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, and the like. The haloalkyl group can be substituted at any available point of attachment, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0033] “C 1-6 "Alkoxy" refers to a -OR group, wherein R is as described above in "C 1-6 Alkyl" and "C 1-6 "Haloalkyl" is defined.

[0034] “C 3-10 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 4-7 Cycloalkyl and C3-6 Cycloalkyl is particularly preferred, more preferably C 5-6 Cycloalkyl. Cycloalkyl also includes a ring system in which the above-mentioned cycloalkyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such a case, the number of carbons continues to represent the number of carbons in the cycloalkyl system. Exemplary cycloalkyls include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. The cycloalkyl group may be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0035] "3-10 membered heterocyclyl" refers to a group of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. In some embodiments, a 4-10 membered heterocyclyl is preferred, which is a 4-10 membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, a 3-8 membered heterocyclyl is preferred, which is a 3-8 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms; a 3-6 membered heterocyclyl is preferred, which is a 3-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; a 4-7 membered heterocyclyl is preferred, which is a 4-7 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms; and a 5-6 membered heterocyclyl is more preferred, which is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes a ring system in which the above-mentioned heterocyclyl ring is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl ring, or a ring system in which the above-mentioned heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such a case, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyls containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiorenyl. Exemplary 4-membered heterocyclyls containing one heteroatom include, but are not limited to, azetidinyl, oxetane, and thiidine. Exemplary 5-membered heterocyclyls containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, hexahydrotriazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thienyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. The heterocyclyl group may be optionally substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0036] “C 6-10 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having 6-10 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C 10 Aryl also includes ring systems in which an aryl ring as described above is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to refer to the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0037] "5-14 membered heteroaryl" refers to a group of a 5-14 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the above-mentioned heteroaryl rings are fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is preferred, which is a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms. In other embodiments, 5-6 membered heteroaryls are particularly preferred and are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryls containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryls containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryls containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryls containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryls containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azacycloheptatrienyl, oxepinyl, and thieptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indanyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. A heteroaryl group can be optionally substituted with one or more substituents, for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0038] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, and the like are defined herein as optionally substituted groups.

[0039] Exemplary substituents on carbon atoms include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )2, -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(R bb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2, -SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa)3、-OSi(R aa )3、-C(=S)N(R bb )2, -C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)2R aa 、 -OP(=O)2R aa 、-P(=O)(R aa )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)2N(R bb )2、-OP(=O)2N(R bb )2、-P(=O)(NR bb )2、-OP(=O)(NR bb )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(NR bb )2、-P(R cc )2、-P(R cc )3、-OP(R cc )2、-OP(R cc )3、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0040] Or the two geminal hydrogen atoms on the carbon atom are replaced by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NR bb or = NOR cc replace;

[0041] R aa Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R aa The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0042] R bb Each of the following is independently selected from: hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R bb The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0043] R cc Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R cc The groups are combined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substitution;

[0044] R ddEach of the is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2,、-N(R ff )3 + X - 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )R ee 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)2Ree 、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently substituted by 0, 1, 2, 3, 4 or 5 R gg Group substitution, or two geminal R dd Substituents may combine to form =O or =S;

[0045] R ee Each of R is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0046] R ff Each of R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R ff The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R gg group substitution;

[0047] R gg Each of the independently: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)2, -N(C 1-6 Alkyl)3 + X - 、-NH(C 1-6 Alkyl)2 + X - 、-NH2(C 1-6 alkyl) + X - 、-NH3 + X - 、-N(OC 1-6 Alkyl)(C 1-6 Alkyl), -N(OH)(C 1-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C 1-6 Alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 Alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 Alkyl, -C(=NH)N(C 1-6 Alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(NH)NH(C 1-6 alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 Alkyl, -SO2OC 1-6 Alkyl, -OSO2C 1-6 Alkyl, -SOC 1-6 Alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2、C(=S)NH(C 1-6 alkyl), C(=S)NH2, -C(=O)S(C 1-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6alkyl)2, -OP(=O)(OC 1-6 Alkyl)2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 cycloalkyl, C6-C 10 Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two geminal R gg Substituents may combine to form =O or =S; wherein X - For the counter ion.

[0048] Exemplary substituents on nitrogen atoms include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)2R aa 、-P(=O)(R aa )2、-P(=O)2N(R cc )2、-P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, or two R attached to the nitrogen atom cc The groups are combined to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4 or 5 R dd group substituted, and wherein R aa 、R bb 、R cc and R dd As mentioned above.

[0049] Other definitions

[0050] The term "siRNA" herein refers to a class of double-stranded RNA molecules that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a gene encoding a protein) that is complementary thereto. siRNA is typically double-stranded, comprising an antisense strand complementary to the target RNA and a sense strand complementary to the antisense strand. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNA (e.g., tRNA) and viral RNA other than mRNA can also be targeted.

[0051] The term "antisense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a target sequence. The term "sense strand" refers to a strand of an siRNA that includes a region that is completely, fully, or substantially complementary to a region that is, as the term is defined herein, an antisense strand.

[0052] The term "complementary region" refers to a region on the antisense strand that is completely, fully or substantially complementary to the target mRNA sequence. In the case where the complementary region is not completely complementary to the target sequence, mispairing can be located in the interior or terminal regions of the molecule. Typically, the most tolerated mispairing is located in the terminal regions, for example, in 5' and / or 3' ends within 5, 4, 3, 2 or 1 nucleotide. The antisense strand portion that is most sensitive to mispairing is referred to as a "seed region." For example, in a siRNA comprising a 19nt chain, the 19th position (from 5' to 3') can tolerate some mispairing.

[0053] The term "complementary" refers to the ability of a first polynucleotide to hybridize to a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50° C. or 70° C. for 12-16 hours. In terms of meeting the above requirements relative to their ability to hybridize, "complementary" sequences may also include or be completely formed from non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogstein base pairing.

[0054] A polynucleotide that is "at least partially complementary," "sufficiently complementary," or "substantially complementary" to a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, a polynucleotide is complementary to at least a portion of a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. The terms "complementary," "fully complementary," "sufficiently complementary," and "substantially complementary" as used herein can be used with respect to base pairing between the sense and antisense strands of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0055] "Fully complementary" refers to the extent to which the sense strand only needs to be complementary to the antisense strand in order to maintain the overall double-stranded nature of the molecule. In other words, while perfect complementarity is generally desired, in some cases, particularly in the antisense strand, one or more, for example, 6, 5, 4, 3, 2, or 1 mismatches (relative to the target mRNA) may be included, but the sense and antisense strands can still maintain the overall double-stranded nature of the molecule.

[0056] "shRNA" stands for short hairpin RNA. shRNA consists of two short inverted repeats. When cloned into an shRNA expression vector, the two short inverted repeats are separated by a stem-loop sequence, forming a hairpin structure controlled by a Pol III promoter. Five to six Ts are then attached to serve as a transcriptional terminator for RNA polymerase III.

[0057] A "nucleoside" is a compound composed of a purine or pyrimidine base and ribose or deoxyribose; a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate; an "oligonucleotide" is a nucleic acid molecule (RNA or DNA) having, for example, fewer than 100, 200, 300, or 400 nucleotides in length.

[0058] "Bases" are the fundamental building blocks of nucleosides, nucleotides, and nucleic acids. They contain nitrogen and are also called "nitrogenous bases." Unless otherwise specified, the capital letters A, U, T, G, and C represent the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0059] The "modification" of nucleotides herein includes, but is not limited to, methoxy modification, fluorination, phosphorothioate linkage, or conventional protecting group protection. For example, the fluorination-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0060] " modified nucleotide " herein includes but is not limited to 2 '-O-methyl modified nucleotides, 2 '-fluoro modified nucleotides, 2 '-deoxy-modified nucleotides, inosine ribonucleotides, abasic nucleotides, reverse abasic deoxyribonucleotides, nucleotides comprising thiophosphate groups, vinyl phosphate modified nucleotides, locked nucleotides, 2 '-amino-modified nucleotides, 2 '-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, the non-natural bases comprising nucleotides and the terminal nucleotides, deoxyribonucleotides or conventional protecting group protections on a cholesterol derivative or a dodecanoic acid didecylamide group. For example, the 2 '-fluoro modified nucleotides refer to nucleotides in which the hydroxyl group at the ribose group 2 ' position is replaced by fluorine. The 2 '-deoxy-modified nucleotides refer to nucleotides in which the 2 '-hydroxyl group at the ribose group is replaced by methoxy and formed.

