Modifications of polynucleic acid molecules and uses thereof

Abstract

Disclosed herein are modifications of polynucleic acid molecules, such as inhibitory polynucleic acid molecules, and pharmaceutical compositions comprising the modified polynucleic acid molecules. In addition, provided herein are methods of utilizing the modified polynucleic acid molecules, as well as methods of repressing expression of a gene targeted by the modified polynucleic acid molecules.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 612,254, filed December 19, 2023; U.S. Provisional Application No. 63 / 619,633, filed January 10, 2024; and U.S. Provisional Application No. 63 / 549,871, filed February 5, 2024, which are incorporated herein by reference in their entirety. Background Technology

[0003] Chemical modifications to repressive polynucleotides (PNUs) are designed and optimized to increase their stability and efficacy, as well as reduce miRNA-like off-target effects. Various modification motifs or patterns with ribose modifications (e.g., 2'- modifications on the ribose moiety), base modifications, and / or nucleotide inter-bond modifications have been designed and tested; however, efforts are underway to develop more modification motifs or patterns that reduce off-target effects without diminishing the efficacy of repressive PNUs, increasing cytotoxicity, or reducing in vivo stability.

[0004] Incorporation

[0005] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is expressly and individually indicated to be incorporated by reference.

[0006] Where any publications and patents or patent applications incorporated herein by reference contradict the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such contradictory material. Summary of the Invention

[0007] To meet the need for more effective modification of polynucleotide molecules, this article discloses, in some respects, modified polynucleotide molecules for regulating target gene expression, wherein the modifications of polynucleotide molecules include nucleotide analogs.

[0008] In one aspect, this disclosure provides a repressive polynucleotide molecule comprising a guest strand (sense strand) and a guide strand (antisense strand), wherein the guide strand comprises a nucleotide analog selected from: acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc). 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0009] On the other hand, this disclosure provides a repressive polynucleotide molecule comprising a guest strand (sense strand) and a guide strand (antisense strand), wherein the guide strand comprises the following formula:

[0010] 5'﹣ (A) n1 -(B) n2 -(C) n3 -(D) n4 -(E) n5 -3'

[0011] in

[0012] A includes one or more nucleotides selected from the following: 2'-O-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-halogen modified nucleotides, DNA, RNA, ENA, BNA, LNA, UNA, and n1 is 1-7.

[0013] B includes one or more nucleotide analogs selected from the following: acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymine... Glycoside-glycol-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and n2 is 1-5;

[0014] C is selected from DNA, RNA, 2'-O-alkyl modified nucleotides and 2'-halogen modified nucleotides and is composed of unmodified nucleotides or nucleotides with the same modification, and n3 is 0-6;

[0015] D is selected from nucleotides modified with 2'-O-alkyl and nucleotides modified with 2'-halogen, including nucleotides that are modified differently from their adjacent nucleotides, and n4 is 4-8; and

[0016] E is selected from nucleotides modified with 2'-O-alkyl, nucleotides modified with 2'-alkoxy, nucleotides modified with 2'-halogen, DNA, RNA, ENA, BNA, LNA, and UNA, and n5 is 3-10.

[0017] In some cases, the repressive polynucleotide molecule is siRNA.

[0018] In some cases, the nucleotide analog is located at the seed region (positions 2-8) of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at any of positions 4-8 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at any of positions 5-8 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 6 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 6 of the guide strand starting from the 5' end.

[0019] In some cases, the nucleotide analog is located at position 6 of the guide strand starting from the 5' end, and the guide strand contains nucleotides modified with 2'-F at at least three of positions 2, 7, 12, 14, and 16 starting from the 5' end.

[0020] In some cases, the nucleotide analog is located at position 7 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 7 of the guide strand starting from the 5' end, and the guide strand contains nucleotides modified with 2'-F at at least three of positions 2, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 7 of the guide strand starting from the 5' end, and the guide strand contains nucleotides modified with 2'-F at positions 2, 12, 14, and 16 starting from the 5' end.

[0021] In some cases, the nucleotide analog is located at position 8 starting from the 5' end of the guide strand. In some cases, the nucleotide analog is located at position 8 starting from the 5' end of the guide strand, and the guide strand contains nucleotides modified with 2'-F at at least three of positions 2, 7, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 8 starting from the 5' end of the guide strand, and the guide strand contains nucleotides modified with 2'-F at positions 2, 7, 12, 14, and 16 starting from the 5' end.

[0022] In some cases, the nucleotide in the guiding strand that is not a nucleotide analog or a 2'-F modified nucleotide is selected from DNA, RNA, and 2'-O-alkyl modified nucleotides. In some cases, the nucleotide in the guiding strand that is not a nucleotide analog or a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

[0023] In some cases, the guiding strand contains at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the guiding strand contains two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end.

[0024] In some cases, the guide strand contains a 5'-mFmmmXFmmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid- 3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl-L-... Threonine-3-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-mFmmmmXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid- 3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).In some cases, the guide strand contains a 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic N-acetyl-L-... Threonine-3-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid- 3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic N-acetyl-L-... Threonine-3-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0025] In some cases, the guest chain contains at least two, at least three, or at least four nucleotides modified with 2'-F. In some cases, the guest chain contains three nucleotides modified with 2'-F. In some cases, the guest chain contains at least one nucleotide modified with 2'-F at positions 7, 9, and 11 starting from the 5' end. In some cases, the guest chain contains nucleotides modified with 2'-F at positions 7, 9, and 11 starting from the 5' end.

[0026] In some cases, the nucleotide in the guest chain that is not a 2'-F modified nucleotide is any one of the following: 2'-O-alkyl modified nucleotide, 2'-alkoxy modified nucleotide, 2'-halogen modified nucleotide, DNA, RNA, ENA, BNA, LNA, and UNA. In some cases, the nucleotide in the guest chain that is not a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

[0027] In some cases, the transfer chain contains at least one phosphate-thioester modified nucleotide bond. In some cases, the transfer chain contains at least one phosphate-thioester modified nucleotide bond at the 5' end. In some cases, the transfer chain contains two phosphate-thioester modified nucleotide bonds at the 5' end.

[0028] In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A). Acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T), and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T). -A), acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A). Acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T), and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T). '-phosphate (T-A), acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A). Acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T), and acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-T). '-phosphate (T-A), acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0029] In some cases, the transit chain is conjugated to the target moiety. In some cases, the target moiety is the desialylate glycoprotein receptor target moiety. In some cases, the desialylate glycoprotein receptor target moiety contains N-acetylgalactosamine (GalNAc) or galactose.

[0030] In some cases, the target portion is joined via a connector. In some cases, the connector comprises the following formula (IV).

[0031] Wherein at least one of Y1 and Y2 is a nucleotide in the polynucleotide molecule. In some cases, the linker of the last nucleotide at the 3' end of the transit chain of the polynucleotide molecule and the desialylated glycoprotein receptor targeting portion are as follows:

[0032] (V')

[0033] (V'''') (V''''') or (V''''''), where Z in formula (V'), (V''''), (V''''') or (V''''') is -H, -OH, -O-methyl, -F or -O-methoxyethyl, and R in formula (V'), (V''''), (V'''') or (V''''') is adenine, uracil, guanine, cytosine, thymine, debasement or other.

[0034] In one aspect, this disclosure provides a method for regulating the mRNA or protein expression level of a target gene in a cell or a desired object, comprising: contacting a cell with or administering an inhibitory polynucleotide molecule described herein to a object, thereby regulating the mRNA or protein expression level of the target gene in the object.

