Oligonucleotide, oligonucleotide conjugate, composition, and use

Abstract

A single-stranded oligonucleotide having a length of 16-30 nucleotides. The composition of the single-stranded oligonucleotide enables the single-stranded oligonucleotide to inhibit the expression of target mRNA by means of an RNAi mechanism. Each nucleotide in the single-stranded oligonucleotide is independently a modified or unmodified nucleotide, wherein at least one nucleotide in the single-stranded oligonucleotide is a nucleotide X; at least one nucleotide is a fluoro-modified nucleotide; and, in the 5' to 3' direction, the 13th nucleotide of the single-stranded oligonucleotide is a substituted alkoxy-modified nucleotide, the 14th nucleotide of the single-stranded oligonucleotide is a nucleotide X, and each of the 15th nucleotide and all subsequent nucleotides of the single-stranded oligonucleotide is independently a modified nucleotide. A double-stranded oligonucleotide comprising the single-stranded oligonucleotide as an antisense strand, an oligonucleotide conjugate and a pharmaceutical composition.

Description

Oligonucleotides, oligonucleotide conjugates and compositions and their uses Technical Field This disclosure relates to a single-stranded oligonucleotide, a double-stranded oligonucleotide, an oligonucleotide conjugate, a pharmaceutically acceptable salt, a pharmaceutical composition, and the uses and preparation methods thereof. Background Technology In the drug development of oligonucleotides, including single-stranded and double-stranded oligonucleotides, the improvement of oligonucleotide modifications has never ceased. Depending on the type, location, and amount of modification, the modification of single-stranded oligonucleotides (such as ASO and ssRNAi) and double-stranded oligonucleotides (such as the antisense strand of siRNA) can significantly affect key properties such as pharmacodynamic activity, stability, and long-lasting effect. Although numerous oligonucleotide modification schemes have been disclosed in the prior art, how to improve the modification of oligonucleotides, especially the antisense strands of single-stranded and double-stranded oligonucleotides, to obtain oligonucleotides with higher activity, higher stability, and / or long-lasting effect remains a research direction in this field. Summary of the Invention This invention provides a single-stranded oligonucleotide, a double-stranded oligonucleotide containing the single-stranded oligonucleotide of this invention as the antisense strand, and oligonucleotide conjugates, which exhibit good pharmaceutical activity and stability when the nucleotide sequence is different and the target site is different. In one aspect, the present invention provides a single-stranded oligonucleotide of 16-30 nucleotides in length, wherein the single-stranded oligonucleotide is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to a first nucleotide sequence, wherein the first nucleotide sequence is a nucleotide sequence of equal length to the single-stranded oligonucleotide and is identical to the target mRNA for at least 16 consecutive nucleotides; each nucleotide in the single-stranded oligonucleotide is a modified or unmodified nucleotide, wherein at least one nucleotide in the single-stranded oligonucleotide is nucleotide X, and at least one nucleotide is a fluorinated nucleotide; furthermore, in the 5'-3' direction, the 13th nucleotide of the single-stranded oligonucleotide is a substituted alkoxy-modified nucleotide; the 14th nucleotide of the single-stranded oligonucleotide is nucleotide X; and all nucleotides thereafter of the single-stranded oligonucleotide are modified nucleotides; each nucleotide X is a deoxynucleotide or an unmodified nucleotide. In another aspect, this disclosure provides a double-stranded oligonucleotide containing a sense strand and an antisense strand, each nucleotide in the sense strand being a modified or unmodified nucleotide, the sense strand and the antisense strand being at least partially anticomplementary to form a double-stranded region, wherein the antisense strand is a single-stranded oligonucleotide as described in this disclosure. In another aspect, this disclosure also provides an oligonucleotide conjugate containing an oligonucleotide group and a delivery group conjugated to the oligonucleotide group, the oligonucleotide group being independently formed by removing one or more atoms or groups of atoms from a single-stranded or double-stranded oligonucleotide as described in this disclosure. In another aspect, this disclosure also provides pharmaceutically acceptable salts of the single-stranded oligonucleotides, double-stranded oligonucleotides, or oligonucleotide conjugates described herein. In another aspect, this disclosure also provides a pharmaceutical composition comprising one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, and pharmaceutically acceptable salts described herein, as well as pharmaceutically acceptable excipients. In another aspect, this disclosure also provides the use of the single-stranded oligonucleotide, double-stranded oligonucleotide, oligonucleotide conjugate, pharmaceutically acceptable salt, or pharmaceutical composition of this disclosure in the preparation of a medicament for treating and / or preventing diseases or symptoms associated with target mRNA levels. In another aspect, this disclosure also provides a method for treating and / or preventing diseases or symptoms associated with target mRNA levels, the method comprising administering to a subject in need an effective amount of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions of this disclosure. In another aspect, this disclosure also provides a method for regulating the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of one or more of the single-stranded oligonucleotide, double-stranded oligonucleotide, oligonucleotide conjugate, pharmaceutically acceptable salt, and pharmaceutical composition of this disclosure. In another aspect, this disclosure also provides one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions of this disclosure for use as pharmaceuticals. In another aspect, this disclosure also provides a cell expressing a target mRNA, the cell further comprising one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions of this disclosure. In addition, this disclosure also provides a kit comprising one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions of this disclosure. Beneficial effects One or more of the following, including single-stranded oligonucleotides as antisense strands, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and / or pharmaceutical compositions, contain the single-stranded oligonucleotides of this disclosure as antisense strands and have high activity in regulating target mRNAs and high long-lasting effects. On the one hand, double-stranded oligonucleotides (e.g., siRNA) containing the single-stranded oligonucleotides of this disclosure as the antisense strand, oligonucleotide conjugates of this disclosure, and / or pharmaceutical compositions exhibit excellent target mRNA inhibitory effects. For example, in in vitro Huh7 cells, the siRNA conjugates of this disclosure and reference conjugates with the same sequence but without the antisense strand of this disclosure inhibited the IC50 of human AGT mRNA. 50 The values ​​were 0.021 nM and 0.027 nM, respectively, indicating that the siRNA conjugate disclosed in this invention has a smaller IC50 value. 50 The values ​​indicate that, compared to the reference conjugate, the siRNA conjugate of this disclosure exhibits higher AGT mRNA inhibition efficiency and superior pharmaceutical activity. For example, in in vitro experiments, in primary mouse liver cells, at a concentration of 0.2 nM, the conjugate provided by this disclosure can achieve an inhibition rate of over 72% against the target gene AGT mRNA, and at a concentration of 1 nM, it can reach 98.5%, or even 99%, demonstrating excellent in vitro inhibitory activity. Furthermore, double-stranded oligonucleotides (e.g., siRNA) containing the single-stranded oligonucleotide as the antisense strand of this disclosure, the oligonucleotide conjugates of this disclosure, and / or pharmaceutical compositions have shown excellent mRNA inhibitory effects on APOE4 gene expression. For example, compared to the blank control group, in in vitro HepG2 human liver cancer cells, at a concentration of 50 nM, the siRNA conjugates disclosed herein all exhibited good inhibitory effects, with inhibition rates of APOE4 mRNA all greater than 85%, and some conjugates reaching an inhibition rate of 90%, with the highest reaching 94%, indicating that the siRNA conjugates disclosed herein have excellent inhibitory effects on APOE4 mRNA levels. Furthermore, in in vitro experiments, the conjugates provided herein exhibit good inhibitory activity against the target sequence, with an IC50 value of [missing information]. 50The value was 0.021 nM. For example, after 48, 72, and 96 hours of transfection into HepG2.2.15 cells, the HBsAg content in the cell supernatant was significantly reduced: at a conjugate concentration (calculated based on the siRNA group in the conjugate, the same below) of 50 nM, HBsAg was 0.28 IU / mL after 48 hours of transfection, 0.29 IU / mL after 72 hours, and 0.30 IU / mL after 96 hours; at a conjugate concentration of 5 nM, HBsAg was 0.30 IU / mL after 48 hours of transfection, and 0.021 nM after 72 hours... The HBsAg concentration was 0.31 IU / mL, and after 96 hours of transfection, it was 0.33 IU / mL. In contrast, reference conjugate 1, at a concentration of 50 nM, showed HBsAg levels of 0.50 IU / mL after 48 hours of transfection, 0.68 IU / mL after 72 hours, and 1.11 IU / mL after 96 hours; at a concentration of 5 nM, the HBsAg concentration was 0.46 IU / mL after 48 hours, 0.57 IU / mL after 72 hours, and 0.70 IU / mL after 96 hours. Furthermore, in in vitro psiCHECK assays, the conjugates disclosed herein exhibited high inhibitory activity against the target NTCP sequence, with inhibition rates exceeding 76% even at a low concentration of 0.1 nM, sometimes reaching 91.59%. For example, the conjugates disclosed herein exhibit high inhibitory activity against NTCP mRNA expression. In primary human liver cells, at concentrations of 5 nM and 50 nM, the conjugates provided herein show high NTCP mRNA inhibitory activity, with inhibition rates exceeding 75%, and even reaching 91%, significantly higher than the reference conjugates. Furthermore, the conjugates provided herein can achieve an inhibition rate of over 86% against NTCP mRNA in HepG2 cells. Moreover, at different concentrations, the conjugates provided herein show high NTCP mRNA inhibitory activity in primary human liver cells, with inhibition rates at least 83.3%, and even reaching 92.1%. For example, the conjugates disclosed herein exhibit high inhibitory activity against PNPLA3 mRNA expression. In primary human liver cells, at concentrations of 5 nM and 50 nM, the conjugates provided herein showed high PNPLA3 mRNA inhibitory activity. At a concentration of 50 nM, some conjugates showed the highest inhibition rate against hPNPLA3 mRNA, reaching 82.2%, demonstrating significant in vitro activity. Furthermore, the conjugates provided herein also showed significant inhibitory effects on PNPLA3 mRNA in primary monkey liver cells. At a concentration of 10 nM, the inhibition rate against PNPLA3 mRNA exceeded 73%, with the highest reaching 86.4%.For example, the conjugates disclosed herein exhibit high inhibitory activity against LPA mRNA expression, with an inhibition rate of at least 57.6% and even up to 96.3% in mouse primary liver cells at a concentration of 50 nM; and for example, the conjugates provided herein have a significant inhibitory effect on LPA mRNA in monkey primary liver cells, with an inhibition rate of up to 74.7% at a concentration of 50 nM. On the other hand, the double-stranded oligonucleotides (e.g., siRNA) containing the single-stranded oligonucleotides of this disclosure as the antisense strand, the conjugates of this disclosure, and / or pharmaceutical compositions have shown highly efficient and long-lasting inhibitory effects on target mRNA and protein expression in in vivo experiments in animal models. For example, in a 43-day experimental period, hAGT transgenic mice administered the same concentration of the conjugate showed a higher inhibition rate of AGT protein expression than mice administered the reference conjugate, maintaining a high inhibition rate throughout the entire experimental period. On day 43, the inhibition rate of AGT protein expression remained close to 80%, which is significantly higher than the inhibition efficiency of one of the most preferred compounds in the art. Furthermore, in multi-dose experiments, different doses of the conjugates of this disclosure all showed excellent hAGT protein inhibition effects, with inhibition rates reaching up to 96.5%, exhibiting a dose-dependent effect. The inhibition rate remained at a high level of up to 90.3% on day 43 after administration. For example, during a 71-day experimental period, mice given the conjugates of this disclosure exhibited high inhibition rates of hAGT protein expression. For instance, on day 15, the inhibition rate of hAGT protein expression was above 90%, and on day 71, the inhibition rate remained close to or reached 70%. Furthermore, the conjugates provided by this disclosure can maintain inhibitory activity against PCSK9 mRNA in vivo for a prolonged period. In mice given the various conjugates of this disclosure, good inhibition rates against PCSK9 mRNA were observed throughout the entire experimental period. The conjugates provided by this disclosure showed inhibition rates above 75% on day 8, and even exceeding 89%; and inhibition rates above 79% on day 15, and even exceeding 89%. For example, in the human PNPLA3 I148M mouse model, all conjugates and dosage combinations exhibited sustained inhibitory effects during the experiment, with inhibition rates reaching 76.4%, and even reaching 91.5% at a prolonged period on day 43, indicating that the conjugates disclosed herein have the potential for long-term inhibition of hPNPLA3 mRNA. Furthermore, throughout the entire experimental period, in in vivo experiments with hLPA transgenic mice, the conjugates disclosed herein maintained a high inhibition rate against Apo(a) protein, retaining an inhibition rate of over 64% at day 85. For example, in in vivo experiments on hLPA transgenic mice, at a dose of 3 mg / kg, the different conjugates disclosed herein consistently maintained an inhibition rate of at least 60% or higher against Apo(a) protein, reaching a maximum of 92.82%, and still maintained a high inhibition rate of up to 86.88% on day 113; even at a lower dose of 1 mg / kg, the conjugates still maintained an inhibition rate of over 54% against Apo(a) protein, reaching a maximum of 80.76%, and still maintained an inhibition rate of 59.40% on day 71.The above results indicate that the conjugate disclosed herein can maintain excellent LPA mRNA inhibition in animal models with high efficiency and long duration, thereby continuously and effectively inhibiting serum Apo(a) protein levels. Therefore, by using the single-stranded oligonucleotide of this disclosure as the antisense strand, the siRNA conjugate of this disclosure has a significantly enhanced inhibitory activity against the target mRNA. The above results demonstrate that the conjugates disclosed herein can efficiently inhibit the levels of target mRNA and target protein over a prolonged period in in vivo animal models. This indicates that the conjugates disclosed herein can stably and efficiently inhibit the expression of target mRNA over a long period. The conjugates disclosed herein exhibit significant and long-lasting pharmaceutical activity in the preparation of drugs for the treatment and / or prevention of diseases or symptoms related to target mRNA expression, and possess excellent development prospects. Incorporate by reference All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually incorporated herein by reference. Detailed Implementation The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure. In this disclosure, AGT mRNA refers to mRNA having the sequence shown in GenBank accession number NM_001384479.1. APOE4 mRNA, or "mRNA expressing the APOE4 gene," specifically refers to mRNA having the sequence shown in GenBank accession number NM_000041.4. NTCP mRNA refers to mRNA with the sequence shown in GenBank accession number NM_003049.4. PCSK9 mRNA refers to mRNA with the sequence shown in GenBank accession number NM_174936.3. PNPLA3 mRNA refers to the mRNA with the sequence shown in one of the following GenBank accession numbers: NM_025225.3, MP146987.1, MP146988.1, MP146989.1, MP146990.1, MP146991.1, MP146992.1, MP146993.1, and MP146994.1. LPA mRNA refers to the mRNA with the sequence shown in GenBank accession number NM_005577.4. definition In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxynucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group. In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, each base of one strand forms a hydrogen bond with a base of the other strand in a complementary manner, achieving base pairing and forming a Watson-Crick base pair. A "base pair" refers to the two bases that form a base pair. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair consists of one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand, and guanine always pairs with cytosine, the two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. When bases are modified, as long as the pairing relationship of purines and pyrimidines (including but not limited to the number and strength of hydrogen bonds between bases) is not affected, the modified bases are considered to be able to form complementary pairs. Correspondingly, in this field, "mismatch" means that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary manner; when the corresponding position includes a baseless nucleotide, it is also considered to have formed a mismatch with the bases on the other strand. In the preceding and following text, "at least partially anti-complementary," "substantially anti-complementary," "truly anti-complementary," and "completely anti-complementary" refer to the base pairing between two single-stranded nucleotide sequences: between a single-stranded oligonucleotide and the target mRNA; between a single-stranded oligonucleotide and the nucleotide sequence m; between the sense and antisense strands of a double-stranded oligonucleotide; and between the antisense strand of a double-stranded oligonucleotide and the target mRNA. Unless otherwise specified, "at least partially anti-complementary" means that within a hypothetical or actual double-stranded region, there is no more than 50% base mismatch between the two nucleotide sequences capable of forming a double-stranded region; "substantially anti-complementary" means that within a hypothetical or actual double-stranded region, there are no more than 3 base mismatches between the two nucleotide sequences capable of forming a double-stranded region; "truly anti-complementary" means that within a hypothetical or actual double-stranded region, there are no more than 1 base mismatch between the two nucleotide sequences capable of forming a double-stranded region; and "completely anti-complementary" means that within a hypothetical or actual double-stranded region, there are no base mismatches between the two nucleotide sequences capable of forming a double-stranded region. When the two nucleotide sequences are “at least partially reverse complementary,” “substantially reverse complementary,” “truly reverse complementary,” or “completely reverse complementary,” they can form a double-stranded hybrid consisting of Watson-Crick base pairs after annealing. Unless otherwise stated, when referring to a shorter nucleotide sequence as "completely anticomplementary" to a longer nucleotide sequence, it means that the two nucleotide sequences are completely anticomplementary along the entire length of the shorter nucleotide sequence. For example, the complete anticomplementary relationship between the single-stranded oligonucleotide and AGT mRNA described in this disclosure means that the single-stranded oligonucleotide is completely anticomplementary along its entire length; in other words, each nucleotide in the single-stranded oligonucleotide forms a Watson-Crick base pair with a corresponding nucleotide on the AGT mRNA to form a double-stranded hybrid. In the context of this disclosure, a "double-stranded region" is a double-stranded structure formed between the shortest nucleotide sequences comprising all base pairs on each single strand of a hypothetical or actual double-stranded nucleic acid structure. Therefore, a double-stranded region consists of all base pairs in the double-stranded nucleic acid structure and all base mismatches between those base pairs. In some embodiments, the double-stranded nucleic acid structure includes a double-stranded region and one or more overhanging ends, said overhanging ends consisting of all nucleotides outside the double-stranded region in one or both single strands of the double-stranded nucleic acid structure that do not form base pairs. In some embodiments, the double-stranded nucleic acid structure includes only a double-stranded region. The two nucleotide sequences that can form a double-stranded region can be of the same or different lengths. In some embodiments, the double-stranded nucleic acid structure includes only the double-stranded region, in which case the two nucleotide sequences forming the double-stranded nucleic acid structure are of the same length. "At least partially anticomplementary" means that there is no more than 50% base mismatch between the two nucleotide sequences; "substantially anticomplementary" means that there is no more than 3 base mismatches between the two nucleotide sequences; "substantially anticomplementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there is no base mismatch between the two nucleotide sequences. In some embodiments, the two nucleotide sequences forming the double-stranded nucleic acid structure are of the same length, the double-stranded nucleic acid structure includes a double-stranded region and one or both overhanging ends of the longer nucleotide sequence. In some embodiments, the two nucleotide sequences forming the double-stranded nucleic acid structure are of different lengths, the double-stranded nucleic acid structure includes a double-stranded region and one or both overhanging ends of the longer nucleotide sequence. For example, in some embodiments, the sense and antisense strands of a double-stranded oligonucleotide are of different lengths. For example, the double-stranded oligonucleotide is siRNA, in which case the sense strand is usually shorter and is a shorter nucleotide sequence, while the antisense strand is longer and is the longer nucleotide sequence. The double-stranded nucleic acid structure includes a double-stranded region and a dangling end in the antisense strand. In the preceding and following text, "nucleotide sequence A and nucleotide sequence B are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary across a length of X nucleotides" means that there exists a continuous nucleotide sequence A' of length X in nucleotide sequence A, which is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to a continuous nucleotide sequence B' of length X in nucleotide sequence B. In the foregoing and hereinafter, particularly in the description of methods for preparing single-stranded oligonucleotides, double-stranded oligonucleotides, pharmaceutical compositions, or oligonucleotide conjugates of this disclosure, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the desired double-stranded oligonucleotide or oligonucleotide conjugate. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable. As used herein, “alkyl” refers to a saturated straight-chain and / or branched hydrocarbon group having a specified number of carbon atoms, typically from 1 to 20 carbon atoms, such as from 1 to 10 carbon atoms, or from 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl comprises straight-chain and branched alkyl groups having 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to cover all branched and straight-chain forms having that number of carbons; thus, for example, “butyl” means including n-butyl, sec-butyl, isobutyl, and tert-butyl; “propyl” includes n-propyl and isopropyl. Alkylenes are subsets of alkyl, referring to residues that are identical to alkyl but have two connection sites. As used herein, "alkoxy" refers to an alkyl group with a specified number of carbon atoms connected by oxygen bridges, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentooxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms connected by oxygen bridges. As used in this article, "alkoxy-modified alkyl" refers to a group formed by replacing one or more hydrogen atoms in an alkyl group with an alkoxy group, such as methoxymethyl (CH3OCH2-), ethoxymethyl (CH3CH2OCH2-), methoxyethyl (CH3OCH2CH2-), etc. As used herein, "alkenyl" refers to an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon double bond obtained by removing a hydrogen molecule from an adjacent carbon atom of a parent alkyl group. The group can be in either a cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as propyl-1-en-1-yl, propyl-1-en-2-yl, propyl-2-en-1-yl (allyl), propyl-2-en-2-yl; butenyl, such as buten-1-en-1-yl, buten-1-en-2-yl, 2-methylpropen-1-en-1-yl, buten-2-en-1-yl, buten-2-en-2-yl, buten-1,3-dien-1-yl, buten-1,3-dien-2-yl, etc. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Subalkenyl groups are a subset of alkenyl groups, referring to residues that are identical to alkenyl groups but have two connection points. As used herein, "alkynyl" refers to an unsaturated branched or straight-chain hydrocarbon group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In some embodiments, the alkynyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Ionynyl is a subset of alkynyl, referring to residues that are identical to alkynyl but have two connection sites. As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. This aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms of 6 to 18, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2)π-electron system according to Hückel's theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl groups. Alearyl groups are a subset of aryl groups, referring to residues identical to aryl groups but with two connection points. As used herein, a "heterocyclic group" refers to a group derived from a monocyclic saturated or partially unsaturated, non-aromatic or bicyclic saturated or partially unsaturated heterocyclic hydrocarbon group, wherein the bicyclic ring system is non-aromatic, the monocyclic or bicyclic ring has, for example, 3 to 10 members or 5 to 10 members, wherein at least one member and up to five members, particularly one, two or three ring members, are heteroatoms selected from N, O and S, and the remaining ring atoms are carbon atoms in a stable combination known to those skilled in the art. The heterocyclic nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. As used herein, the heterocycle can form a bicyclic ring with another ring system, i.e., one or two of the atoms constituting the heterocycle are shared with another ring system. The heterocyclic group can be linked to the rest of the molecule via carbon or heteroatoms; and, in the case of a bicyclic group, the above-mentioned linking can be made via a ring containing heteroatoms or a fused ring. Examples of heterocyclic groups include, but are not limited to: aziridine, pyrrolidinyl, piperidinyl, aziridine-heptyl, diaziridine-heptyl, dihydrofuranyl (e.g., 2,3-dihydrofuranyl, 2,5-dihydrofuranyl), dioxacyclopentyl, morpholinyl, oxazolyl, oxazinyl, indololinyl, isoindolinyl, piperazinyl, tetrahydrofuranyl, thiomorpholinyl, and dihydropyranyl (e.g., 3,4-dihydropyranyl). 3,6-dihydropyranyl), piperazineyl, dioxane, hexahydropyrimidinyl, pyrazolinyl, pyrazolylylene, 4H-quinazinyl, quininecycloyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiopheneyl, thiazolyl, benzopyranyl, tetrahydroquinolinyl, dihydropyrrolopyridinyl, dihydrobenzoxazinyl, pyrrolopyridinyl, dihydronaphthidinyl, dihydroisoquinolinyl, and tetrahydroisoquinolinyl. Subheterocyclic groups are a subset of heterocyclic groups, referring to residues identical to heterocyclic groups but with two connection sites. As used herein, "heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, comprising 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprising a cyclic delocalized (4n+2) π-electron system according to Hückel's theory. Heteroaryls include fused ring or bridged ring systems. In some embodiments, the heteroatoms in the heteroaryl are oxidized heteroatoms. In some embodiments, the heteroaryl contains one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl are quaternized nitrogen atoms. The heteroaryl is attached to the remainder of the molecule via any ring atom. Examples of heteroaryl groups include, but are not limited to: aziridine, heptatrienyl, acridine, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzene benzo[a]furanyl, benzo[a]oxazolyl, benzo[a]dioxolyl, benzo[a]dioxinyl, benzo[a]pyranyl, benzo[a]pyranoneyl, benzo[a]furanyl, benzo[a]furanoneyl, benzo[a]thiophenyl, benzo[a]thiophene[3,2-d]pyrimidinyl, benzo[a]triazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazoleyl, cinnolinyl, cyclopentano[d]pyrimidinyl, 6,7-dihydro-5H-cyclopentano[4,5] Thiophene[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cycloheptano[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanoneyl, furano[3,2-c]pyridinyl, 5,6,7,8 9,10-Hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-Hexahydrocyclooctano[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocyclooctano[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indole, isoyindolyl, dihydroindolyl, isodihydroindolyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrroleyl, phenazinyl, phenothiazinyl, phenotoxazinyl, phthalazinyl, pteridinyl, purine, pyrroleyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl Pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrroleyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptano[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyridano[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-c]pyridinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl / thienyl. Hypo-heteroaryl groups are a subset of heteroaryl groups, referring to residues that are identical to heteroaryl groups but have two linkage sites. In the foregoing or hereinafter, "substituted," "substituted," or "substituted" groups refer to substituted amino, substituted alkyl, or substituted aryl groups. Unless otherwise specified, a "substituted" or "substituted" group means a group formed by replacing one or more hydrogen atoms in the group with a substituent. For example, "substituted alkyl" means a group formed by replacing one or more hydrogen atoms in an alkyl group with a substituent. Those skilled in the art will understand that compounds usable in this disclosure may contain various substituents, as long as the introduction of such substituents does not affect the function of this disclosure and achieves the purpose of this disclosure. In some embodiments, the substituents are selected from the group consisting of C1-C1 groups. 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), N(C1-C) 10 Alkyl) (C1-C 10 alkylphenyl), NH(C1-C 10 Alkylphenyl), -CN, -NO2, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (Halogenated alkyl). In some embodiments, the substituent is one of -C1-C3 alkyl, -C6-C8 aryl, -O-C1-C3 alkyl, -O-(C1-C3 alkyl)phenyl, halogen, -OH, -NH2, -CN, or -NO2. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable. As used herein, “halogenated” or “halogenated” refers to fluorinated, chlorinated, bromine, and iodinated substances, and the term “halogen” includes fluorine, chlorine, bromine, and iodine. As used herein, “haloalkyl” means an alkyl group as defined above in which a specified number of carbon atoms are replaced by one or more, up to a maximum permissible number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl. Various protecting groups, such as amino and hydroxyl protecting groups, may be used in this disclosure. Generally, protecting groups make a chemical function insensitive to specific reaction conditions and can be added to and removed from that function in a molecule without substantially impairing the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Peter GMWuts, GREENE's Protective Groups in Organic Synthesis, Chapter 2, 5th edition, John Wiley & Sons, Inc., New Jersey, 2014, all of which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl). The term “subject” as used herein refers to any animal, such as a mammal or marsupial. Subjects in this disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any kind of poultry. In some embodiments, “subject” refers to a mammal, such as a rodent or primate. In some embodiments, “subject” refers to a mouse, rat, or non-human primate. In some embodiments, “subject” refers to a human subject. As used herein, “treatment” refers to a method of achieving a beneficial or desired outcome, including but not limited to treatment benefits. A “treatment benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a treatment benefit is achieved by eradicating or improving one or more physical symptoms associated with the underlying disorder, thereby observing improvement in the subject, even though the subject may still be suffering from the underlying disorder. As used herein, “prevention” refers to methods for obtaining a beneficial or desired outcome, including but not limited to preventative benefits. To obtain a “preventative benefit,” a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate may be administered to a subject at risk of developing a specific disease, or to a subject reporting one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made. In some embodiments, “prevention” includes, prior to the progression of disease risk associated with a target mRNA level to a defined disease process, intervening in the level of the target mRNA or the protein corresponding to the target mRNA to reduce or eliminate the disease risk by administering a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate to a subject at risk of developing a specific disease. Unless otherwise specified, in the context of any application or method provided in this disclosure, referring solely to an oligonucleotide and / or oligonucleotide conjugate, including but not limited to any oligonucleotide and / or oligonucleotide conjugate represented by a structural formula as described in the application or method provided in this disclosure, also refers to a pharmaceutically acceptable salt of that oligonucleotide and / or oligonucleotide conjugate, depending on the context. The single-stranded oligonucleotides disclosed herein In one aspect, this disclosure provides a single-stranded oligonucleotide of 16-30 nucleotides in length, the composition of which enables the single-stranded oligonucleotide to inhibit the expression of target mRNA via an RNAi mechanism; each nucleotide in the single-stranded oligonucleotide is independently modified or unmodified, wherein at least one nucleotide in the single-stranded oligonucleotide is nucleotide X, and at least one nucleotide is a fluorinated nucleotide; furthermore, along the 5'-3' direction, the 13th nucleotide of the single-stranded oligonucleotide is a substituted alkoxy-modified nucleotide; the 14th nucleotide of the single-stranded oligonucleotide is nucleotide X; and each of the 15th nucleotide and all subsequent nucleotides of the single-stranded oligonucleotide is independently modified; each nucleotide X is independently deoxyribonucleotide or unmodified nucleotide. In the context of this disclosure, unless otherwise stated, "target gene" refers to a gene whose expression level is desired to be modulated by administering the single-stranded oligonucleotide, double-stranded oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition described in this disclosure. In this disclosure, diseases associated with the expression level and / or protein level of the gene are treated or prevented by regulating the expression level of the gene and / or altering the corresponding protein level. In the context, unless otherwise stated, "target mRNA" refers to the mRNA transcribed from the target gene. "Regulating gene expression level" means regulating the level of target mRNA in cells. The inventors unexpectedly discovered that the single-stranded oligonucleotides described in this disclosure, double-stranded oligonucleotides containing the single-stranded oligonucleotides described in this disclosure as antisense strands, and oligonucleotide conjugates have good stability and target mRNA inhibitory activity in cells and / or in subjects, and therefore have good application prospects. To achieve RNAi activity, the single-stranded oligonucleotides described in this disclosure are 16-30 nucleotides in length. In some embodiments, the single-stranded oligonucleotides described in this disclosure are 17-28, 19-27, or 20-25 nucleotides in length. In some embodiments, the single-stranded oligonucleotides described in this disclosure are 19, 21, or 23 nucleotides in length. In these cases, the single-stranded oligonucleotides described in this disclosure, the double-stranded oligonucleotides containing the single-stranded oligonucleotide as the antisense strand, and the oligonucleotide conjugates exhibit a better balance between stability and RNAi activity. In the single-stranded oligonucleotides of this disclosure, at least one nucleotide is nucleotide X, at least one nucleotide is a fluorinated nucleotide, and the 14th nucleotide of the single-stranded oligonucleotide is nucleotide X in a 5'-3' orientation, the 13th nucleotide of the single-stranded oligonucleotide is a substituted alkoxy-modified nucleotide, and the 15th nucleotide and all subsequent nucleotides of the single-stranded oligonucleotide are modified nucleotides. The inventors have particularly discovered that the single-stranded oligonucleotides of this disclosure, by comprising the aforementioned fluorinated nucleotide, substituted alkoxy-modified nucleotide, and nucleotide X, can effectively maintain the high inhibitory activity of double-stranded oligonucleotides and oligonucleotide conjugates against target mRNA while maintaining stability. In some embodiments, the number of nucleotide X is 1-3, for example, 1, 2, or 3. In some embodiments, the 12th and 14th nucleotides are each independently nucleotide X. In some embodiments, only the 14th nucleotide is nucleotide X. Each nucleotide X is independently selected from deoxyribonucleotides or unmodified nucleotides. In this context, "unmodified nucleotide" refers to an unmodified ribonucleotide (RNA), i.e., the 2' position of the ribose sugar is an unprotected hydroxyl group (2'-OH). Correspondingly, "modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose sugar is replaced by another atom or group, or refers to a nucleotide analog. In some embodiments, the 14th nucleotide or the 12th and 14th nucleotides in the single-stranded oligonucleotide, with the 5'-3' orientation, is nucleotide X, and the other nucleotides are modified nucleotides. In some embodiments, the number of unmodified nucleotides in the single-stranded oligonucleotide of this disclosure is no more than 3, no more than 2, or no more than 1. In some embodiments, the number of unmodified nucleotides in the single-stranded oligonucleotide is 2 or 1. In some embodiments, each nucleotide in the single-stranded oligonucleotide is independently modified. As previously described, in addition to nucleotide X and substituted alkoxy-modified nucleotides, the single-chain oligonucleotides of this disclosure also include fluorinated nucleotides. In some embodiments, the number of fluorinated nucleotides is 2-7. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to 2-5 of the 2nd, 5th, 6th, 7th, 12th, 16th, 18th, and 19th nucleotides of the single-chain oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to 1 or 2 of the 2nd and 12th nucleotides, 1 or 2 of the 5th-7th nucleotides, and 0-2 of the 16th-19th nucleotides of the single-chain oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to the 2nd and 6th nucleotides of the single-chain oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to the 2nd, 6th, and 16th nucleotides of the single-chain oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to the 2nd, 5th, 7th, 12th, and 16th nucleotides of the single-stranded oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to the 2nd, 7th, 12th, 16th, and 19th nucleotides of the single-stranded oligonucleotide. In some embodiments, with respect to the 5'-3' orientation, the fluorinated nucleotides refer to the 2nd, 6th, 12th, 16th, and 19th nucleotides of the single-stranded oligonucleotide. In some embodiments, each modified nucleotide in the single-stranded oligonucleotide, except for the 13th and 14th nucleotides in the 5'-3' orientation and fluorinated nucleotides, is independently selected from one of alkoxy-modified nucleotides, substituted alkoxy-modified nucleotides, alkyl-modified nucleotides, substituted alkyl-modified nucleotides, amine-modified nucleotides, heat-labile nucleotides, and BNA. In some embodiments, each modified nucleotide in the single-stranded oligonucleotide, except for the 13th and 14th nucleotides in the 5'-3' orientation and fluorinated nucleotides, is independently selected from alkoxy-modified nucleotides, substituted alkoxy-modified nucleotides, or heat-labile nucleotides. In some embodiments, the number of substituted alkoxy-modified nucleotides in the single-stranded oligonucleotide does not exceed three. In some embodiments, the number of substituted alkoxy-modified nucleotides in the single-stranded oligonucleotide does not exceed two. In some embodiments, the number of substituted alkoxy-modified nucleotides in the single-stranded oligonucleotide is one. In some embodiments, the single-stranded oligonucleotide does not contain heat-labile nucleotides. In some embodiments, the number of heat-labile nucleotides does not exceed two. In some embodiments, the number of heat-labile nucleotides is one or two. In some embodiments, each modified nucleotide other than the substituted alkoxy-modified nucleotide, nucleotide X, fluorinated nucleotide, and heat-labile nucleotide is independently an alkoxy-modified nucleotide. In this context, "thermally unstable nucleotide" refers to a nucleotide with a thermally unstable modification, wherein the thermally unstable modification is a modification that lowers the thermal dissociation temperature of the oligonucleotide duplex by at least 0.5 °C compared to an oligonucleotide duplex with an unmodified nucleotide at the corresponding position. Exemplary thermally unstable modifications can be found in the specification in PCT Publication WO2018 / 098328A1.