[0061] " reactive phosphorus group " refers to the phosphorus-containing group contained in the nucleotide unit or in the nucleotide analog unit, and it can react with the hydroxyl or the amido reaction contained in another molecule, especially in another nucleotide unit or in another nucleotide analog by nucleophilic attack reaction.Usually, such reaction produces the ester type internucleoside bond that the first nucleotide unit or the first nucleotide analog unit are connected with the second nucleotide unit or the second nucleotide analog unit.Reactive phosphorus group can be selected from phosphoramidites, H-phosphonates, alkyl-phosphonates, phosphate or phosphate analogs, include but not limited to: natural phosphate, thiophosphate, phosphorodithioate, borane phosphate, borane thiophosphate, phosphonate, halogen-substituted phosphonate and phosphate, phosphoramidate, phosphodiester, phosphotriester, thiophosphate diester, thiophosphate triester, diphosphate and triphosphate, preferably-P(OCH2CH2CN)(N(iPr)2).

[0062] A "protecting group," also known as a "blocking group," refers to any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. A "protecting group" may be an unstable chemical moiety known in the art that is used to protect reactive groups, such as hydroxyl, amino, and thiol groups, from undesirable or inappropriate reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites and can then be removed to leave the unprotected group intact or available for further reactions.

[0063] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butyloxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy] methyl or tert-butyldiphenylsilyl); esters (e.g., benzoate); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein), and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr); 4,4′,4″-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, neopentyl (Vl), methylthiomethyl ether, ... Valeryl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-iso-propylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE) N,N-dimethylformamidine and 2-cyanoethyl (CE).

[0064] A "hydroxyl protecting group" is a group that protects the hydroxyl group from chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These groups primarily include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred:

[0065] Trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc) , benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl, more preferably -C(O)CH2CH2C(O)OH.

[0066] As used herein, the term "pharmaceutically acceptable salt" refers to those carboxylate salts, amino acid addition salts of the compounds of the present invention that are suitable for use in contact with patient tissues within the scope of sound medical judgment, do not produce undue toxicity, irritation, allergic response, etc., are commensurate with a reasonable benefit / risk ratio, and are effective for their intended use, including (where possible) zwitterionic forms of the compounds of the present invention.

[0067] The present invention includes tautomers, which are functional isomers produced by the rapid movement of an atom in two positions in a molecule. Compounds that exist in different tautomeric forms are not limited to any specific tautomer, but are intended to cover all tautomeric forms.

[0068] The compounds of the present invention may include one or more asymmetric centers and may therefore exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0069] The present invention also includes isotopically labeled compounds (isotopic variants) which are identical to those described in formula (I) but for which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, for example 2 H. 3 H. 13 C. 11 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Compounds of the present invention containing the above-mentioned isotopes and / or other isotopes of other atoms, their prodrugs and pharmaceutically acceptable salts of the compounds or prodrugs are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, such as those in which radioactive isotopes (e.g. 3 H and 14 C) can be used in drug and / or substrate tissue distribution assays. 3 H and carbon-14, i.e. 14 C isotopes are particularly preferred because they are easy to prepare and detect. 2 H, because greater metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents when carrying out the processes disclosed in the following schemes and / or the Examples and Preparations.

[0070] Compounds of the present invention

[0071] The present invention specifically relates to a nucleotide dimer represented by formula (A):

[0072] in,

[0073] One of Q1 and Q2 is R4 and the other is O-L2;

[0074] L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0075] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0076] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0077] Y1 is O, S or NR;

[0078] Y2 is O, S or a chemical bond;

[0079] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0080] R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0081] R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe.

[0082] P1 is a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxy Carbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr;

[0083] P2 is a reactive phosphorus group such as a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic, such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranophosphate, a boranophosphothioate, a phosphonate, a halogen-substituted phosphonate and phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2);

[0084] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0085] R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0086] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-14 membered heteroaryl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a Rb 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-S(O) n R a 、-S(O) n OR a 、-S(O) n NR a R b 、-OS(O) n R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b 、-NR a -S(O) n R b or -NR a C(O)NR a R b ;

[0087] R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group;

[0088] m is selected from 1, 2, 3, 4 or 5;

[0089] n is independently selected from 1 or 2.

[0090] The present invention also relates to a double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand comprises one or more nucleotide monomers represented by formula (III) or (IV):

[0091] in,

[0092] The nucleotide monomers are shown in the order 5'=>3' from to connect;

[0093] Each group is as defined above and below;

[0094] Preferably, the nucleotide monomer is selected from:

[0095] Among them, Base is selected from

[0096] Q1 and Q2

[0097] In one embodiment, Q1 is R4 and Q2 is O-L2; in another embodiment, Q1 is O-L2 and Q2 is R4.

[0098] X

[0099] In one embodiment, X is -(CR1R2) m -; In another embodiment, X is -CR1=CR2-.

[0100] In a more specific embodiment, X is -CH2-; in another more specific embodiment, X is -CH(OH)-; in another more specific embodiment, X is -CH2-CH2-; in another more specific embodiment, X is -CH=CH-.

[0101] Y1 and Y2

[0102] In one embodiment, Y1 is O; in another embodiment, Y1 is S; in another embodiment, Y1 is NR.

[0103] In one embodiment, Y2 is O; in another embodiment, Y2 is S; in another embodiment, Y2 is a chemical bond.

[0104] L1 and L2

[0105] In one embodiment, L1 is H; in another embodiment, L1 is P1; in another embodiment, L1 is a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose sugar of another nucleotide or oligonucleotide.

[0106] In one embodiment, L2 is H; in another embodiment, L2 is P2; in another embodiment, L2 is a chemical bond to the phosphate P atom at the 5' end of the ribose sugar of another nucleotide or oligonucleotide.

[0107] R1 and R2

[0108] In one embodiment, R1 is H; in another embodiment, R1 is D; in another embodiment, R1 is halogen; in another embodiment, R1 is OH; in another embodiment, R1 is CN; in another embodiment, R1 is C 1-6 Alkyl; in another embodiment, R1 is C 1-4 Alkyl; in another embodiment, R1 is C 1-6 In another embodiment, R1 is C 1-4 In another embodiment, R1 is C 2-6 In another embodiment, R1 is C 2-6 Alkynyl; in another embodiment, R1 is C 3-10 In another embodiment, R1 is a 3-10 membered heterocyclyl; in another embodiment, R1 is C 6-10 Aryl; in another embodiment, R1 is 5-14 membered heteroaryl.

[0109] In one embodiment, R1 is unsubstituted; in another embodiment, R1 is substituted with 1 R'; in another embodiment, R1 is substituted with 2 R'; in another embodiment, R1 is substituted with 3 R'; in another embodiment, R1 is substituted with 4 R'; in another embodiment, R1 is substituted with 5 R'; in another embodiment, R1 is substituted with 6 R'; in another embodiment, R1 is substituted with 7 R'; in another embodiment, R1 is substituted with 8 R'; in another embodiment, R1 is substituted with multiple R'.

[0110] In one embodiment, R2 is H; in another embodiment, R2 is D; in another embodiment, R2 is halogen; in another embodiment, R2 is OH; in another embodiment, R2 is CN; in another embodiment, R2 is C 1-6 Alkyl; in another embodiment, R2 is C 1-4 Alkyl; in another embodiment, R2 is C 1-6 In another embodiment, R2 is C 1-4 In another embodiment, R2 is C 2-6 In another embodiment, R2 is C 2-6 Alkynyl; in another embodiment, R2 is C 3-10 In another embodiment, R2 is a 3-10 membered heterocyclyl; in another embodiment, R2 is C 6-10 Aryl; in another embodiment, R2 is a 5-14 membered heteroaryl.

[0111] In one embodiment, R2 is unsubstituted; in another embodiment, R2 is substituted with 1 R'; in another embodiment, R2 is substituted with 2 R'; in another embodiment, R2 is substituted with 3 R'; in another embodiment, R2 is substituted with 4 R'; in another embodiment, R2 is substituted with 5 R'; in another embodiment, R2 is substituted with 6 R'; in another embodiment, R2 is substituted with 7 R'; in another embodiment, R2 is substituted with 8 R'; in another embodiment, R2 is substituted with multiple R'.

[0112] R3

[0113] In one embodiment, R3 is H; in another embodiment, R3 is C 1-6 Alkyl; in another embodiment, R3 is C 1-4 Alkyl, such as Me; in another embodiment, R3 is C 1-6 In another embodiment, R3 is C 1-4 In another embodiment, R3 is C 2-6 In another embodiment, R3 is C 2-6 Alkynyl; in another embodiment, R3 is C 3-10 In another embodiment, R3 is a 3-10 membered heterocyclyl; in another embodiment, R3 is C 6-10 Aryl; in another embodiment, R3 is 5-14 membered heteroaryl.