[0035] In another aspect, this disclosure provides a method for inhibiting off-target effects caused by the guide strand of an inhibitory polynucleotide molecule in a cell, comprising introducing the inhibitory polynucleotide molecule described herein into the cell. Attached Figure Description

[0036] Various aspects of this disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by referring to the following detailed description illustrating the principles of this disclosure and the accompanying drawings.

[0037] Figure 1 The in vivo efficacy of modified siRNA targeting AGT mRNA in HuAoGen mice was characterized. Results are shown as relative human AGT mRNA expression on day 35 after siRNA treatment.

[0038] Figure 2 The in vivo efficacy of additionally modified siRNAs targeting AGT mRNA in HuAoGen mice was characterized. Results are shown as relative human AGT mRNA expression on day 35 after siRNA treatment.

[0039] Figure 3 The in vivo efficacy of modified siRNA targeting AGT mRNA in HuAoGen mice was characterized. Results are shown as relative human AGT mRNA expression on day 42 after siRNA treatment.

[0040] Figure 4The in vivo efficacy of modified siRNA targeting AGT mRNA in cynomolgus monkeys was depicted. Results are shown as the change in serum AGT after siRNA treatment on day 1.

[0041] Figure 5 The inhibitory efficacy of the selected ANGPTL3 siRNA used in vivo testing in mice in Example 5 was depicted. Results are shown as the mean % change in serum hANGPTL3 protein levels relative to the mediator control group. These results correspond to the data in Table 10.

[0042] Figure 6 The in vivo efficacy of a modified siRNA conjugated with GalNAc targeting Lp(a) mRNA at a dose of 1 mg / kg was depicted in cynomolgus monkeys. Results are shown as percentage changes in serum Lp(a) protein levels relative to pre-dose (day 1) time points. These results correspond to the data in Table 12.

[0043] Figure 7 The in vivo efficacy of a modified siRNA conjugated with GalNAc targeting Lp(a) mRNA at a dose of 0.5 mg / kg was depicted in cynomolgus monkeys. Results are shown as percentage changes in serum Lp(a) protein levels relative to pre-dose (day 1) time points. These results correspond to the data in Table 14.

[0044] Figure 8 The in vivo efficacy of a modified siRNA targeting Lp(a) mRNA at a dose of 0.5 mg / kg was depicted in cynomolgus monkeys. Results are shown as percentage changes in serum Lp(a) protein levels relative to pre-dose (day 1) time points. These results correspond to the data in Table 16.

[0045] Figure 9 The in vivo efficacy of modified siRNA targeting ApoC3 mRNA at a dose of 0.5 mg / kg was characterized in transgenic mice. Results are shown as plasma hApoC3 protein levels relative to the mediator group. These results correspond to the data in Table 18. Detailed Implementation

[0046] Polynucleotide molecules, such as repressive polynucleotide molecules, can be used to repress the expression of target genes. For example, in some cases, repressive polynucleotide molecules, such as short interfering RNA (siRNA), can function in the RNA interference (RNAi) pathway. Short interfering RNA, or siRNA, is a double-stranded synthetic RNA containing two RNA strands: an antisense (or guide) strand and a sense (or guest) strand. These two RNA strands can form a double-stranded siRNA, and in some cases, one or both strands have overhangs at one end. When siRNA is present in a cell, siRNA-induced gene silencing is initiated by the assembly of an RNA-induced silencing complex (RISC), which contains the guide strand and other proteins, such as enzymes. The guide strand directs the RISC to the complementary sequence region of the target RNA, which in some cases triggers the cleavage of the phosphodiester bond between two nucleotides on the target RNA, generating RNA fragments that are further degraded by cellular enzymes, such as exonucleases.

[0047] Chemical modifications to repressive polynucleotide molecules are designed and optimized to increase their stability and efficacy, as well as reduce miRNA-like off-target effects. In one aspect, this disclosure provides modifications to the polynucleotide molecules described herein by incorporating nucleotide analogs into them. In some cases, the polynucleotide molecule includes a repressive polynucleotide molecule. In some cases, the repressive polynucleotide molecule includes siRNA. In some cases, the repressive polynucleotide molecule includes a single-stranded repressive polynucleotide molecule. In some cases, the single-stranded repressive polynucleotide molecule is an antisense oligonucleotide (ASO). In some cases, the repressive polynucleotide molecule includes a double-stranded repressive polynucleotide molecule. In some cases, the double-stranded repressive polynucleotide molecule contains a guiding strand (antisense strand) or a guest strand (sense strand).

[0048] In some cases, modification of polynucleotide molecules can inhibit off-target effects caused by the guide strand (or antisense strand) of repressive polynucleotide molecules. In some cases, modification of polynucleotide molecules can improve their stability. In some cases, modification of polynucleotide molecules can improve their efficacy. In some cases, modification of polynucleotide molecules can inhibit off-target effects without impairing the efficacy of repressive polynucleotide molecules. In some cases, modification of polynucleotide molecules can inhibit off-target effects without increasing the cytotoxicity of repressive polynucleotide molecules. In some cases, modification of polynucleotide molecules can inhibit off-target effects without reducing the stability of repressive polynucleotide molecules.

[0049] In some respects, this document provides conjugates of the repressive polynucleotide molecules described herein. In some cases, the repressive polynucleotide molecule is coupled to one or more targeting moieties that target or deliver the polynucleotide molecule to preferred tissues, organs, or cells within a target body. In some cases, the targeting moieties include a galactose moiety. In some cases, the targeting moieties include a GalNAc moieties. In some cases, the targeting moieties are conjugated or coupled to the repressive polynucleotide molecule via a linker.

[0050] In another aspect, this disclosure provides a method for modulating the expression levels of target mRNAs or proteins in cells or desired subjects by contacting or administering the polynucleotide molecules described herein to cells or subjects. Furthermore, this disclosure provides a method for suppressing off-target effects caused by the guide strand of the inhibitory polynucleotide molecules described herein. In another aspect, this disclosure provides a method for improving the stability of the inhibitory polynucleotide molecules described herein. In yet another aspect, this disclosure provides a method for improving the efficacy of the inhibitory polynucleotide molecules described herein.

[0051] definition

[0052] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references. For example, the term “cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0053] When a range of values ​​is provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other stated or intermediate value within the stated range (unless otherwise expressly specified by the context, one-tenth of a unit down to the lower limit) is included within this disclosure. The upper and lower limits of these smaller ranges may be independently included within that smaller range and are also included within this disclosure, but are affected by any limits specifically excluded from the stated range. When a stated range includes one or both limits, the range excluding any one or both of the included limits is also included in this disclosure.

[0054] Certain ranges are presented herein with numerical values ​​preceded by the term "about". The term "about" is used herein to provide literal support for the precise figures that follow, as well as figures that are close to or approximate to the figures that follow the term. In determining whether a figure is close to or approximates a specifically recorded figure, a close to or approximate unrecorded figure may be a figure that provides a substantially equal value to the specifically recorded figure in the context in which it is presented.

[0055] The “percentage of sequence identity (%)” or “percentage of identity (%)” for the nucleic acid sequences identified in this paper is defined as the percentage of nucleic acids in the candidate sequences that are identical to the compared nucleic acid sequence after sequence alignment in which any conserved substitutions are considered part of sequence identity.

[0056] All scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any enumerated scope may be considered sufficient to describe and implement the same scope being divided into at least two, three, four, five, ten, etc., parts. As a non-limiting example, each scope discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all terms such as “at most,” “at least,” “greater than,” “less than,” etc., include the stated numbers and refer to a scope that can subsequently be divided into subscopes as discussed above. Finally, those skilled in the art will understand that a scope includes each individual member. Thus, for example, a group having 1–3 items means a group having 1, 2, or 3 items. Similarly, a group having 1–5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which polynucleotide molecules, polynucleotide conjugates, pharmaceutical compositions, methods and other aspects pertain.