[0236] -

[0251] The thermal instability modification described in the paragraph. In some embodiments, the heat-labile nucleotide is a type of acyclic nucleotide or heteronucleotide. Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unblocking nucleic acids (UNA) or glycerol nucleic acids (GNA), wherein UNA is shown in formula (15) and GNA is shown in formula (16): In formulas (15) and (16) above, R is selected from H, OH or alkoxy (O-alkyl), and Base represents a nucleic acid base, such as A, U, G, C or T. Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position on the ribose ring, as shown in formula (17) or (18). In the compounds of formulas (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorinated modifications as described above. In some embodiments, the heat-labile nucleotide is selected from one of the following: GNA as shown in formula (27A), 2'-OMe abasic nucleotide as shown in formula (27B), 3'-OMe modified nucleotide as shown in formula (27C), 5'-Me modified nucleotide as shown in formula (27D), SNA as shown in formula (27E), hGNA as shown in formula (27F), hhGNA as shown in formula (27G), mGNA as shown in formula (27H), TNA as shown in formula (27I), h'GNA as shown in formula (27J), UNA as shown in formula (27K), or a hyperspacer as shown in formula (27L). In the compounds of formulas (27A)-(27L) above, Base represents a nucleic acid base, such as A, U, G, C, or T; R 27 Selected from H, OH, F, alkoxy, alkyl, or alkoxy-substituted alkyl groups. * indicates that the carbon atom is chiral, and the compound can be an R configuration, an S configuration, or a racemic mixture of R and S configurations. In some embodiments, each thermally unstable nucleotide is independently a GNA as shown in formula (27A). In some embodiments, for ease of synthesis, each alkoxy-modified nucleotide is a 2'-methoxy-modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (9). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (10). In equations (7) to (10) above, Base represents a nucleic acid base, such as A, U, G, C or T. In the preceding and following text, “fluorinated nucleotides,” “2’-fluorinated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by fluorine,” and “nucleotides with a 2’-fluorinated ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the nucleotide is replaced by fluorine, resulting in compounds with the structure shown in formula (7); “methoxylated nucleotides,” “2’-methoxylated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by methoxyl,” and “nucleotides with a 2’-methoxy ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the ribose group of the nucleotide is replaced by methoxyl, resulting in compounds with the structure shown in formula (8). In some embodiments, the single-stranded oligonucleotides of this disclosure are 19-23 nucleotides in length, and in a 5'-3' orientation, the 13th nucleotide is a substituted alkoxy-modified nucleotide, the 14th nucleotide is nucleotide X, one of the 5th-7th nucleotides, and the 2nd and 16th nucleotides are fluorinated nucleotides, the 3rd nucleotide is an alkoxy-modified nucleotide or a substituted alkoxy-modified nucleotide, the 5th nucleotide is a fluorinated nucleotide, an alkoxy-modified nucleotide, or a substituted alkoxy-modified nucleotide, and each of the remaining nucleotides in the single-stranded oligonucleotide is independently an alkoxy-modified nucleotide. In some embodiments, the single-stranded oligonucleotide of this disclosure is 21 nucleotides in length, and in a 5'-3' orientation, the 13th nucleotide is a substituted alkoxy-modified nucleotide, the 14th nucleotide is nucleotide X, the 2nd, 6th and 16th nucleotides are fluorinated nucleotides, the 3rd or 5th nucleotide is an alkoxy-modified nucleotide or a substituted alkoxy-modified nucleotide, and each of the remaining nucleotides in the single-stranded oligonucleotide is an alkoxy-modified nucleotide. In some embodiments, each nucleotide X in the single-chain oligonucleotides of this disclosure refers to a deoxynucleotide. Each alkoxy-modified nucleotide refers to a methoxy-modified nucleotide. In some embodiments, each substituted alkoxy-modified nucleotide in the single-chain oligonucleotides of this disclosure refers to a 2'-O-methoxyethyl-modified nucleotide. In some embodiments, each BNA in the single-chain oligonucleotides of this disclosure refers to an LNA or cET BNA. In some embodiments, each heat-labile nucleotide in the single-chain oligonucleotides of this disclosure refers to a GNA. In some embodiments, at least two of the linking groups between adjacent nucleotides in the single-stranded oligonucleotide are phosphate groups with modifying groups. In some embodiments, in the single-stranded oligonucleotide of this disclosure, one to four of the linking groups between adjacent nucleotides in the 5' end (1-5 nucleotides) are each independently a phosphate group with a modifying group. In some embodiments, in the single-stranded oligonucleotide of this disclosure, one to four of the linking groups between adjacent nucleotides in the 3' end (1-5 nucleotides) are phosphate groups with modifying groups. In some embodiments, in the single-stranded oligonucleotide of this disclosure, two linking groups or four linking groups between adjacent nucleotides in the 5' end (1-3 nucleotides) are phosphate groups with modifying groups. In some embodiments, in the single-stranded oligonucleotide of this disclosure, each of the two linking groups between adjacent nucleotides in the 3' end (1-3 nucleotides) or four linking groups between adjacent nucleotides in the 3' end (1-5 nucleotides) is independently a phosphate group with a modifying group. In some embodiments, in the single-stranded oligonucleotides of this disclosure, if unmodified nucleotides are present, each of the two linking groups between each unmodified nucleotide and its adjacent nucleotide is independently a phosphate ester group with a modifying group. The modified phosphate ester group can enhance the resistance of the single-stranded oligonucleotides of this disclosure to exonuclease activity and improve their stability in the body. In some embodiments, each of the 2-6 linking groups between adjacent nucleotides in the single-stranded oligonucleotide is independently a phosphate ester group with a modifying group. In some embodiments, three or four linking groups between adjacent nucleotides in the single-stranded oligonucleotide are each independently a phosphate ester group with a modifying group. In some embodiments, each of the linking groups between adjacent nucleotides in the first to third nucleotides at the 5' end and between adjacent nucleotides in the first to third nucleotides at the 3' end of the single-stranded oligonucleotide is independently a phosphate ester group with a modifying group. In some embodiments, if unmodified nucleotides are present in the single-stranded oligonucleotide, each of one or both of the two linking groups between each unmodified nucleotide and its adjacent nucleotide is independently a phosphate group with a modifying group. In some embodiments, each phosphate group with a modifying group is independently a thiophosphate group having the structure shown in formula (28): In some embodiments, the 5'-terminal nucleotide of the single-stranded oligonucleotide is a 5'-hydroxynucleotide, a 5'-phosphate nucleotide, or a 5'-phosphate analog-modified nucleotide, wherein the 5'-hydroxynucleotide has the structure shown in formula (29); the 5'-phosphate nucleotide has the structure shown in formula (30); and the 5'-phosphate analog-modified nucleotide has a structure selected from those shown in formulas (31) to (34). R is selected from H, OH, OCH3 and F; Base represents a nucleic acid base, selected from A, U, C, G or T. In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (30), the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (31), or a phosphate thioester modified nucleotide as shown in formula (33). In some embodiments, the 5'-terminal nucleotide of the single-stranded oligonucleotide is a 5'-hydroxy nucleotide or a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification. In some embodiments, the 5'-terminal nucleotide being a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide can further increase one or more of the stability, in vivo pharmacodynamic activity, and long-lasting effect of the single-stranded oligonucleotide, double-stranded oligonucleotide containing the single-stranded oligonucleotide, and oligonucleotide conjugates described herein. As previously stated, the single-stranded oligonucleotides described in this disclosure have sufficient complementarity with the target mRNA to produce RNAi effects. In some embodiments, the single-stranded oligonucleotides described in this disclosure are fully complementary to the target mRNA. In the context of this disclosure, "fully complementary" means that the complementarity between the single-stranded oligonucleotides described in this disclosure and the target mRNA is sufficient for the single-stranded oligonucleotides to reduce or eliminate the production of the protein encoded by the target mRNA through RNAi. In some embodiments, "fully complementary" means that the single-stranded oligonucleotides described in this disclosure are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the target mRNA across at least 16 nucleotides, for example, across 16-25 nucleotides, across 18-23 nucleotides, or across 19-21 nucleotides. In some embodiments, the single-stranded oligonucleotides described in this disclosure are completely anticomplementary to the target mRNA. In some embodiments, the two nucleotide sequences that are “fully complementary” may include completely anticomplementary internal regions (e.g., completely anticomplementary across a length of at least 6, 8, or 10 nucleotides). In some embodiments, the single-stranded oligonucleotide of this disclosure is completely anticomplementary to the target mRNA at least within a seed region. The “seed region” refers to the region of nucleotides 2-8 of the single-stranded oligonucleotide of this disclosure, in which the single-stranded oligonucleotide of this disclosure can better mediate RNAi action and suppress the level of the target mRNA. In some embodiments, the single-stranded oligonucleotide is substantially anticomplementary or completely anticomplementary to the target mRNA across a length of at least 16 nucleotides. In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide from position 2 to 19, in the 5'-3' orientation, is completely anticomplementary to the target mRNA. In some embodiments, the nucleotide sequence of the single-stranded oligonucleotide other than position 1 at the 5' end, in the 5'-3' orientation, is completely anticomplementary to the target mRNA. In some embodiments, all nucleotides of the single-stranded oligonucleotide are completely anticomplementary to the target mRNA. In some embodiments, the single-stranded oligonucleotide is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to a continuous nucleotide sequence m in the target mRNA; the length of the nucleotide sequence m is not greater than the length of the single-stranded oligonucleotide, and the length of the nucleotide sequence m is the same as, differs from, the length of the single-stranded oligonucleotide by no more than 8 nucleotides, or differs from, the length of the single-stranded oligonucleotide by 1-5 nucleotides. In some embodiments, the length of the nucleotide sequence m is at least 16 nucleotides, or 16-25 nucleotides, or 18-23 nucleotides, or 19-21 nucleotides. In some embodiments, the length of the single-stranded oligonucleotide is the same as the nucleotide sequence m, and at least the nucleotide sequence other than the terminal nucleotide of the single-stranded oligonucleotide is completely anticomplementary to the nucleotide sequence m, thus, the single-stranded oligonucleotide of this disclosure can further improve the inhibitory effect on the target mRNA. In some embodiments, the nucleotide sequence other than the first position of the single-stranded oligonucleotide is completely anticomplementary to the nucleotide sequence m in the 5'-3' direction. In some embodiments, all nucleotides of the single-stranded oligonucleotide are completely anticomplementary to the nucleotide sequence m. In some embodiments, the target mRNA is the mRNA transcribed from the target gene.The target genes are selected from the group consisting of the following genes: angiotensin-converting enzyme 2 (ACE2), angiotensinogen (AGT), aldehyde dehydrogenase 2 (ALDH2), angiopoietin-like protein 3 (ANGPTL3), lipoprotein A (LPA), apolipoprotein B (ApoB), apolipoprotein C3 (ApoC3), apolipoprotein E4 (ApoE4), amyloid-β precursor (APP), androgen receptor (AR), apoptosis signal-regulated kinase (ASK1), arginine vasopressin receptor 2 (AVPR2), complement C3 (C3 or CC3), complement C5 (C5), complement factor B (CFB), type □ collagen α1 (Col1A1), connective tissue growth factor (CTGF), dual homologous box protein 4 (DUX4), Ebola virus, endothelin receptor type A (EDNRA), epidermal growth factor receptor (EGFR), fatty acid synthase (FASN), fibrinogen-like 1 (FGL1), forkhead box protein 1 (FOXO1), obesity gene-associated protein (FTO), coagulation factor VII (FVII), coagulation factor XI (FXI), coagulation factor FXII (FXII), glucagon receptor (GCGR), hepatitis C virus gene (HCV), hepatitis D virus gene (HDV), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3β-hydroxysteroid dehydrogenase (HSD), huntingtin protein (HTT), cytosolic adaptor protein (KEAP1), kininogen gene (KNG), leukocyte immunoglobulin-like receptor B3 (LilrB3), microtubule-associated protein tau (MAPT), serine protease 2 (MASP2), sodium taurocholate cotransporter polypeptide (NTCP) Human tumor suppressor gene p53 (p53), proprotein convertase isoform 9 (PCSK9), programmed cell death-ligand 1 (PD-L1), protein kinase K (PKK), plasminogen (PLG), nucleoside phosphorylase (PNP), phospholipase-like domain-containing protein 3 (PNPLA3), phosphatidylinositol-3-kinase (PSD3), receptor for advanced glycation end products (RAGE), target of rapamycin (mTOR) regulatory-associated protein complex (RPTOR), SARS-CoV-2 gene, desaturated fat Enzyme 1 (SCD1), the α subunit of the epithelial cell sodium channel (SCNN1A), sodium-glucose cotransporter 2 (SGLT2), synuclein α (SNCA), superoxide dismutase (SOD1), signal transduction and transcription activator 3 (STAT3), matrix metalloproteinase inhibitor-1 (TIMP-1), transmembrane serine protease 6 (TMPRSS6), xanthine dehydrogenase (XO), urate transporter 1 (URAT1), urate transporter 2 (URAT2), and glucose facilitated transporter 9 (URATv1 or SLC2A9). In some embodiments, the single-stranded oligonucleotide of this disclosure is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to at least 15, 16, 17, 18, or 19 consecutive nucleotides in the sense strand sequence of any of the siRNAs listed in Tables 1A-1F below. In some embodiments, the unmodified equivalent sequence of the single-stranded oligonucleotide has at least 17, 18, 19, 20, or 21 consecutive identical nucleotides with the antisense strand sequence of any of the siRNAs listed in Tables 1A-1F, and these consecutive identical nucleotides include no more than 3 base differences, no more than 1 base difference, or no base difference. In some embodiments, at least nucleotides 2-19 of the unmodified equivalent sequence of the single-stranded oligonucleotide have no more than 1 base difference or no base difference with the sense strand sequence of any of the siRNAs listed in Tables 1A-1F, in the 5'-3' direction. In some embodiments, the single-stranded oligonucleotide further includes a second nucleotide sequence, 1-3 nucleotides in length, located at the 3' end of the single-stranded oligonucleotide, forming the 3' overhang of the antisense strand of the double-stranded oligonucleotide. In some embodiments, the second nucleotide sequence is 2 nucleotides in length, wherein the 2 nucleotides are two consecutive thymine deoxynucleotides, two consecutive uracil nucleotides, or completely anticomplementary to the target mRNA. In some embodiments, the single-stranded oligonucleotide of this disclosure can exert pharmacological activity independently. In some embodiments, the single-stranded oligonucleotide of this disclosure is an antisense oligonucleotide (ASO). In some embodiments, the single-stranded oligonucleotide of this disclosure is a single-stranded RNAi (ssRNAi) compound. In some embodiments, the single-stranded oligonucleotide of this disclosure exerts pharmacological activity as a single strand (e.g., the antisense strand) of a double-stranded oligonucleotide. The double-stranded oligonucleotides disclosed herein In another aspect, this disclosure also provides a double-stranded oligonucleotide containing a sense strand and an antisense strand, each nucleotide in the sense strand being a modified or unmodified nucleotide, the sense strand and the antisense strand being at least partially anticomplementary to form a double-stranded region, wherein the antisense strand is a single-stranded oligonucleotide as described above in this disclosure. In the double-stranded oligonucleotides disclosed herein, the length of the sense strand is 15-26, 17-24, or 19-23 nucleotides. In some embodiments, the length of the sense strand is 19-21 nucleotides. In some embodiments, the length difference between the sense and antisense strands is 0-5 nucleotides. In some embodiments, the length of the sense strand is not greater than the length of the antisense strand. In some embodiments, for ease of synthesis, the length of the sense strand is 19-21 nucleotides, and the length of the antisense strand is 19-24 nucleotides. In some embodiments, the length of the antisense strand is 1-3 nucleotides longer than the length of the sense strand; or the length of the antisense strand is 2 nucleotides longer than the length of the sense strand. In some embodiments, the length of the sense strand is 19-23 nucleotides. Therefore, the length ratio of the sense strand to the antisense strand of the double-stranded oligonucleotide disclosed herein can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 23 / 23, 23 / 24, 23 / 25, or 23 / 26. In some embodiments, the length of the sense strand is 15-26 or 17-24 nucleotides. In some embodiments, the length of the sense strand is 19-21 nucleotides. In some embodiments, the lengths of the sense strand and the antisense strand are the same, both being 19, 20, or 21 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides and the length of the antisense strand is 21 nucleotides; or the length of the sense strand is 21 nucleotides and the length of the antisense strand is 21 nucleotides; or the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides. In some embodiments, in the positive strand of the double-stranded oligonucleotide of this disclosure, 2-3 of the 11th-13th nucleotides of the positive strand, in the 3'-5' direction, are fluorinated nucleotides, and the first nucleotide of the positive strand is an alkoxylated nucleotide or an inverted abasic deoxyribonucleotide (abbreviated as invab or ia, having the structure shown in formula (35)). In some embodiments, in the 3'-5' direction, the last nucleotide of the positive strand is an alkoxylated nucleotide or an inverted abasic deoxyribonucleotide. In some embodiments, apart from the above-mentioned fluorinated and inverted abasic deoxyribonucleotides, the nucleotides at the remaining positions in the positive strand are each independently non-fluorinated nucleotides, and each non-fluorinated nucleotide is independently selected from one of alkoxylated nucleotides, alkylated nucleotides, amine-modified nucleotides, and thermally unstable nucleotides. In some embodiments, the oxygen atom directly attached to the ribose ring as shown in formula (35) may be attached to the 3' phosphate group of the penultimate nucleotide of the positive strand. In some embodiments, the oxygen atom directly attached to the ribose ring as shown in formula (35) may be attached to the 3' phosphate group of the 3' terminal nucleotide of the positive strand, and the oxygen atom attached to the ribose ring via a methylene group as shown in formula (35) may be attached to a hydrogen atom, a hydroxyl protecting group, or a delivery group as described below. In some embodiments, the oxygen atom of formula (35) connected to the ribose ring via a methylene group may be attached to the 5' phosphate group of the penultimate nucleotide at the 5' end of the positive strand. In some embodiments, the oxygen atom of formula (35) connected to the ribose ring via a methylene group may be attached to the 5' phosphate group of the penultimate nucleotide at the 5' end of the positive strand, and the oxygen atom of formula (35) directly attached to the ribose ring may be attached to a hydrogen atom, a hydroxyl protecting group, or a delivery group as described below. In some embodiments, the sense strand contains 19-21 nucleotides, and the antisense strand contains 21-23 nucleotides. In some embodiments, along the 3'-5' direction, the 11th and 13th nucleotides, or the 11th-13th nucleotides, of the sense strand are fluorinated nucleotides, the first nucleotide and / or the last nucleotide of the sense strand is an alkoxylated nucleotide or a reverse debased deoxynucleotide, and the nucleotides at the remaining positions are alkoxylated nucleotides. In some embodiments, in the positive strand, at least one of the linking groups connecting two adjacent nucleotides is a phosphate ester group with a modifying group, and the phosphate ester group with the modifying group is present at least once between two adjacent nucleotides in the first to fifth nucleotides at the 5' end of the positive strand and between two adjacent nucleotides in the first to fifth nucleotides at the 3' end. In some embodiments, 1 to 4 of the linking groups between any two adjacent nucleotides in the first to fifth nucleotides at the 5' end of the positive strand, and / or 1 to 4 of the linking groups between any two adjacent nucleotides in the first to fifth nucleotides at the 3' end of the positive strand are phosphate ester groups with modifying groups. In some embodiments, all four of the linking groups between adjacent nucleotides in the first to fifth nucleotides at the 5' end of the positive strand are phosphate ester groups with modifying groups. In some embodiments, all four of the linking groups between adjacent nucleotides in the first to fifth nucleotides at the 3' end of the positive strand are phosphate ester groups with modifying groups. In some embodiments, the linking group connecting two adjacent nucleotides in the 1-3, 1-4, or 1-5th nucleotides at the 5' and / or 3' ends of the sense strand is a phosphate ester group with a modifying group. The definition and selection range of the phosphate ester group with a modifying group are the same as those described above for the antisense strand of this disclosure. In some embodiments, each phosphate ester group with a modifying group is a thiophosphate ester group having the structure shown in formula (28). In some embodiments, the sense strand comprises 19-21 nucleotides, and the antisense strand comprises 21-23 nucleotides; in the 3'-5' direction, the 11th and 13th nucleotides, or the 11th-13th nucleotides, of the sense strand are fluorinated nucleotides; the first and / or last nucleotide of the sense strand is a methoxylated nucleotide or a reverse debased deoxynucleotide; and the remaining nucleotides are methoxylated nucleotides; 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 5' end of the sense strand, and / or 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 3' end of the sense strand are thiophosphate groups. In some embodiments, the linking group between every two adjacent nucleotides in the 1st-2nd, 1st-3rd, 1st-4th, or 1st-5th nucleotides at the 5' end and / or 3' end of the sense strand is a thiophosphate group, and the linking groups between the remaining adjacent nucleotides in the sense strand are phosphate groups. In some embodiments, the linker between any two adjacent nucleotides in the 1st-3rd, 1st-4th, or 1st-5th nucleotides at the 5' end of the positive strand is a phosphate ester group, and the linker between the remaining adjacent nucleotides in the positive strand is a phosphate ester group. In some embodiments, the linker between any two adjacent nucleotides in the 1st-2nd, 1st-3rd, 1st-4th, or 1st-5th nucleotides at the 3' end of the positive strand is a phosphate ester group, and the linker between the remaining adjacent nucleotides in the positive strand is a phosphate ester group. In some embodiments, the double-stranded oligonucleotide of this disclosure comprises a sense strand containing 19 nucleotides and an antisense strand containing 21 nucleotides. In the sense strand, the 11th and 13th nucleotides, or nucleotides 11-13, are fluorinated nucleotides in the 3'-5' direction; the 1st and / or the last nucleotide is a reverse debased deoxynucleotide; and the remaining nucleotides are alkoxy-modified nucleotides. In the sense strand, 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 5' end, and / or 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 3' end, are phosphate thioester groups. In some embodiments, in the antisense strand, the 13th nucleotide in the 5'-3' direction is a 2'-O-methoxyethyl modified nucleotide, the 14th nucleotide is a deoxynucleotide, and the 2nd, 6th, and 16th nucleotides are fluorinated nucleotides. Each remaining nucleotide in the antisense strand is an alkoxy-modified nucleotide. In some embodiments, each ribose 2'-alkoxy modified nucleotide is a ribose 2'-C1-C3 alkoxy modified nucleotide, such as a ribose 2'-methoxy modified nucleotide. In some embodiments, each ribose 2'-substituted alkoxy modified nucleotide is independently a ribose 2'-C1-C3 alkoxy modified nucleotide with substituents, each substituent being independently selected from C1-C3 alkoxy groups. In some embodiments, each ribose 2'-alkoxy modified nucleotide refers to a ribose modified nucleotide. In some embodiments, each ribose 2'-substituted alkoxy modified nucleotide refers to a 2'-O-methoxyethyl (MOE) modified nucleotide. In some embodiments, each BNA refers to an LNA or cET. In some embodiments, in the antisense strand, the linking groups between any two adjacent nucleotides in the first to third nucleotides at the 5' end and between any two adjacent nucleotides in the first to third nucleotides at the 3' end are phosphate ester groups with modifying groups, and if unmodified nucleotides are present, one to two of the two linking groups between each unmodified nucleotide and its adjacent nucleotides are phosphate ester groups with modifying groups. In some embodiments, the 5' terminal nucleotide of the antisense strand is a 5'-hydroxy nucleotide of formula (29) or a 5'-vinyl phosphate-modified nucleotide of formula (31). The double-stranded oligonucleotides disclosed herein, through the aforementioned modification schemes, achieve a good balance between gene expression regulatory activity and in vivo stability. In the context of this disclosure, "modification scheme" refers to a combination of nucleotide ribose modifications, phosphate modifications, 5' end modifications, and / or base modifications of different numbers, positions, and types that are unrelated to or weakly related to a specific sequence. In some embodiments, the double-stranded oligonucleotides of this disclosure, through the aforementioned modification schemes, can maintain excellent stability without significantly reducing the original drug activity of the double-stranded oligonucleotide, thereby achieving a good balance between gene expression regulatory activity and in vivo stability. In some embodiments, the double-stranded oligonucleotides of this disclosure are siRNAs. The double-stranded oligonucleotides of this disclosure, through the aforementioned modification schemes, can maintain excellent stability without significantly reducing the original RNAi activity of the siRNA, thereby achieving a good balance between target mRNA inhibitory activity and in vivo stability. In some embodiments, the double-stranded oligonucleotide of this disclosure comprises a substantially anticomplementary or completely anticomplementary double-stranded region, and one or two dangling ends of the sense strand and / or one or two dangling ends of the antisense strand. In some embodiments, the double-stranded oligonucleotide of this disclosure comprises a substantially anticomplementary or completely anticomplementary double-stranded region and one dangling end of the antisense strand. In some embodiments, the length of the double-stranded region formed by the sense and antisense strands is at least 16 nucleotides. In some embodiments, the length of the double-stranded region formed by the sense and antisense strands is 16-23 nucleotides. In some embodiments, the length of the double-stranded region formed by the sense and antisense strands is 18, 19, 20, or 21 nucleotides. In some embodiments, the sense strand and the antisense strand are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary. In some embodiments, the sense strand and the antisense strand are substantially anticomplementary or completely anticomplementary within the double-stranded region. In some embodiments, along the 5'-3' direction, at least the nucleotide sequence of the sense strand other than the first and last positions is substantially anticomplementary or completely anticomplementary to the antisense strand. In some embodiments, along the 5'-3' direction, the nucleotide sequence of the sense strand other than the last position is completely anticomplementary to the antisense strand; or all nucleotides of the sense strand are completely anticomplementary to the antisense strand. In some embodiments, the unmodified equivalent sequence of the positive strand of the double-stranded oligonucleotide described in this disclosure comprises a nucleotide sequence of the same length as nucleotide sequence m, and differing by no more than 3 bases, no more than 1 base, or having no base difference. The definition and selection of nucleotide sequence m are as described above. In the preceding and following text, a "base difference" between one nucleotide sequence and another means that the base type of the nucleotide at the same position has changed compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, then a base difference exists between the two nucleotide sequences at that position. When bases are modified, as long as the purine-pyrimidine pairing relationship in forming the above-mentioned double-stranded nucleic acid structure is not affected, it is also considered that there is no base difference between the modified base and the original base. In some embodiments, it is considered that there is no base difference between U and T. In some embodiments, it is considered that there is no base difference between C and 5-methylcytosine (5mC). In some implementations, a base difference at a given position can also be considered to have occurred when a baseless nucleotide or its equivalent is replaced. When comparing two nucleotide sequences to determine the number of base differences, alignment is performed using the method that minimizes the number of base differences among all alignment methods, and the base differences are determined based on this alignment. In this case, "identical positions" refers to the corresponding positions between the two nucleotide sequences in that alignment. For example, when positions 1-5 of nucleotide sequence A are aligned with positions 2-6 of nucleotide sequence B in the same direction, the number of base differences is minimized compared to other alignment methods. Therefore, "identical positions" means that position 1 of nucleotide sequence A is aligned with position 2 of nucleotide sequence B, position 2 of nucleotide sequence A is aligned