[0114] In one embodiment, R3 is unsubstituted; in another embodiment, R3 is substituted with 1 R'; in another embodiment, R3 is substituted with 2 R'; in another embodiment, R3 is substituted with 3 R'; in another embodiment, R3 is substituted with 4 R'; in another embodiment, R3 is substituted with 5 R'; in another embodiment, R3 is substituted with 6 R'; in another embodiment, R3 is substituted with 7 R'; in another embodiment, R3 is substituted with 8 R'; in another embodiment, R3 is substituted with multiple R'.

[0115] R4 and R5

[0116] In one embodiment, R4 is H; in another embodiment, R4 is D; in another embodiment, R4 is OH; in another embodiment, R4 is halogen; in another embodiment, R4 is C 1-6 Alkyl; in another embodiment, R4 is C 1-4 Alkyl; in another embodiment, R4 is C 1-6In another embodiment, R4 is C 1-4 In another embodiment, R4 is C 1-6 Alkoxy; in another embodiment, R4 is C 1-4 Alkoxy, such as OMe; in another embodiment, R4 is C 1-6 In another embodiment, R4 is C 1-4 Halogenated alkoxy.

[0117] In one embodiment, R5 is H; in another embodiment, R5 is D; in another embodiment, R5 is OH; in another embodiment, R5 is halogen, such as F; in another embodiment, R5 is C 1-6 Alkyl; in another embodiment, R5 is C 1-4 Alkyl; in another embodiment, R5 is C 1-6 In another embodiment, R5 is C 1-4 In another embodiment, R5 is C 1-6 Alkoxy; in another embodiment, R5 is C 1-4 Alkoxy, such as OMe; in another embodiment, R5 is C 1-6 In another embodiment, R5 is C 1-4 Halogenated alkoxy.

[0118] P1 and P2

[0119] In one embodiment, P1 is a protecting group; in another embodiment, P1 is a hydroxy protecting group, such as trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloro Ethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytrityl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), 4,4'-dimethoxytrityl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH, preferably DMTr.

[0120] In one embodiment, P2 is a reactive phosphorus group such as a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate or a phosphate mimetic such as a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranephosphate, a boranephosphorothioate, a phosphonate, a halogen-substituted phosphonate and a phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorothioate diester, a phosphorothioate triester, a diphosphate or a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

[0121] Base and Base'

[0122] In one embodiment, Base is H; in another embodiment, Base is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G, and C.

[0123] In one embodiment, Base' is H; in another embodiment, Base' is a modified or unmodified base or a leaving group, such as, preferably, modified or unmodified A, U, T, G, and C.

[0124] In a more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is In another more specific embodiment, Base is

[0125] In a more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is In another more specific embodiment, Base' is

[0126] R

[0127] In one embodiment, R is H; in another embodiment, R is C 1-6 Alkyl; in another embodiment, R is C 1-6 Halogenated alkyl.

[0128] R'

[0129] In one embodiment, R' is D; in another embodiment, R' is halogen; in another embodiment, R' is CN; in another embodiment, R' is C 1-6 Alkyl; in another embodiment, R' is C 1-6 In another embodiment, R' is C 2-6 In another embodiment, R' is C 2-6 Alkynyl; in another embodiment, R' is C 3-10In another embodiment, R' is a 3-10 membered heterocyclyl; in another embodiment, R' is C 6-10 Aryl; in another embodiment, R' is 5-14 membered heteroaryl; in another embodiment, R' is -OR a , such as OH; in another embodiment, R' is -OC(O)R a In another embodiment, R' is -OC(O)OR b In another embodiment, R' is -OC(O)NR a R b In another embodiment, R' is -C(O)R a In another embodiment, R' is -C(O)OR a In another embodiment, R' is -C(O)NR a R b In another embodiment, R' is -S(O) n R a In another embodiment, R' is -S(O) n OR a In another embodiment, R' is -S(O) n NR a R b In another embodiment, R' is -OS(O) n R b In another embodiment, R' is -NR a R b In another embodiment, R' is -NR a C(O)R b In another embodiment, R' is -NR a -C(O)OR b In another embodiment, R' is -NR a -S(O) n R b In another embodiment, R' is -NR a C(O)NR a R b .

[0130] Among them, each R a Independently selected from: H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or Ra and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group;

[0131] Each R b Independently selected from: H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group.

[0132] m

[0133] In one embodiment, m is 0; in another embodiment, m is 1; in another embodiment, m is 2; in another embodiment, m is 3; in another embodiment, m is 4; in another embodiment, m is 5.

[0134] n

[0135] In one embodiment, n is 0; in another embodiment, n is 1; in another embodiment, n is 2; in another embodiment, n is 3; in another embodiment, n is 4; in another embodiment, n is 5.

[0136] GalNAc

[0137] In one embodiment, GalNAc is a conjugated group represented by formula (X):

[0138] in,

[0139] Indicates the location of attachment to a biomolecule;

[0140] Q is independently H,

[0141] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0142] L G2 is a chemical bond or -CH2CH2C(O)-;

[0143] LG3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0144] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0145] Where a = 0, 1, 2 or 3;

[0146] b = 1, 2, 3, 4, or 5;

[0147] c = 1, 2, 3, 4, or 5;

[0148] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0149] A is a chemical bond, -CH2O- or -NHC(O)-;

[0150] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0151] Where e is 1, 2, 3, 4 or 5;

[0152] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0153] Where M is

[0154] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0155] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0156] where R G For-OR G ', -CH2OR G'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0157] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0158] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0159] In another embodiment, GalNAc is a conjugate group represented by formula (I'):

[0160] in,

[0161] Indicates the location of attachment to a biomolecule;

[0162] Q is independently H,

[0163] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0164] L G2 is a chemical bond or -CH2CH2C(O)-;

[0165] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0166] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0167] Where a = 0, 1, 2 or 3;

[0168] b = 1, 2, 3, 4, or 5;

[0169] c = 1, 2, 3, 4, or 5;

[0170] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0171] A is -CH2O- or -NHC(O)-;

[0172] A' is a chemical bond, -C(O)NH- or -NHC(O)-;

[0173] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0174] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0175] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0176] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0177] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0178] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein,

[0179] Q is independently H,

[0180] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0181] L G2 is a chemical bond or -CH2CH2C(O)-;

[0182] L G3For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0183] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0184] Where a = 0, 1, 2 or 3;

[0185] b = 1, 2, 3, 4, or 5;

[0186] c = 1, 2, 3, 4, or 5;

[0187] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0188] A is a chemical bond, -CH2O- or -NHC(O)-;

[0189] A' is a chemical bond, -C(O)NH-, -NHC(O)- or -O(CH2CH2O) e -;

[0190] Where e is 1, 2, 3, 4 or 5;

[0191] B is a chemical bond, -CH2-, -M-, -CH2-M- or -C(O)-M-;

[0192] Where M is

[0193] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0194] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0195] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0196] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0197] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0198] In another embodiment, GalNAc is a conjugated group represented by formula (X), wherein:

[0199] Q is independently H,

[0200] Among them L G1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0201] L G2 is a chemical bond or -CH2CH2C(O)-;

[0202] L G3 For chemical bonds, -(NHCH2CH2) b -、-(NHCH2CH2CH2) b - or -C(O)CH2-;

[0203] L G4 -(OCH2CH2) c -、-(OCH2CH2CH2) c -、-(OCH2CH2CH2CH2) c -、-(OCH2CH2CH2CH2CH2) c -or-NHC(O)-(CH2) d -;

[0204] Where a = 0, 1, 2 or 3;

[0205] b = 1, 2, 3, 4, or 5;

[0206] c = 1, 2, 3, 4, or 5;

[0207] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0208] A is a chemical bond, -CH2O- or -NHC(O)-;

[0209] A' is -O(CH2CH2O) e -;

[0210] Where e is 1, 2, 3, 4 or 5;

[0211] B is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M- or -C(O)-M-;

[0212] Where M is

[0213] R G1 and R G2 Together to form -CH2CH2O- or -CH2CH(R G )-O-, and R G3 is H;

[0214] or R G1 and R G3 Together they form -C 1-2 Alkylene-, and R G2 is H;

[0215] where R G For-OR G ', -CH2OR G 'or-CH2CH2OR G ', where R G ' is H, a hydroxyl protecting group or a solid phase support, wherein the hydroxyl protecting group is preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0216] m1 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0217] n1=0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0218] Any technical solution or any combination thereof in any of the above specific embodiments may be combined with any technical solution or any combination thereof in any other specific embodiment. For example, any technical solution or any combination thereof of X may be combined with any technical solution or any combination thereof of Q1, Q2, Y1, Y2, L1, L2, R1, R2, R3, R4, R5, Base, and Base', etc., or any combination thereof. The present invention is intended to include all such combinations of technical solutions, which are not listed here one by one due to space limitations.