[0058] As used in this article, the term “complementary” indicates a sufficient degree of complementarity between two nucleic acid molecules that bind stably and specifically to avoid nonspecific binding.

[0059] As used herein, the terms “polynucleotide” and “polynucleotide” are used interchangeably to refer to nucleotide chains. The term “nucleotide” includes the sequences “G,” “C,” “A,” “T,” and “U,” each typically representing a nucleotide containing guanine, cytosine, adenine, thymidine, and uracil as a base. In some cases, “nucleotide” can refer to a modified nucleotide (e.g., having a modified sugar moiety, a modified base, a modified internucleotide bond, or a combination thereof, including but not limited to 2'-modified nucleotides, LNA, ENA, BNA, UNA, GNA, etc.). In some cases, “nucleotide” can refer to a modified nucleotide having unconventional bases (e.g., including but not limited to 2-thiouridine, 2-thiothymidine, inosine, 2-aminopurine, 2,6-diaminopurine, dihydrouridine, 4-thiouridine, 4-thiothymidine, 2-thiocytidine).

[0060] As described herein, "phosphoramidites" or "amidites" refers to building blocks used in the chemical synthesis of oligonucleotides, such as the polynucleotide molecules described herein. In the synthesis of oligonucleotides, phosphoramidites are used in a series of chemical reactions to produce nucleotide chains or polynucleotide molecules.

[0061] As used in this article, “object” can be any mammal, including humans and non-human primates.

[0062] As used herein, the term "treatment" for any disease or condition refers in one context to improving the disease or condition (i.e., slowing or halting or reducing the development of the disease or at least one of its clinical symptoms). In another context, "treatment" refers to reducing or improving at least one bodily parameter, including those that the patient may not be aware of. In yet another context, "treatment" refers to regulating the disease or condition physically (e.g., stabilizing noticeable symptoms), physiologically (e.g., stabilizing bodily parameters), or both.

[0063] As used herein, the term "prevention" refers to reducing the occurrence of pathological changes in an object that does not have a disease or condition but is at risk of developing one or is susceptible to it. Prevention can be complete, for example, the complete absence of pathological changes in the object. Prevention can also be partial, such that the occurrence of pathological changes in the object is less than the pathological changes that would occur without this disclosure.

[0064] As used herein, the term "administration" and its grammatical equivalents can refer to the administration of the pharmaceutical composition described herein to a subject or patient. The composition may be administered to a subject using conventional methods known to those skilled in the art of medicine, depending on the type or site of the disease to be treated. For example, the composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, via an implantable reservoir, or via infusion. One or more of these routes may be used.

[0065] As used herein, the term "pharmaceutical composition" and its grammatical equivalents may refer to a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient and one or more pharmaceutically acceptable excipients, carriers, and / or therapeutic agents to be administered to a subject of need (e.g., a human).

[0066] As used herein, the term "pharmaceutically acceptable" and its grammatical equivalents can refer to the properties of materials that are generally safe and non-toxic, neither biologically nor otherwise adverse, and acceptable for veterinary and human pharmaceutical use. "Pharmaceutically acceptable" can refer to materials such as carriers or diluents that do not eliminate the biological activity or properties of the compound and are relatively non-toxic, meaning that the material can be administered to a patient without causing adverse biological effects or interacting harmfully with any component of a pharmaceutical composition containing the material.

[0067] "Pharmaceutical acceptable excipients" are excipients that can be applied to the target body together with the agent without destroying the agent's pharmacological activity and are non-toxic when applied in a dose sufficient to deliver a therapeutic amount of the agent.

[0068] The term "therapeutic agent" can refer to any agent that, when administered to a subject, has a therapeutic, diagnostic, and / or preventative effect, and / or induces the desired biological and / or pharmacological effect. Therapeutic agents may also be referred to as "active substances" or "active agents." Such agents include, but are not limited to, cytotoxic agents, radioactive ions, chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids.

[0069] As used herein, the term "sense strand" is used interchangeably with the term "passenger strand," and the term "antisense strand" is used interchangeably with the term "guide strand." In some cases, the nucleic acid sequences described herein for sense strands and passenger strands are used interchangeably. Furthermore, in some cases, the nucleic acid sequences described herein for antisense strands and guide strands are used interchangeably.

[0070] As used herein, the term "continuous sequence" refers to a sequence containing multiple consecutive nucleotides derived from a reference sequence. For example, if the reference sequence is N1N2N3N4N5N6N7, the continuous sequence can be N1N2N3N4 or N3N4N5N6, but the sequence N1N3N4N5 or N3N4N7 cannot be a continuous sequence.

[0071] As used in this article, the term "negative control" refers to a subject or cell that did not receive treatment or a placebo.

[0072] It should be understood that certain features of polynucleotide molecules and / or polynucleotide conjugates, pharmaceutical compositions comprising polynucleotide molecules or polynucleotide conjugates, methods, and other aspects described for clarity in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, various features of polynucleotide molecules and / or polynucleotide conjugates, pharmaceutical compositions comprising polynucleotide molecules or polynucleotide conjugates, methods, and other aspects described for brevity in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments are specifically included in this disclosure and are disclosed herein as if each combination were disclosed individually and explicitly, to the extent that such combinations encompass operable processes and / or compositions. Furthermore, all sub-combinations listed in embodiments describing such variables are also specifically included in this disclosure of polynucleotide molecules and / or polynucleotide conjugates, pharmaceutical compositions comprising polynucleotide molecules or polynucleotide conjugates, methods, and other aspects, and are disclosed herein as if each such sub-combination were disclosed herein as if each such sub-combination were disclosed herein individually and explicitly.

[0073] Nucleotide analogues

[0074] In some respects, this disclosure provides an inhibitory polynucleotide molecule incorporating nucleotide analogs.

[0075] In some cases, modification of nucleotides with the nucleotide analogs described herein can alter base pairing and structural changes in repressive polynucleotide molecules. In some cases, nucleotide analogs can be incorporated into repressive polynucleotide molecules, thereby inhibiting off-target effects. In some cases, nucleotide analogs can be incorporated into repressive polynucleotide molecules, thereby improving the stability and / or efficacy of the polynucleotide molecule. In some cases, the nucleotide analogs described herein can be incorporated into the guide strand, the transit strand, or a combination thereof.

[0076] In some cases, nucleotide analogs can be placed within repressive polynucleotide molecules, or can be substitutes for nucleotides in repressive polynucleotide molecules, thereby inhibiting off-target effects. In some cases, nucleotide analogs can replace nucleotides in repressive polynucleotide molecules, thereby improving the stability and / or efficacy of the repressive polynucleotide molecules. In some cases, the nucleotide analogs described herein can be placed in the guide strand, the transit strand, or both.

[0077] In some aspects, this disclosure provides a repressive polynucleotide molecule comprising a guest strand (sense strand) and a guide strand (antisense strand), wherein the guide strand comprises a nucleotide analog as described herein. In some cases, nucleotide analogs include acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), and 2'-fluoro-2-thiouridine-3'-phosphate (U3f). 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2), 2',3'-open-cyclic uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), or 2'-O-methylinosine-3'-phosphate (i). In some cases, the nucleotide analogue is selected from the following nucleotide analogues: acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate. Acid (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2), 2',3'-open-cyclic uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).In some cases, nucleotide analogs are selected from the following nucleotide analogs: acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glyoxalool... GNA S-isomers (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0078] In some cases, acyclic L-threonine nucleic acids-3'-phosphate (T-T) have a general representation as shown below, where the base can be any suitable base or modified base that can bind to a base on the opposite strand in a Watson-Crick-like manner:

[0079] .