with position 3 of nucleotide sequence B, and so on. In the preceding and following text, "unmodified equivalent sequence" refers to an oligonucleotide sequence that does not contain any ribose ring modifications, base modifications, or phosphate backbone modifications compared to the original sequence used as the basis for alignment. For example, the unmodified equivalent sequence of VPAmsCfsdTGmsUmia is ACUGUN, where N is A, C, G, or U. In some embodiments, the number of base differences between two nucleotide sequences of different lengths refers to the number of base differences calculated from the first nucleotide without a base difference to the last nucleotide without a base difference in the alignment with the minimum number of base differences. In some embodiments, the number of base differences between two nucleotide sequences of the same length refers to the total number of base differences between the first to last nucleotides of one nucleotide sequence and the first to last nucleotides of the other nucleotide sequence, in the same direction. In some embodiments, the absence of base differences between two nucleotide sequences of different lengths means that, in the same direction, there are no base differences between the first to last nucleotides of the shorter nucleotide sequence and each nucleotide at the same position in the other nucleotide sequence. In some embodiments, the absence of base differences between two nucleotide sequences of the same length means that, in the same direction, there are no base differences between the first to last nucleotides of one nucleotide sequence and the first to last nucleotides of the other nucleotide sequence. The double-stranded oligonucleotides disclosed herein can be various double-stranded oligonucleotides that regulate gene expression. In some embodiments, they can be double-stranded oligonucleotides that inhibit or downregulate gene expression, such as siRNA; in some embodiments, they can be double-stranded oligonucleotides that activate or upregulate gene expression, such as saRNA. In some embodiments, the double-stranded oligonucleotide is siRNA. In some embodiments, the target mRNA is the mRNA transcribed from a target gene, and the target gene is selected from the group consisting of the following genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, ApoE4, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCG. R, HCV, HDV, HMGCR, HSD, HTT, KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PCSK9, PD-L1, PKK, PLG, PNP, PNPLA3, PSD 3. RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1. In some embodiments, the target mRNA is AGT mRNA, APOE4 mRNA, NTCP mRNA, PCSK9 mRNA, PNPLA3 mRNA, or LPA mRNA, wherein the unmodified equivalent sequences of the sense and antisense strands independently share at least 15, 16, 17, 18, or 19 consecutive nucleotides with any of the unmodified equivalent sequences of the sense and antisense strands of any of the siRNAs listed in Tables 1A-1F, and wherein there are no more than 3 base differences, no more than 1 base difference, or no base differences. In some embodiments, the double-stranded oligonucleotide is one of the siRNAs shown in Tables 1A-1F below: Table 1A: Disclosed siRNA sequences Table 1B: Disclosed siRNA sequences Table 1C shows the disclosed siRNA sequences. Table 1D contains the disclosed siRNA sequences. Table 1E: Disclosed siRNA sequences Table 1F discloses the siRNA sequences. In this diagram, uppercase letters C, G, U, A, and T represent the base composition of a nucleotide; lowercase letter o indicates that the uppercase letter to the left of o represents an alkoxy-modified nucleotide; lowercase letter f indicates that the uppercase letter to the left of f represents a fluorinated nucleotide; lowercase letter e indicates that the uppercase letter to the left of e represents a substituted alkoxy-modified nucleotide; uppercase letter combinations enclosed in parentheses (NS) indicate that the uppercase letter to the left of S represents a substituted alkoxy-modified nucleotide N, where N is C, G, U, A, or T; and lowercase letter s indicates the base composition of a nucleotide. The two closest uppercase letters on either side of the nucleotide are linked by a phosphate thioester group, or the two closest uppercase letters on either side of the nucleotide are linked by a phosphate thioester group. The lowercase letter x indicates that the two closest nucleotides on either side of that letter are linked by a phosphate thioester group. The lowercase letter d indicates that the uppercase letter to the right of that letter represents a deoxynucleotide. P1 indicates that the uppercase letter to the right of that letter represents a 5'-hydroxynucleotide or a 5'-vinyl phosphate (VP) modified nucleotide. ia indicates a reverse debased deoxynucleotide. Furthermore, each X in the above sequence independently represents U or T, and / or each Y independently represents C or 5mC. In some embodiments, each substituted alkoxy-modified nucleotide is independently selected from the 2'-O-methoxyethyl modified nucleotide represented by moe. In some embodiments, each alkoxy-modified nucleotide is independently selected from the 2'-O-methoxy modified nucleotide. In some embodiments, each lowercase letter x independently indicates that the two closest nucleotides on either side of that letter are linked by a phosphate thioester group. The single-stranded and / or double-stranded oligonucleotides provided in this disclosure can be obtained using conventional oligonucleotide preparation methods in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is already available as a commercially available custom service. Modified nucleotide groups can be introduced into the single-stranded and / or double-stranded oligonucleotides described in this disclosure using appropriately modified nucleoside monomers. Methods for preparing appropriately modified nucleoside monomers and for introducing modified nucleotide groups into single-stranded and / or double-stranded oligonucleotides are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared using known methods. The double-stranded oligonucleotides provided in this disclosure can be used alone, or in combination with a pharmaceutically acceptable carrier to form a pharmaceutical composition, or in combination with a delivery group to form an oligonucleotide conjugate, or in any other suitable form. Contacting cells with an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate can modulate the expression of a target gene, or administering an effective amount of the double-stranded oligonucleotide, the oligonucleotide conjugate, or the pharmaceutical composition to a subject can modulate the expression of a target gene to achieve the purpose of treating a pathological condition or disease associated with the level of expression of the target gene. Oligonucleotide conjugates In another aspect, this disclosure provides an oligonucleotide conjugate comprising a double-stranded oligonucleotide group and a delivery group conjugated to the double-stranded oligonucleotide group, wherein the double-stranded oligonucleotide group is independently formed by removing one or more atoms or groups of atoms from the double-stranded oligonucleotide provided in this disclosure. Those skilled in the art will understand that the RNAi activity of the double-stranded oligonucleotide group formed by this removal is at least the same as or equivalent to the RNAi activity of the double-stranded oligonucleotide itself. In some embodiments, the removal of one or more atoms or groups of atoms does not impair the inhibitory activity or stability of the double-stranded oligonucleotide (e.g., siRNA) against the target mRNA. In some embodiments, the double-stranded oligonucleotide group is formed by removing one atom or group of atoms (e.g., a hydrogen atom, a hydroxyl group, or a phosphate ester group) from the double-stranded oligonucleotide provided in this disclosure. For example, the siRNA group may be a chemical portion formed by removing a hydrogen atom from a phosphate ester bond in siRNA, or a chemical portion formed by removing a hydrogen atom from the 5' hydroxyl group of the 5' terminal nucleotide of the sense or antisense strand in siRNA, or a chemical portion formed by removing a hydrogen atom from the 3' hydroxyl group of the 3' terminal nucleotide of the sense or antisense strand in siRNA. In the context of this disclosure, unless otherwise stated, "conjugation" means the covalent connection between two or more chemical parts, each having a specific function (however, without theoretical limitation, the individual components within the functional chemical part—such as a double-stranded oligonucleotide or a metal ion-ligand chelate—may not necessarily be covalently connected); correspondingly, "conjugation" refers to a compound formed by the covalent connection of the individual chemical parts. Further, "oligonucleotide conjugation" refers to a compound formed by the covalent attachment of one or more functional chemical parts to an oligonucleotide. Oligonucleotide conjugation should be understood, depending on the context, as a collective term for multiple oligonucleotide conjugations or an oligonucleotide conjugation represented by a particular chemical formula. In the context of this disclosure, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to an oligonucleotide to ultimately form the oligonucleotide conjugation of this disclosure. The delivery group is a group used to deliver a double-stranded oligonucleotide group into a cell expressing a target mRNA. In some embodiments, the delivery group comprises a linker group and a pharmaceutically acceptable target group, and the double-stranded oligonucleotide group, the linker group, and the target group are covalently or non-covalently linked in sequence, each target group being selected from ligands capable of binding to cell surface receptors or groups capable of increasing tissue compatibility. In some embodiments, each target group independently targets one or more of the central nervous system, liver, kidney, lung, muscle, and eye. In some embodiments, there are 1-6 target groups. In one embodiment, there are 2-4 target groups. The double-stranded oligonucleotide group can be non-covalently or covalently conjugated to the delivery group, for example, it can be covalently conjugated to the delivery group. The conjugation site between the double-stranded oligonucleotide group and the delivery group can be at the 3' or 5' end of the sense strand of the double-stranded oligonucleotide, at the 3' or 5' end of the antisense strand, or within the internal sequence of the double-stranded oligonucleotide. In some embodiments, the conjugation site between the double-stranded oligonucleotide group and the delivery group is at the 3' end of the sense strand of the double-stranded oligonucleotide. In some embodiments, the delivery group can be attached to any position on the nucleotide, such as a phosphate group, a 2', 3', or 5'-hydroxyl group of the ribose, or a base. When the delivery group is attached to the 3' or 5' end of the double-stranded oligonucleotide chain, it is typically attached to the oxygen atom formed by removing a hydrogen atom from the 3' or 5'-hydroxyl group of the nucleotide; when the delivery group is attached to the inner sequence of the double-stranded oligonucleotide, it is typically attached to a phosphate group, a ribose ring, or a base. In some embodiments, the delivery group can be attached to the 3'-hydroxyl group of an inner sequence nucleotide of the double-stranded oligonucleotide, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. Various connection methods can be found in the following non-patent literature: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7. The entire contents of this article are incorporated herein by reference. In some embodiments, the double-stranded oligonucleotide and the delivery group are linked by acid-labile or reducible chemical bonds that can degrade in the acidic environment of the endosome, thereby converting the double-stranded oligonucleotide group into a free state. For non-degradable conjugations, the delivery group can be attached to the positive and negative strands of the double-stranded oligonucleotide group to minimize the impact of the conjugation on the activity of the double-stranded oligonucleotide group. The targeting group can be linked to the oligonucleotide group via a suitable linker. Those skilled in the art can select a suitable linker based on the specific type of the targeting group. For details on these linkers, the types of targeting groups, and the methods of linking them to oligonucleotides, please refer to the disclosure of WO2015006740A2, the entire disclosure of which is incorporated herein by reference. In some embodiments, the targeting group may be a ligand commonly used in the field of double-stranded oligonucleotide drug delivery, such as the various ligands described in WO2009082607A2, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to cell surface receptors expressing the target mRNA. In some embodiments, at least one or each of the target groups is selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in the central nervous system. Depending on the system, organ, tissue, or cell targeted, the targeting group can be selected from groups with different targeting properties. For example, all targeting groups in each delivery group have the same targeting property and are independently selected from one of the following groups: lipophilic groups or targeting peptide groups formed by targeting peptides that can improve the compatibility of the conjugate in the central nervous system; targeting peptide groups, antibody groups, or small molecule groups formed by targeting peptides, antibodies, or small molecules with affinity for receptors on the surface of muscle tissue or muscle cells; targeting peptide groups or antibody groups formed by targeting peptides or antibodies with affinity for transferrin receptor (TfR); targeting peptide groups formed by targeting peptides with affinity for receptors on the surface of the lungs, lung tissue, or lung cells; small molecule ligand groups with affinity for αvβ6 receptors; targeting peptide groups, aptamers, or antibodies formed by targeting peptides, aptamer groups, or antibody groups with affinity for receptors on the surface of tumor cells; targeting peptide groups with affinity for receptors on the surface of kidney cells; and small molecule ligand groups with affinity for desialyl glycoprotein receptors on the surface of liver parenchymal cells. In some embodiments, at least one or each of the target groups is selected from a lipophilic group or a target peptide group. In some embodiments, each of the target groups is C5-C. 18The double-chain oligonucleotide group is selected from one of the following: straight-chain, branched-chain hydrocarbon, steroidal compound, or target peptide that can accumulate in the central nervous system. The double-chain oligonucleotide group is capable of regulating the expression level of target mRNA in central nervous system cells, wherein the target mRNA is selected from one of the following mRNAs transcribed from genes: ApoE4, RPTOR, SOD1, APP, HTT, MAPT, SNCA, and LilrB3. In some embodiments, at least one or each of the target groups is selected from one of the target peptide groups or antibody groups formed by target peptides or antibodies with affinity for transferrin receptor (TfR). In some embodiments, the double-stranded oligonucleotide group is capable of regulating the expression level of a target mRNA in muscle tissue cells, the target mRNA being selected from one of the mRNAs transcribed from the following genes: DUX4, RPTOR. In some embodiments, at least one or each of the target groups is selected from small molecule ligand groups with affinity for the αvβ6 receptor. In some embodiments, the double-stranded oligonucleotide group is capable of regulating the expression level of a target mRNA in lung cells, the target mRNA being selected from mRNAs transcribed from the following genes: SCNN1A, RAGE, RPTOR, SARS-CoV-2, ACE2. In some embodiments, at least one or each of the target groups is selected from one of the following: a target peptide group, an aptamer group, or an antibody group that has an affinity for a receptor on the surface of tumor cells. In some embodiments, the double-stranded oligonucleotide group is capable of regulating the expression level of a target mRNA in tumor tissue cells, wherein the target mRNA is selected from one of the following mRNAs transcribed from genes: AR, STAT3, and PD-L1. In some embodiments, at least one or each of the target groups is selected from a group of target peptide groups that have affinity for receptors on the surface of kidney cells. In some embodiments, the double-stranded oligonucleotide group is capable of regulating the expression level of a target mRNA in kidney tissue cells, the target mRNA being selected from mRNAs transcribed from the following genes: SGLT2, KEAP1, URAT1, URATv1, EGFR, CC3, RPTOR. In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a ligand with affinity for desialyl glycoprotein receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a desialyl glycoprotein or a sugar. In some embodiments, each of the target groups is independently selected from D-mannose pyranoyl ... α-D-Furfural, β-D-Furfural, α-D-Fructose, α-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, Glucosamine, Sialic acid, Galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2 -Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4 One of the following: tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-alosulfonyl, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, at least one or each of the target groups is galactose or N-acetylgalactosamine.In some embodiments, the double-stranded oligonucleotide group can regulate the expression level of target mRNA in hepatocytes, wherein the target mRNA is selected from one of the mRNAs transcribed from the following genes: AGT, ANGPTL3, LPA, ApoB, ApoC3, ApoE4, ASK1, CC3, C5, CFB, Col1A1, CTGF, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HSD, NTCP, p53, PCSK9, PNP, PLG, PKK, PNPLA3, KNG, RPTOR, MASP2, SCD1, SOD1, TIMP-1, TMPRSS6, XO, PSD3, FGL1, NTCP, ALDH2, PD-L1, HMGCR, FASN. The delivery group in the oligonucleotide conjugates disclosed herein can be any of the various delivery groups known to those skilled in the art of oligonucleotide pharmaceuticals. In some embodiments, the linker group in the oligonucleotide conjugate of this disclosure has a structure as shown in formula (301): Where k is an integer from 1 to 5, Indicates the site of covalent linkage of groups; all L A Connect to L C The same atom in; or, each L A Independently connected to L C Different atoms in it. In some implementations, L C It has -NH-C(H) n301 (CH2O-) k The structure shown is such that k is an integer from 1 to 3, and n301 = 3 - k; L B The length is 5-20 atoms. In some implementations, each L A Independently, it is a straight-chain alkylene group with a length of 5-20 carbon atoms, wherein one or more methylene groups are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, 1,2,3-triazolyl, butadieneyl. In some implementations, L A Having a structure containing amide bonds as shown in formula (302), L B It has a structure as shown in equation (303): Where, n 302 q 302 and p 302 Each is an independent integer from 2 to 6; optionally, n 302 q302 and p 302 Each can be 2 or 3 independently; n 303 n is an integer between 4 and 16, optionally n 303 For integers between 8 and 12, This indicates the site where the group is covalently linked. In some embodiments, the linking group has a structure as shown in formula (304) or formula (305): In the linking group, each L A Each of the target groups is connected via an ether bond and via L C The oxygen atom of the hydroxyl group in some of the middle groups is related to L. C Partially linked by ether bonds; L B Through the carbonyl group in formula (303) and L C The nitrogen atom of the amino group in some of them forms an amide bond and is connected to the double-stranded oligonucleotide group through the oxygen atom in formula (303) by forming a phosphate ester bond or a thiophosphate ester bond. In some embodiments, the oligonucleotide conjugates provided in this disclosure have a structure as shown in formula (305A): Wherein, Nu represents the double-stranded oligonucleotide group formed from the double-stranded oligonucleotides provided in this disclosure. In some embodiments, the linker group in the oligonucleotide conjugate of this disclosure has the structure shown in formula (306): Where, n 306 For each p, the integer is between 0 and 3. 306 Independently, integers from 1 to 6. The site indicates a covalently linked group; the linking group is connected to the target group by an ether bond formed by an oxygen atom marked with *; the linking group is connected to the double-stranded oligonucleotide by at least one of the oxygen atoms marked with # forming a phosphate ester bond or a thiophosphate ester bond, and the remaining oxygen atoms marked with # are connected to hydrogen atoms to form hydroxyl groups, or connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups; In some embodiments, the oligonucleotide conjugates of this disclosure have a structure as shown in formula (307): Wherein, Nu represents the double-stranded oligonucleotide group formed from the double-stranded oligonucleotides provided in this disclosure. In some embodiments, the oligonucleotide conjugates of this disclosure have the structure shown in formula (308): in, n308 The integers are selected from 2 to 4; Each m 308 Independently selected as an integer from 2 to 5; Each R 308 Independently, it can be a hydrogen atom, a methyl group, or an ethyl group, or two R atoms attached to the same carbon atom. 308 Together with this carbon atom, they form a carbonyl group; One of the groups independently represented by A0 is a double-stranded oligonucleotide group, which is a group formed by removing an atom or group of atoms from the double-stranded oligonucleotide described in this disclosure; the remaining A0s are all targeting groups, each of which may be the same or different, and their definition and selection range are as described above. In some embodiments, each targeting group is independently selected from a ligand that has an affinity for the desialylate glycoprotein receptor on the surface of mammalian hepatocytes. Each L1 is independently a divalent linker with a length of 1-70 atoms; This indicates the site where the group is covalently linked. In some embodiments, each L1 is independently a straight-chain alkylene group with a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, OP(O)2, OP(O)(S), C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and the linear alkylene group may optionally have substituents of any one or more of the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C)10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), N(C1-C) 10 Alkyl) (C1-C 10 alkylphenyl), NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl groups, -CONH2, -NHC(O) (C1-C) 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (Halogenated alkyl groups). Those skilled in the art will understand that, although for convenience, L1 is defined as a linking group formed by substitution or substitution of a linear alkylene group, it may not be a linear group or may have a different name, such as an amine or alkenyl group resulting from the aforementioned substitution and / or substitution. Unless otherwise stated, in the chemical structural formulas described in this disclosure, the “length” of any group refers to the number of atoms in the longest atomic chain of that group, excluding hydrogen atoms; in the calculation of group length, when multiple connections are involved between two atoms (e.g., two atoms belong to the same cyclic group, and thus at least two atomic chains contain the two atoms), the length is calculated according to the shortest atomic chain between the two atoms. For example, 1,4-cyclohexanediyl, 1,4-piperidinediyl, 1,4-phenylene, and 1,4-piperazindiyl are all calculated as having a length of 4 atoms, while 1,2-cyclopentadiyl is calculated as having a length of only 2 atoms. The function of L1, covalently linked to A0 representing a double-stranded oligonucleotide group, is to covalently link the double-stranded oligonucleotide group to the target group. This allows the oligonucleotide conjugate containing the double-stranded oligonucleotide group to enter the cell expressing mRNA through the targeting effect of the target group, without affecting the regulatory effect of the double-stranded oligonucleotide group on the target mRNA level after entering the cell. Therefore, in some embodiments, the length of L1 covalently linked to A0 representing the double-stranded oligonucleotide group is 3-20 atoms, 4-15 atoms, or 5-12 atoms. In some embodiments, L1 covalently linked to A0 representing the double-stranded oligonucleotide group is selected from one or more of A1, A2, A4, A5, A10, A16, A18, and A19, combined with a phosphate ester group or a modified phosphate ester group. Where j1 is an integer between 2 and 10; This indicates the site where the group is covalently linked. In some embodiments, R2 is selected from at least two of A1, A2, A4, A10 and A16 connected with a phosphate ester group or a modified phosphate ester group; in some embodiments, R2 is selected from at least two of A1, A2, A10 connected with a phosphate ester group or a modified phosphate ester group. In some embodiments, L1, covalently linked to A0 representing a double-stranded oligonucleotide group, has a structure as shown in formulas (B1), (B2), (B3), or (B4): in, L represents the site where groups are covalently linked. B1 and L B2 Whether identical or different, independently selected from one of the following groups or any combination thereof: -(CH2) q1 -、-CH(OH)-、-CH(CH2OH)-、-NH-、-O-、-S-、1,4-cyclohexanediyl、1,4-piperidinidyl、1,4-phenylene、1,4-piperazinidyl、pyrrolidinediyl、where q1 is an integer from 1 to 6, L B1 and L B2 The length of each is independently 1-20 atoms. In some embodiments, L B1 and L B2 The length of each is independently 1-10 atoms. In some embodiments, L B1 and L B2 The length of each atom is 1-6 atoms. L B3A group selected from phosphate ester groups, thiophosphate ester groups, and dithiophosphate ester groups is covalently linked to the 5' hydroxyl group at the 5' position of the ribose of the sense or antisense strand of the double-stranded oligonucleotide group, or the oxygen atom remaining after removing one hydrogen atom from the 3' hydroxyl group at the 3' position of the ribose of the 3' terminal nucleotide. In some embodiments, L B3 It is a phosphate ester group, covalently linked to the 5' hydroxyl group of the ribose at the 5' end of the positive strand of the double-stranded oligonucleotide group, or the oxygen atom remaining after removing one hydrogen atom from the 3' hydroxyl group of the ribose at the 3' end of the positive strand of the double-stranded oligonucleotide group. In some embodiments, when the oligonucleotide conjugates of this disclosure are prepared by a solid-phase synthesis process, L1, covalently linked to A0 representing a double-stranded oligonucleotide group, needs to simultaneously contain a linking site for N-linking to a nitrogen-containing backbone, a linking site for linking to a double-stranded oligonucleotide group, and a functional group capable of linking to a solid-phase support. In some embodiments, the N-linking site on the nitrogen-containing backbone in L1, covalently linked to A0 representing a double-stranded oligonucleotide group, forms an amide bond with N, is covalently linked to the double-stranded oligonucleotide group via a phosphate ester bond, and the functional group capable of linking to the solid-phase support is a hydroxyl or amino group. In some embodiments, R2 is B5, B6, B5', or B6'. in, This indicates the site where a group is covalently bonded. The value of q2 can be an integer from 1 to 10. In some implementations, q2 is an integer from 1 to 5. The function of L1 covalently linked to A0, representing the target group, is to position the target group in a suitable spatial location, thereby better binding to receptors on the surface of mammalian hepatocytes, and thus specifically targeting and entering the hepatocytes. Therefore, any L1 covalently linked to A0, representing the target group, can be used in this disclosure as long as it has an appropriate length and its chemical properties do not significantly affect delivery. In some embodiments, each L1 covalently linked to the target group is independently a divalent linker with a length of 3-25 atoms. In some embodiments, each L1 covalently linked to A0, representing the target group, has an independent length of 4-15 atoms. In some embodiments, each L1 covalently linked to A0, representing the target group, has a length of 5-10 atoms. In some embodiments, the length of each L1 covalently linked to A0, representing the target group, is the same. In some embodiments, each L1 covalently linked to A0 representing a target group may be the same or different, and is independently selected from the group consisting of the groups represented by formulas (L3)-(L18) and any combination thereof: Where each j1 is an integer from 2 to 10; each R' is independently a hydrogen atom or a C1-C3 alkyl group. This indicates the site where the group is covalently linked. For ease of synthesis and / or chemical stability, in some embodiments, each L1 covalently linked to A0 representing a target group is independently a combination of at least two linking units, each linking unit independently having a structure shown in any one of formulas (L3)-(L7). In some embodiments, each linking unit independently has a structure shown in any one of formulas (L3), (L4), and (L7). For ease of synthesis, in some embodiments, each L1 covalently linked to A0 representing a target group includes a carbonyl group bonded to a nitrogen atom shown in formula (308). In some embodiments, each L1 covalently linked to A0 representing a target group independently has the structure shown in formula (L20) or (L21): Where j2 is an integer from 4 to 9, and j3 is 1 or 2. In some embodiments, j2 is 5, 6, or 7, and j3 is 1. In some embodiments, each L1 covalently linked to A0 representing the target group is identical. In the conjugates disclosed herein, the number of targeting groups and the spacing between them are the number and spacing that provide a suitable spatial configuration of multiple targeting groups. For this purpose, n308 and each m308 are independently integers selected from 2 to 4. In some embodiments, n308 is 3 or 4, so that the number of targeting groups in the conjugates of this disclosure is 3 or 4, which enables better binding to hepatocyte surface receptors. In some embodiments, n308 is 3, and each m308 is independently 3 or 4. Those skilled in the art will understand that each R 308 When the individual atoms are hydrogen atoms, methyl groups, or ethyl groups, the delivery effect of the oligonucleotide conjugate is not affected, and the objectives of this disclosure can still be achieved. In some embodiments, for ease of synthesis, each R... 308 All are hydrogen atoms. In the conjugates disclosed herein, each targeting group may be the same or different, and is independently selected from a ligand having an affinity for a receptor on the surface of mammalian hepatocytes. In some embodiments, at least one or each targeting group is a ligand having an affinity for the desialylate glycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each targeting group is a galactose group or an N-acetylgalactosamine group formed by removing an atom or group from galactose or N-acetylgalactosamine (GalNAc). In some embodiments, the