[0219] The present invention also provides a vector comprising a nucleotide sequence encoding the siRNA of the present invention. The vector of the present invention is capable of amplifying or expressing the nucleotide sequence encoding the siRNA of the present invention linked thereto.

[0220] For example, siRNA targeting the PCSK9 gene can be expressed from a transcription unit inserted into a DNA or RNA vector. Expression can be short-lived (a few hours to a few weeks) or continuous (a few weeks to a few months or longer), depending on the specific construct and target tissue or cell type used. The coding nucleotides of the siRNA can be introduced into a linear construct, a circular plasmid or a viral vector. The nucleotides of the siRNA can be integrated into the cell genome for stable expression, or expressed in an extrachromosomal stable inheritance. In general, siRNA expression vectors are typically DNA plasmids or viral vectors.

[0221] Viral vector systems containing siRNA coding sequences include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenovirus or gut-free adenovirus.

[0222] The present invention also provides a cell containing the siRNA or vector of the present invention, wherein the siRNA or vector of the present invention can be transcribed in the cell.

[0223] The present invention specifically relates to the following technical solutions:

[0224] 1. A nucleotide dimer represented by formula (A):

[0225] in,

[0226] One of Q1 and Q2 is R4 and the other is O-L2;

[0227] L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0228] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0229] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0230] Y1 is O, S or NR;

[0231] Y2 is O, S or a chemical bond;

[0232] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0233] R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R';

[0234] R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe.

[0235] P1 is a hydroxyl protecting group, preferably DMTr;

[0236] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0237] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0238] R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0239] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-14 membered heteroaryl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-S(O)n R a 、-S(O) n OR a 、-S(O) n NR a R b 、-OS(O) n R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b 、-NR a -S(O) n R b or -NR a C(O)NR a R b ;

[0240] R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group;

[0241] m is selected from 1, 2, 3, 4 or 5;

[0242] n is independently selected from 1 or 2.

[0243] 2. The nucleotide duplex of technical solution 1 has a structure of formula (I) or (II):

[0244] Wherein, each group is as defined in Technical Solution 1.

[0245] 3. The nucleotide dimer of technical solution 2, wherein,

[0246] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0247] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R';

[0248] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b or -NR a C(O)NR a R b ;

[0249] R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0250] m is selected from 1, 2, 3, 4 or 5;

[0251] Preferably,

[0252] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0253] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's;

[0254] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a or -NR a R b ;

[0255] R a and R b Independently selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0256] m is selected from 1, 2 or 3;

[0257] More preferably,

[0258] X is selected from -CH2-, -CH(OH)-, -CH2-CH2- or -CH=CH-.

[0259] 4. The nucleotide dimer of technical solution 2 or 3, wherein:

[0260] Y1 is O or NR;

[0261] Y2 is O, S or a chemical bond;

[0262] R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0263] Preferably,

[0264] Y1 is O or NR;

[0265] Y2 is O, S or a chemical bond;

[0266] R is selected from H or C 1-6 alkyl;

[0267] More preferably,

[0268] Y1 is O;

[0269] Y2 is O.

[0270] 5. The nucleotide duplex according to any one of technical solutions 2 to 4, wherein:

[0271] R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R';

[0272] Preferably,

[0273] R3 is selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's;

[0274] More preferably,

[0275] R3 is C 1-4Alkyl, preferably Me.

[0276] 6. The nucleotide dimer according to any one of technical solutions 2 to 5, wherein:

[0277] R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 alkoxy;

[0278] Preferably,

[0279] R4 and R5 are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F, OH or OMe;

[0280] More preferably,

[0281] R4 is selected from H, OH or C 1-4 Alkoxy, preferably H or OMe;

[0282] R5 is selected from halogen, OH or C 1-4 Alkoxy, preferably F or OMe.

[0283] 7. The nucleotide dimer according to any one of technical solutions 2 to 6, wherein:

[0284] Base and Base' are independently selected from

[0285] 8. The nucleotide dimer according to any one of technical solutions 2 to 7, wherein:

[0286] L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0287] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0288] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0289] Y1 is O, S or NR;

[0290] Y2 is O, S or a chemical bond;

[0291] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R';

[0292] R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R';

[0293] R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe;

[0294] P1 is a hydroxyl protecting group, preferably DMTr;

[0295] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0296] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0297] R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0298] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b or -NR a C(O)NR a R b ;

[0299] Ra and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl;

[0300] m is selected from 1, 2, 3, 4 or 5.

[0301] 9. The nucleotide duplex according to any one of technical solutions 2 to 8, wherein:

[0302] L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0303] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0304] X is selected from -(CR1R2) m -or-CR1=CR2-;

[0305] Y1 is O or NR;

[0306] Y2 is O, S or a chemical bond;

[0307] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's;

[0308] R3 is selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's;

[0309] R4 and R5 are independently selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe;

[0310] P1 is a hydroxyl protecting group, preferably DMTr;

[0311] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0312] Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C;

[0313] R is selected from H, C 1-6 Alkyl or C1-6 alkyl halide;

[0314] R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a or -NR a R b ;

[0315] R a and R b Independently selected from H, C 1-6 Alkyl or C 1-6 alkyl halide;

[0316] m is selected from 1, 2 or 3.

[0317] 10. The nucleotide duplex according to any one of technical solutions 2 to 9, wherein:

[0318] L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1;

[0319] L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2;

[0320] X is selected from -(CR1R2) m - or -CR1=CR2-, preferably -CH2-, -CH(OH)-, -CH2-CH2- or -CH=CH-;

[0321] Y1 is O;

[0322] Y2 is O;

[0323] R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-4 Alkyl or C 1-4 alkyl halide;

[0324] R3 is C 1-4 Alkyl, preferably Me;

[0325] R4 is selected from H, OH or C 1-4 Alkoxy, preferably H or OMe;

[0326] R5 is selected from halogen, OH or C 1-4 Alkoxy, preferably F or OMe;

[0327] P1 is a hydroxyl protecting group, preferably DMTr;

[0328] P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0329] Base and Base' are independently selected from

[0330] m is selected from 1, 2 or 3.

[0331] 11. The nucleotide duplex of any one of technical solutions 1-10, selected from:

[0332] Wherein, Base and Base' are as defined in any one of technical solutions 1-10, preferably

[0333] R5 is as defined in any one of technical solutions 1-10, preferably F or OMe.

[0334] 12. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand comprises one or more nucleotide monomers represented by formula (III) or (IV):

[0335] in,

[0336] The nucleotide monomers are shown in the order 5'=>3' from to connect;

[0337] Each group is as defined in any one of technical solutions 1-11;

[0338] Preferably, the nucleotide monomer is selected from:

[0339] Among them, Base is selected from

[0340] 13. The double-stranded RNA molecule of technical solution 12, wherein the sense strand and the antisense strand each have 20 to 25 nucleotides.

[0341] 14. The double-stranded RNA molecule of technical solution 12 or 13, wherein the nucleotide monomer is located at position 2-8 of the 5' end of the antisense strand, preferably position 6 or 7, more preferably position 7.

[0342] 15. The double-stranded RNA molecule of any one of technical solutions 12-14, wherein the double-stranded RNA shows enhanced stability compared to a double-stranded RNA having the same sequence but not comprising the nucleotide monomer described in technical solution 12.

[0343] 16. The double-stranded RNA molecule of any one of technical solutions 12-15, wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably about 55°C to 67°C.

[0344] 17. The double-stranded RNA molecule of any one of technical solutions 12-16, wherein the double-stranded RNA shows reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not containing the nucleotide monomer described in technical solution 12.

[0345] 18. The double-stranded RNA molecule of any one of technical solutions 12-17, wherein the double-stranded RNA shows enhanced effectiveness compared to a double-stranded RNA having the same sequence but not comprising the nucleotide monomer described in technical solution 12.

[0346] 19. The double-stranded RNA molecule of any one of technical solutions 12-18, wherein the antisense strand has a sequence that is fully complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.

[0347] 20. The double-stranded RNA molecule of technical solutions 12-19, wherein the target mRNA is encoded by an endogenous gene or a pathogen gene.

[0348] 21. The double-stranded RNA molecule of any one of technical solutions 12-20, wherein the sense strand and / or antisense strand comprises a 3' and / or 5' overhang.

[0349] 22. The double-stranded RNA molecule of any one of technical solutions 12-21, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

[0350] 23. A nucleic acid molecule, the nucleotide sequence of which contains one or more nucleotide monomers as described in technical solution 12.