[0080] In some cases, the phosphoramidite structure of acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is shown below:

[0081] .

[0082] In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is incorporated into repressive polynucleotide molecules. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is incorporated into siRNA. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is incorporated into the guide strand. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is incorporated into the transit strand. In some cases, the incorporated acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) has the structure shown below:

[0083] .

[0084] In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) is incorporated into repressive polynucleotide molecules. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) replaces one or more nucleotides in the siRNA. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) replaces one or more nucleotides in the guest strand. In some cases, acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) replaces one or more nucleotides in the guide strand.

[0085] In some cases, the phosphoramidite structure of acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is shown below:

[0086] .

[0087] In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is incorporated into repressive polynucleotide molecules. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is incorporated into siRNA. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is incorporated into the guide strand. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is incorporated into the transit strand. In some cases, the incorporated acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) has the structure shown below:

[0088] .

[0089] In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) is incorporated into repressive polynucleotide molecules. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) replaces one or more nucleotides in the siRNA. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) replaces one or more nucleotides in the guest strand. In some cases, acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A) replaces one or more nucleotides in the guide strand.

[0090] In some cases, the phosphoramidite structure of acyclic N-acetyl-L-threonol debased nucleic acid -3'-phosphate (T-NAc) is shown below:

[0091] .

[0092] In some cases, acyclic N-acetyl-L-threonol abasic nucleic acid 3'-phosphate (T-NAc) is incorporated into repressive polynucleotide molecules. In some cases, acyclic N-acetyl-L-threonol abasic nucleic acid 3'-phosphate (T-NAc) is incorporated into siRNA. In some cases, acyclic N-acetyl-L-threonol abasic nucleic acid 3'-phosphate (T-NAc) is incorporated into the guide strand. In some cases, acyclic N-acetyl-L-threonol abasic nucleic acid 3'-phosphate (T-NAc) is incorporated into the transit strand. In some cases, the incorporated acyclic N-acetyl-L-threonol abasic nucleic acid 3'-phosphate (T-NAc) has the structure shown below:

[0093] .

[0094] In some cases, acyclic N-acetyl-L-threonol debased nucleic acid (3'-phosphate) (T-NAc) is incorporated into repressive polynucleotide molecules. In some cases, acyclic N-acetyl-L-threonol debased nucleic acid (3'-phosphate) (T-NAc) replaces one or more nucleotides in the siRNA. In some cases, acyclic N-acetyl-L-threonol debased nucleic acid (3'-phosphate) (T-NAc) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, acyclic N-acetyl-L-threonol debased nucleic acid (3'-phosphate) (T-NAc) replaces one or more nucleotides in the guest strand. In some cases, acyclic N-acetyl-L-threonol debased nucleic acid (3'-phosphate) (T-NAc) replaces one or more nucleotides in the guide strand.

[0095] In some cases, the phosphorous amide structure of 1',2'-dideoxyribose-3'-phosphate (dAB) is shown below:

[0096] .

[0097] In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) is incorporated into repressive polynucleotide molecules. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) is incorporated into siRNA. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) is incorporated into the guide strand. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) is incorporated into the transit strand. In some cases, the incorporated 1',2'-dideoxyribose-3'-phosphate (dAB) has the structure shown below:

[0098] .

[0099] In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) is incorporated into repressive polynucleotide molecules. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) replaces one or more nucleotides in siRNA. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) replaces one or more nucleotides in the guest strand. In some cases, 1',2'-dideoxyribose-3'-phosphate (dAB) replaces one or more nucleotides in the guide strand.

[0100] In some cases, the phosphoramide structure of the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is shown below:

[0101] .

[0102] In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into repressive polynucleotide molecules. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into siRNA. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into the director strand. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is incorporated into the transit strand. In some cases, the incorporated thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) has the structure shown below:

[0103] .

[0104] In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) is placed in the repressive polynucleotide molecule. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) replaces one or more nucleotides in the siRNA. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) replaces one or more nucleotides in the guest strand. In some cases, the thymidine-glycol nucleic acid (GNA) S-isomer (Tgn) replaces one or more nucleotides in the guide strand.

[0105] In some cases, the phosphoramide structure of 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is shown below:

[0106] .

[0107] In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is incorporated into repressive polynucleotide molecules. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is incorporated into siRNA. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is incorporated into the guide strand. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is incorporated into the transit strand. In some cases, the incorporated 2'-O-methyl-2-thiouridine-3'-phosphate (u3) has the structure shown below:

[0108] .

[0109] In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) is incorporated into the repressive polynucleotide molecule. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) replaces one or more nucleotides in the siRNA. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) replaces one or more nucleotides in the guest strand. In some cases, 2'-O-methyl-2-thiouridine-3'-phosphate (u3) replaces one or more nucleotides in the guide strand.

[0110] In some cases, the phosphoramidic structure of 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is shown below:

[0111] .

[0112] In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is incorporated into repressive polynucleotide molecules. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is incorporated into siRNA. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is incorporated into the director strand. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is incorporated into the transit strand. In some cases, the incorporated 2'-fluoro-2-thiouridine-3'-phosphate (U3f) has the structure shown below: .

[0113] In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) is incorporated into repressive polynucleotide molecules. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) replaces one or more nucleotides in the siRNA. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) replaces one or more nucleotides in the guest strand. In some cases, 2'-fluoro-2-thiouridine-3'-phosphate (U3f) replaces one or more nucleotides in the guide strand.

[0114] In some cases, the phosphoramidic structure of 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is shown below:

[0115] .

[0116] In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is incorporated into repressive polynucleotide molecules. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is incorporated into siRNA. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is incorporated into the director strand. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is incorporated into the transit strand. In some cases, the incorporated 2-amino-2'-O-methyladenosine-3'-phosphate (a1) has the structure shown below: .

[0117] In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) is placed in the repressive polynucleotide molecule. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) replaces one or more nucleotides in the siRNA. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) replaces one or more nucleotides in the guest strand. In some cases, 2-amino-2'-O-methyladenosine-3'-phosphate (a1) replaces one or more nucleotides in the guide strand.

[0118] In some cases, the phosphoramidite structure of the C3﹣ spacer (Pr) is shown below:

[0119] .

[0120] In some cases, the C3-spacer (Pr) is incorporated into polynucleotide molecules. In some cases, the C3-spacer (Pr) is incorporated into siRNA. In some cases, the C3-spacer (Pr) is incorporated into the guide strand. In some cases, the C3-spacer (Pr) is incorporated into the transit strand. In some cases, the incorporated C3-spacer (Pr) has the structure shown below:

[0121] .

[0122] In some cases, the C3-spacer (Pr) is placed within a polynucleotide molecule. In some cases, the C3-spacer (Pr) replaces one or more nucleotides in the siRNA. In some cases, the C3-spacer (Pr) replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, the C3-spacer (Pr) replaces one or more nucleotides in the guest strand. In some cases, the C3-spacer (Pr) replaces one or more nucleotides in the guide strand.

[0123] In some cases, 2',3'-octane adenosine-3'-phosphate (A2), 2',3'-octane guanosine-3'-phosphate (G2), 2',3'-octane cytidine-3'-phosphate (C2), and 2',3'-octane uridine-3'-phosphate (U2) are shown below:

[0124] (A2) (G2) (C2) (U2).