oligonucleotide conjugates of this disclosure have the structures shown in formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422): In this embodiment, Nu represents a double-stranded oligonucleotide group. The P atom shown in the above structural formula is covalently linked to the 3' terminal nucleotide of the positive strand of the double-stranded oligonucleotide group. In some embodiments, the 3' terminal nucleotide of the positive strand of the double-stranded oligonucleotide group is a reverse deoxy-debasic nucleotide, and the P atom shown in the above structural formula is covalently linked to the double-stranded oligonucleotide group by substituting the hydrogen atom in the hydroxyl group of the reverse deoxy-debasic nucleotide of the positive strand of the double-stranded oligonucleotide group represented by Nu, which is connected to the ribose ring via a methylene group. In some embodiments, the P atom shown in the above structural formula is covalently linked to the oxygen atom remaining after removing one hydrogen atom from the 3' position hydroxyl group of the ribose ring of the 3' terminal nucleotide of the positive strand of the double-stranded oligonucleotide group represented by Nu. In some embodiments, the P atom shown in the above structural formula is covalently linked to the oxygen atom of the reverse deoxy-debasic nucleotide ia shown in formula (35) at the 3' terminal of the positive strand of the siRNA represented by Nu, which is connected to the ribose ring via a methylene group, thereby covalently linking it to the positive strand of the siRNA. In some embodiments, the oligonucleotide conjugates of this disclosure have the structure shown in formula (403). In some embodiments, the oligonucleotide conjugates of this disclosure may contain siRNA groups formed by removing an atom or group of atoms from siRNA; in this case, the oligonucleotide conjugates of this disclosure are also referred to as siRNA conjugates. In some embodiments, the double-stranded oligonucleotide groups contained in the oligonucleotide conjugates of this disclosure may be siRNA groups formed from, for example, the siRNAs listed in Table 1. siRNA conjugates containing these siRNA groups exhibit excellent stability and high target mRNA inhibitory activity. This disclosure relates to the preparation of oligonucleotide conjugates. Those skilled in the art can prepare the oligonucleotide conjugates described herein using various suitable methods. For example, by solid-phase synthesis, when linking nucleoside monomers one by one according to the sequence and modification scheme of the sense and antisense strands of the double-stranded oligonucleotides described herein, a delivery group can be introduced using methods already described in detail in the prior art to synthesize the oligonucleotide conjugates described herein. For example, WO2015006740A2 describes in detail various methods for preparing oligonucleotide conjugates. When the double-stranded oligonucleotide is siRNA, the oligonucleotide conjugates of this disclosure can also be obtained using methods well known to those skilled in the art. For example, WO2014025805A1 describes a method for preparing the structure shown in formula (305A), and Rajeev et al. describe a method for preparing the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail a method for preparing the oligonucleotide conjugate shown in formula (308). The contents of the above-mentioned documents are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts In another aspect, this disclosure also provides pharmaceutically acceptable salts of the single-stranded oligonucleotides, double-stranded oligonucleotides, or oligonucleotide conjugates described herein. Pharmaceutically acceptable salts are known to those skilled in the art. By forming salts, the pharmaceutically acceptable salts of the single-stranded oligonucleotides, double-stranded oligonucleotides, or oligonucleotide conjugates described herein may exhibit better solubility, bioavailability, or stability than the single-stranded oligonucleotides, double-stranded oligonucleotides, or oligonucleotide conjugates themselves. In some embodiments, in the double-stranded oligonucleotides or oligonucleotide conjugates described herein, each adjacent nucleotide is linked by a phosphodiester bond or a thiophosphate diester bond, the non-bridging oxygen or sulfur atom in the phosphodiester bond or thiophosphate diester bond being negatively charged. This phosphodiester bond or thiophosphate diester bond may be present in the form of a hydroxyl or mercapto group, and the hydrogen ion in the hydroxyl or mercapto group may be partially or completely replaced by a cation. The cation may be any cation, such as a metal cation, ammonium ion (NH4+). +The delivery group may contain one of the organic ammonium cations. Further, the delivery group may also contain a salt-forming group, such as a phosphate group. For the purpose of improving solubility and / or bioavailability, in some embodiments, the pharmaceutically acceptable salt is a partial or complete water-soluble salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate. In some embodiments, the water-soluble salt may be one or more of an amine salt, an alkali metal salt, or an alkaline earth metal salt. In some embodiments, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts; the alkali metal salt is selected from potassium or sodium salts; and the alkaline earth metal salt is selected from calcium or magnesium salts. In some embodiments, the tertiary amine salt is one or more of triethylamine salt, triisopropylamine salt, or N,N-diisopropylethylamine salt. In some embodiments, the pharmaceutically acceptable salt of the single-stranded oligonucleotide, double-stranded oligonucleotide, or oligonucleotide conjugate is a mixture of methylamine salt and ammonium salt of the single-stranded oligonucleotide, double-stranded oligonucleotide, or oligonucleotide conjugate. In some embodiments, the pharmaceutically acceptable salt is a salt or a portion of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate, wherein the salt is one or more of a methylamine salt, a triethylamine salt, a sodium salt, a calcium salt, or a magnesium salt. In some embodiments, the pharmaceutically acceptable salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate is a sodium salt or a portion of a sodium salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate. In some embodiments, the pharmaceutically acceptable salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate is a calcium salt or a portion of a calcium salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate. Pharmaceutical Composition In another aspect, this disclosure also provides a pharmaceutical composition comprising one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates and pharmaceutically acceptable salts provided in this disclosure, and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients are one or more of the various components commonly used in the art, such as solvents, protectants, osmotic pressure regulators, and one or more other pharmaceutically acceptable carriers. For example, when the pharmaceutical composition is an injection, the pharmaceutically acceptable excipient is a solvent, such as deionized water, water for injection, physiological saline, ethanol, aqueous ethanol solution, or a pH buffer. The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5. The amount of solvent used is adjusted according to the required solution concentration. Based on the oligonucleotide groups in the double-stranded oligonucleotide, the concentration of the oligonucleotide conjugate in the injection solution can be 0.01 mg / mL-20 mg / mL, 0.1 mg / mL-10 mg / mL, or 0.5 mg / mL-5 mg / mL. The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight. The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure. In some embodiments, the dosage of the formulation made from the pharmaceutical composition may be adjusted during administration depending on the route of administration. In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be administered subcutaneously, intramuscularly, or intravenously, or may be delivered via a spray to the lungs, or via a spray to other organs (such as the liver), or orally. In some embodiments, the pharmaceutical composition is administered via subcutaneous injection. Other pharmaceutically acceptable carriers may be carriers conventionally used in the field of double-stranded oligonucleotide drug delivery, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), and poly(2-aminoethyl ethylene) phosphate. One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethyl methacrylate), PDMAEMA, and their derivatives. In some embodiments, there are no particular requirements for the content of double-stranded oligonucleotides and pharmaceutically acceptable carriers in the pharmaceutical composition. In some embodiments, the weight ratio of double-stranded oligonucleotides to pharmaceutically acceptable carriers can be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50). In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid. The organic amine, cofactor lipid, and polyethylene glycol-modified lipid may be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, cofactor lipids, and polyethylene glycol-modified lipids described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety). In some embodiments, the organic amine may be a compound of formula (201) as described in Chinese patent application CN103380113A, or a pharmaceutically acceptable salt thereof: in: X 101 and X 102 Each can be independently O, S, NA, or CA, where A is hydrogen or C1-C. 20 hydrocarbon chain; Y 101 and Z 101 Each can be independently C=O, C=S, S=O, CH-OH, or SO2; R 101 R 102 R 103 R 104 R 105 R 106 and R 107 Each is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight heteroaliphatic group, substituted or unsubstituted, branched or straight acyl group, substituted or unsubstituted, branched or straight aryl group, substituted or unsubstituted, branched or straight heteroaryl group; x is an integer from 1 to 10; n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; where, if m = p = 0, then R 102 It is hydrogen; Furthermore, if at least one of n or m is 2, then R 103 The nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203): In this context, g, e, and f are each an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201). In some implementations, R 103 It is a polyamine. In other embodiments, R 103 It is a ketal. In some embodiments, R in formula (201) 101 and R 102 Each of them is independently an arbitrary substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to 20 carbon atoms, such as 8 to 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds. In some implementations, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following equations (204)-(213): In equations (204)-(213), g, e, and f are each independent integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R. 103 Possible connection points with nitrogen atoms in equation (201), wherein each H at any * position can be replaced to achieve connection with nitrogen atoms in equation (201). Those skilled in the art can obtain the compound represented by formula (201) by any reasonable method. In some embodiments, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A. In some embodiments, the organic amine is an organic amine as shown in formula (214) and / or an organic amine as shown in formula (215): The auxiliary lipid is cholesterol, cholesterol analogues and / or cholesterol derivatives; The PEGylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000. In some embodiments, the molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid in the pharmaceutical composition is (19.7-80):(19.7-80):(0.3-50), for example, (50-70):(20-40):(3-20). In some embodiments, the pharmaceutical composition particles formed from the double-stranded oligonucleotide of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm. For example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm. In some embodiments, in the pharmaceutical composition formed from the oligonucleotide or oligonucleotide conjugate of this disclosure and the aforementioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the oligonucleotide or oligonucleotide conjugate (based on oligonucleotide groups) to all lipids (e.g., organic amines, auxiliary lipids, and / or polyethylene glycol-modified lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:20, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:20, from about 1:3 to about 1:20, from about 1:4 to about 1:20, from about 1:3 ...30, from about 1:3 to about 1:30, from about 1:3 to about 1:30, from The ratio of oligonucleotides or oligonucleotide conjugates (based on oligonucleotide groups) of this disclosure to total lipids is in the range of about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10. For example, the weight ratio of the oligonucleotides or oligonucleotide conjugates (based on oligonucleotide groups) of this disclosure to total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18. In some embodiments, the components of the pharmaceutical composition may exist independently when sold, and may be in liquid form when used. In some embodiments, the pharmaceutical composition formed by the oligonucleotide or oligonucleotide conjugate provided in this disclosure and the above-described pharmaceutically acceptable carrier can be prepared according to various known methods, simply by replacing existing oligonucleotides with the oligonucleotide or oligonucleotide conjugate provided in this disclosure; in some embodiments, it can be prepared according to the following method: An organic amine, auxiliary lipid, and polyethylene glycol-modified lipid are suspended in an alcohol at the above molar ratio and mixed to obtain a lipid solution. The amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol. The oligonucleotide or oligonucleotide conjugate provided in this disclosure is dissolved in a buffer salt solution to obtain an aqueous solution of the oligonucleotide or oligonucleotide conjugate. The concentration of the buffer salt solution is 0.05-0.5M, for example, 0.1-0.2M. The pH of the buffer salt solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffer salt solution used is such that the concentration of oligonucleotide groups in the oligonucleotide or oligonucleotide conjugate does not exceed 0.6 mg / mL, for example, 0.2-0.4 mg / mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and / or potassium acetate. The lipid solution and the aqueous solution of the oligonucleotide or oligonucleotide conjugate are mixed, and the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the incubated liposome formulation. The volume ratio of the lipid solution to the aqueous solution of the oligonucleotide or oligonucleotide conjugate is 1:(2-5), for example, 1:4. The incubated liposome formulation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided in this disclosure. Its physicochemical parameters are: pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters can be: pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400 mOsm / kg. Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be achieved using various existing methods, such as ultrafiltration at 100 kDa using a tangential flow system or hollow fiber column, with the ultrafiltration exchange solution being phosphate-buffered saline (PBS) at pH 7.4. Sterilization can be achieved using various existing methods, such as filtration sterilization through a 0.22 μm filter. The use of single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions disclosed herein. This disclosure also provides for the use of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described herein in a medicament for treating and / or preventing diseases or symptoms associated with the level of target mRNA expression of a target gene. This disclosure also provides a method for treating and / or preventing diseases or symptoms associated with target mRNA levels, the method comprising administering to a subject in need an effective amount of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described in this disclosure. In another aspect, this disclosure also provides one or more of single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions for use as pharmaceuticals. Furthermore, this disclosure also provides a method for regulating the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of one or more of the single-stranded oligonucleotide, double-stranded oligonucleotide, oligonucleotide conjugate, pharmaceutically acceptable salt, and pharmaceutical composition described in this disclosure. In another aspect, this disclosure also provides a cell expressing a target mRNA, the cell further comprising one or more of the single-stranded oligonucleotide, double-stranded oligonucleotide, oligonucleotide conjugate, pharmaceutically acceptable salt, and pharmaceutical composition of this disclosure. In some embodiments, the target mRNA is selected from mRNAs transcribed from the following genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, ApoE4, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, EDNRA, EGFR, Ebola, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV , HDV, HSD, HTT, KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PCSK9, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAG E, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1. As used herein, the term "administration" refers to the delivery of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described herein to a subject via a method or route that at least partially targets one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described herein to a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration results in the delivery of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and / or pharmaceutical compositions to a specific site compared to the entire body of the subject; while systemic administration results in the delivery of one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions to substantially the entire body of the subject. The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration). Administration frequency may be once or more daily, weekly, bi-weekly, bi-weekly, monthly, or annually. The dosages of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and / or pharmaceutical compositions described in this disclosure are conventional dosages in the art, determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to die) and ED50 (the dose that elicits 50% of the maximum response intensity in a quantitative response, and the dose that elicits a positive response in 50% of the subjects in a qualitative response). Ranges of human dosages can be derived based on data obtained from cell culture analysis and animal studies. When administering one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described in this disclosure, for example, to male or female C57BL / 6J or C3H / HeNCrlVr mice, aged 6-12 weeks and weighing 18-25 g, the amount of oligonucleotide used, based on the amount of single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, and / or pharmaceutical compositions, can be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in further embodiments 0.05-20 mg / kg body weight, in even further embodiments 0.1-15 mg / kg body weight, and in still further embodiments 0.1-10 mg / kg body weight. The above-mentioned amounts are preferred when administering one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described in this disclosure. The methods provided in this disclosure for inhibiting the expression of specific genes in cells involve single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and / or oligonucleotides in pharmaceutical compositions, the amounts of which are readily determined by those skilled in the art based on the desired effect. For example, in some embodiments, the oligonucleotide conjugate is an siRNA conjugate, and the amount of siRNA in the provided siRNA conjugate is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or about 5 nM at the surface of the target cells. The amount required to achieve this local concentration will vary with various factors, including the delivery method, the delivery site, the number of cell layers between the delivery site and the target cells or tissue, and whether the delivery is local or systemic. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue. Reagent test kit This disclosure provides a kit comprising one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions disclosed herein. In some embodiments, the kit described herein may provide single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and / or pharmaceutical compositions in a single container. In some embodiments, the kit described herein may include a container providing pharmaceutically acceptable excipients. In some embodiments, the kit may also contain other components, such as stabilizers or preservatives. In some embodiments, the kit described herein may contain at least one other therapeutic agent in a container other than the container providing one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions described herein. In some embodiments, the kit may include instructions for mixing one or more of single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions with pharmaceutically acceptable carriers and / or excipients or other ingredients (if any). In the kits disclosed herein, one or more of the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions, along with pharmaceutically acceptable carriers and / or excipients, may be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the single-stranded oligonucleotides, double-stranded oligonucleotides, oligonucleotide conjugates, pharmaceutically acceptable salts, and pharmaceutical compositions, along with pharmaceutically acceptable carriers and / or excipients, are substantially pure and / or sterile. In some embodiments, sterile water may be provided in the kits disclosed herein. The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto. Without intending to be limiting, the invention is further described in detail below in the embodiments and in exemplary embodiments relating to small interfering RNA (siRNA) in pharmaceutical compositions and / or oligonucleotide conjugates of this disclosure. In this context, the double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates of this disclosure are siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, respectively. In the context of this disclosure, for ease of description, the siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates in these embodiments are also referred to as the siRNA, pharmaceutical compositions, and siRNA conjugates of this disclosure. This does not mean that the double-stranded oligonucleotides of this disclosure can only be siRNA; rather, the double-stranded oligonucleotides can be other variants disclosed herein or known to those skilled in the art, such as small activating RNA (saRNA), etc. It is contemplated that, based on the detailed description of siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, other double-stranded oligonucleotides will similarly function when used alone or in the formation of the pharmaceutical compositions and / or oligonucleotide conjugates described in this disclosure. Example Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed in accordance with the methods described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)). For the conjugates containing siRNA sequences in the following examples, unless otherwise specified, the obtained conjugates are sodium salts of conjugates in which all hydroxyl hydrogen ions in all phosphate groups are replaced by sodium ions. The following describes an example where the target mRNA is AGT mRNA. Preparation Example A1: Synthesis of siRNA conjugates A1-A4 disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates A1-A4 as shown in Table 2A were prepared, differing only in that the sense and antisense strands of the siRNA contained in the conjugates are as shown in Table 2A. For nucleic acid sequences containing the sense and antisense strand sequences of conjugates A1-A4 siRNA as shown in Table 2A, nucleoside phosphoramide monomers were linked one by one to synthesize the sense and antisense strands of the siRNA. After synthesis, the siRNA was purified by centrifugation and ultrafiltration using a 3K (MWCO) ultrafiltration tube. Conjugates 1-4 are mixtures of methylamine and ammonium salts of compounds with the structure shown in formula (403), wherein the P atom shown in formula (403) is covalently linked to the inverse debased deoxynucleotide ia shown in formula (35) at the 3' end of the positive strand of the siRNA represented by Nu, and the oxygen atom is linked to the ribose ring via a methylene group, thereby covalently linking to the positive strand of the siRNA. Furthermore, the siRNA contained in the siRNA conjugate has the siRNA sequence corresponding to conjugates 1-4 in Table 2A. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)). Molecular weight was then determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The theoretical molecular weight of the positive strand of conjugate 1 was 7324.22, and the measured molecular weight was 7323.60; the theoretical molecular weight of the antisense strand was 7194.91, and the measured molecular weight was 7194.27. For conjugate 2, the theoretical molecular weight of the positive strand was 7292.11, and the measured molecular weight was 7291.54; the theoretical molecular weight of the antisense strand was 7194.91, and the measured molecular weight was 7194.37. The theoretical molecular weight of the positive strand of conjugate 3 was 735... The measured molecular weight of the conjugate 1-4 was 7355.83, while the theoretical molecular weight of the antisense strand was 7194.91 and the measured molecular weight was 7194.26. The theoretical molecular weight of the sense strand of conjugate 4 was 7324.23 and the measured molecular weight was 7323.58, while the theoretical molecular weight of the antisense strand was 7141.96 and the measured molecular weight was 7141.23. The measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugates 1-4 contained the target double-stranded nucleic acid sequence. Table 2A siRNA sequences in siRNA conjugates In this context, uppercase letters C, G, U, A, and T represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide represented by the uppercase letter to the left of f is 2'-fluoro modified; lowercase letter s indicates that the linking group between the two uppercase letters to the left and right of s is a thiophosphate group; lowercase letter d indicates that the nucleotide represented by the uppercase letter to the right of d is a deoxynucleotide; the letter combination in parentheses (moe) indicates that the nucleotide represented by the uppercase letter to the left of moe is 2'-methoxyethyl modified; the letter combination VP indicates that the nucleotide represented by the uppercase letter to the right of VP is vinyl phosphate modified; and ia indicates a reverse debased deoxynucleotide. Synthesis of the reference conjugate in Comparative Preparation Example A1 Following the same method as in Preparation Example A1, the reference conjugate A1, numbered in Table 2A, was synthesized by solid-phase synthesis. Reference conjugate A1 is a conjugate having a sense chain very similar to the conjugate sequence of this disclosure, but not the antisense chain of this disclosure. Experimental Example 1: In vitro inhibitory activity of the disclosed conjugate. This experimental example investigated the inhibitory activity of conjugate A4 and reference conjugate A1 on the AGT gene in Huh-7 cells in vitro. Reference conjugate A1 is a conjugate having substantially the same sense strand as conjugate A4 of this disclosure, but without the antisense strand of this disclosure. The specific steps are as follows: [1] Cell culture Huh-7 human hepatocellular carcinoma cells (Cells-0120) were cultured at 37°C in an incubator containing 5% CO2 / 95% air using DMEM complete medium (purchased from Zhongke Maichen (Beijing) Technology Co., Ltd., catalog number: CM15019) supplemented with 10% fetal bovine serum (FBS, purchased from GIBCO, catalog number 10099-141C). Huh-7 cells were fed at a concentration of 0.5 × 10⁻⁶. 5 Cells were seeded into 24-well plates at 0.5 mL of cell culture per well. After culturing for 16 h, the culture medium in the wells was aspirated, and 0.5 mL of Opti-MEM medium (purchased from GIBCO, catalog number 31985-070) was added to each well. The cells were then cultured for another 1.5 h. [2] Transfection For each siRNA conjugate to be tested, a 20 μM solution of each siRNA conjugate was prepared using PBS. Starting from this solution, the solutions were sequentially diluted 4-fold to obtain 11 different concentrations of siRNA conjugate working solutions: 5 μM, 1.25 μM, 0.312 μM, 0.0781 μM, 0.0195 μM, 0.00488 μM, 0.00122 μM, 0.000305 μM, 0.0000763 μM, 0.0000191 μM, and 0.00000477 μM (based on the amount of siRNA in the siRNA conjugate). The siRNA conjugates used were conjugate A4 and reference conjugate A1. Prepare A3A solutions, each containing 3 μL of siRNA conjugate working solution and 97 μL of cell maintenance culture medium, to make 11 different concentrations of A3A1-A3A11 solutions. Prepare A3B solution, each serving containing 2 μL of Lipofectamine. TM 2000 (Invitrogen) and 98 μL Opti-MEM medium. For conjugate A4, one part of solution A3A and one part of solution A3B were mixed to obtain transfection complex A3X1. In culture wells (all containing Huh-7 cells and 1 mL of Opti-MEM medium, hereinafter the same), transfection complex A3X1 for each siRNA conjugate was added and mixed thoroughly at a volume of 100 μL / well, resulting in transfection mixtures with concentrations of 50 nM (based on siRNA), 12.5 nM, 3.13 nM, 0.781 nM, 0.195 nM, 0.0488 nM, 0.0122 nM, 0.00305 nM, 0.000763 nM, 0.000191 nM, and 0.0000477 nM, respectively. Each siRNA conjugate transfection complex A3X1 was transfected into two culture wells to obtain a transfection mixture containing siRNA conjugate A4, denoted as test group A2X1-A2X11. For reference conjugate A1, one part of solution A3A and one part of solution A3B were mixed to obtain transfection complex AY1. In each culture well (all containing Huh-7 cells and 1 mL of Opti-MEM medium, hereinafter the same), 100 μL of transfection complex AY1 for each reference conjugate A1 was added and mixed thoroughly, yielding transfection mixtures with concentrations of 50 nM (based on siRNA), 12.5 nM, 3.13 nM, 0.781 nM, 0.195 nM, 0.0488 nM, 0.0122 nM, 0.00305 nM, 0.000763 nM, 0.000191 nM, and 0.0000477 nM, respectively. Each reference conjugate A1 was transfected into two culture wells with transfection complex AY1 to obtain a transfection mixture containing reference conjugate A1, denoted as test group A2Y1-A2Y11. Mix one portion of A3B solution with 100 μL of cell maintenance medium to obtain blank transfection mixture AB. Add 100 μL of blank transfection mixture AB to another culture well to obtain a transfection mixture without siRNA conjugate, which is designated as blank control group A2B. The test groups A2X1-A2X11, A2Y1-A2Y11 and the blank control group A2B were placed in an incubator with an air atmosphere containing 5% CO2 and cultured at 37°C for 24 hours. [3] Detection Total RNA was extracted from cells in each well using TRIZOL reagent (purchased from Sigma, catalog number: T9424-200 mL). For each well of cells, 1 μg of total RNA was taken and reverse transcribed into cDNA using the Reverse Transcription System (Promega, catalog number A3500) according to the manufacturer's instructions. The reagents provided included Oligo(dT)15 as the primer. A 20 μL reverse transcription reaction system was prepared according to the kit's instructions. The reverse transcription conditions were as follows: for each reaction system, the system was incubated at 70°C for 10 min, then at 42°C for 30 min, and finally at 95°C for 5 min. After the reaction, 80 μL of RNase-free water was added to the system to obtain the cDNA-containing solution. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided in the SYBR Select Master Mix kit (Applied Biosystems, catalog number 4472908). The PCR primer sequences for amplifying the target gene AGT and the internal reference gene GAPDH are shown in Table 3A, with a final concentration of 10 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument (Thermo Fisher), and a three-step amplification method was used. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product AW1 containing amplified target gene AGT and internal reference gene GAPDH. The product AW1 was then subjected to a gradient temperature increase to 95℃ for 15s, 60℃ for 1min, and then to 95℃ with fluorescence signals collected every 0.3℃. After 15s at 95℃, the melting curves of the target gene and the internal reference gene GAPDH in the product AW1 were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene AGT and the internal reference gene GAPDH. Table 3A Primer Information The relative quantitative calculation of the expression level of the target gene AGT mRNA in each test group was performed using the Ct(ΔΔCt) method. The calculation method is as follows: ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the two culture wells in the control group. Therefore, each culture well in both the test and control groups corresponds to a ΔCt value. The expression level of AGT mRNA in the test group was normalized based on the average value of the control group, with the average value of AGT mRNA expression in the control group defined as 100%. The relative expression level of AGT mRNA in the test group = 2 - ΔΔCt(test group) × 100% AGT mRNA inhibition rate in the test group = (1 - relative expression level of AGT mRNA in the test group) × 100% Based on the relative expression levels of human AGT mRNA in HEK293A cells after transfection with different concentrations of the target siRNA, the nonlinear regression analysis function of Graphpad Prism 8.0 software was used to fit the log (inhibitor) vs. response-variable slope (four parameters) dose-response curve, and the values ​​of Bot, Top, and Hillslope parameters were obtained. Based on the function corresponding to the fitted dose-response curve, calculate the IC50 of the target sequence of the siRNA to be tested. 50 The value, the function is as follows, In the formula: Y is the ratio R, which represents the relative residual activity of Renilla. X represents the logarithm of the siRNA transfection concentration. Bot is the Y value at the bottom of the steady-state period. Top is the Y value at the peak of the steady-state period. X' is the X value when Y is halfway between the bottom and the top, while HillSlope is the slope of the curve at X'. Based on the dose-response curve and the corresponding function, determine X when Y = 50%. 50 The IC50 value of each siRNA was calculated. 50 Value = 10^X 50 (nM). As a result, the IC50 values ​​of the siRNA conjugate A4 of this disclosure and the reference conjugate A1, which has a substantially identical sequence but does not have the antisense strand modification scheme of this disclosure, are significantly higher. 50 The values ​​were 0.021 nM and 0.027 nM, respectively, indicating that the siRNA conjugate disclosed in this invention has a smaller IC50 value. 50 The value indicates that the siRNA conjugate disclosed herein has a higher AGT mRNA inhibition efficiency compared to the reference conjugate. Experimental Example A2: In vitro inhibitory activity of the disclosed conjugate. This experiment investigated the activity of the conjugate A1-A3 in primary hepatocytes of hAGT mice. [1] Cell culture Primary hepatocytes were obtained from fresh liver tissue of human AGT transgenic mice (B6.Cg-Tg(hAGT)2041Sig / J, grade: SPF, age: 6-8 weeks, purchased from Jackson Lab). The density of the primary hepatocytes was adjusted to 2x10⁻⁶ cells in Opti-MEM (1X) medium (GIBCO, catalog number 31985-70). 5To obtain a suspension of primary mouse liver cells, the cells were measured at 1 x 10⁶ cells / mL. 5 Cells were seeded into 12-well plates with 1 mL of cell culture per well. After culturing for 16 h, the culture medium in the wells was aspirated, and 1 mL of Opti-MEM medium (GIBCO) was added to each well. The cells were then cultured for another 1.5 h. [2] Transfection Working solutions of conjugates A1-A3 at concentrations of 0.8 μM and 4 μM were prepared using PBS buffer. 0.3 μL of each concentration of working solution and PBS were added to each culture well (each containing primary mouse liver cells and Opti-MEM as described above). Two wells of each working solution were added separately. Then, 99.7 μL of DMEM was added to each well, resulting in transfection mixtures with final concentrations of 0.2 nM and 1 nM, respectively. The group with 0.2 nM working solution was designated as test group A1, the group with 1 nM working solution as test group A2, and the group with PBS as the blank control group. The 12-well plates were incubated in a CO2 incubator for 24 hours. [3] Detection Total RNA was extracted from cells in each well using TRIZOL reagent (purchased from Sigma, catalog number: T9424-200 mL). For each well of cells, 1 μg of total RNA was taken and reverse transcribed into cDNA using the Reverse Transcription System (Promega, catalog number: A3500) according to its instructions, yielding a solution containing cDNA, of which Oligo(dT) was selected. 15 As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions to reverse transcribe total RNA from cells in each well. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 70°C for 10 min, then at 42°C for 30 min, and finally at 95°C for 5 min. After the reaction was completed, 80 μL of RNase-free water was added to the reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided in the SYBR Select Master Mix kit (Applied Biosystems, catalog number 4472908). The PCR primer sequences for amplifying the target gene hAGT and the internal reference gene mGAPDH are shown in Table 4A, with a final concentration of 10 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument (Thermo Fisher), and a three-step amplification method was used. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product AW1 containing amplified target gene AGT and internal reference gene GAPDH. The product AW1 was then subjected to a gradient temperature increase to 95℃ for 15s, 60℃ for 1min, and then to 95℃ with fluorescence signals collected every 0.3℃. After 15s at 95℃, the melting curves of the target gene and the internal reference gene GAPDH in the product AW1 were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene AGT and the internal reference gene GAPDH. Table 4A Primer Sequence Information The relative quantification of the target gene AGT in each test group was performed using the comparison Ct(ΔΔCt) method, as follows: ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the two culture wells in the blank control group. Therefore, each culture well in both the test group and the blank control group corresponds to a ΔCt value. The mRNA expression levels in the test group were normalized using the mean value of the blank control group as a baseline, with the mean mRNA expression level of the blank control group defined as 100%. The relative mRNA expression level in the test group = 2 - ΔΔCt(test group) × 100% The mRNA inhibition rate of the test group = (1 - the relative expression level of mRNA in the test group) × 100%. The experimental results are shown in Table 5A below. Table 5A shows the inhibition rate of AGT mRNA in primary mouse liver cells after transfection with 0.2 nM and 1 nM concentrations of the conjugates A1-A3 disclosed herein. Table 5A shows the inhibition rate of AGT mRNA in primary mouse liver cells. As shown in Table 5A, in primary liver cells of hAGT transgenic mice, the conjugate provided in this disclosure can inhibit the target gene AGT mRNA by more than 72% at a concentration of 0.2 nM, and can reach 98.5% or even 99% at a concentration of 1 nM, demonstrating excellent in vitro inhibitory activity. Experimental Example: Activity Assay of A3 siRNA Conjugate in Mice (in vivo) This experimental example investigated the use of the same concentrations of the disclosed conjugate A4 and reference conjugate A2 dissolved in PBS to prepare solutions with a concentration of 0.6 mg / mL (based on the amount of siRNA). Reference conjugate A2 is an siRNA conjugate, designated AD85481 in Table 5 of the specification of US Patent Publication US11,015,201B2, obtained by the method described in Example 1 of the patent publication. This siRNA conjugate has the structure shown in formula (305), wherein the conjugation group is linked to the 3' position of the ribose of the 3' terminal nucleotide of the sense strand of the siRNA represented by Nu. Furthermore, the siRNA contained in this siRNA conjugate has the sense and antisense strand sequences shown in Table 6A below: Table 6A siRNA sequence of reference conjugate A2 Wherein, T(gn) represents GNA with base T, having the structure shown in formula (27A). Twenty mice (human AGT transgenic mice (B6.Cg-Tg(hAGT)2041Sig / J); grade: SPF; age: 6-8 weeks, purchased from Jackson Lab) were randomly divided into three groups of 6 mice each, with half males and half females in each group: test group A1, control group A2, and blank group. Mice in test group A1 and control group A2 were administered 0.6 mg / mL of conjugate A4 solution or reference conjugate A2 solution via subcutaneous injection in the abdomen. Mice in the blank group were administered PBS solution. The weight of mice in each group was recorded before administration and the dosage was based on body weight, with a single dose of 5 mL / kg of mouse body weight. Using the drug administration time point as day 1, blood samples were collected from mice on days 8, 15, 22, 29, and 43 post-administration. The expression levels of hAGT protein in the serum of the test and control groups were detected using the Abcam hAGT ELISA kit (ab108823) (purchased from Xi'an Zhuangzhi Biotechnology Co., Ltd.) according to the instructions. The decrease rate of hAGT protein expression in mouse serum was calculated by normalization with the expression level of hAGT protein in the blank control group at the corresponding time point as 100%. The results are shown in Table 7A. Table 7A shows the inhibition rate of hAGT protein in mice. As shown in Table 7A, during the 43-day experimental period, mice treated with conjugate A4 exhibited a higher inhibition rate of hAGT protein expression than mice treated with reference conjugate A2. This high inhibition rate was maintained throughout the entire experimental period, remaining close to 80% on day 43. Considering that reference conjugate A2 is the most effective siRNA conjugate in the prior art (US11,015,201B2) and is protected by patent, the above results indicate that the conjugates containing the antisense strand of this disclosure can inhibit AGT mRNA expression for a prolonged period, thereby reducing serum hAGT protein expression, and exhibiting significantly higher inhibition efficiency than one of the most preferred compounds in the art. Experimental Example: Activity test of A4 siRNA conjugate in mice (in vivo) This experimental example investigated the activity of different concentrations of the disclosed conjugate A4 in mice. The experiment was conducted according to the method in Experiment Example A3, with the only difference being that the siRNA conjugate used was conjugate A4. Two concentrations of solutions, 0.6 mg / mL and 1.8 mg / mL, were prepared and administered to mice in different test groups, while the blank group was given PBS solution. The single dose was 5 mL / kg mouse body weight, meaning the dosage for different test groups was 3 mg / kg or 9 mg / kg mouse body weight, respectively. Blood samples were collected from mice at the time of administration and on days 8, 15, 22, 36, and 43 post-administration for analysis. The results are shown in Table 8A. Table 8A shows the inhibition rate of hAGT protein in mice. As shown in Table 8A, during the 43-day experimental period, mice treated with the disclosed conjugate A4 exhibited a high inhibition rate of hAGT protein expression, with the highest inhibition rate reaching 96.5%. Furthermore, this high inhibition rate was maintained throughout the entire experimental period, reaching a maximum of 90.3% on day 43. This indicates that the disclosed conjugate can reduce serum hAGT protein expression for a prolonged period, exhibiting a dose-dependent effect. The disclosed conjugate demonstrates significant and long-lasting pharmaceutical activity in the preparation of drugs for the treatment and / or prevention of diseases or symptoms related to AGT mRNA expression, showing excellent development potential. Experimental Example A5: Activity test of the disclosed conjugate in mice (in vivo) The conjugates A1 and A3 prepared above were dissolved in PBS to obtain solutions with a concentration of 0.2 mg / mL (based on the amount of siRNA). Human AGT transgenic mice (B6.Cg-Tg(hAGT)2041Sig / J) (grade: SPF; age: 6-8 weeks, purchased from Jackson Lab) were randomly divided into 4 groups of 6 mice each, with half males and half females in each group: test group A1, test group A2, and blank control group. Mice in test group A1 were administered 0.2 mg / mL of conjugate A1 solution via subcutaneous abdominal injection; mice in test group A2 were administered 0.2 mg / mL of conjugate A3 solution; and mice in the blank control group were administered PBS solution. Mice in all groups were weighed and their body weight was recorded before administration. The dosage was 5 mL / kg of mouse body weight per administration. Using the drug administration time point as day 1, blood samples were collected from mice on days 15, 43, and 71 post-administration. The expression levels of hAGT protein in the serum of the test group and the blank control group were detected using the Abcam hAGT ELISA kit (ab108823) (purchased from Xi'an Zhuangzhi Biotechnology Co., Ltd.) according to the instructions. The decrease rate of hAGT protein expression in mouse serum was calculated by normalization with the expression level of hAGT protein in the blank control group at the corresponding time point as 100%. The results are shown in Table 9A. Table 9A shows the inhibition rate of hAGT protein in mice. As shown in Table 9A, during the 71-day experimental period, mice given the conjugate of this disclosure exhibited a high inhibition rate of hAGT protein expression. On day 15, the inhibition rate of hAGT protein expression was above 90%, and on day 71, the inhibition rate of hAGT protein expression was still close to or reached 70%. This indicates that the conjugate of this disclosure containing the antisense strand of this disclosure can inhibit the expression of AGT mRNA for a long time, thereby reducing the level of hAGT protein expression in serum. The following describes an example where the target mRNA is APOE4 mRNA. Preparation Example B1: Synthesis of siRNA conjugates B1-B4 disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates B1-B4 as shown in Table 2B were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in the siRNA conjugates are as shown in Table 2B. For nucleic acid sequences containing the sense and antisense strand sequences of the siRNA in conjugates B1-B4 as shown in Table 2B, nucleoside phosphoramide monomers were linked one by one to synthesize the sense and antisense strands of the siRNA. After synthesis, the siRNA was purified by centrifugation and ultrafiltration using a 3K (MWCO) ultrafiltration tube. Conjugates B1-B4 are each independently a mixture of methylamine and ammonium salts of compounds having the structure shown in formula (403), wherein the conjugate group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the positive strand of the siRNA represented by Nu. Furthermore, the siRNA contained in the siRNA conjugate has the siRNA sequences corresponding to conjugates B1-B4 in Table 2B. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)). Molecular weight was then determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The measured values ​​were consistent with the theoretical values, indicating that the synthesized conjugates B1-B4 contained the designed double-stranded nucleic acid sequence. For example, for conjugate B3, the theoretical value for the sense strand was 7394.462; the measured value for the sense strand was 7393.50; the theoretical value for the antisense strand was 7010.744; and the measured value for the antisense strand was 7009.92. Table 2B siRNA sequences in siRNA conjugates In this context, uppercase letters C, G, U, A, and T represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide adjacent to the left of f is 2'-fluoro modified; (moe) indicates that the nucleotide adjacent to its left is ribose 2'-O-methoxyethyl modified; lowercase letter s indicates that the linking group between the two nucleotides to its left and right is a thiophosphate group; lowercase letter d indicates that the nucleotide to its right is a deoxynucleotide; and ia indicates a reverse debased deoxynucleotide. In vitro inhibitory activity of the B1 conjugate B1-B4 (Example B1) This experiment investigated the inhibitory activity of conjugate B1-B4 and reference conjugate NC on APOE4 mRNA in HepG2 human hepatocellular carcinoma cells in vitro. The specific steps are as follows: [1] Cell culture HepG2 human liver cancer cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM medium (M&C GENE) supplemented with 10% fetal bovine serum (FBS, RMBIO) at 37°C in an incubator containing 5% CO2 / 95% air. HepG2 cells were loaded at 1.5 x 10⁻⁶ cm⁻¹. 5 Cells were seeded into 12-well plates with 1 mL of cell culture per well. After culturing for 16 h, the culture medium in the wells was aspirated, and 1 mL of Opti-MEM medium (GIBCO) was added to each well. The cells were then cultured for another 1.5 h. [2] Transfection For each siRNA conjugate to be tested, a 20 μM (based on the amount of siRNA in the conjugate) working solution was prepared using PBS. The siRNA conjugates used were conjugates B1-B4 and reference conjugate NC. For each siRNA conjugate to be tested, a B1A solution was prepared, with each B1A solution containing 3 μL of siRNA conjugate working solution and 97 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, prepare a B1B solution, with each B1B solution containing 2 μL of Lipofectamine. TM RNAiMAX transfection reagent (Invitrogen, catalog number: 13778150) and 98 μL Opti-MEM medium (GIBCO). For each siRNA conjugate to be tested, one portion of solution B1A and one portion of solution B1B were mixed and incubated at room temperature for 20 min to obtain transfection complex BX1. In each culture well (each containing HepG2 cells and 1 mL of Opti-MEM medium, hereinafter the same), 200 μL of transfection complex BX1 for each siRNA conjugate was added and mixed thoroughly to obtain a transfection mixture with a concentration of 50 nM (based on the amount of siRNA). Each siRNA conjugate transfection complex BX1 was transfected into two culture wells to obtain transfection mixtures containing the siRNA conjugate, designated as test group BX1-BX4 and negative control group BX1', respectively. One portion of B1B solution was mixed with 100 μL of cell maintenance medium to obtain blank transfection mixture B. Blank transfection mixture B was added to two other wells at a volume of 200 μL / well to obtain transfection mixtures without siRNA conjugates, which were designated as the blank control group. The test groups BX1-BX4, the negative control group BX1', and the blank control group B were placed in an incubator with an air atmosphere containing 5% CO2 and cultured at 37°C for 24 hours. [3] Detection Total RNA was extracted from cells in each well using the UNIQ-10 column-based total RNA extraction kit (purchased from Sangon Biotech, model: Sangon Biotech) according to the instructions. For each well of cells, 1 μg of total RNA was collected and used to reverse transcribe the RNA using the Goldenstar reverse transcription kit. TM The reagents provided in the RT6 cDNA Synthesis Kit (purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd., catalog number TSK301M) included Goldenstar. TM Oligo(dT) 15 As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions, and the total RNA from each well was reverse transcribed. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 70°C for 10 min, then at 42°C for 30 min, and finally at 95°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, take 5 μL of the above solution containing cDNA as a template, and use... 20 μL of qPCR reaction system was prepared using the reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., catalog number E096-01B). The PCR primer sequences for amplifying the target gene APOE4 and the internal reference gene GAPDH are shown in Table 3B, with a final concentration of 10 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument (purchased from Thermo Fisher) and amplified using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product BW1 containing amplified target gene APOE4 and internal reference gene GAPDH. Product BW1 was then subjected to a gradient temperature increase to 95℃ for 15s, 60℃ for 1min, and then to 95℃ with fluorescence signals collected every 0.3℃. After 15s at 95℃, the melting curves of the target gene and the internal reference gene GAPDH in product BW1 were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene APOE4 and the internal reference gene GAPDH. Table 3B Primer Information The relative quantitative calculation of the expression level of the target gene APOE4 mRNA in each test group was performed using the Ct(ΔΔCt) method. The calculation method is as follows: ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the two culture wells in the control group. Therefore, each culture well in both the test and control groups corresponds to a ΔCt value. The expression level of APOE4 mRNA in the test group was normalized based on the mean of the control group, and the mean of the APOE4 mRNA expression level in the control group was defined as 100%. The relative expression level of APOE4 mRNA in the test group = 2 - ΔΔCt(test group) × 100% APOE4 mRNA inhibition rate in the test group = (1 - relative expression level of APOE4 mRNA in the test group) × 100% The experimental results are shown in Table 4B below. Table 4B Inhibitory activity of siRNA conjugates in HepG2 cells Table 4B shows the relative inhibition rate of APOE4 mRNA in HepG2 human hepatocellular carcinoma cells in vitro after transfection with 50 nM of the disclosed siRNA conjugates. The results indicate that all tested siRNA conjugates exhibited good inhibitory effects at a concentration of 50 nM in HepG2 human hepatocellular carcinoma cells in vitro. Conjugates B1-B4 showed inhibition rates of over 85% on APOE4 mRNA, while conjugates B2-B4 achieved inhibition rates exceeding 90%, even reaching 94%. This result demonstrates the good inhibitory effect of the disclosed siRNA conjugates on APOE4 mRNA expression. The following describes examples where the target mRNA is LPA mRNA. Preparation Examples C1-C30: Synthesis of the siRNA conjugates provided in this disclosure. Following the preparation method described in Example 13 of CN110959011A, conjugates C1-C30 as shown in Table 2C were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in each siRNA conjugate are as shown in Table 2C. For nucleic acid sequences containing the sense and antisense strand sequences of siRNAs numbered C1-C30 in Table 2C, nucleoside phosphoramide monomers were ligated one by one to synthesize the sense and antisense strands of the siRNA. After synthesis, the siRNAs were purified by centrifugation and ultrafiltration using a 3K (MWCO) ultrafiltration tube. Conjugates C1-C30 are each independently a mixture of methylamine and ammonium salts of compounds having the structure shown in formula (403), wherein the P atom shown in formula (403) is covalently linked to the inverted deoxy-debasic nucleotide (ia) at the 3' end of the positive strand of the siRNA represented by Nu, as shown in formula (35), and an oxygen atom is linked to the ribose ring via a methylene group, thereby covalently linking to the positive strand of the siRNA. Furthermore, the siRNA contained in this siRNA conjugate has the siRNA sequences corresponding to conjugates C1-C30 in Table 2C. Table 2C shows the siRNA sequences in the siRNA conjugates. In this context, uppercase letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide represented by the uppercase letter to the left of f is 2'-fluoro modified; lowercase letter s indicates that the nucleotides represented by the two uppercase letters to its left and right are linked by a thiophosphate group; lowercase letter d indicates that the nucleotide represented by the uppercase letter to its right is a deoxynucleotide; the letter combination moe indicates that the nucleotide represented by the uppercase letter to its right is 2'-O-methoxyethyl modified; the letter combination VP indicates that the nucleotide represented by the uppercase letter to its right is 5'-vinyl phosphate (VP) modified; and ia indicates a reverse debase deoxynucleotide. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)). Molecular weight was then determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). The molecular weight results for some conjugates are shown in Table 3C. The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates are the designed double-stranded nucleic acid sequences. Table 3 shows the molecular weight of the C-conjugates. Comparative preparation example: Synthesis of C1-C2 reference conjugate C1-C2 Following the same method as in Preparation Example C1, reference conjugates C1-C2 in Table 2C were prepared by solid-phase synthesis. Each of the reference conjugates C1-C2 is independently a mixture of methylamine and ammonium salts of a compound having the structure shown in formula (403), wherein the conjugation group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the positive strand of the siRNA represented by Nu. Furthermore, the siRNA contained in the reference conjugates C1-C2 has the siRNA sequences corresponding to those in Table 2C, all of which are negative control sequences dissimilar to known mRNAs. Preparation Examples C31-C38: Synthesis of the siRNA conjugates C31-C38 disclosed herein Conjugates 31-38 were prepared by solid-phase synthesis using the same method as in Preparation Example C1. The difference was that after synthesis, conjugates C31-C38 were first purified by self-packed column ion exchange using strong anion exchange packing material, and then purified by desalting using a HiPrep 26 / 10 desalting pre-packed column. Conjugates C31-C38 are sodium salts of compounds having the structure shown in formula (403). Preparation Examples C39-C40: Synthesis of siRNA conjugates C1A-C2A disclosed herein Following the same method as in Preparation Example 1, conjugates C1A and C2A were prepared by solid-phase synthesis. Conjugate C1A and C1A have the same siRNA positive and negative strand sequences as shown in Table 2C, and their structures are both as shown in formula (403). Conjugate C2A and C2A have the same siRNA positive and negative strand sequences as shown in Table 2C, and their structures are both as shown in formula (403). The difference lies in that, after synthesis, conjugates 1A and 2A were first purified by self-packed column ionization using strong anion exchange packing material, and then purified by desalting using a HiPrep 26 / 10 desalting pre-packed column. Both conjugates C1A and C2A are sodium salts of compounds having the structure shown in formula (403). Experimental Example C1: In vitro inhibitory activity of the disclosed conjugate. Experimental Example C1-1: Inhibitory Activity of siRNA Conjugate Transfected in Primary Mouse Hepatocytes This experimental example determined the effect of Lipofectamine. TM The activity of siRNA conjugates transfected with 2000 in primary liver cells of transgenic mice expressing human transgenic LPA. Primary hepatocytes from mice were extracted from fresh liver tissue of pAlb-hLPA-Tg mice (strain: LPA pAlb-hLPA-Tg, grade: SPF; sex: male, age: 12-14 weeks, weight: 25±5g, purchased from Shanghai Southern Model Biotechnology Co., Ltd.). The density of the isolated primary hepatocytes was adjusted to 1×10⁻⁶ using Opti-MEM (1X) (GIBCO, Cat. No. 31985-070). 5 Cells / mL. The cell suspension was prepared at 1×10⁻⁶. 5 Cells were seeded in 12-well plates, with 3 replicates per sample and 1.0 mL of cell culture per well. For each siRNA conjugate to be tested, a 20 μM stock solution (based on the amount of siRNA in the conjugate) was prepared using PBS. The siRNA conjugates used were conjugates C3-C11, C13-C14, C17-C20, C25, C28, and reference conjugates C1-C2. Preparation of C1A solution: The above conjugate stock solution was diluted with Opti-MEM medium to a working solution of 0.6 μM, denoted as C1A solution, with each C1A solution being 100 μL. Preparation of C1B solution: Each serving contains 2 μL of Lipofectamine TM The solutions of 2000 and 98.0 μL of Opti-MEM culture medium are denoted as C1B solutions. Mix one part C1A solution with one part C1B solution and incubate at room temperature for 20 min to obtain a transfection mixture, denoted as C1X1. Mix one part C1B solution with 100 μL of Opti-MEM culture medium and incubate at room temperature for 20 min to obtain a blank transfection mixture, denoted as C1X'. For each siRNA conjugate, transfection mixture C1X1 was added to three wells containing primary mouse liver cells and Opti-MEM, and mixed thoroughly at a rate of 200 μL per well to obtain a transfection mixture with a concentration of 50 nM (based on the amount of siRNA), designated as the test group. Transfection complex C1X' was added to three other wells at a rate of 200 μL per well to obtain a transfection mixture without the siRNA conjugate, serving as the blank control group. After transfecting the siRNA conjugate mixture and the transfection complex without the siRNA conjugate into separate wells for 4 h, 1000 μL of complete culture medium was added to each well. The 12-well plate was then incubated at 37 °C for 24 h in a CO2 incubator. After culturing, total RNA was extracted from the cells using TRIZOL (purchased from SIGMA, Cat. No. T9424) according to the method described in the instructions. For each well of cells, 1 μg of total RNA was taken and transduced using the Reverse Transcription System kit (Promega, catalog number: A3500), with Goldenstar selected as the primary RNA source. TM Oligo(dT) 17As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions, and the total RNA from each well was reverse transcribed. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 70°C for 10 min, then at 42°C for 30 min, and finally at 95°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided in the TaqMan Fast Advanced Master MIX(2x) kit (Thermo Fisher, catalog number: 4444556). The PCR primer sequences for amplifying the target gene hLPA and the internal reference gene mGAPDH are shown in Table 4C, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method. The amplification program was: 50℃ pre-denaturation for 20 s, 95℃ pre-denaturation for 20 s, then 95℃ denaturation for 1 s, and 60℃ annealing / extension for 20 s. This denaturation and annealing / extension process was repeated 40 times to obtain the product CW containing amplified target gene LPA and internal reference gene mGAPDH. The Ct values ​​of the target gene hLPA and the internal reference gene β-actin were also obtained. The relative quantification of the target gene LPA in each test group was performed using the comparison Ct(ΔΔCt) method, as follows: ΔΔCt=(Ct 测试组目标基因 -Ct 测试组内参基因 )-(Ct 空白对照组目标基因 -Ct 空白对照组内参基因 mean Relative expression level = 2 -ΔΔCt ×100% mRNA inhibition rate = (blank control group 2) -ΔΔCt Mean - Test Group 2 -ΔΔCt ) / Blank control group 2 -ΔΔCt Mean × 100% The results are shown in Table 5C. Table 4C: Primer Sequence Information Table 5 shows the inhibition rate of LPA mRNA by C siRNA conjugates in primary mouse liver cells. As can be seen from Table 5C, the conjugates disclosed herein maintain a high inhibition rate in primary mouse liver cells. Among the tested conjugates, the inhibition rate is at least 57.6%, and can even reach 96.3%. Experimental Example: Inhibitory Activity of C2 siRNA Conjugate in Primary Monkey Liver Cells In this embodiment, Lipofectamine is used. TM The conjugates prepared in the preparation example were transfected into primary monkey liver cells using the 2000 transfection reagent. The final concentrations of each conjugate were 50 nM and 1 nM (based on siRNA levels). Each conjugate was transfected into two wells of cells, with untreated cells serving as a blank control. Twenty-four hours after transfection, cellular RNA was harvested, and the expression level of the target gene LPA mRNA in primary monkey liver cells transfected with each conjugate was detected using real-time quantitative PCR. The specific procedures are as follows: Primary monkey liver cells were purchased from Myosun (Shanghai) Biotechnology Co., Ltd. (Catalog No.: CCH-100CYS-PQ). Cell resuscitation was performed according to the provided culture medium and reagents, following the manufacturer's instructions. 120 mL of resuscitation medium (Catalog No. HTS-R-120) was preheated to 37°C. Cryopreservation tubes containing cells were placed in a 37°C water bath and gently agitated until some ice crystals thawed. The cell suspension was then poured into the preheated resuscitation medium, rinsed, and mixed thoroughly. The cell suspension was centrifuged at 800 rpm for 5 min at room temperature, the supernatant was discarded, and the cells were resuspended in 20 mL of plating medium (Catalog No. HPM-R-120). After cell counting, the cells were plated. Dilute cells to 1×10⁻⁶ using plating medium. 5 Cells / mL (live cell count), add 1 mL of cell culture to each well of a 12-well plate, shake well, and incubate at 37°C in an incubator containing 5% CO2 / 95% air. After overnight cell attachment, discard the plating medium, add 1 mL of maintenance medium (catalog number: HMM-R-120) to each well, and incubate at 37°C in an incubator containing 5% CO2 / 95% air until transfection. For each conjugate to be tested, prepare a 10 μM (based on the amount of siRNA in the conjugate) stock solution using 1×PBS. The above conjugate stock solution was prepared by diluting it with Opti-MEM medium to prepare 0.6 μM and 0.012 μM working solutions of the conjugate, denoted as C2A solutions, with each C2A solution having a volume of 100 μL. Each serving contains 2 μL of Lipofectamine TM A solution of 2000 μL (purchased from Thermo Fisher Scientific, catalog number: 11668-019) and 98 μL of Opti-MEM medium is designated as solution C2B. After mixing, it is incubated at room temperature for 5 min. For each conjugate to be tested, one portion of C2B solution and one portion of C2A solution were mixed and incubated at room temperature for 20 min to obtain a transfection complex, denoted as C2X. Two portions of each transfection complex C2X were prepared for transfecting two wells of cells. In the culture wells (all containing primary monkey liver cells and 1 mL of maintenance medium, the same below), 200 μL of the transfection complex C2X for each conjugate was added and mixed thoroughly to obtain transfection mixtures with final concentrations of 50 nM and 1 nM (based on the amount of siRNA), which were denoted as the test group. Take 200 μL of Opti-MEM medium as the blank transfection mixture, denoted as C2X'. Prepare two portions of each C2X' to add to two wells of cells. Add one portion of the blank transfection mixture C2X' to each of the other two culture wells, at a volume of 200 μL / well, to obtain a transfection mixture without the conjugate, denoted as the blank control group. The 12-well plates were placed in an incubator containing 5% CO2 / 95% air and cultured at 37°C for 24 hours. After culture, total RNA was extracted from the cells using TRIZOL (purchased from SIGMA, Cat. No. T9424) according to the method described in the manufacturer's instructions. For each well of cells, 0.25 μg of total RNA was taken and reverse transcribed into cDNA using the Reverse Transcription System kit (Promega, Cat. No. A3500) according to its instructions, yielding a solution containing cDNA. Goldenstar cells were selected from this solution. TM Oligo(dT) 17 As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions, and the total RNA from each well was reverse transcribed. The reverse transcription conditions were as follows: the reverse transcription reaction system was incubated at 70°C for 10 min, then at 42°C for 30 min, and finally at 95°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided in the TaqMan Fast Advanced Master MIX(2x) kit (Thermo Fisher, catalog number: 4444556). The PCR primer sequences for amplifying the target gene LPA and the internal reference gene cβ-actin are shown in Table 6C, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step method. The amplification program was: 50℃ pre-denaturation for 20 s, 95℃ pre-denaturation for 20 s, then 95℃ denaturation for 1 s, and 60℃ annealing / extension for 20 s. This denaturation and annealing / extension process was repeated 40 times to obtain the product CW containing amplified target gene LPA and internal reference gene cβ-actin. The Ct values ​​of the target gene LPA and the internal reference gene cβ-actin were also obtained. The relative quantification of the target gene LPA in each test group was performed using the same comparison Ct (ΔΔCt) method as in experimental example C1. The results are shown in Table 7C. Table 6. Primer sequence information for C Table 7. Inhibition rate of C siRNA conjugates in untransfected monkey liver primary cells. As can be seen from Table 7C, the conjugates disclosed herein have good LPA mRNA inhibitory activity in primary monkey liver cells. Among them, conjugate C1 has an inhibition rate of 74.7% at 50 nM, and even at a lower 1 nM, it still maintains a high inhibition rate of 69.1%. Experimental Example C3: In vivo inhibitory activity of the disclosed conjugate. The prepared conjugate C31-conjugate C38 was dissolved in PBS to prepare an injection solution of 5.0 mg / mL, and then diluted to a 0.6 mg / mL injection solution. Forty-five mice (strain: LPA pAlb-hLPA-Tg, grade: SPF; sex: male, age: 12-14 weeks, weight: 25±5g, purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were randomly divided into two groups of 5 mice each: the test group (C1-C8) and the blank control group. The mice in the test group (C1-C8) were administered the conjugate C31-conjugate C38 via subcutaneous abdominal injection at a dose of 3 mg / kg body weight. The mice in the blank control group were administered PBS solution at a dose of 5 mL / kg body weight. Using the drug administration date as day 1, blood samples were collected from mice before drug administration and on days 8, 15, 29, 43, 57, 71, and 85 after drug administration. The expression levels of Apo(a) protein in the serum of the test group and the blank control group were detected at each time point using the Abcam Human Lipoprotein A ELISA Kit (catalog number ab212165) according to the instructions. The Apo(a) protein levels in the serum of mice before drug administration were normalized to 100% after drug administration, with the pre-drug Apo(a) protein level defined as 100%. For conjugates C32-C34 and C36-C38, data were collected up to day 43. The results are shown in Table 8C. The inhibition rate of Apo(a) protein expression in mouse serum at different time points after drug administration was: Inhibition rate of Apo(a) protein expression = (1 - Apo(a) protein content after administration / Apo(a) protein content before administration) × 100%. Table 8. Apo(a) expression levels of C siRNA conjugates in mice. As shown in Table 8C, the inhibitory rates of the conjugates disclosed herein against Apo(a) protein remained at a high level throughout the entire experimental period, maintaining an inhibition rate of over 64% on day 85. Conjugate C35, in particular, achieved an inhibition rate of 69.3% on day 85. Conjugates C31, C32, C33, C35, C36, and C38 consistently maintained inhibition rates above 60% throughout the experimental period, with conjugate C35 consistently maintaining an inhibition rate above 69%. Conjugate C31 maintained an inhibition rate above 89% on day 43. These results demonstrate that the conjugates disclosed herein exhibit highly effective and long-lasting inhibition of Apo(a) protein in the subjects. Experimental Example C4: In vivo inhibitory activity of the disclosed conjugate. The inhibitory activity of siRNA conjugates against Apo(a) protein in mice was detected using the method described in Experiment C3, except that conjugates C1A and C2A were used, with corresponding dosages of 3 mg / kg and 1 mg / kg, respectively. Blood samples were collected from mice before administration and on days 8, 15, 29, 43, 57, 71, 86, 113, and 141 after administration. The inhibition rate of Apo(a) protein expression in mouse serum was measured using the same method as in Experiment C3. Blood collection for the 1 mg / kg group was only conducted up to day 71. The results are shown in Table 9C. Table 9. Inhibition rate of Apo(a) protein by C siRNA conjugates in mice. As shown in Table 9C, the conjugates of this disclosure exhibit a long-lasting inhibitory effect on Apo(a) protein, and the inhibition rate of Apo(a) protein remains at a high level throughout the entire experimental period. At a dosage of 3 mg / kg, the different conjugates of this disclosure consistently maintained an inhibition rate of at least 60% or higher against Apo(a) protein, reaching a maximum of 92.82%, and still maintained a high inhibition rate of up to 86.88% on day 113. Even at a lower dosage of 1 mg / kg, the conjugates still maintained an inhibition rate of over 54% against Apo(a) protein, reaching a maximum of 80.76%, and still maintained an inhibition rate of 59.40% on day 71. These results indicate that the conjugates of this disclosure can maintain excellent LPA mRNA inhibition effects efficiently and for a long time in animal models, thereby continuously and effectively inhibiting serum Apo(a) protein levels. The following describes an example where the target mRNA is NTCP mRNA. Preparation Examples D1-D8: Synthesis of the siRNA conjugates disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates D1-D8 as shown in Table 2D were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in the conjugates are as shown in Table 2D. For nucleic acid sequences containing the sense and antisense strand sequences of the siRNAs numbered as conjugates D1-D8 in Table 2D, nucleoside phosphoramide monomers were ligated one by one to synthesize the sense and antisense strands of the siRNA. After synthesis, the siRNAs were purified by centrifugation and ultrafiltration using a 3K (MWCO) ultrafiltration tube. Conjugates D1-D8 are mixtures of methylamine and ammonium salts of compounds having the structure shown in formula (403). The P atom shown in formula (403) is covalently linked to the oxygen atom at the 3' end of the positive strand of the siRNA represented by Nu, as shown in formula (35), and connected to the ribose ring via a methylene group, thereby covalently linking it to the positive strand of the siRNA. Furthermore, the siRNA contained in these siRNA conjugates has the siRNA sequences corresponding to conjugates D1-D8 in Table 2D. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)), and then the molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Among them, conjugate D1: theoretical value of the positive chain is 7338.314, measured value is 7337.14, theoretical value of the negative chain is 7111.868, measured value is 7110.64; conjugate D2: theoretical value of the positive chain is 7313.304, measured value is 7312.09, theoretical value of the negative chain is 7074.799, measured value is 7073.57; conjugate D3: theoretical value of the positive chain is 7227.195, measured value is 7226.08, theoretical value of the negative chain is 7159.924, measured value is 7158.82; conjugate D4: theoretical value of the positive chain is 7340.227, measured value is 7338.78, theoretical value of the negative chain is 7139.001, measured value is 7137.8 0; Conjugate D5: Theoretical value of the positive chain is 7424.423, measured value is 7423.07, theoretical value of the negative chain is 6969.735, measured value is 6968.39; Conjugate D6: Theoretical value of the positive chain is 7423.439, measured value is 7422.25, theoretical value of the negative chain is 7027.731, measured value is 7026.51; Conjugate D7: Theoretical value of the positive chain is 7321.331, measured value is 7320.23, theoretical value of the negative chain is 7050.769, measured value is 7049.61; Conjugate D8: Theoretical value of the positive chain is 7316.256, measured value is 7315.12, theoretical value of the negative chain is 7056.828, measured value is 7055.72. The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates D1-D8 contain the target double-stranded nucleic acid sequence. Preparation Examples D9-D14: Synthesis of the siRNA conjugates disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates D9-D14 as shown in Table 2D were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in the conjugates are as shown in Table 2D. For nucleic acid sequences containing the sense and antisense strand sequences of the siRNAs numbered as conjugates D9-D14 in Table 2D, nucleoside phosphoramide monomers were sequentially ligated to synthesize the sense and antisense strands of the siRNAs. After synthesis, conjugates D9-D14 were first purified using a self-packed column with Source15Q strong anion exchange packing material, and then desalted using a HiPrep 26 / 10 desalting pre-packed column. Conjugates D9-D14 are sodium salts of compounds having the structure shown in formula (403), wherein the P atom shown in formula (403) is covalently linked to the reverse debased deoxynucleotide (ia) at the 3' end of the positive strand of the siRNA represented by Nu, as shown in formula (35), and the oxygen atom is linked to the ribose ring via a methylene group, thereby covalently linking to the positive strand of the siRNA. Furthermore, the siRNAs contained in these siRNA conjugates each have the siRNA sequences corresponding to conjugates D9-D14 in Table 2D. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)). Molecular weight was then determined using liquid chromatography-mass spectrometry (LC-MS, Waters Corporation, model: LCT Premier). Specifically, conjugate D9 had a theoretical positive molecular weight of 7370.05 for the sense strand and a measured value of 7369 for the antisense strand; conjugate D10 had a theoretical positive molecular weight of 7371.97 for the sense strand and a measured value of 7369 for the antisense strand; and conjugate D11 had a theoretical positive molecular weight of 7455.17 for the sense strand and a measured value of 7453 for the antisense strand. 25; Conjugate D12: Theoretical value for the sense strand is 7353.06, measured value is 7351; theoretical value for the antisense strand is 7050.65, measured value is 7049; Conjugate D13: Theoretical value for the sense strand is 7348, measured value is 7346; theoretical value for the antisense strand is 7056.7, measured value is 7054; Conjugate D14: Theoretical value for the sense strand is 7378.08, measured value is 7376; theoretical value for the antisense strand is 7020.63, measured value is 7019. The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates D9-D14 contain the target designed double-stranded nucleic acid sequence. Table 2D shows the siRNA sequences in siRNA conjugates. In this context, uppercase letters C, G, U, A, and T represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide represented by the uppercase letter to the left of f is 2'-fluoro modified; lowercase letter s indicates that the linking group between the two uppercase letters to the left and right of s is a thiophosphate group; lowercase letter d indicates that the nucleotide represented by the uppercase letter to the right of d is a deoxynucleotide; ia indicates a reverse debased deoxynucleotide; and (moe) indicates that the nucleotide represented by the uppercase letter to the left is 2'-O-methoxyethyl modified. Synthesis of Reference Conjugate D1 (Comparative Preparation Example D1) Following the same method as Preparation Example D1, reference conjugate D1 in Table 2D was prepared by solid-phase synthesis. The theoretical value for the sense strand of reference conjugate D1 was 7485.396, and the measured value was 7484.61; the theoretical value for the antisense strand was 7045.784, and the measured value was 7044.92. Reference conjugate 1 is a mixture of methylamine and ammonium salts of compounds having the structure shown in formula (403), wherein the conjugation group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the sense strand of the siRNA represented by Nu. Furthermore, the siRNA contained in this siRNA conjugate has the siRNA sequence corresponding to reference conjugate D1 in Table 2D, which is a negative control sequence not associated with NTCP mRNA. The D1 conjugate exhibited inhibitory activity against the NTCP target sequence in the in vitro psiCHCEK system. This experiment investigated the on-target activity of conjugates D1-D8 in the in vitro psiCHECK system, specifically measuring the activity of the eight conjugates targeting perfectly matched target sequences. Following the method described in Kumico Ui-Tei et al., Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect. Nucleic Acids Research, 2008, 36(7), 2136-2151, a detection plasmid was constructed and co-transfected with the siRNA conjugate into HEK293A cells. The on-target activity of the siRNA conjugate was reflected by the expression level of the dual luciferase reporter gene. The specific steps are as follows: [1] Constructing detection plasmids Using psiCHECK TM -2(Promega TM The detection plasmid was constructed containing a target sequence that is fully complementary to all 21 nucleotides of the antisense strand in the siRNA conjugate to be tested and has no base differences from a portion of the NTCP mRNA. Clone the target sequence into psiCHECK TM -2 Xho I / Not I sites on the plasmid. [2] Transfection HEK293A (purchased from Nanjing Kebai Biotechnology Co., Ltd.) was cultured in H-DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone) and 0.2% penicillin-streptomycin (Gibco, Invitrogen) at 37°C in an incubator containing 5% CO2 / 95% air. The HEK293A cells cultured above were then subjected to a culturing rate of 8 × 10⁻⁶ cells / mL. 3 Cells were seeded in 96-well plates. After 24 hours, when the cell growth density reached 70-80%, the H-DMEM medium in the culture wells was aspirated, and 80 μL of Opti-MEM medium (GIBCO) was added to each well for further culture for 1.5 hours. Prepare a 0.01 μM working solution of each of the conjugates D1-D8 using PBS. Dilute the test plasmid with DEPC-treated water to a working solution of 200 ng / μL. In the culture wells of a 96-well plate containing the aforementioned cells, according to Lipofectamine TM Following the instructions for use of RNAiMAX transfection reagent (Invitrogen, catalog number: 13778150), co-transfect the siRNA conjugate and the plasmid described above. Specifically, add the following to each well containing the cells: 1) 0.05 μL of plasmid working solution (containing 10 ng of test plasmid) diluted in 9 μL of Opti-MEM medium; 2) 1 μL of siRNA conjugate working solution; and 3) 0.2 μL of Lipofectamine. TM The RNAiMAX transfection reagent solution was prepared in 9.8 μL of Opti-MEM medium, with a final concentration of 0.1 nM of the conjugate in each well. The conjugates were one of the aforementioned conjugates D1-D8. Each group had 3 replicates. [3] Detection Twenty-four hours after co-transfection, HEK293A cells were lysed using a Dual luciferase reporter gene assay kit (Promega, cat. E2940) according to the instructions, and the expression level of the dual luciferase reporter gene was detected. Each siRNA treatment group was used as a control (con.) with a specific concentration of siRNA. Ren luciferase (Ren) protein levels were normalized relative to firefly luciferase (Fir) protein levels. Results are shown in Table 3D. Table 3D shows the inhibitory activity of siRNA conjugates against target sequences. As shown in Table 3D, the conjugates disclosed herein exhibit high inhibitory activity against the target sequence in the in vitro psiCHECK system. At a concentration of 0.1 nM, the inhibition rate is above 76%, and can even reach 91.59%. Experimental study: Detection of the inhibitory efficiency of D2 siRNA conjugate on NTCP mRNA expression in HepG2 cells. Use Lipofectamine according to the instructions provided by the supplier. TM RNAiMAX transfection reagent (Invitrogen, catalog number: 13778150) is used to transfect HepG2 cells with the test siRNA conjugates (siRNA conjugates D3-D5 and reference conjugate D1). The final concentration of each siRNA conjugate is 50 nM, and each concentration is tested in duplicate. Cells without any siRNA conjugate treatment serve as a blank control. The expression level of NTCP mRNA in HepG2 cells transfected with each siRNA conjugate was detected by quantitative real-time PCR. The specific steps were as follows: After culturing transfected cells for 24 hours, total RNA was extracted from the cells using Trizol (Thermo Fisher Scientific) according to the standard operating procedure for total RNA extraction. 1 μg of total RNA was taken and reverse transcribed into cDNA using a reverse transcription kit (Promega, catalog number A3500) according to the manufacturer's instructions. The expression level of NTCP mRNA was detected using a 2×Ultra SYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used for amplifying NTCP and for GAPDH as an internal control gene are shown in Table 4D. Table 4D Primer Information The relative quantitative calculation of the expression level of the target gene FASN mRNA in each test group was performed using the Ct(ΔΔCt) method. The calculation method is as follows: ΔΔCt=(Ct 测试组目标基因 -Ct 测试组内参基因 )-(Ct 空白对照组目标基因 -Ct 空白对照组内参基因 mean Relative expression level = 2 -ΔΔCt ×100% mRNA inhibition rate = (blank control group 2) -ΔΔCt Mean - Test Group 2 -ΔΔCt ) / Blank control group 2 -ΔΔCt Mean × 100% In this study, each test group consisted of HepG2 cells treated with different concentrations of siRNA, while the control group consisted of cells not treated with siRNA. The inhibition rate of the conjugates on NTCP mRNA is shown in Table 5D. Table 5. Inhibition rate of NTCP mRNA in HepG2 cells. As can be seen from the results in Table 5D, the conjugates provided in this disclosure all showed high NTCP mRNA inhibitory activity in HepG2 cells, with an inhibition rate of at least 75.3%, and even reaching over 86%. Experimental study: Detection of the inhibitory efficiency of D3 siRNA conjugate on NTCP mRNA expression in human primary liver cells. Use Lipofectamine according to the instructions provided by the supplier. TMRNAiMAX transfection reagent (Invitrogen, catalog number: 13778150) is used to transfect human primary liver cells with the test siRNA conjugates (siRNA conjugates D1-D8 and reference conjugate D1). The final concentrations of each siRNA conjugate are 50 nM and 0.5 nM, with two replicates per concentration. Cells not treated with any siRNA conjugates serve as a blank control. The expression level of NTCP mRNA in primary human liver cells transfected with each siRNA conjugate was detected by quantitative real-time PCR. The specific steps were as follows: After culturing transfected cells for 24 hours, total RNA was extracted from the cells using Trizol (Thermo Fisher Scientific) according to the standard operating procedure for total RNA extraction. 1 μg of total RNA was taken and reverse transcribed into cDNA using a reverse transcription kit (Promega, catalog number A3500) according to the manufacturer's instructions. The expression level of NTCP mRNA was detected using a 2×Ultra SYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used for amplifying NTCP and for GAPDH as an internal control gene are shown in Table 4D. NTCP mRNA expression level is calculated using the following equation: NTCP mRNA expression level = (NTCP mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (NTCP mRNA expression level in the control group / GAPDH mRNA expression level in the control group) × 100%. mRNA inhibition rate = (1 - NTCP mRNA expression level) × 100%. The test groups consisted of HepG2 cells treated with different concentrations of siRNA, while the control group consisted of cells not treated with siRNA. The results are shown in Table 6D. Table 6. Inhibition rate of NTCP mRNA in primary human liver cells. As can be seen from the results in Table 6D, the negative control reference conjugate showed almost no inhibitory effect on NTCP mRNA in human primary liver cells; in contrast, the conjugate provided in this disclosure showed high NTCP mRNA inhibitory activity in human primary liver cells, with inhibition rates of over 75% at a low concentration of 0.5 nM, and even reaching 91%. Experimental study: Detection of the inhibitory efficiency of D4 siRNA conjugate on NTCP mRNA expression in human primary liver cells. The inhibitory effects of conjugates D2, D4, D6, and D9-D14 on NTCP mRNA expression in primary human liver cells were tested according to the method in Experiment D3, with the only difference being that the compounds used were conjugates D2, D4, D6, and D9-D14, respectively. The results are shown in Table 7D. Table 7. Inhibition rate of NTCP mRNA in primary human liver cells. As can be seen from the results in Table 7D, the conjugates provided in this disclosure showed high NTCP mRNA inhibitory activity in human primary liver cells at different concentrations, with inhibition rates of at least 83.3% and even reaching 92.1%. The following describes an example where the target mRNA is PCSK9 mRNA. Preparation Examples E1-E5: Synthesis of siRNA conjugates disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates E1-E5 as shown in Table 2E were prepared, with the only difference being that the sense and antisense strands of the siRNA contained in the siRNA conjugates are as shown in Table 2E. For nucleic acid sequences containing the sense and antisense strand sequences of siRNAs numbered E1-E5 in Table 2E, nucleoside phosphoramide monomers were ligated one by one to synthesize the sense and antisense strands of the siRNA. After synthesis, the siRNA was purified by centrifugation and ultrafiltration using a 3K (MWCO) ultrafiltration tube. Conjugates E1-E5 are mixtures of methylamine and ammonium salts of compounds having the structure shown in formula (403), wherein the conjugate group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the positive strand of the siRNA represented by Nu. Furthermore, the siRNA contained in the siRNA conjugate has the siRNA sequences corresponding to conjugates E1-E5 in Table 2E. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)), and then the molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Among them, conjugate E1: theoretical value of the positive chain is 7319.277, measured value is 7318.02, theoretical value of the negative chain is 7133.985, measured value is 7132.61; conjugate E2: theoretical value of the positive chain is 7412.429, measured value is 7411.04, theoretical value of the negative chain is 6999.820, measured value is 6998.54; conjugate E3: theoretical value of the positive chain is 7323.368, measured value is 7322.04, theoretical value of the negative chain is 7119.899, measured value is 7118.48; conjugate E4: theoretical value of the positive chain is 7322.384, measured value is 7321.06, theoretical value of the negative chain is 7096.857, measured value is 7095.59. The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates E1-E5 contain the target double-stranded nucleic acid sequence. Table 2E siRNA sequences in siRNA conjugates In this context, uppercase letters C, G, U, A, and T represent the base composition of the nucleotide; lowercase letter m indicates that the uppercase letter to the left of m represents a nucleotide modified with 2'-methoxy; lowercase letter f indicates that the uppercase letter to the left of f represents a nucleotide modified with 2'-fluoro; lowercase letter s indicates that the linking group between the two uppercase letters to the left and right of s is a phosphate thioester group; lowercase letter d indicates that the uppercase letter to the right of d represents a deoxynucleotide; ia represents a reverse debased deoxynucleotide; VP indicates that the uppercase letter to the right of this letter combination represents a nucleotide modified with 5'-VP; and the letter combination (moe) indicates that the uppercase letter to the left of this letter combination represents a nucleotide modified with 2'-O-methoxyethyl. Synthesis of Reference Conjugate E1 (Comparative Preparation Example) Following the same method as Preparation Example E1, reference conjugate E1 in Table 2E was prepared by solid-phase synthesis. The theoretical value for the sense strand of reference conjugate E1 was 7426.265, and the measured value was 7425.05; the theoretical value for the antisense strand was 7084.880, and the measured value was 7083.90. Reference conjugate E1 is a mixture of methylamine and ammonium salts of a compound having the structure shown in formula (403), wherein the conjugation group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the sense strand of the siRNA represented by Nu. Furthermore, the siRNA contained in this siRNA conjugate has the siRNA sequence corresponding to reference conjugate E1 in Table 2E. Reference conjugate E1 is an siRNA conjugate with the same sequence as conjugate E1, differing only in its modification scheme. Experimental Example E1: Inhibitory activity of the disclosed conjugate in vivo. This experimental example investigated the activity of the disclosed conjugates E1-E4 in mice. The prepared conjugates E1-E4 and the reference conjugate E1 were dissolved in PBS to prepare an injection solution of 5.0 mg / mL. Thirty mice (strain: hPCSK9-C57BL / 6j, grade: SPF, sex: female, purchased from SPAF (Beijing) Biotechnology Co., Ltd.) were randomly divided into groups of 5 mice each, designated as test groups 1-5 and a blank control group. Mice in test groups E1-E5 were administered conjugates E1-E4 and reference conjugate E1 via subcutaneous abdominal injection at a dose of 3 mg / kg mouse body weight. Mice in the blank control group were administered PBS solution at a dose of 5 mL / kg mouse body weight. Using the time of drug administration as day 1, five mice from each group were sacrificed on day 8 and day 15 after drug administration, and liver tissue was collected and preserved using RNAlater. The expression level of PCSK9 mRNA in each siRNA conjugate was detected by quantitative real-time PCR. The specific steps were as follows: After culturing transfected cells for 24 hours, total RNA was extracted from liver tissue using Trizol (Thermo Fisher Scientific) according to the standard operating procedure for total RNA extraction. 