[0351] 24. The nucleic acid molecule of technical solution 23, wherein the nucleic acid is selected from DNA, RNA and DNA / RNA hybrid.

[0352] 25. The nucleic acid molecule of technical solution 24, wherein the nucleic acid molecule is single-stranded or double-stranded.

[0353] 26. The nucleic acid molecule of any one of technical solutions 23-25, wherein the nucleic acid molecule is selected from small interfering RNA (siRNA) and short hairpin RNA (shRNA).

[0354] 27. A vector comprising a nucleotide sequence encoding the double-stranded RNA according to any one of the aforementioned technical solutions 12-22.

[0355] 28. A cell containing the double-stranded RNA according to any one of technical solutions 12 to 22 or the vector according to technical solution 27.

[0356] 29. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of technical solutions 12 to 22, and a pharmaceutically acceptable carrier or excipient.

[0357] 30. A kit comprising the double-stranded RNA molecule according to any one of technical solutions 12-22.

[0358] 31. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing the double-stranded RNA molecule described in any one of technical solutions 12-22 into the cell.

[0359] 32. A method for inhibiting the expression of a target gene in a cell, comprising expressing the double-stranded RNA molecule described in any one of technical solutions 12-22 in the cell.

[0360] 33. A method for reducing off-target toxicity in cells, comprising the step of introducing the double-stranded RNA molecule described in any one of technical solutions 12-22 into the cells.

[0361] 34. A method for reducing off-target toxicity in a cell, comprising expressing the double-stranded RNA molecule described in any one of technical solutions 12-22 in the cell. Example

[0362] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0363] Example 1 Preparation of Compound E1

[0364] 1. Preparation of Compound 2

[0365] Compound 1 (50.0 g, 263 mmol) was dissolved in DCM (800 mL) at 25°C, followed by the addition of imidazole (26.9 g, 394 mmol) and TBDPSCl (75.2 mL, 289 mmol). The reaction was stirred at 25°C for 18 hours. Thin-layer chromatography (DCM / MeOH = 10 / 1, PE / EA = 3 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction solution was dried to give the crude product, which was then purified by MPLC (PE / EA = 1 / 0-5 / 1) to afford compound 2 (95.0 g, 84.31% yield) as a colorless oil.

[0366] 1H NMR (400MHz, CDCl3) δ7.70(dd,J=7.6,1.6Hz,4H),7.35-7.46(m,6H),5.86(d,J=3.6Hz,1H),4.61(dd,J=4.8,4.0H z,1H),4.15(td,J=8.8,5.2Hz,1H),3.93-4.01(m,1H),3.81-3.92(m,2H),1.60(s,3H),1.39(s,3H),1.06(s,9H).

[0367] 2. Preparation of Compound 3

[0368] Compound 2 (95.0 g, 222 mmol) was dissolved in toluene (1.50 L) at 25°C. Imidazole (30.2 g, 443 mmol), triphenylphosphine (116 g, 443 mmol), and iodine (84.4 g, 322 mmol) were added sequentially. The reaction mixture was stirred at 100°C for 18 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 20.0 mL of saturated NaHSO₃ solution and 500 mL of water were added to the reaction mixture. The layers were separated, and the organic phase was washed with saturated NaCl solution (50.0 mL x 3), dried over anhydrous Na₂SO₄, and spin-dried to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to afford compound 3 (115 g, 96.35% yield) as a colorless oil.

[0369] 1 H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.37-7.49(m,6H),5.95(d,J=3.6Hz,1H),5.06(d,J=3.6Hz,1H),4.42(d,J=3.2Hz,1H),3. 87(dd,J=10.4,5.6Hz,1H),3.66(dd,J=10.4,6.4Hz,1H),3.53(td,J=6.0,3.2Hz,1H),1.44(s,3H),1.29(s,3H),1.02-1.07(m,9H).

[0370] 3. Preparation of Compound 4

[0371] Compound 3 (12.5 g, 23.2 mmol) was dissolved in a mixture of MeOH (225 mL) and EtOAc (25 mL) at 25°C. TEA (6.45 mL, 46.4 mmol) and 10% Pd / C (2.00 g, 18.8 mmol) were added. The reaction mixture was stirred at 25°C under a hydrogen atmosphere (14.696 psi) for 14 hours. Thin-layer chromatography (PE / EA = 10 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was filtered, and the filtrate was dried to give the crude product, which was then purified by MPLC (PE / EA = 1 / 0 to 10 / 1) to afford compound 4 (8.75 g, 83.06% yield) as a colorless oil.

[0372] 1 H NMR (400MHz, CD3OD) δ7.64-7.72(m,4H),7.36-7.47(m,6H),5.80(d,J=3.6Hz,1H),4.77(t,J=4.4Hz,1H),4.25-4.32 (m,1H),3.68-3.82(m,2H),2.00(dd,J=13.6,4.8Hz,1H),1.78-1.87(m,1H),1.45(s,3H),1.30(s,3H),1.04(s,9H).

[0373] 4. Preparation of Compound 5

[0374] Compound 4 (35.0 g, 84.8 mmol) was dissolved in THF (500 mL) at 25°C, and tetraethylammonium fluoride (63.3 g, 424 mmol) was added. The reaction was stirred at 25°C for 18 hours. Thin-layer chromatography (PE / EA = 10 / 1, DCM / MeOH = 10 / 1) showed complete consumption of the reactants and the formation of new spots. The reaction mixture was concentrated in vacuo to afford the crude product, which was then purified by MPLC (DCM / MeOH = 1 / 0 to 10 / 1) to afford Compound 5 (12 g, 81.21% yield) as a white solid.

[0375] 1 H NMR (400MHz, CD3OD) δ5.79(d,J=3.6Hz,1H),4.77(t,J=4.0Hz,1H),4.21-4.28(m,1H),3.70(dd,J=12.0,3.6Hz,1H ),3.54(dd,J=12.0,4.8Hz,1H),1.95-2.02(m,1H),1.74(ddd,J=13.2,10.8,4.8Hz,1H),1.46(s,3H),1.30(s,3H).

[0376] 5. Preparation of Compound 6

[0377] Compound 5 (12.0 g, 68.9 mmol) was dissolved in toluene (500 mL) at 25°C. Imidazole (9.38 g, 138 mmol), triphenylphosphine (36.1 g, 138 mmol), and iodine (26.2 g, 103 mmol) were added sequentially. The reaction mixture was stirred at 100°C for 3 hours. Thin-layer chromatography (PE / EA = 5 / 1) showed complete consumption of the reactants and the formation of new spots. 100 mL of saturated NaHSO₃ solution and 100 mL of water were added to the reaction mixture. The layers were separated, and the organic phase was washed with saturated NaCl solution (100 mL x 3), dried over anhydrous Na₂SO₄, and spin-dried to obtain the crude product. The crude product was purified by MPLC (PE / EA = 1 / 0-10 / 1) to afford Compound 6 (16.6 g, 84.82% yield) as a white solid.

[0378] 1 H NMR(400MHz, CDCl3)δ5.88(d,J=3.6Hz,1H),4.77(t,J=4.2Hz,1H),4.13-4.21(m,1H),3.2 5-3.38(m,2H),2.31(dd,J=13.6,4.4Hz,1H),1.61-1.67(m,1H),1.52(s,3H),1.33(s,3H).

[0379] 6. Preparation of Compound 7

[0380] Compound 6 (40.0 g, 141 mmol) was dissolved in trimethyl phosphite (500 mL) at 25°C, and the reaction mixture was stirred at 120°C for 11 hours. Thin-layer chromatography (ethyl acetate / acetone = 3 / 1, PE / EA = 10 / 1) showed residual starting material and the formation of new spots. The reaction mixture was spin-dried to obtain a crude product, which was purified by MPLC (ethyl acetate / acetone = 1 / 0-20 / 1) to afford compound 7 (8.90 g, 23.74% yield) as a pale yellow oil and recovered as a white solid (30.0 g).

[0381] 1H NMR(400MHz, CDCl3) δ5.81(d,J=4.0Hz,1H),4.74(t,J=4.4Hz,1H),4.40-4.52(m,1H),3.76(dd,J=10.8,0.8H z,6H),2.23-2.35(m,2H),1.95-2.07(m,1H),1.63(ddd,J=13.6,10.8,4.8Hz,1H),1.52(s,3H),1.32(s,3H).