[0125] In some cases, A2, G2, C2, or U2 are incorporated into polynucleotide molecules. In some cases, A2, G2, C2, or U2 are incorporated into siRNA. In some cases, A2, G2, C2, or U2 are incorporated into the guide strand. In some cases, A2, G2, C2, or U2 are incorporated into the transit strand. In some cases, the incorporated A2, G2, C2, or U2 has the structure shown below:

[0126] (A2) (G2)

[0127] (C2) (U2).

[0128] In some cases, A2, G2, C2, or U2 is placed in a polynucleotide molecule. In some cases, A2, G2, C2, or U2 replaces one or more nucleotides in the siRNA. In some cases, A2, G2, C2, or U2 replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, A2, G2, C2, or U2 replaces one or more nucleotides in the guest strand. In some cases, A2, G2, C2, or U2 replaces one or more nucleotides in the guide strand.

[0129] In some cases, the phosphoramidic structures of 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3) and 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) are shown below:

[0130] (dA3) (dT3).

[0131] In some cases, 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3) or 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) is incorporated into polynucleotide molecules. In some cases, dA3 or dT3 is incorporated into siRNA. In some cases, dA3 or dT3 is incorporated into the guide strand. In some cases, dA3 or dT3 is incorporated into the transit strand. In some cases, the incorporated dA3 or dT3 has the structure shown below:

[0132] (dA3) (dT3).

[0133] In some cases, dA3 or dT3 is placed in polynucleotide molecules. In some cases, dA3 or dT3 replaces one or more nucleotides in siRNA. In some cases, dA3 or dT3 replaces one or more nucleotides in the guest strand and / or the guide strand. In some cases, dA3 or dT3 replaces one or more nucleotides in the guest strand. In some cases, dA3 or dT3 replaces one or more nucleotides in the guide strand.

[0134] In some cases, nucleotide analogs include nucleosides containing inosine nucleobases (e.g., inosine). In some cases, nucleotide analogs contain 2'-O-methylinosine-3'-phosphate (“i”).

[0135] In some aspects, this disclosure provides a repressive polynucleotide molecule comprising a guest strand (sense strand) and a guide strand (antisense strand). In some cases, the guide strand comprises the following formula:

[0136] 5'﹣ (A) n1 -(B) n2-(C) n3 -(D) n4 -(E) n5 -3'

[0137] Wherein A includes one or more nucleotides selected from the following: 2'-O-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-halogen modified nucleotides, DNA, RNA, ENA, BNA, LNA, UNA, and in some cases, n1 is 1, 2, 3, 4, 5, 6 or 7 (1-7).

[0138] B comprises one or more nucleotide analogs selected from the following: said nucleotide analogs are selected from acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino -2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2), 2',3'-open-cyclic uridine -3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i), in some cases, n2 is 1, 2, 3, 4, or 5 (1-5);

[0139] C is selected from DNA, RNA, 2'-O-alkyl modified nucleotides and 2'-halogen modified nucleotides and is composed of unmodified or identically modified nucleotides. In some cases, n3 is 0, 1, 2, 3, 4, 5 or 6 (0-6).

[0140] D is selected from nucleotides modified with 2'-O alkyl and 2'-halogen, and includes nucleotides modified differently from their adjacent nucleotides. In some cases, n4 is 4, 5, 6, 7, or 8 (4-8); and

[0141] E is selected from 2'-O-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-halogen modified nucleotides, DNA, RNA, ENA, BNA, LNA, UNA, and in some cases, n5 is 3, 4, 5, 6, 7, 8, 9 or 10 (3-10).

[0142] In some cases, B includes one or more nucleotide analogs selected from: acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glyoxalool... GNA S-isomers (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0143] In some cases, n1 is 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, or 6 to 7. In other cases, n1 is 1, 2, 3, 4, 5, 6, or 7.

[0144] In some cases, n² is 1 to 5, 2 to 5, 3 to 5, or 4 to 5.

[0145] In some cases, n3 is 0 to 6, 1 to 6, 2 to 6, 3 to 6, 4 to 6, or 5 to 6.

[0146] In some cases, n4 is 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8.

[0147] In some cases, n5 is 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, or 9 to 10.

[0148] Modification of multiple nucleic acid molecules

[0149] In some cases, the repressive polynucleotide molecule is modified at one or more different structures of the polynucleotide molecule described herein (e.g., modifications to the sugar ring, backbone, or internucleotide bonds, or bases). In some cases, the repressive polynucleotide molecule contains modified nucleotides, which are nucleotides with modifications to the sugar ring, backbone, or internucleotide bonds and / or bases. In some cases, the repressive polynucleotide molecule is further modified to incorporate the nucleotide analogs described herein. In some cases, the repressive polynucleotide molecule contains modified nucleotides, which are nucleotides with modifications to the sugar ring, backbone, or internucleotide bonds and / or bases, and the nucleotide analogs described herein. In some cases, the modifications described herein involve incorporating the nucleotide analogs described herein into the repressive polynucleotide molecule. In some cases, the modifications described herein involve replacing one or more nucleotides in the repressive polynucleotide molecule with the nucleotide analogs described herein.

[0150] In some respects, repressive polynucleotide molecules are single-stranded nucleic acid molecules that hybridize with certain regions of target RNA (e.g., mRNA or lncRNA). In some respects, repressive polynucleotide molecules are double-stranded nucleic acid molecules. In some cases, repressive polynucleotide molecules are siRNAs. In some cases, repressive polynucleotide molecules contain a guide strand and a transit strand, wherein the guide strand hybridizes with certain regions of the target RNA.

[0151] In some respects, the repressive polynucleotide molecules described herein are 100% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are not 100% complementary to the target region of the target RNA. In some cases, the repressive polynucleotide molecules described herein are approximately 95% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are approximately 90% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are approximately 85% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are approximately 80% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are approximately 75% complementary to the target region of the target RNA. In some respects, the repressive polynucleotide molecules described herein are approximately 70% complementary to the target region of the target RNA.

[0152] In some respects, the repressive polynucleotide molecules described herein comprise lengths of about 15–30, 16–30, 17–30, 18–30, 18–27, 18–25, 18–23, 19–23, 20–23, or 21–23 nucleotides. In some respects, the repressive polynucleotide molecules described herein comprise lengths of about 15, 16, 17, 18, 19, or 20 nucleotides. In some respects, the repressive polynucleotide molecules described herein comprise lengths of about 21, 22, 23, 24, or 25 nucleotides. In some respects, the repressive polynucleotide molecules described herein comprise lengths of about 26, 27, 28, 29, or 30 nucleotides. In some respects, the repressive polynucleotide molecules described herein comprise lengths of 19 nucleotides. In some respects, the repressive polynucleotide molecules described herein comprise lengths of 21 nucleotides. In some respects, the repressive polynucleotide molecule described in this article contains 23 nucleotides in length.

[0153] In some respects, repressive polynucleotide molecules are double-stranded and, in some cases, contain a guide strand and a guest strand. In some cases, the guide strand (or antisense strand) described herein is 100% complementary to the target region of the target RNA. In some respects, the guide strand described herein is not 100% complementary to the target region of the target RNA. In some cases, the guide strand described herein is approximately 95% complementary to the target region of the target RNA. In some respects, the guide strand described herein is approximately 90% complementary to the target region of the target RNA. In some respects, the guide strand described herein is approximately 85% complementary to the target region of the target RNA. In some respects, the guide strand described herein is approximately 80% complementary to the target region of the target RNA. In some respects, the guide strand described herein is approximately 75% complementary to the target region of the target RNA. In some respects, the guide strand described herein is approximately 70% complementary to the target region of the target RNA.