1 μg of total RNA was taken and reverse transcribed into cDNA using a reverse transcription kit (Promega, catalog number A3500) according to the manufacturer's instructions. The expression level of PCSK9 mRNA was detected using a 2×Ultra SYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used for amplifying PCSK9 and GAPDH as an internal control gene are shown in Table 3E. Table 3E Primer Information PCSK9 mRNA expression level is calculated using the following equation: PCSK9 mRNA expression level = (PCSK9 mRNA expression level in the test group / GAPDH mRNA expression level in the test group) / (PCSK9 mRNA expression level in the control group / GAPDH mRNA expression level in the control group) × 100%. PCSK9 mRNA inhibition rate = (1 - PCSK9 mRNA expression level) × 100%. The results are summarized in Table 4E. Table 4 shows the inhibition rate of PCSK9 mRNA in E mice. As can be seen from the results in Table 4E, the conjugates provided in this disclosure can maintain inhibitory activity against PCSK9 mRNA in vivo for a relatively long period of time. Compared with the reference conjugate, the conjugates provided in this disclosure showed higher inhibition rates on both day 8 and day 15. The inhibition rate on day 8 was above 75%, and even reached above 89%; the inhibition rate on day 15 was above 79%, and even reached above 89%. In particular, the inhibition rate of conjugate E1 was higher than that of the reference conjugate E1, which had a basically the same sequence but a different modification scheme. The following describes an example where the target mRNA is PNPLA3 mRNA. Preparation Examples F1-F13 Synthesis of the siRNA conjugates F1-F13 disclosed herein Following the preparation method described in Example 13 of CN110959011A, conjugates F1-F13 as shown in Table 2F were prepared, differing only in that the sense and antisense strands of the siRNA contained in the conjugates are as shown in Table 2F. For nucleic acid sequences containing the sense and antisense strand sequences of the siRNAs in conjugates F1-F13 as shown in Table 2F, nucleoside phosphoramide monomers were sequentially linked to synthesize the sense and antisense strands of the siRNA. After preparation, conjugates F1-F8 were purified by centrifugation and ultrafiltration using 3K (MWCO) ultrafiltration tubes. Conjugates F9-F13 were first purified by self-packed column ionization using strong anion exchange packing material, and then purified by desalting using a HiPrep 26 / 10 desalting pre-packed column. Conjugates F1-F8 are each a mixture of methylamine and ammonium salts of compounds having the structure shown in formula (403), and conjugates F9-F13 are each sodium salts of compounds having the structure shown in formula (403). In conjugates F1-F13, the conjugating group is attached to the 3' position of the ribose of the 3' terminal nucleotide of the positive strand of the siRNA represented by Nu. Furthermore, the siRNA contained in this siRNA conjugate has the siRNA sequence corresponding to conjugates F1-F13 in Table 2F. Each siRNA conjugate was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm (25℃)), and then the molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Among them, the theoretical molecular weight of the positive chain of conjugate F1 is 7336.35, and the measured molecular weight is 7336.42; the theoretical molecular weight of the antisense chain is 7066.77, and the measured molecular weight is 7066.82; the theoretical molecular weight of the positive chain of conjugate F2 is 7503.49, and the measured molecular weight is 7502.23; the theoretical molecular weight of the antisense chain is 6873.63, and the measured molecular weight is 6873.5; the theoretical molecular weight of the positive chain of conjugate F3 is 7433.38, and the measured molecular weight is 7433.20; the theoretical molecular weight of the antisense chain is 6930.69, and the measured molecular weight is 6930.96; the theoretical molecular weight of the positive chain of conjugate F4 is 7... The theoretical molecular weight of the antisense chain is 7104.83, and the measured molecular weight is 7104.84. For conjugate F5, the theoretical molecular weight of the positive chain is 7355.29, and the measured molecular weight is 7355.37; for the antisense chain, the theoretical molecular weight is 7032.80, and the measured molecular weight is 7032.85. For conjugate F6, the theoretical molecular weight of the positive chain is 7313.30, and the measured molecular weight is 7313.16; for the antisense chain, the theoretical molecular weight is 7096.85, and the measured molecular weight is 7096.93. For conjugate F7, the theoretical molecular weight of the positive chain is 7345.30, and the measured molecular weight is 7344.5. 5. The theoretical molecular weight of the antisense chain is 7125.89, and the measured molecular weight is 7125.35; the theoretical molecular weight of the positive chain of conjugate F8 is 7396.30, and the measured molecular weight is 7396.47, while the theoretical molecular weight of the antisense chain is 6999.82, and the measured molecular weight is 6999.89; the theoretical molecular weight of the positive chain of conjugate F9 is 7535.61, and the measured molecular weight is 7535.36, while the theoretical molecular weight of the antisense chain is 6873.63, and the measured molecular weight is 6873.36; the theoretical molecular weight of the positive chain of conjugate F10 is 7387.42, and the measured molecular weight is 7387.11, while the theoretical molecular weight of the antisense chain is 7032.8. The theoretical molecular weight of the positive chain of conjugate F11 is 7535.61, and the measured molecular weight is 7535.14. The theoretical molecular weight of the negative chain is 6950.60, and the measured molecular weight is 6950.09. The theoretical molecular weight of the positive chain of conjugate F12 is 7387.41, and the measured molecular weight is 7387.06. The theoretical molecular weight of the negative chain is 7108.79, and the measured molecular weight is 7108.34. The theoretical molecular weight of the positive chain of conjugate F13 is 7535.60, and the measured molecular weight is 7535.95. The theoretical molecular weight of the negative chain is 6949.62, and the measured molecular weight is 6949.89. The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates F1-F13 contain the target double-stranded nucleic acid sequence. Table 2F shows the siRNA sequences in siRNA conjugates. In this context, uppercase letters C, G, U, A, and T represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide represented by the uppercase letter to the left of m is 2'-methoxy modified; lowercase letter f indicates that the nucleotide represented by the uppercase letter to the left of f is 2'-fluoro modified; (moe) indicates that the nucleotide represented by the uppercase letter to its left is ribose 2'-O-methoxyethyl modified; lowercase letter s indicates that the linking group between the two uppercase letters to its left and right is a thiophosphate group; lowercase letter d indicates that the nucleotide represented by the uppercase letter to its right is a deoxynucleotide; VP indicates that the nucleotide represented by the uppercase letter to its right is 5'-vinyl phosphate (VP) modified; and ia indicates a reverse debase deoxynucleotide. Experimental Example F1: In vitro inhibitory activity of the disclosed conjugate. This experiment investigated the inhibitory activity of conjugates F1, F2, F5, F7, and F8 on the hPNPLA3 gene in HepG2 human hepatocellular carcinoma cells in vitro at concentrations of 50 nM, 5 nM, and 0.5 nM. The specific steps are as follows: [1] Cell culture HepG2 human liver cancer cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured in DMEM medium (M&C GENE) supplemented with 10% fetal bovine serum (FBS, GIBCO), 1% NEAA, and 1 mM NAP at 37°C in an incubator containing 5% CO2 / 95% air. HepG2 cells were loaded at 5 x 10 4 Cells were seeded into 24-well plates with 0.5 mL of cell culture per well. After culturing for 16 h, the culture medium in the wells was aspirated, and 0.5 mL of Opti-MEM medium (GIBCO) was added to each well. The cells were then cultured for another 1.5 h. [2] Transfection For each siRNA conjugate to be tested, working solutions of 20 μM, 2 μM, and 0.2 μM were prepared using PBS. The siRNA conjugates used were conjugates F1, F2, F5, F7, and F8, respectively. For each siRNA conjugate to be tested, an F1A1 solution was prepared, each F1A1 solution containing 1.5 μL of 20 μM working solution of the conjugate and 48.5 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, an F1A2 solution was prepared, each F1A2 solution containing 1.5 μL of 2 μM working solution of the conjugate and 48.5 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, an F1A3 solution was prepared, each F1A3 solution containing 1.5 μL of 0.2 μM working solution of the conjugate and 48.5 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, prepare an F1B solution, each containing 1.5 μL of Lipofectamine. TM RNAiMAX transfection reagent (Invitrogen, catalog number: 13778150) and 48.5 μL Opti-MEM medium (GIBCO). For each siRNA conjugate, one portion of F1A1 solution and one portion of F1B solution were mixed and incubated at room temperature for 20 min to obtain transfection complex FX1. In two culture wells (both containing HepG2 human liver cancer cells and 0.5 mL Opti-MEM medium, hereinafter the same), 100 μL of transfection complex FX1 for each siRNA conjugate was added and mixed thoroughly to obtain a transfection mixture with a concentration of 50 nM (based on the amount of siRNA). Each siRNA conjugate transfection complex FX1 was transfected into two culture wells to obtain a transfection mixture containing the siRNA conjugate, denoted as test group FX. a1 -FX a5 . For each siRNA conjugate, one portion of F1A2 solution and one portion of F1B solution were mixed and incubated at room temperature for 20 min to obtain transfection complex FX2. In two culture wells (both containing HepG2 human liver cancer cells and 0.5 mL Opti-MEM medium, hereinafter the same), 100 μL of transfection complex FX2 for each siRNA conjugate was added and mixed thoroughly to obtain a transfection mixture with a concentration of 5 nM (based on the amount of siRNA). Each siRNA conjugate transfection complex FX2 was transfected into two culture wells to obtain a transfection mixture containing the siRNA conjugate, denoted as test group FX. b1 -FX b5 . For each siRNA conjugate, one portion of F1A3 solution and one portion of F1B solution were mixed and incubated at room temperature for 20 min to obtain transfection complex FX3. In two culture wells (both containing HepG2 human liver cancer cells and 0.5 mL Opti-MEM medium, hereinafter the same), FX3 transfection complex for each siRNA conjugate was added and mixed thoroughly at a volume of 100 μL / well to obtain a transfection mixture with a concentration of 0.5 nM (based on the amount of siRNA, hereinafter the same). Each siRNA conjugate transfection complex FX3 was transfected into two culture wells to obtain a transfection mixture containing the siRNA conjugate, denoted as test group FX. c1 -FX c5 . The blank control group was treated with 100 μL of Opti-MEM per well, for a total of two culture wells. Test group FX mentioned above a1 -FX a5 FX b1 -FX b5 FX c1 -FX c5 Both the control and blank control groups were placed in an incubator with an air atmosphere containing 5% CO2 and cultured at 37°C for 24 hours. [3] Detection Total RNA was extracted from cells in each well using Trizol (Sigma-Aldrich) according to the method described in the instruction manual. For each well of cells, 1 μg of total RNA was extracted and reverse transcribed into cDNA using the Reverse Transcription System kit (Promega) according to its instructions, yielding a solution containing cDNA. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 70°C for 10 min, then at 42°C for 30 min, at 95°C for 5 min, and finally at 4°C for 5 min or more. After the reaction, 80 μL of DEPC water was added to the reverse transcription system to obtain a solution containing cDNA. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using reagents provided by SYBR Select Master Mix (Thermo Fisher Scientific). The PCR primer sequences for amplifying the target gene hPNPLA3 and the internal reference gene GAPDH are shown in Table 3F, with a final concentration of 10 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument (Thermo Fisher Scientific) and amplified using a three-step method. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product A-W1 containing amplified target gene hPNPLA3 and internal reference gene GAPDH. Product A-W1 was then subjected to a gradient temperature increase to 95℃ for 15s, 60℃ for 1min, and then to 95℃ with fluorescence signals collected every 0.3℃. After 15s at 95℃, the melting curves of the target gene and the internal reference gene GAPDH in product A-W1 were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene hPNPLA3 and the internal reference gene GAPDH. Table 3F Primer Information The relative quantitative calculation of the expression level of the target gene hPNPLA3 mRNA in each test group was performed using the Ct(ΔΔCt) method. The calculation method is as follows: ΔCt(test group) = Ct(target gene in test group) - Ct(internal reference gene in test group) ΔCt(control group) = Ct(target gene in control group) - Ct(internal reference gene in control group) ΔCt(test group) = ΔCt(test group) - ΔCt(control group average) ΔCt(control group) = ΔCt(control group) - ΔCt(control group average) Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) from each of the two culture wells in the blank control group. Therefore, each culture well in the test group and the control group corresponds to a ΔCt value. Using the mean value of the blank control group as a baseline, the expression level of hPNPLA3 mRNA in the test group was normalized, and the mean value of hPNPLA3 mRNA expression level in the blank control group was defined as 100%. The relative expression level of hPNPLA3 mRNA in the test group = 2 - ΔΔCt(test group) × 100% The inhibition rate of hPNPLA3 mRNA in the test group = (1 - relative expression level of hPNPLA3 mRNA in the test group) × 100%. The inhibition rates of each conjugate on hPNPLA3 mRNA are summarized in Table 4F below: Table 4 shows the inhibition rates of each conjugate of the target gene hPNPLA3 mRNA. The results in Table 4F show that, in HepG2 cells in vitro, the various siRNA conjugates provided in this disclosure all exhibited good inhibitory effects against the target gene hPNPLA3 mRNA. At a concentration of 50 nM, conjugate F2 showed the highest inhibition rate against hPNPLA3 mRNA, reaching 82.2%, demonstrating significant in vitro activity. Even when the concentration was reduced to 5 nM and 0.5 nM, the conjugates maintained a certain level of inhibition, especially conjugate F2, which showed inhibition rates of 67.3% and 68.3% at 5 nM and 0.5 nM, respectively, while conjugate F5 also reached 73.0% at 5 nM. Experimental Example F2: Inhibition of hPNPLA3 mRNA expression by conjugates F1-F8 in mice. This experiment investigated the inhibitory activity of the conjugate F1F-8 on the hPNPLA3 gene in humanized PNPLA3 I148M transgenic mice. Humanized PNPLA3 I148M transgenic mice (all female, purchased from Jicui Pharmaceutical Co., Ltd.) were randomly divided into groups of 5 mice each, and each group was numbered. Each group of mice (F1-F8) was administered the test conjugates (siRNA) subcutaneously at a dose of 9 mg / kg. The siRNA conjugates were provided as a 0.9% sodium chloride aqueous solution containing 1.8 mg / mL of the siRNA conjugate, with an administration volume of 5 mL / kg. One group of mice was given 1×PBS at a volume of 5 mL / kg as the control group. The animals were sacrificed on day 8 after administration, with the time of administration taken as day 1. Liver tissue was collected from each mouse and preserved using RNA later (Sigma-Aldrich). The liver tissue was homogenized using a tissue homogenizer and then total RNA was extracted using Trizol (Sigma-Aldrich) according to the procedure described in the manufacturer's instructions. For each mouse liver tissue, 1 μg of total RNA was extracted and reverse transcribed into cDNA using the Reverse Transcription System kit (Promega) according to its instructions, yielding a solution containing cDNA. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 70°C for 10 min, then at 42°C for 30 min, at 95°C for 5 min, and finally at 4°C for 5 min or more. After the reaction, 80 μL of DEPC water was added to the reverse transcription system to obtain a solution containing cDNA. For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using reagents provided by the Real-Time PCR Kit (Beijing Kangwei Century Biotechnology Co., Ltd.). The PCR primer sequences for amplifying the target gene PNPLA3 and the internal reference gene GAPDH are shown in Table 3F, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument (Thermo Fisher), and a three-step amplification method was used. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product FW1 containing amplified target gene PNPLA3 and internal reference gene GAPDH. The product FW1 was then incubated sequentially at 95℃ for 15s, 60℃ for 1min, and 95℃ for 15s. The melting curves of the target gene PNPLA3 and the internal reference gene GAPDH in the product FW1 were collected by a real-time fluorescence quantitative PCR instrument, and the Ct values ​​of the target gene PNPLA3 and the internal reference gene GAPDH were obtained. In the calculation of PNPLA3 mRNA expression level and inhibition rate, the control group consisted of mice administered PBS in this experiment, and the test group consisted of mice administered different conjugates F1-F8. The PNPLA3 mRNA expression level in the control group was recorded as 100%, and the corresponding inhibition rate of PNPLA3 mRNA expression level was recorded as 0%. The test results were standardized based on the PNPLA3 mRNA expression level in the control group. Primer information and calculation process are shown in Experiment F1, and the results are shown in Table 5F below. Table 5F: PNPLA3 mRNA inhibition rate of different conjugates According to the results shown in Table 5F, the conjugates provided in this disclosure all exhibited significant inhibitory effects on PNPLA3 mRNA at a concentration of 9 mg / kg siRNA. Specifically, the inhibition rates of these conjugates ranged from 81.3% to 97.1%, with most conjugates showing inhibition rates close to or exceeding 90%. In vitro inhibitory activity of F3 conjugate F9-F12 This experiment investigated the inhibitory activity of conjugates F9, F10, F11, and F12 on the hPNPLA3 gene in HepG2 human hepatocellular carcinoma cells in vitro at concentrations of 50 nM and 5 nM. The specific steps are as follows: HepG2 cells were cultured using the same method as in experimental case F1, at a density of 5 x 10⁻⁶ cells / year. 4 Cells were seeded into 24-well plates with 0.5 mL of cell culture per well. After culturing for about 16 h, the culture medium in the wells was aspirated, and 0.5 mL of Opti-MEM medium (GIBCO) was added to each well. The cells were then cultured for another 1.5 h. For each siRNA conjugate to be tested, working solutions of 20 μM and 2 μM were prepared using PBS. The siRNA conjugates used were conjugates F9, F10, F11, and F12. For each siRNA conjugate to be tested, an F3A1 solution was prepared, each F3A1 solution containing 1.5 μL of 20 μM working solution of the conjugate and 48.5 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, an F3A2 solution was prepared, each containing 1.5 μL of 2 μM working solution of the conjugate and 48.5 μL of cell maintenance culture medium. For each siRNA conjugate to be tested, prepare an F3B solution, each containing 1.5 μL of Lipofectamine. TM RNAiMAX transfection reagent (Invitrogen, catalog number: 13778150) and 48.5 μL Opti-MEM medium (GIBCO). For each siRNA conjugate, one portion of F3A1 solution and one portion of F3B solution were mixed and incubated at room temperature for 20 min to obtain transfection complex F3X1. In each culture well (all containing HepG2 human liver cancer cells and 0.5 mL Opti-MEM medium, the same applies below), 100 μL of transfection complex F3X1 for each siRNA conjugate was added and mixed thoroughly to obtain a transfection mixture with a concentration of 50 nM (based on the amount of siRNA, the same applies below). Two culture wells were transfected with each siRNA conjugate's transfection complex F3X1 to obtain a transfection mixture containing the siRNA conjugate, denoted as test group F3X. a1 -F3X a4 . For each siRNA conjugate, one portion of solution F3A2 and one portion of solution F3B were mixed and incubated at room temperature for 20 min to obtain transfection complex F3X2. In two culture wells (both containing HepG2 human liver cancer cells and 1 mL of Opti-MEM medium, the same applies below), 100 μL of transfection complex FX2 for each siRNA conjugate was added and mixed thoroughly to obtain a transfection mixture with a concentration of 5 nM (based on the amount of siRNA, the same applies below). Each siRNA conjugate transfection complex FX2 was transfected into two culture wells to obtain a transfection mixture containing the siRNA conjugate, denoted as test group F3X. b1 -F3X b4 . The blank control group was treated with 100 μL of Opti-MEM per well, for a total of two culture wells. The above test group, test group F3X a1 -F3X a4 F3X b1 -F3X b4 Both the control and blank control groups were placed in an incubator with an air atmosphere containing 5% CO2 and cultured at 37°C for 24 hours. Total RNA and reverse transcription were extracted from cells in each well using the same method as in Experiment F1, and the relative quantification of the target gene PNPLA3 mRNA in each test group was calculated. The results are shown in Table 6F below. Table 6 shows the inhibition rates of each F conjugate on the target gene hPNPLA3 mRNA. The results in Table 6F show that, in HepG2 cells in vitro, the various siRNA conjugates provided in this disclosure all exhibited good inhibitory effects, with inhibition rates ranging from a minimum of 55.5% to a maximum of 68.0% at 5 nM; at a concentration of 50 nM, the inhibition rate ranged from a minimum of 66.0% to as high as 73.5%. Inhibitory activity of F4 conjugates F11-F13 in primary monkey liver cells (Example) This experiment investigated the inhibitory activity of conjugates F11, F12, and F13 on the hPNPLA3 gene in primary monkey liver cells at concentrations of 10 nM, 1 nM, and 0.1 nM. The specific steps are as follows: [1] Cell plating Primary monkey liver cells were revived (purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.). The primary monkey liver cells were cultured at 1x10⁶ cells / cells using the pre-contained plating medium. 5 Cells were seeded into 12-well plates with 1 mL of cell culture per well. After culturing for 16 h, the culture medium in the wells was aspirated, and 1 mL of maintenance medium was added to each well. The cells were then cultured for another 1.5 h. [2] Transfection For each siRNA conjugate to be tested, a working solution of the conjugate was prepared with PBS at a concentration of 20 μM (based on the amount of siRNA in the conjugate). The siRNA conjugates used were conjugates F11, F12, and F13. Prepare F4A solutions, each containing 3 μL of the conjugate working solution and 97 μL of Opti-MEM medium. Prepare F4B solutions, each containing 3 μL of Lipofectamine. TM RNAMAX transfection reagent (Invitrogen, catalog number: 13778150) and 97 μL Opti-MEM medium (GIBCO). For each conjugate, one part F4A solution and one part F4B solution were mixed to obtain transfection complex F4X1. In each culture well (all containing primary monkey liver cells and 1 mL Opti-MEM medium, hereinafter the same), 3X1 of each siRNA conjugate transfection complex was added and mixed thoroughly. The addition volume was 200 μL / well, resulting in a transfection complex concentration of 50 nM (based on the amount of siRNA, hereinafter the same). Each siRNA conjugate transfection complex F4X1 was transfected into two culture wells to obtain a transfection mixture containing the siRNA conjugate, denoted as test group F4X. a1 -F4X a4 This is designated as the test group. The blank control group was treated with 200 μL of Opti-MEM per well, for a total of two culture wells. Total RNA was extracted from cells in each well using the same method as in Experiment F1, and reverse transcription was performed to calculate the relative quantification of the target gene PNPLA3 mRNA in each test group. The difference is that the primer sequences used in this experiment are shown in Table 7F below: Table 7F Primer Information The inhibition rates of each conjugate on PNPLA3 mRNA are shown in Table 8F below: Table 8F shows the inhibition rate of the conjugates disclosed herein against PNPLA3 mRNA. The results in Table 8F show that the siRNA conjugates provided in this disclosure have a significant inhibitory effect on PNPLA3 mRNA in primary monkey liver cells. All tested conjugates showed an inhibition rate of over 73% against PNPLA3 mRNA at a concentration of 10 nM, with conjugates F11 and F13 even achieving an inhibition rate of 86.4%. Even at a low concentration of 0.1 nM, the inhibition rate against PNPLA3 mRNA still reached 67%. Inhibitory activity of F5 conjugate F9-F12 in AAV-hPNPLA3 148M mice This experiment investigated the inhibitory efficiency of conjugates F9, F10, F11, and F12 on hPNPLA3 148M mRNA expression in an AAV-hPNPLA3 148M mouse model. hPNPLA3 148M AAV8 adeno-associated virus was prepared by Shandong Weizhen Biotechnology Co., Ltd. The adeno-associated virus vector pAV-TBG was selected, and the transfected fragment was a 1446 bp sequence (mRNA NCBI reference: NM_025225.3) from the CDS region (108-1553) of the human PNPLA3 148M gene, with the C at position 444 changed to G, resulting in hPNPLA3 148M AAV8. C57BL / 6J mice (all male) were randomly divided into groups of six, and each group was numbered. Each group of mice was administered the test conjugates F9-F12 subcutaneously at a dose of 9 mg / kg (based on siRNA). The siRNA conjugates were provided as a 0.9% sodium chloride aqueous solution containing 1.8 mg / mL of the siRNA conjugate, with an administration volume of 5 mL / kg. One group of mice was given 1×PBS at a volume of 5 mL / kg as the control group. Three days after administration, each mouse was given 1 × 10⁻⁶ hPNPLA3 148MAAV8 adeno-associated virus via tail vein administration. 11 vg. hPNPLA3 148M AAV8 at 5×10 11 The titer is provided in vg / ml, and the administration volume is 200μL / animal. Using the AAV injection time point as day 1, animals were sacrificed on day 8, and liver tissue from each mouse was collected and preserved using RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using Trizol (Sigma Aldrich) according to the manufacturer's instructions. Following the procedure in Experiment F2, the inhibitory activity of PNPLA3 mRNA was measured and calculated. The results are shown in Table 9F below. Table 9 shows the inhibition rates of each F conjugate on the target gene PNPLA3 mRNA. As shown in Table 9F, in the AAV-hPNPLA3 148M mouse model, the conjugates provided in this disclosure all showed good inhibition rates at a siRNA dosage of 9 mg / kg, and the conjugates F9, F11 and F12 could inhibit PNPLA3 mRNA by more than 80%. Inhibitory activity of F6 conjugates F11 and F13 in AAV-hPNPLA3 148M mice This experiment investigated the inhibitory efficiency of conjugates 11 and 13 prepared in the preparation example on the expression of hPNPLA3 148M mRNA in AAV-induced humanized mice. C57BL / 6J mice were randomly divided into two groups (all males): a PBS group of 20 mice and other groups of 15 mice each, and were numbered accordingly. hPNPLA3 148M AAV8 adeno-associated virus was prepared by Shandong Weizhen Biotechnology Co., Ltd., and the plasmid construction method was as described in Experiment F5. Each mouse was administered 1 × 10⁻⁶ hPNPLA3 148M AAV8 adeno-associated virus via tail vein. 11 vg. hPNPLA3 148M AAV8 at 5×10 11 The titer is provided in vg / ml, and the administration volume is 200μL / animal. Fourteen days after AAV injection, two groups of mice were administered the test conjugates F11 and F13 subcutaneously at a dose of 9 mg / kg (based on siRNA). The siRNA conjugates were provided as a 0.9% sodium chloride aqueous solution of 1.8 mg / mL siRNA conjugate, with a dosage of 5 mL / kg per mouse. The test conjugates F11 and F13 were administered subcutaneously to each of the other two groups of mice at a dose of 3 mg / kg (based on siRNA). The siRNA conjugates were provided as a 0.9% sodium chloride aqueous solution of 0.6 mg / mL siRNA conjugate, with a dosage of 5 mL / kg per mouse. One group of mice was given 1×PBS, with each mouse receiving 5 mL / kg of the PBS, serving as a blank control group. Using the drug administration point as Day 1, before administration on Day 1, five animals in the PBS group were sacrificed, and liver tissue from each mouse was collected and preserved using RNA later (Sigma Aldrich). On days 15, 29, and 43 after drug administration, five animals in each group were sacrificed, and liver tissue was collected and preserved using RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using Trizol (Sigma Aldrich) according to the instructions. The inhibitory activity of PNPLA3 mRNA was measured and calculated following the procedure in Experiment F2. The results are shown in Table 10F. Table 10F mouse hPNPLA3 mRNA inhibition rate As shown in Table 10F, all conjugates and dosage combinations exhibited sustained inhibitory effects during the experiment, with inhibition rates of at least 76.4%, and even reaching 91.5% at a prolonged period on day 43. These results indicate that the conjugates disclosed herein have the potential for long-term inhibition of hPNPLA3 mRNA. Inhibitory activity of the F7 conjugate in AAV-hPNPLA3 148M mice Using the same method as in Example F6, the inhibitory efficiency of conjugate F13 on hPNPLA3 148M mRNA expression in AAV-hPNPLA3 148M mice was determined, except that mice were administered conjugate F13 at doses of 1 mg / kg and 3 mg / kg (both calculated as siRNA), respectively. The inhibition rate of hPNPLA3 mRNA in mouse liver tissue was measured on days 15, 29, and 43 post-administration. Following the procedure in Example F2, the inhibitory activity of hPNPLA3 148M mRNA was measured and calculated. The results are shown in Table 11F. Table 1. Inhibition rate of hPNPLA3 148M mRNA in 11F mice According to the results in Table 11F, the dose of the conjugate was positively correlated with the inhibitory effect, with the inhibition rate of the 3 mg / kg group being higher than that of the 1 mg / kg group at all time points. Throughout the experiment, the 1 mg / kg dose group maintained an inhibition rate of 57.6% at D43, while the 3 mg / kg dose group reached 73.0%, both demonstrating sustained inhibitory effects, indicating that the conjugate disclosed in this study has the potential for long-lasting in vivo inhibition of target mRNA levels. Some embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure. It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately. Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A single-stranded oligonucleotide having a length of 16-30 nucleotides, said single-stranded oligonucleotide having a nucleotide composition capable of inhibiting the expression of a target mRNA via an RNAi mechanism; each nucleotide in said single-stranded oligonucleotide is independently modified or unmodified, wherein, At least one nucleotide in the single-stranded oligonucleotide is nucleotide X, and at least one nucleotide is a fluorinated nucleotide. Furthermore, in the 5'-3' direction, the 13th nucleotide of the single-stranded oligonucleotide is a substituted alkoxy-modified nucleotide; the 14th nucleotide of the single-stranded oligonucleotide is nucleotide X; and each of the 15th nucleotide and all subsequent nucleotides of the single-stranded oligonucleotide is independently a modified nucleotide. Each nucleotide X is independently a deoxynucleotide or an unmodified nucleotide.