[0382] 7. Preparation of Compound 8

[0383] Compound 7 (13 g, 48.830 mmol), Ac2O (23.036 mL, 244.150 mmol), and H2SO4 (2.615 mL, 48.830 mmol) were added sequentially to AcOH (260 mL). The reaction was allowed to proceed at 25°C for 6 hours. A new spot was detected by TLC (ethyl acetate:acetone = 3:1). The reaction solution was quenched with ice water, extracted with DCM (500 mL x 3), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (ethyl acetate:acetone = 50:1-3:1) to obtain compound 8 (7.9 g, 25.464 mmol, 52.15%) as a yellow oil.

[0384] 1 H NMR (400MHz, CDCl3) δ6.10(d,J=1.2Hz,1H),5.18(d,J=5.2Hz,1H),4.58-4.72(m,1H),3.75-3 .79(m,3H),3.71-3.74(m,3H)2.16-2.38(m,3H),2.07(s,3H),2.05(s,3H),1.96-2.04(m,1H).

[0385] 8. Preparation of Compound 9

[0386] Compound 8 (7.9 g, 25.464 mmol) and compound 8A (6.09 g, 25.464 mmol) were added to CHCN (316 mL). SnCl (8.781 mL, 76.392 mmol) was then slowly added dropwise at 0°C. The mixture was allowed to react at 25°C for 2 hours. TLC (ethyl acetate:acetone = 10:1, PMA) showed the disappearance of the starting compound 8 and the formation of a new spot. LCMS (RW0006-267-P1A) indicated a 31.3% yield of product. The reaction solution was cooled to 0°C and adjusted to pH 8 with saturated aqueous NaHCO3 solution. The aqueous phase was then extracted with DCM (200 mL x 3). The organic phase was dried over anhydrous Na2SO4, filtered, and spin-dried to give a crude product, which was purified by column chromatography (ethyl acetate:acetone = 20:1-3:1) to give compound 9 (6.1 g, 12.464 mmol, 48.95%) as a yellow oil.

[0387] 1 H NMR(400MHz, CDCl3)δ8.75-8.84(m,1H),8.17(s,1H),8.01-8.10(m,2H), 7.59-7.70(m,1H),7.50-7.57(m,2H),6.07(s,1H),5.66-5.73(m,1H),4.9 8-5.12(m,1H),4.70-4.81(m,1H),3.71-3.82(m,12H),2.70-2.81(m,1H), 2.34-2.52(m,2H),2.21-2.27(m,1H),2.16(s,3H),2.06(d,J=4.4Hz,4H).

[0388] 9. Preparation of Compound 10

[0389] Compound 9 (6.1 g, 12.259 mmol) was dissolved in pyridine (30 mL) and water (20 mL) and reacted at 60°C for 12 hours. TLC (DCM:MeOH = 10:1) showed the disappearance of the starting material. The reaction solution was directly spin-dried to obtain the crude product, which was then purified by Prep-HPLC (MeCN / H2O = 30 / 1 to 80 / 1; flow rate: 30 mL / min) to afford Compound 10 (3.85 g, 8.098 mmol, 66.06%) as a yellow oil.

[0390] 10. Preparation of Compound 11

[0391] Compound 10 (3.7 g, 7.783 mmol) and compound SM2 (10.24 g, 15.566 mmol) were dissolved in pyridine (37 mL). 2,4,6-Triisopropylbenzenesulfonyl chloride (14.14 g, 46.698 mmol) was added at 0°C. After 60 minutes of reaction at 0°C, N-methylimidazole (5.11 g, 62.263 mmol) was slowly added dropwise to the reaction solution. The reaction was continued at 25°C for 13 hours. LCMS (RW0006-284-P1C) revealed the product. TLC (DCM:MeOH = 10:1, UV) revealed the formation of a new spot. The reaction solution was cooled to 0°C and quenched by the addition of 50 mL of saturated sodium carbonate solution. The layers were separated, and the organic phase was diluted with 300 mL of DCM and washed with water (50 mL x 3) and saturated aqueous NaCl solution (50 mL x 1). The organic phase was dried over anhydrous Na2SO4, filtered, and spin-dried to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH=2%-4%, TEA) to give compound 11 (2100 mg, 1.883 mmol, 24.20%) as a brown oil.

[0392] 11. Preparation of Compound 12

[0393] Compound 11 (2.4 g, 2.152 mmol) was dissolved in MeOH (36 mL), and NH3 / MeOH (12.299 mL, 7 M) was added. The reaction was carried out at 0°C for 3 hours. LCMS (RW0006-290-P1A) showed that a product was generated. The reaction solution was diluted with 150 mL of DCM and washed with 30 mL of water. The organic phase was dried over anhydrous Na2SO4, filtered and dried to give a crude product. The crude product was purified by Prep-HPLC (column: 01-Waters Xbridge BEH C18 19*150 mm; mobile phase: TEAA-ACN; gradient: 33%-58.5% / 15 min; flow rate: 15 ml / min) to give a yellow solid compound 12 (380 mg, 0.354 mmol, 16.45%).

[0394] 12. Preparation of E1

[0395] Compound 12 (380 mg, 0.354 mmol) was washed three times with acetonitrile and dissolved in DCM (6.0 mL). 5A molecular sieves, DCI (41.82 mg, 0.354 mmol), and compound 13 (426.95 mg, 1.417 mmol) were then added sequentially. The atmosphere was purged with nitrogen three times and the reaction was allowed to proceed at 25°C for 1 hour. LCMS (RW00006-291-P1A) indicated 5.4% starting material remaining. The reaction mixture was quenched by adding dropwise to 20 mL of ice-cold saturated aqueous NaHCO₃ solution, diluted with 30 mL of DCM, and separated. The organic phase was washed with 20 mL of saturated aqueous NaHCO₃ and 20 mL of saturated aqueous NaCl. The organic phase was dried over anhydrous Na2SO4, filtered, and dried to give a crude product, which was purified by reverse phase purification (acetonitrile / water: 20-80%, 30 min, 20 mL / min) to give a yellow solid E1 (210 mg, 0.165 mmol, 46.57%).

[0396] 1 H NMR (400MHz, CDCl3) δ11.96-12.03(m,1H),10.07-10.20(m,1H),8.99-9.12(m,1H),8.62-8.80(m,1H),8.07-8.16(m,1H),8.01-8.05(m,2 H),7.78(d,J=1.2Hz,1H),7.60-7.66(m,1H),7.51-7.58(m,2H),7.21-7.26(m,2H),7.09-7.20(m,7H),6.66-6.75(m,4H),6.37-6.56(m,1 H),5.75-6.11(m,3H),5.07-5.19(m,1H),4.60-4.74(m,1H),4.16-4.29(m,1H),3.81-3.89(m,1H),3.76-3.81(m,3H),3.70(d,J=1.6Hz,7 H),3.54-3.69(m,3H),3.05-3.15(m,1H),2.54-2.65(m,2H),2.03-2.53(m,5H),1.43-1.50(m,1H),1.14-1.21(m,11H),1.01-1.13(m,6H).

[0397] Example 2 Preparation of siRNA

[0398] The siRNA of the present invention is prepared using the solid phase phosphoramidite method well known in the art. Specific methods can be found in, for example, PCT Publication Nos. WO2016081444 and WO2019105419, and are briefly described below.

[0399] 1 Synthesis of positive sense chain (SS chain)

[0400] Using solid-phase phosphoramidite synthesis, a blank CPG solid support or a solid support connected to L96 is used as the starting cycle. Nucleoside monomers are attached one by one in the 3'-5' direction according to the order of the positive chain nucleotides. Each ligation of a nucleoside monomer involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide are as follows:

[0401] The nucleoside monomer was provided in a 0.05 mol / L acetonitrile solution. The reaction conditions for each step were the same, i.e., the temperature was 25°C, and deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution. The coupling reaction was performed twice using a 0.25 mol / L ETT-acetonitrile solution as the activator. Capping was performed twice using 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v). Oxidation was performed twice using 0.05 mol / L iodine / tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v). Thiolysis was performed twice using 0.2 mol / L PADS in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0402] 2 Synthesis of antisense strand (AS strand)

[0403] Using solid-phase phosphoramidite synthesis, a blank CPG solid support is used as the starting cycle. Nucleoside monomers or nucleotide duplexes of the present invention are linked one by one in the 3'-5' direction according to the order of the antisense nucleotides. Each ligation involves four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for a 5 μmol oligonucleotide of the antisense strand are the same as those for the sense strand.

[0404] 3. Oligonucleotide Purification and Annealing

[0405] 3.1 Ammonolysis

[0406] The synthesized solid phase carrier (sense chain or antisense chain) was added to a 5 mL centrifuge tube, 3% diethylamine / ammonia water (v / v) was added, and the reaction was carried out in a constant temperature water bath at 35°C (or 55°C) for 16 hours (or 8 hours). After filtering, the solid phase carrier was washed three times with ethanol / water, each time with 1 mL. The filtrate was centrifuged and concentrated, and the crude product was purified.