[0154] In some aspects, the repressive polynucleotide molecules described herein comprise a transit chain and a guide chain of approximately 15–30, 16–30, 17–30, 18–30, 18–27, 18–25, 18–23, 19–23, 20–23, or 21–23 nucleotides in length. In some aspects, the repressive polynucleotide molecules described herein comprise a transit chain and a guide chain of approximately 15, 16, 17, 18, 19, or 20 nucleotides in length. In some aspects, the repressive polynucleotide molecules described herein comprise a transit chain and a guide chain of approximately 21, 22, 23, 24, or 25 nucleotides in length. In some aspects, the repressive polynucleotide molecules described herein comprise a transit chain and a guide chain of approximately 26, 27, 28, 29, or 30 nucleotides in length. In some respects, the repressive polynucleotide molecule described herein comprises a 19-nucleotide-long transit chain and a guide chain of approximately 21 nucleotides. In other respects, the repressive polynucleotide molecule described herein comprises a 21-nucleotide-long transit chain and a guide chain of approximately 23 nucleotides.

[0155] In some aspects, the transit chain and the guiding chain described herein are anti-complementary to each other, forming a double chain with a 3' protrusion on the guiding chain. In some aspects, the transit chain and the guiding chain described herein are anti-complementary to each other, forming a double chain with a 5' protrusion on the guiding chain. In some aspects, the transit chain and the guiding chain described herein are anti-complementary to each other, forming a double chain with a 3' protrusion on the transit chain. In some aspects, the transit chain and the guiding chain described herein are anti-complementary to each other, forming a double chain with a 5' protrusion on the transit chain.

[0156] In some respects, polynucleotide molecules are modified with the nucleotide analogs described herein by incorporating nucleotide analogs into the seed region of the guiding strand (positions 2–8 of the guiding strand from the 5' end). As described herein, a “seed region” refers to a region on a polynucleotide molecule containing sequences essential for the binding of the polynucleotide molecule described herein to the target RNA.

[0157] In some cases, polynucleotide molecules are modified with nucleotide analogs described herein at positions 3-8, 4-8, 5-8, 6-8, or 7-8 starting from the 5' end of the guiding strand. In some cases, polynucleotide molecules are modified with nucleotide analogs described herein at positions 2, 3, 4, 5, 6, 7, 8, or combinations thereof starting from the 5' end of the guiding strand. In some cases, polynucleotide molecules are modified with one, two, three, four, five, six, or seven nucleotide analogs. In some cases, two or more consecutive positions in the guiding strand of the polynucleotide molecule are modified with nucleotide analogs. In some cases, two or more nucleotide analog modifications may be disposed in alternating positions (e.g., positions 3 and 5, positions 4 and 6, positions 5 and 7, etc.) in the polynucleotide molecule.

[0158] In some cases, polynucleotide molecules include repressive polynucleotide molecules. In some cases, repressive polynucleotide molecules are siRNAs containing a guide strand and a guest strand. In some cases, the nucleotide analog is located in the guide strand seed region at positions 2-8 from the 5' end. In some cases, the nucleotide analog is located at positions 3-8 from the 5' end. In some cases, the nucleotide analog is located at positions 4-8 from the 5' end. In some cases, the nucleotide analog is located at positions 5-8 from the 5' end. In some cases, the nucleotide analog is located at positions 6-8 from the 5' end. In some cases, the nucleotide analog is located at positions 6-7 from the 5' end. In some cases, the nucleotide analog is located at positions 7-8 from the 5' end. In some cases, the nucleotide analog is located at positions 10-14, 10-13, 11-14, or 11-13 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at position 12 of the guiding strand starting from the 5' end. In some cases, at least one nucleotide analog is located in the guiding strand seed region at positions 2-8 starting from the 5' end, and another nucleotide analog is located at positions 10-14, 10-13, 11-14, or 11-13 starting from the 5' end of the guiding strand. In some cases, at least one nucleotide analog is located in the guiding strand seed region at positions 6-8 starting from the 5' end, and another nucleotide analog is located at position 12 of the guiding strand starting from the 5' end. In some cases, one nucleotide analog is located in the guiding strand seed region at position 7 starting from the 5' end, and another nucleotide analog is located at position 12 of the guiding strand starting from the 5' end.

[0159] In some cases, the nucleotide analog is located at any of positions 2-8 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 3-8 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 4-8 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 5-8 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 6-8 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 6-7 from the 5' end of the guide strand. In some cases, the nucleotide analog is located at any of positions 7-8 from the 5' end of the guide strand.

[0160] In some cases, the nucleotide analog is located at position 1 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 2 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 3 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 4 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 5 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 6 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 7 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 9 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 10 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 11 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 12 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 13, starting from the 5' end of the guide strand. In other cases, the nucleotide analog is located at position 14, starting from the 5' end of the guide strand.

[0161] Sugar-modified nucleotides

[0162] In some cases, the sugar-modified nucleotide includes a modification at the 2' hydroxyl group of the ribose moiety. In some cases, the sugar-modified nucleotide includes a modification using H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. In some aspects, the sugar-modified nucleotide includes 2'-O-alkyl-modified nucleotides (e.g., 2'-O-methyl-modified nucleotides). In some aspects, the sugar-modified nucleotide is a 2'-O-methyl-modified nucleotide or a 2'-alkoxy-modified nucleotide (e.g., 2'-methoxy-modified nucleotide). In some cases, 2'-hydroxyl modification includes 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In some cases, the alkyl moiety contains heterosubstitution. In some cases, the carbon atom of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some aspects, the sugar-modified nucleotide is a 2'-amino-modified nucleotide. In some aspects, the sugar-modified nucleotide is a 2'-azido-modified nucleotide. In some respects, the sugar-modified nucleotide is a 2'-deoxy-modified nucleotide. In some respects, the sugar-modified nucleotide is 2'-O-methoxyethyl (2'-MOE). In some respects, the sugar-modified nucleotide is a locked nucleic acid (LNA). In some respects, the sugar-modified nucleotide is a bridged nucleic acid (BNA). In some respects, the sugar-modified nucleotide is an ethylene-bridged nucleic acid (ENA). In some respects, the sugar-modified nucleotide is (S)-constrained ethyl (cEt). In some respects, the sugar-modified nucleotide is tricyclic DNA (tcDNA). In some respects, the sugar-modified nucleotide is a 2'-NH2 nucleic acid. In some respects, the sugar-modified nucleotide is an unlocked nucleic acid (UNA). In some respects, sugar-modified nucleotides include 2'-O-alkyl-modified nucleotides, 2'-alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-halogen-modified nucleotides, ENA, BNA, LNA, or UNA.

[0163] In some respects, the polynucleotide molecules described herein contain modified nucleotides, which include one or more sugar phosphate-modified nucleotides. In some respects, the modified sugar phosphate is phosphorodiamidate morpholino (PMO). In some respects, the modified sugar phosphate is an aminophosphate. In some cases, the heterocyclic substitution includes imidazole and pyrrolidine. In some respects, the modified sugar phosphate is a thioaminophosphate. In some respects, the modified sugar phosphate is a peptide nucleic acid (PNA).

[0164] In some respects, the polynucleotide molecules described herein contain modified nucleotides, which include one or more sugar-modified nucleotides. In some respects, the sugar-modified nucleotides are 2'-fluoro (2'-F) modified nucleotides. In some cases, the 2'-F modified nucleotides include nucleotides containing a thiomodified base, such as 2'-fluoro-2-thiouridine-3'-phosphate (U3f). In some cases, the polynucleotide molecule contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight 2'-fluoro (2'-F) modified nucleotides. In some cases, the polynucleotide molecule contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight 2'-fluoro (2'-F) modified nucleotides. In some cases, polynucleotide molecules contain one to eight, two to eight, three to eight, four to eight, five to eight, six to eight, or seven to eight 2'-fluoro (2'-F) modified nucleotides. In some cases, polynucleotide molecules contain one, two, three, four, five, six, seven, or eight 2'-fluoro (2'-F) modified nucleotides. In some cases, polynucleotide molecules contain three 2'-F modified nucleotides. In some cases, polynucleotide molecules contain five 2'-F modified nucleotides.