2. The single-chain oligonucleotide as described in claim 1, wherein, The single-stranded oligonucleotide has a length of 17-28, 19-27, or 20-25 nucleotides; or, the single-stranded oligonucleotide has a length of 19, 21, or 23 nucleotides.

3. The single-stranded oligonucleotide as described in claim 1 or 2, wherein, The number of nucleotides X is 1-3.

4. The single-stranded oligonucleotide according to any one of claims 1-3, wherein, In the single-stranded oligonucleotide, the 12th and 14th nucleotides are each independently nucleotide X in the 5'-3' orientation; or, only the 14th nucleotide is nucleotide X.

5. The single-chain oligonucleotide according to any one of claims 1-4, wherein, The number of unmodified nucleotides does not exceed 3, 2, or 1; or, each nucleotide in the single-stranded oligonucleotide is independently a modified nucleotide.

6. The single-stranded oligonucleotide according to any one of claims 1-5, wherein, The number of fluorinated nucleotides is 2-7.

7. The single-chain oligonucleotide of claim 6, wherein, In the 5'-3' orientation, fluorinated nucleotides refer to 2-5 of the 2nd, 5th, 6th, 7th, 12th, 16th, 18th and 19th nucleotides in the single-stranded oligonucleotide.

8. The single-chain oligonucleotide of claim 7, wherein, In the 5'-3' direction, fluorinated nucleotides refer to one or two of the 2nd and 12th nucleotides, one or two of the 5th-7th nucleotides, and 0-2 of the 16th-19th nucleotides in the single-stranded oligonucleotide; Alternatively, in the 5'-3' orientation, the fluorinated nucleotide refers to the 2nd and 6th nucleotides of the single-stranded oligonucleotide; the 2nd, 6th and 16th nucleotides; the 2nd, 5th, 7th, 12th and 16th nucleotides; the 2nd, 7th, 12th, 16th and 19th nucleotides; or the 2nd, 6th, 12th, 16th and 19th nucleotides.

9. The single-chain oligonucleotide of claim 8, wherein, In the single-stranded oligonucleotide, except for the 13th and 14th nucleotides in the 5'-3' orientation and the fluorinated nucleotides, each modified nucleotide is independently selected from one of alkoxy-modified nucleotides, substituted alkoxy-modified nucleotides, alkyl-modified nucleotides, substituted alkyl-modified nucleotides, amine-modified nucleotides, heat-labile nucleotides, and BNA.

10. The single-chain oligonucleotide of claim 9, wherein, Except for the 13th and 14th nucleotides in the 5'-3' orientation and fluorinated nucleotides, each modified nucleotide is independently selected from alkoxy-modified nucleotides, substituted alkoxy-modified nucleotides, or heat-labile nucleotides, and the number of substituted alkoxy-modified nucleotides does not exceed 3 and the number of heat-labile nucleotides does not exceed 2.

11. The single-chain oligonucleotide of claim 10, wherein, The single-stranded oligonucleotide is 19-23 nucleotides in length, and In the 5'-3' orientation, the 13th nucleotide is a substituted alkoxy-modified nucleotide, the 14th nucleotide is nucleotide X, one of the 5th-7th nucleotides, and the 2nd and 16th nucleotides of the single-stranded oligonucleotide are fluorinated nucleotides, the 3rd nucleotide is an alkoxy-modified nucleotide or a substituted alkoxy-modified nucleotide, and the 5th nucleotide, if not a fluorinated nucleotide, is an alkoxy-modified nucleotide or a substituted alkoxy-modified nucleotide, and each of the remaining nucleotides in the single-stranded oligonucleotide is independently an alkoxy-modified nucleotide.

12. The single-stranded oligonucleotide of claim 11, wherein, The single-stranded oligonucleotide is 21 nucleotides in length, and In the 5'-3' orientation, the 13th nucleotide is a substituted alkoxy-modified nucleotide, the 14th nucleotide is nucleotide X, the 2nd, 6th and 16th nucleotides are fluorinated nucleotides, the 3rd or 5th nucleotide is an alkoxy-modified nucleotide or a substituted alkoxy-modified nucleotide, and each of the remaining nucleotides in the single-stranded oligonucleotide is independently an alkoxy-modified nucleotide.

13. The single-chain oligonucleotide according to any one of claims 9-12, wherein, Each nucleotide X refers to a deoxynucleotide; Each alkoxy-modified nucleotide is a nucleotide modified with an alkoxy group; Each substituted alkoxy-modified nucleotide refers to a nucleotide modified with 2'-O-methoxyethyl; and / or Each thermally unstable nucleotide refers to GNA.

14. The single-stranded oligonucleotide according to any one of claims 1-13, wherein, At least two of the linking groups between adjacent nucleotides in the single-stranded oligonucleotide are phosphate groups with modifying groups.

15. The single-stranded oligonucleotide of claim 14, wherein, One to four linking groups between adjacent nucleotides in the 5' end of the single-stranded oligonucleotide, and / or one to four linking groups between adjacent nucleotides in the 3' end of the single-stranded oligonucleotide, are each independently a phosphate ester group with a modifying group; and / or if unmodified nucleotides are present in the single-stranded oligonucleotide, one or all two linking groups between each unmodified nucleotide and its adjacent nucleotide are independently a phosphate ester group with a modifying group; and / or In the single-stranded oligonucleotide, 2-6, or 3 or 4 of the linking groups between adjacent nucleotides are phosphate ester groups that are each independently modified.

16. The single-stranded oligonucleotide of claim 14 or 15, wherein, Each of the linking groups between adjacent nucleotides in the 5' end of the first to third nucleotides and between adjacent nucleotides in the 3' end of the first to third nucleotides is independently a phosphate ester group with a modifying group; and / or if there are unmodified nucleotides in the single-stranded oligonucleotide, each of the two linking groups between each unmodified nucleotide and its adjacent nucleotide is independently a phosphate ester group with a modifying group.

17. The single-stranded oligonucleotide according to any one of claims 14-16, wherein, Each of the phosphate groups having the modified group is independently a thiophosphate group having the structure shown in formula (28):

18. The single-stranded oligonucleotide according to any one of claims 1-17, wherein, The 5' terminal nucleotide of the single-stranded oligonucleotide is a 5'-hydroxynucleotide, a 5'-phosphate nucleotide, or a 5'-phosphate analog modified nucleotide, wherein the 5'-hydroxynucleotide has the structure shown in formula (29); the 5'-phosphate nucleotide has the structure shown in formula (30); and the 5'-phosphate analog modified nucleotide is selected from one of the nucleotides shown in formulas (31) to (34). R is selected from H, OH, OCH3 and F; Base represents a nucleic acid base, selected from A, U, C, G or T.

19. The single-stranded oligonucleotide according to any one of claims 1-18, wherein, The single-stranded oligonucleotide is substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to a continuous nucleotide sequence m in the target mRNA; the length of the nucleotide sequence m is not greater than the length of the single-stranded oligonucleotide, and the length of the nucleotide sequence m is the same as the length of the single-stranded oligonucleotide, or differs by no more than 8 nucleotides, or differs by 1-5 nucleotides. Alternatively, the length of the nucleotide sequence m is at least 16 nucleotides, or 16-25 nucleotides, or 18-23 nucleotides, or 19-21 nucleotides; Alternatively, the single-stranded oligonucleotide has the same length as the nucleotide sequence m, and at least the nucleotide sequence of the single-stranded oligonucleotide other than the terminal nucleotide is completely anticomplementary to the nucleotide sequence m; Alternatively, in the 5'-3' direction, the nucleotide sequence of the single-stranded oligonucleotide other than the first position is completely anticomplementary to the nucleotide sequence m; or all nucleotides of the single-stranded oligonucleotide are completely anticomplementary to the nucleotide sequence m. Alternatively, the target mRNA may be selected from one of the following mRNAs transcribed from genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HMGCR, HSD, HTT , KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1.

20. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each nucleotide in the sense strand being a modified or unmodified nucleotide, wherein the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region, wherein, The antisense strand is a single-stranded oligonucleotide as described in any one of claims 1-19.

21. The double-stranded oligonucleotide of claim 20, wherein, The length of the positive chain is 15-26, 17-24, or 19-23 nucleotides; Alternatively, the length of the justice chain is 19-21 nucleotides.

22. The double-stranded oligonucleotide of claim 21, wherein, The length difference between the sense and antisense strands is 0-5 nucleotides. Alternatively, the length of the justice chain is not greater than the length of the antisense chain; Alternatively, the sense and antisense strands may be of the same length, either 19, 20, or 21 nucleotides. Alternatively, the length of the sense strand is 19-21 nucleotides, the length of the antisense strand is 20-24 nucleotides, and the length of the antisense strand is 1-3 nucleotides longer than the length of the sense strand; or the length of the antisense strand is 2 nucleotides longer than the length of the sense strand. Alternatively, the length of the sense strand is 19 nucleotides and the length of the antisense strand is 21 nucleotides; or the length of the sense strand is 21 nucleotides and the length of the antisense strand is 21 nucleotides; or the length of the sense strand is 21 nucleotides and the length of the antisense strand is 23 nucleotides.

23. The double-stranded oligonucleotide according to any one of claims 20-22, wherein, In the 3'-5' orientation, 2-3 of the 11th-13th nucleotides of the positive strand are fluorinated nucleotides, the first and / or last nucleotide is an alkoxylated nucleotide or a reverse debased deoxynucleotide, and the remaining nucleotides in the positive strand are non-fluorinated nucleotides. Each non-fluorinated nucleotide is independently selected from one of alkoxylated nucleotides, alkylated nucleotides, amine-modified nucleotides, and heat-labile nucleotides.

24. The double-stranded oligonucleotide of claim 23, wherein, In the 3'-5' direction, the 11th and 13th nucleotides, or the 11th-13th nucleotides, of the positive strand are fluorinated nucleotides, the 1st and / or the last nucleotide is a reverse debased deoxynucleotide, and each nucleotide at the remaining position of the positive strand is independently an alkoxylated nucleotide.

25. The double-stranded oligonucleotide of claim 23 or 24, wherein, Each of the alkoxy-modified nucleotides is independently a methoxy-modified nucleotide.

26. The double-stranded oligonucleotide according to any one of claims 20-25, wherein, In the positive chain, at least one of the linking groups connecting two adjacent nucleotides is a phosphate group with a modifying group, and the phosphate group with the modifying group is present at least once between two adjacent nucleotides in the first to fifth nucleotides at the 5' end of the positive chain and between two adjacent nucleotides in the first to fifth nucleotides at the 3' end.

27. The double-stranded oligonucleotide of claim 26, wherein, One to four, or all four, of the linking groups connecting any two adjacent nucleotides from the first to the fifth nucleotide at the 5' end of the positive strand are independently phosphate groups with a modifying group; and / or, one to four, or all four, of the linking groups connecting any two adjacent nucleotides from the first to the fifth nucleotide at the 3' end of the positive strand are independently phosphate groups with a modifying group; or, each phosphate group with a modifying group is independently a thiophosphate group having the structure shown in formula (28).

28. The double-stranded oligonucleotide according to any one of claims 20-27, wherein, The sense strand contains 19-21 nucleotides, and the antisense strand contains 21-23 nucleotides; In the positive chain, the 11th and 13th nucleotides, or the 11th to 13th nucleotides, are fluorinated nucleotides in the 3'-5' direction, the 1st and / or the last nucleotide is a methoxylated nucleotide or a reverse debased deoxynucleotide, and the nucleotides at the remaining positions are each independently methoxylated nucleotides. Each of 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 5' end of the positive strand, and / or 1-4 of the linking groups between adjacent nucleotides in the 1st-5th nucleotides at the 3' end of the positive strand, is an independently phosphate thioester group.

29. The double-stranded oligonucleotide of claim 28, wherein, The sense strand contains 19 nucleotides, and the antisense strand contains 21 nucleotides; In the positive strand, the 11th and 13th nucleotides, or the 11th to 13th nucleotides, are fluorinated nucleotides in the 3'-5' direction; the 1st and / or the last nucleotide is a reverse debased deoxynucleotide; and the nucleotides at the remaining positions are each independently methoxylated nucleotides. In the positive strand, 1-4 of the linking groups between adjacent nucleotides in the 1st to 5th nucleotides at the 5' end, and / or 1-4 of the linking groups between adjacent nucleotides in the 1st to 5th nucleotides at the 3' end, are each independently a phosphate thioester group. In the antisense strand, in the 5'-3' direction, the 13th nucleotide is a 2'-O-methoxyethyl modified nucleotide, the 14th nucleotide is a deoxynucleotide, and the 2nd, 6th and 16th nucleotides are fluorinated modified nucleotides. Each of the remaining nucleotides in the antisense strand is independently an alkoxy modified nucleotide. The linking groups between any two adjacent nucleotides of the 5' end of the first to third nucleotides of the antisense strand, and between any two adjacent nucleotides of the 3' end of the first to third nucleotides, are independently phosphate groups with modified groups; and if unmodified nucleotides are present, one or two of the linking groups between each unmodified nucleotide and its two adjacent nucleotides are independently phosphate groups with modified groups; and The 5' terminal nucleotide of the antisense strand is a 5'-hydroxy nucleotide as shown in formula (29) or a nucleotide modified with 5'-vinyl phosphate as shown in formula (31).

30. The double-stranded oligonucleotide according to any one of claims 20-29, wherein, The justice chain and the antisense chain are essentially opposite complementary, substantially opposite complementary, or completely opposite complementary. Alternatively, in the 5'-3' direction, at least the nucleotide sequence other than the first and last positions of the sense strand is substantially anticomplementary or completely anticomplementary to the antisense strand; Alternatively, in the 5'-3' direction, the nucleotide sequence of the sense strand except the last nucleotide is completely anticomplementary to the antisense strand; or all nucleotides of the sense strand are completely anticomplementary to the antisense strand. Alternatively, the unmodified equivalent sequence of the positive strand may contain a nucleotide sequence of the same length as nucleotide sequence m and differing by no more than 3 bases, no more than 1 base, or no bases, wherein nucleotide sequence m is a continuous nucleotide sequence in the target mRNA and the length of nucleotide sequence m is at least 16 nucleotides, or 16-25 nucleotides, or 18-23 nucleotides, or 19-21 nucleotides.

31. The double-stranded oligonucleotide of claim 30, wherein, The double-stranded oligonucleotide is siRNA.

32. The double-stranded oligonucleotide according to any one of claims 20-31, wherein, The target mRNA is selected from one of the following mRNAs transcribed from genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HMGCR, HSD, HTT , KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1.

33. An oligonucleotide conjugate comprising an oligonucleotide group and a delivery group conjugated to the oligonucleotide group, wherein the oligonucleotide group is independently formed by removing one or more atoms or groups from a single-stranded oligonucleotide of any one of claims 1-19 or a double-stranded oligonucleotide of any one of claims 20-32.

34. The oligonucleotide conjugate of claim 33, wherein, The delivery group comprises a linker group and a pharmaceutically acceptable target group, and the oligonucleotide group, the linker group and the target group are covalently or non-covalently linked in sequence, each of the target groups being selected from ligands capable of binding to cell surface receptors or groups capable of increasing tissue compatibility.

35. The oligonucleotide conjugate of claim 34, wherein, Each of the said targeting groups independently targets one or more of the central nervous system, liver, kidneys, lungs, muscles, and eyes.

36. A pharmaceutically acceptable salt of a single-stranded oligonucleotide as described in any one of claims 1-19, a double-stranded oligonucleotide as described in any one of claims 20-32, or an oligonucleotide conjugate as described in any one of claims 33-35; Alternatively, the pharmaceutically acceptable salt is a partial or complete water-soluble salt of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate. Alternatively, the water-soluble salt is one or more of an amine salt, an alkali metal salt, or an alkaline earth metal salt; Alternatively, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts, and the alkali metal salt is selected from potassium salts or sodium salts, and the alkaline earth metal salt is selected from sodium salts or magnesium salts; Alternatively, the tertiary amine salt is one or more of triethylamine salt, triisopropylamine salt, or N,N-diisopropylethylamine salt; Alternatively, the pharmaceutically acceptable salt is a salt or a portion of the single-stranded oligonucleotide, the double-stranded oligonucleotide, or the oligonucleotide conjugate, wherein the salt is one or more of a methylamine salt, a triethylamine salt, a sodium salt, a calcium salt, or a magnesium salt.

37. A pharmaceutical composition comprising one or more of the single-stranded oligonucleotide of any one of claims 1-19, the double-stranded oligonucleotide of any one of claims 20-32, the oligonucleotide conjugate of any one of claims 33-35, and the pharmaceutically acceptable salt of claim 36, and a pharmaceutically acceptable excipient; Alternatively, the pharmaceutically acceptable excipient is one or more of solvents, preservatives, osmotic pressure regulators, and other pharmaceutically acceptable carriers; Alternatively, the solvent is one of deionized water, water for injection, pH buffer, physiological saline, ethanol, or an aqueous solution of ethanol.

38. Use of one or more of the single-stranded oligonucleotides of any one of claims 1-19, the double-stranded oligonucleotides of any one of claims 20-32, the oligonucleotide conjugates of any one of claims 33-35, the pharmaceutically acceptable salt of claim 36, and the pharmaceutical composition of claim 37 in the preparation of a medicament for treating and / or preventing diseases or symptoms associated with target mRNA levels.

39. The use as described in claim 38, wherein, The target mRNA is selected from one of the following mRNAs transcribed from genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HMGCR, HSD, HTT, KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1.

40. A method for treating and / or preventing a disease or symptom associated with target mRNA levels, the method comprising administering to a subject in need an effective amount of one or more of the following: a single-stranded oligonucleotide of any one of claims 1-19, a double-stranded oligonucleotide of any one of claims 20-32, an oligonucleotide conjugate of any one of claims 33-35, a pharmaceutically acceptable salt of claim 36, and a pharmaceutical composition of claim 37.

41. The method of claim 40, wherein, The target mRNA is selected from one of the following mRNAs transcribed from genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HMGCR, HSD, HTT, KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1.

42. A method for regulating the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of one or more of the single-stranded oligonucleotide of any one of claims 1-19, the double-stranded oligonucleotide of any one of claims 20-32, the oligonucleotide conjugate of any one of claims 33-35, the pharmaceutically acceptable salt of claim 36, and the pharmaceutical composition of claim 37.

43. The method of claim 42, wherein, The target gene is selected from one of the following genes: ACE2, AGT, ALDH2, ANGPTL3, LPA, ApoB, ApoC3, APP, AR, ASK1, AVPR2, CC3, C5, CFB, Col1A1, CTGF, DUX4, Ebola, EDNRA, EGFR, FASN, FGL1, FOXO1, FTO, FVII, FXI, FXII, GCGR, HCV, HDV, HMGCR, HSD, HTT, KEAP1, KNG, LilrB3, MAPT, MASP2, NTCP, p53, PD-L1, PKK, PLG, PNP, PNPLA3, PSD3, RAGE, RPTOR, SARS-CoV-2, SCD1, SCNN1A, SGLT2, SNCA, SOD1, STAT3, TIMP-1, TMPRSS6, XO, URAT1, URAT2, URATv1.

44. One or more of the following as used as a medicament: the single-stranded oligonucleotide of any one of claims 1-19, the double-stranded oligonucleotide of any one of claims 20-32, the oligonucleotide conjugate of any one of claims 33-35, the pharmaceutically acceptable salt of claim 36, and the pharmaceutical composition of claim 37.

45. A cell expressing a target mRNA, and the cell comprising one or more of the following: a single-stranded oligonucleotide of any one of claims 1-19, a double-stranded oligonucleotide of any one of claims 20-32, an oligonucleotide conjugate of any one of claims 33-35, a pharmaceutically acceptable salt of claim 36, and a pharmaceutical composition of claim 37.

46. ​​A kit comprising one or more of the following: a single-stranded oligonucleotide according to any one of claims 1-19, a double-stranded oligonucleotide according to any one of claims 20-32, an oligonucleotide conjugate according to any one of claims 33-35, a pharmaceutically acceptable salt according to claim 36, and a pharmaceutical composition according to claim 37.

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