[0407] 3.2 Purification

[0408] Purification and desalination methods are well known in the art. For example, a column packed with a strong anion filler can be used, and a sodium chloride-sodium hydroxide system can be used for elution and purification, and the product can be collected and stored in a tube. A gel-filled purification column can be used for desalination, and the elution system can be pure water.

[0409] 3.3 Annealing

[0410] According to Table 6, the sense chain (SS chain) and the antisense chain (AS chain) were mixed at a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95° C., maintained for 3-5 min, naturally cooled to room temperature, and the system was lyophilized to obtain the product.

[0411] The siRNA sequences used in the present invention are as follows:

[0412] In this article, the meanings of the abbreviations are as follows:

[0413] The A, U, G, and C distribution represents the natural adenine, uracil, guanine, and cytosine ribonucleotides.

[0414] m indicates that the nucleotide adjacent to its left is a 2'-OCH3 modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3 modified A, U, G, and C.

[0415] f indicates that the nucleotide adjacent to its left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0416] "s" or "s-" indicates that the two adjacent nucleotides and / or delivery vectors are linked by phosphorothioate.

[0417] L96 represents a GalNAc delivery vector of the following structure well known in the art, wherein The position of the siRNA is indicated by a phosphate group or a phosphorothioate group, as described in, for example, PCT Publication Nos. WO2009073809 and WO2009082607.

[0418] ROR14 represents a nucleotide substitute of the structure described above, wherein Base can be any base, for example, ROR14-A represents that Base is adenine.

[0419] Specifically, the structure of ROR14-A is as follows: Among them, Base is adenine.

[0420] Example 3 Detection of on-target and off-target activities of siRNA compound psi-CHECK2

[0421] 1. Plasmid preparation:

[0422] On-target plasmid: The corresponding antisense strand on-target plasmid was designed according to the compound sequence. The psiCHECK2GSCM recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and diluted to 1000 ng / μL for later use.

[0423] Off-target plasmid: The corresponding antisense chain off-target plasmid was designed according to the compound sequence. The psiCHECK2GSSM-5Hits recombinant plasmid was prepared by Sangon Biotech (Shanghai) Co., Ltd. and the recombinant plasmid was diluted to 1000 ng / μL for use.

[0424] 2. Cell transfection:

[0425] HEK293A cells (Nanjing Kebai, Cat. No. CBP60436) were plated in 96-well plates with 100 μL of cell resuspension. The cell number was 8 × 10 3 cells / well.

[0426] On the second day, the complete culture medium in the wells was first aspirated and discarded, and replaced with 80 μL / well of Opti-MEM culture medium, and the cells were starved for about 1.5 h.

[0427] Plasmid mixture: Single-well preparation volume: plasmid 0.01 μL / well, Opti-MEM 8.99 μL / well.

[0428] Lipo mix: dilute Lipo 2000 (Lipofectamine TM 2000 transfection reagent, Thermo, 11668019), incubate at room temperature for 5 minutes, and prepare the Lipo mixture in the following amounts: Lipo 0.2 μL / well, Opti-MEM 9.8 μL / well.

[0429] Aliquot 22 μL of the prepared Lipo mixture, 2.2 μL of the compound, and 19.8 μL of the plasmid mixture into corresponding wells (designated Well A). Mix thoroughly by pipetting and incubate at room temperature for 20 minutes before co-transfection. Finally, add 20 μL of the Well A mixture to each well of cells, adding 80 μL of Opti-MEM to the original volume, for a final volume of 100 μL per well. After incubation at 37°C in a 5% CO2 incubator for 4 hours, add 100 μL of DMEM supplemented with 20% fetal bovine serum to each well. Incubate at 37°C in a 5% CO2 incubator for 24 hours before testing.

[0430] 3. Result detection:

[0431] Before the experiment, mix the Luciferase( Luciferase Assay System, Promega, E2940) was reconstituted and after equilibration to room temperature, DMEM was added to each tube at a ratio of 1:1 to prepare substrate I for immediate use. Stop& Buffer was reconstituted and then equilibrated to room temperature. Stop& Substrate 100:1 is prepared as Substrate II and is ready for use.

[0432] The vacuum pump sucks away the original culture medium in the 96-well culture plate;

[0433] Add 150 μL of substrate I to each well and incubate on a shaker at room temperature for 10 min;

[0434] 120 μL of substrate I was transferred to a 96-well microplate, and the Firefly chemiluminescence value was read on a microplate reader (Tecan, Infinite 200);

[0435] Then, 60 μL of substrate II was added to each well, and the cells were incubated on a shaker at room temperature for 10 min. The Renilla chemiluminescence value was read on a microplate reader.

[0436] 4. Data Analysis and Processing

[0437] Fluorescence activity was measured by a microplate reader. The collected Renilla signal was normalized by the Firefly signal standard. The inhibitory effect of siRNA was calculated by comparing it with the untreated result (residual inhibitory activity). The calculation process is as follows:

[0438] Normalized Ren / Fir ratio: Ratio = Renilla (Renilla luciferase) / Firefly (Firefly luciferase).

[0439] Residual inhibition rate: 2 replicates (Ratio siRNA / Ratio control )*100% mean: where Ratio control The average value of Ratio of two replicate wells in the control well (without siNRA) was used to calculate the Ratio of two replicate wells. siRNA / Ratio control , and then take the mean value as the residual inhibition rate;

[0440] Graphing: Graphpad Prism

[0441] Half maximal inhibitory concentration (IC50): In this experiment, Top and Bottom plots were used. The IC50 value was calculated using the formula Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where Y = 50 and X = log(concentration).

[0442] In the target activity detection experiment, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was selected for psiCHECK2-GSCM recombinant plasmid transfection, and the starting concentration of the compound was selected as 10 nM, and 11 concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, 0.000169 nM) were diluted 3-fold for siRNA compound activity screening. The results are shown in Table 1.

[0443] Table 1 Results of psi-CHECK2 on-target activity detection experiments

[0444] For the off-target activity detection experiment, HEK293A (Nanjing Kebai, catalog number CBP60436) cell line was transfected with psiCHECK2-GSSM-5Hits recombinant plasmid, and the starting concentration of the compound was 10 nM. Eleven concentration points (10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.123 nM, 0.041 nM, 0.0136 nM, 0.0045 nM, 0.00152 nM, 0.000508 nM, and 0.000169 nM) were selected for siRNA compound activity screening. The results are shown in Table 2.

[0445] Table 2 Results of psi-CHECK2 off-target activity detection experiments

[0446] The results showed that siRNA carrying ROR14 effectively reduced off-target activity while maintaining on-target activity.

[0447] Example 4 Verification of the long-term efficacy of the compound in the C57BL / 6 mouse model

[0448] C57BL / 6 mice (male, 18-21 g, 6-8 weeks) were randomly divided into groups according to Table 3, with 6 animals in each group. The dosage of each animal was calculated based on body weight, and a single subcutaneous injection was used for administration. The siRNA compound was first prepared as a 1 mg / mL solution (0.9% sodium chloride aqueous solution as the solvent). Before the experiment, the siRNA compound was dissolved in 0.9% sodium chloride aqueous solution and the volume was adjusted to the desired solution concentration and volume. The administration volume of physiological saline and siRNA compound was 5 mL / kg.

[0449] Table 3 Animal experiment groups

[0450] Blood was collected from the orbital venous plexus of mice before administration (marked as day 0) and on days 14, 28, 42, and 56 after administration. Serum mTTR protein was detected using an ELISA kit (Abcam, ab282297) at each time point. At the last experimental time point, 10 mg of liver was collected and placed in RNAlater solution to detect liver mTTR mRNA.

[0451] Table 4 Results of long-term efficacy verification of compounds in C57BL / 6 mouse model

[0452] The results showed that siRNA carrying ROR14 could reduce target gene expression for a long time in vivo.

Claims

1. A nucleotide dimer represented by formula (A): in, One of Q1 and Q2 is R4 and the other is O-L2; L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1; L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2; X is selected from -(CR1R2) m -or-CR1=CR2-; Y1 is O, S or NR; Y2 is O, S or a chemical bond; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R'; R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 aryl or 5-14 membered heteroaryl, optionally substituted with 1, 2, 3, 4, 5, 6, 7, 8 or more R'; R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe. P1 is a hydroxyl protecting group, preferably DMTr; P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-14 membered heteroaryl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-S(O) n R a 、-S(O) n OR a 、-S(O) n NR a R b 、-OS(O) n R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b 、-NR a -S(O) n R b or -NR a C(O)NR a R b ; R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 3-10 membered heterocyclic group, C 6-10 Aryl or 5-14 membered heteroaryl; or R a and R b and the nitrogen atom to which they are attached form a 3-10 membered heterocyclic group; m is selected from 1, 2, 3, 4 or 5; n is independently selected from 1 or 2.