[0165] In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 2 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 7 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 12 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 14 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 16 starting from the 5' end. In other aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at positions 2, 12, 14, 16, or combinations thereof starting from the 5' end. In other aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at positions 2, 7, 12, 14, 16, or combinations thereof starting from the 5' end. In other respects, the polynucleotide molecule contains at least three 2'-fluorinated nucleotides at positions 2, 12, 14, and 16 starting from the 5' end.

[0166] In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 7 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 9 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at position 11 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at at least one of positions 7, 9, and 11 starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at positions 7, 9, 11, or a combination thereof starting from the 5' end. In some aspects, the polynucleotide molecule contains a 2'-fluorinated nucleotide at positions 7, 9, and 11 starting from the 5' end.

[0167] Modified nucleotide bonds

[0168] In some cases, the modifications described herein include internucleotide bond modifications. In some aspects, the modified backbone is a methylphosphonate. In some aspects, the modified internucleotide bond is a thiophosphate internucleotide bond. In some aspects, the modified internucleotide bond is a guanidinopropylaminophosphate internucleotide bond. In some aspects, the modified internucleotide bond is a methanesulfonyl-aminophosphate (MsPA) internucleotide bond. In some cases, the modified internucleotide bond comprises one or more of the following: dithiophosphate, methylphosphonate, 5'-alkylphosphonate, 5'-methylphosphonate, 3'-alkylphosphonate, trifluoroborate, borane phosphate with a 3'-5' or 2'-5' bond, selenophosphate, phosphate triester, thiocarbonyl alkyl phosphate triester, phosphonate hydrogen ester, alkylphosphonate, alkylthiophosphonate, arylthiophosphonate, selenophosphate, and aminophosphate.

[0169] In some cases, these modifications involve phosphate-thioester modified nucleotide bonds. As described herein, in some cases, a phosphate-thioester modified nucleotide bond refers to a phosphate-thioester bond connecting two nucleotides or the modified nucleotides described herein, rather than a phosphodiester bond. In some cases, the polynucleotide molecules described herein contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphate-thioester modified nucleotide bonds. In some cases, the polynucleotide molecules described herein contain at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the polynucleotide molecules described herein contain at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphate-thioester modified nucleotide bonds. In some cases, the polynucleotide molecules described herein contain one, two, three, four, five, six, seven, or eight phosphate-thioester modified nucleotide bonds. In some cases, the polynucleotide molecules described herein contain 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 phosphate-thioester modified nucleotide bonds. In other cases, the polynucleotide molecules described herein contain 1 to 4, 2 to 4, or 3 to 4 phosphate-thioester modified nucleotide bonds.

[0170] In some cases, the polynucleotide molecules described herein contain one phosphate-thioester modified nucleotide bond at the 5' end and one phosphate-thioester modified nucleotide bond at the 3' end. In some cases, the polynucleotide molecules described herein contain two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the polynucleotide molecules described herein contain three phosphate-thioester modified nucleotide bonds at the 5' end and three phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the polynucleotide molecules described herein contain four phosphate-thioester modified nucleotide bonds at the 5' end and four phosphate-thioester modified nucleotide bonds at the 3' end.

[0171] In some cases, the guiding chain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains one, two, three, four, five, six, seven, or eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains 1 to 4, 2 to 4, or 3 to 4 phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains at least one phosphate-thioester modified nucleotide internucleotide bond at the 5' end and at least one phosphate-thioester modified nucleotide internucleotide bond at the 3' end. In some cases, the guiding chain contains two phosphate-thioester modified nucleotide internucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide internucleotide bonds at the 3' end. In some cases, the guiding chain contains two consecutive phosphate-thioester modified nucleotide internucleotide bonds at the 5' end and two consecutive phosphate-thioester modified nucleotide internucleotide bonds at the 3' end. In some aspects, the modified backbone is a phosphate-thioester, and the phosphate-thioester is stereochemically enriched. In some aspects, the guiding chain contains at least one stereochemically enriched phosphate-thioester. In some aspects, the guiding chain contains at least one, two, or three stereochemically enriched phosphate-thioesters. In some aspects, the guiding chain contains only one, two, three, or four stereochemically enriched phosphate-thioesters. In a further aspect, at least one (e.g., one or two) stereochemically enriched thiophosphates are located between two consecutive nucleotides of two of the six 5'-terminal nucleotides serving as the guiding chain. In an even further aspect, at least one (e.g., one or two) stereochemically enriched thiophosphates are located between two consecutive nucleotides of two of the six 3'-terminal nucleotides serving as the guiding chain. In still a further aspect, one stereochemically enriched thiophosphate is covalently bonded to a first and second nucleotide of the guiding chain starting from the 5' end. In some aspects, one stereochemically enriched thiophosphate is covalently bonded to a twenty-first and twenty-second nucleotide of the guiding chain starting from the 5' end. In some aspects, one stereochemically enriched thiophosphate is covalently bonded to a twenty-second and twenty-third nucleotide of the guiding chain starting from the 5' end. In a particular aspect, the stereochemically enriched thiophosphate has R P Stereochemical identity. In some respects, stereochemically enriched thiophosphates possess S... P Stereochemical identity.

[0172] In some cases, the guest chain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains one, two, three, four, five, six, seven, or eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains 1 to 4, 2 to 4, or 3 to 4 phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains at least one phosphate-thioester modified nucleotide bond at the 5' end. In some cases, the guest chain contains two phosphate-thioester modified nucleotide internucleotide bonds at the 5' end. In some cases, the guest chain contains two consecutive phosphate-thioester modified nucleotide internucleotide bonds at the 5' end. In some aspects, the modified backbone is a phosphate-thioester, and the phosphate-thioester is stereochemically enriched. In some aspects, the guest chain contains at least one stereochemically enriched phosphate-thioester. In some aspects, the guest chain contains at least one, two, or three stereochemically enriched phosphate-thioesters. In some aspects, the guest chain contains only one, two, three, or four stereochemically enriched phosphate-thioesters. In a further aspect, at least one (e.g., one or two) stereochemically enriched phosphate-thioesters are located between two consecutive nucleotides of two of the six 5'-terminal nucleotides that are the guest chain. In an even further aspect, at least one (e.g., one or two) stereochemically enriched phosphate-thioesters are located between two consecutive nucleotides of two of the six 3'-terminal nucleotides that are the guest chain. Further, a stereochemically enriched thiophosphate is covalently bonded to the first and second nucleotides within the transfer chain, starting from the 5' end. In some aspects, a stereochemically enriched thiophosphate is covalently bonded to the twenty-first and twenty-second nucleotides within the transfer chain, starting from the 5' end. In other aspects, a stereochemically enriched thiophosphate is covalently bonded to the twenty-second and twenty-third nucleotides within the transfer chain, starting from the 5' end. In a particular aspect, the stereochemically enriched thiophosphate has R... P Stereochemical identity. In some respects, stereochemically enriched thiophosphates possess S... P Stereochemical identity.