2. The nucleotide duplex of claim 1, which has a structure of formula (I) or (II): in, Each group is as defined in claim 1.

3. The nucleotide duplex according to claim 2, wherein X is selected from -(CR1R2) m -or-CR1=CR2-; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R'; R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b or -NR a C(O)NR a R b ; R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; m is selected from 1, 2, 3, 4 or 5; Preferably, X is selected from -(CR1R2) m -or-CR1=CR2-; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's; R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a or -NR a R b ; R a and R b Independently selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; m is selected from 1, 2 or 3; More preferably, X is selected from -CH2-, -CH(OH)-, -CH2-CH2- or -CH=CH-.

4. The nucleotide duplex according to claim 2 or 3, wherein Y1 is O or NR; Y2 is O, S or a chemical bond; R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; Preferably, Y1 is O or NR; Y2 is O, S or a chemical bond; R is selected from H or C 1-6 alkyl; More preferably, Y1 is O; Y2 is O.

5. The nucleotide duplex according to any one of claims 2 to 4, wherein R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R'; Preferably, R3 is selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's; More preferably, R3 is C 1-4 Alkyl, preferably Me.

6. The nucleotide duplex according to any one of claims 2 to 5, wherein R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 alkoxy; Preferably, R4 and R5 are independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F, OH or OMe; More preferably, R4 is selected from H, OH or C 1-4 Alkoxy, preferably H or OMe; R5 is selected from halogen, OH or C 1-4 Alkoxy, preferably F or OMe.

7. The nucleotide duplex according to any one of claims 2 to 6, wherein Base and Base' are independently selected from 8. The nucleotide duplex according to any one of claims 2 to 7, wherein L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1; L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2; X is selected from -(CR1R2) m -or-CR1=CR2-; Y1 is O, S or NR; Y2 is O, S or a chemical bond; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R'; R3 is selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 alkynyl, optionally substituted with 1, 2, 3, 4, 5 or more R'; R4 and R5 are independently selected from H, D, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe; P1 is a hydroxyl protecting group, preferably DMTr; P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a 、-OC(O)R a 、-OC(O)OR b 、-OC(O)NR a R b 、-C(O)R a 、-C(O)OR a 、-C(O)NR a R b 、-NR a R b 、-NR a C(O)R b 、-NR a -C(O)OR b or -NR a C(O)NR a R b ; R a and R b Independently selected from H, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl; m is selected from 1, 2, 3, 4 or 5.

9. The nucleotide duplex according to any one of claims 2 to 8, wherein L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1; L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2; X is selected from -(CR1R2) m -or-CR1=CR2-; Y1 is O or NR; Y2 is O, S or a chemical bond; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's; R3 is selected from H, C 1-6 Alkyl or C 1-6 haloalkyl, optionally substituted with 1, 2, 3 or more R's; R4 and R5 are independently selected from H, D, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl or C 1-6 Alkoxy, preferably H, F or OMe; P1 is a hydroxyl protecting group, preferably DMTr; P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base and Base' are independently selected from H, modified or unmodified bases or leaving groups, preferably modified or unmodified A, U, T, G and C; R is selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; R' is selected from D, halogen, CN, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, -OR a or -NR a R b ; R a and R b Independently selected from H, C 1-6 Alkyl or C 1-6 alkyl halide; m is selected from 1, 2 or 3.

10. The nucleotide duplex according to any one of claims 2 to 9, wherein L1 is H or P1, or a chemical bond to the phosphate P atom at the 2' or 3' end of the ribose of another nucleotide or oligonucleotide, preferably P1; L2 is H or P2, or a chemical bond to the phosphate P atom at the 5' end of the ribose of another nucleotide or oligonucleotide, preferably P2; X is selected from -(CR1R2) m - or -CR1=CR2-, preferably -CH2-, -CH(OH)-, -CH2-CH2- or -CH=CH-; Y1 is O; Y2 is O; R1 and R2 are independently selected from H, D, halogen, OH, CN, C 1-4 Alkyl or C 1-4 alkyl halide; R3 is C 1-4 Alkyl, preferably Me; R4 is selected from H, OH or C 1-4 Alkoxy, preferably H or OMe; R5 is selected from halogen, OH or C 1-4 Alkoxy, preferably F or OMe; P1 is a hydroxyl protecting group, preferably DMTr; P2 is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base and Base' are independently selected from m is selected from 1, 2 or 3.

11. The nucleotide duplex according to any one of claims 1 to 10, selected from the group consisting of: in, Base and Base' are as defined in any one of claims 1 to 10, preferably R5 is as defined in any one of claims 1 to 10, preferably F or OMe.

12. A double-stranded RNA molecule or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides, and the antisense strand comprises one or more nucleotide monomers represented by formula (III) or (IV): in, The nucleotide monomers are shown in the order 5'=>3' from to connect; Each group is defined as in any one of claims 1 to 11; Preferably, the nucleotide monomer is selected from: Among them, Base is selected from 13. The double-stranded RNA molecule of claim 12, wherein the sense strand and antisense strand each have 20 to 25 nucleotides. The double-stranded RNA molecule according to claim 12 or 13, wherein the nucleotide monomer is located at position 2-8, preferably position 6 or 7, more preferably position 7, of the 5' end of the antisense strand.

15. The double-stranded RNA molecule of any one of claims 12 to 14, wherein the double-stranded RNA exhibits enhanced stability compared to a double-stranded RNA having the same sequence but not comprising the nucleomonomer of claim 12.

16. The double-stranded RNA molecule of any one of claims 12 to 15, wherein the double-stranded RNA has a melting temperature from about 40°C to about 80°C, preferably about 55°C to 67°C.

17. The double-stranded RNA molecule of any one of claims 12 to 16, wherein the double-stranded RNA exhibits reduced off-target toxicity compared to a double-stranded RNA having the same sequence but not comprising the nucleomonomer of claim 12.

18. The double-stranded RNA molecule of any one of claims 12 to 17, wherein the double-stranded RNA exhibits enhanced effectiveness compared to a double-stranded RNA having the same sequence but not comprising the nucleomonomer of claim 12.

19. The double-stranded RNA molecule according to any one of claims 12 to 18, wherein the antisense strand has a sequence that is sufficiently complementary to the sense strand and the target mRNA, and has the ability to induce degradation of the target mRNA.

20. The double-stranded RNA molecule of claims 12-19, wherein the target mRNA is encoded by an endogenous gene or by a pathogen gene.

21. The double-stranded RNA molecule of any one of claims 12 to 20, wherein the sense strand and / or antisense strand comprises a 3' and / or 5' overhang.

22. The double-stranded RNA molecule according to any one of claims 12 to 21, wherein the double-stranded RNA is further coupled to a ligand, preferably, the ligand comprises one or more GalNAc.

23. A nucleic acid molecule, the nucleotide sequence of which comprises one or more nucleotide monomers according to claim 12.

24. The nucleic acid molecule of claim 23, wherein the nucleic acid is selected from the group consisting of DNA, RNA, and a DNA / RNA hybrid.

25. The nucleic acid molecule of claim 24, which is single-stranded or double-stranded.

26. The nucleic acid molecule of any one of claims 23 to 25, wherein the nucleic acid molecule is selected from the group consisting of small interfering RNA (siRNA) and short hairpin RNA (shRNA).

27. A vector comprising a nucleotide sequence encoding the double-stranded RNA according to any one of claims 12 to 22.

28. A cell comprising the double-stranded RNA according to any one of claims 12 to 22 or the vector according to claim 27.

29. A pharmaceutical composition comprising the double-stranded RNA molecule according to any one of claims 12 to 22, and a pharmaceutically acceptable carrier or excipient.

30. A kit comprising the double-stranded RNA molecule according to any one of claims 12 to 22.

31. A method for inhibiting the expression of a target gene in a cell, comprising the step of introducing the double-stranded RNA molecule according to any one of claims 12 to 22 into the cell.

32. A method for inhibiting the expression of a target gene in a cell, comprising expressing the double-stranded RNA molecule of any one of claims 12 to 22 in the cell.

33. A method for reducing off-target toxicity in a cell, comprising the step of introducing the double-stranded RNA molecule of any one of claims 12 to 22 into the cell.

34. A method for reducing off-target toxicity in a cell, comprising expressing the double-stranded RNA molecule of any one of claims 12-22 in the cell.