[0173] In some respects, the polynucleotide molecules described herein contain one or more (e.g., 1 to 20, 1 to 10, or 1 to 5) stereochemically enriched dithiophosphate nucleotide bonds (e.g., diastereomeric excess of at least 10%, 50%, 60%, 70%, 80%, or 90%, e.g., up to about 99%) at the P-stereoisomer center. The polynucleotide molecules described herein contain one or more (e.g., 1 to 20, 1 to 10, or 1 to 5; e.g., inter-nucleosides) dithiophosphate nucleotide bonds. The dithiophosphate in the polynucleotide molecules described herein may be non-P-stereoisomeric. Thiophosphates and dithiophosphates can enhance the stability of the polynucleotide molecules described herein to serum exonuclease activity. Non-P-stereoisomeric dithiophosphates can simplify the synthesis of the polynucleotide molecules described herein by reducing the number of diastereomers. Typically, thiophosphates or dithiophosphates can link two adjacent nucleotides within the six 3' terminal nucleotides and six 5' terminal nucleotides of the polynucleotide molecules described herein. In some respects, stereochemically enriched thiophosphates (e.g., R...) P -Enriched thiophosphates can be covalently bonded to a first nucleoside (e.g., the 3'-carbon atom of the first nucleoside) and a second nucleoside (e.g., the 5'-carbon atom of the second nucleoside) starting from the 5' end of the guiding chain. Alternatively, stereochemically enriched thiophosphates (e.g., S...) P -Enriched thiophosphates can be covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside) and the 22nd nucleoside (e.g., the 5'-carbon atom of the 22nd nucleoside) starting from the 5' end of the guiding chain. Further, additionally or alternatively, stereochemically enriched thiophosphates (e.g., S... P --enriched thiophosphates or Rp-enriched thiophosphates can be covalently bonded to the 22nd nucleotide (e.g., the 3'-carbon atom of the 22nd nucleotide) and the 23rd nucleotide (e.g., the 5'-carbon atom of the 23rd nucleotide) starting from the 5' end of the guide chain. In the antisense chain, the 5' R... P -Enriched thiophosphates (e.g., R-terminated phosphates covalently bonded to a first nucleoside (e.g., the 3'-carbon atom of the first nucleoside) and a second nucleoside (e.g., the 5'-carbon atom of the second nucleoside) starting from the 5' end). P -Enriched thiophosphates) and 3'S P -Enriched thiophosphates (e.g., S-terminals covalently bonded to the 21st nucleotide (e.g., the 3'-carbon atom of the 21st nucleotide) and the 22nd nucleotide (e.g., the 5'-carbon atom of the 22nd nucleotide) starting from the 5' end). P Combinations of (-enriched thiophosphates) can produce better efficacy and / or duration of action, for example, as by means of 5' R... P - Enriched thiophosphates and 3'S PThe activity of the target is measured by a reference guide chain of stereochemically enriched thiophosphates or combinations of 5' Rp-enriched thiophosphates and 3' Sp and Rp-enriched thiophosphates. In some embodiments, the stereochemically enriched thiophosphates may contain R... p R p S p S p (R at positions 1 and 2 of the guide chain) p R p and the S at positions 21 and 22 of the guide chain p S p ) or R p R p S p R p (R at positions 1 and 2 of the guide chain) p R p and the S at positions 21 and 22 of the guide chain p R pIn some respects, the polynucleotide molecules described herein comprise four stereochemically enriched thiophosphates: (1) Rp-enriched thiophosphates covalently bonded to the first nucleoside (e.g., the 3'-carbon atom of the first nucleoside) and the second nucleoside (e.g., the 5'-carbon atom of the second nucleoside) from the 5' end of the antisense chain; (2) covalently bonded to the second nucleoside (e.g., the 3'-carbon atom of the second nucleoside) and the third nucleoside (e.g., the 5'-carbon atom of the third nucleoside) from the 5' end of the antisense chain. (3) Rp-enriched thiophosphates; and (4) Sp-enriched thiophosphates covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside) and the 22nd nucleoside (e.g., the 5'-carbon atom of the 22nd nucleoside) starting from the 5' end of the antisense chain. In some respects, the polynucleotide molecules described herein contain four stereochemically enriched thiophosphates: (1) Rp-enriched thiophosphates covalently bonded to the first nucleoside (e.g., the 3'-carbon atom of the first nucleoside) and the second nucleoside (e.g., the 5'-carbon atom of the second nucleoside) from the 5' end of the antisense chain; and (2) covalently bonded to the second nucleoside (e.g., the 3'-carbon atom of the second nucleoside) and the third nucleoside (e.g., the 5'-carbon atom of the third nucleoside) from the 5' end of the antisense chain. (3) Rp-enriched thiophosphates; and (4) Sp-enriched thiophosphates covalently bonded to the 21st nucleoside (e.g., the 3'-carbon atom of the 21st nucleoside) and the 22nd nucleoside (e.g., the 5'-carbon atom of the 22nd nucleoside) starting from the 5' end of the antisense chain; and (5) Rp-enriched thiophosphates covalently bonded to the 22nd nucleoside (e.g., the 3'-carbon atom of the 22nd nucleoside) and the 23rd nucleoside (e.g., the 5'-carbon atom of the 23rd nucleoside) starting from the 5' end of the antisense chain.

[0174] Base-modified nucleotides

[0175] In some aspects, the polynucleotide molecules described herein contain modified nucleotides that include one or more purine modifications. In some aspects, the purine modification described herein is 2,6-diaminopurine. In some aspects, the purine modification described herein is 3-denitro-adenine. In some aspects, the purine modification described herein is 7-denitro-guanine. In some aspects, the purine modification described herein is 8-azido-adenine. In some aspects, the modified nucleotides include modified guanine (e.g., inosine) or one or more non-natural nucleic acids of any type.

[0176] In some respects, the polynucleotide molecules described herein contain modified nucleotides, which include one or more pyrimidine modifications. In some respects, the pyrimidine modification described herein is 2-thio-thymidine. In some respects, the pyrimidine modification described herein is 5-formamide-uracil. In some respects, the pyrimidine modification described herein is 5-methyl-cytosine. In some respects, the pyrimidine modification described herein is 5-ethynyluracil.

[0177] Debase-modified nucleotides

[0178] In some aspects, the polynucleotide molecules described herein contain modified nucleotides, including debase-modified nucleotides. Because debase substitutions lack nucleobases capable of participating in base-pairing interactions and alleviate steric hindrance, including one or more (e.g., one or two) debase substitutions in the hybridized polynucleotide construct can reduce or even eliminate miRNA-like off-target effects. Therefore, the polynucleotide molecules disclosed herein may contain one or more (e.g., one or two) debase substitutions. In some aspects, the debase substitution is located at the 5th nucleotide from the 5' end of the guiding strand described herein. In some aspects, the debase substitution is located at the 7th nucleotide from the 5' end of the guiding strand described herein.

[0179] When the polynucleotide molecules disclosed herein contain two or more debase substitutions, their structures may be the same or different. In some aspects, the guest strand contains one debase substitution (e.g., the guiding strand may not have a debase substitution). In other aspects, the guiding strand contains one debase substitution (e.g., the guest strand may not have a debase substitution). In still other aspects, both the guiding strand and the guest strand contain one debase substitution. In a further aspect, the guest strand contains a debase substitution between nucleoside number (x) and nucleoside number (x+1), where x is an integer from 2 to 7. In an even further aspect, the guiding strand contains a debase substitution between nucleoside number (x) and nucleoside number (x+1), where x is an integer from 2 to 7.

[0180] Debasic substitution can be performed using formula (III):

[0181] ,

[0182] (III)

[0183] in

[0184] L is a sugar analogue, or is substituted with a heteroacyl group from A, U, C, or G, or is any other substituted nucleic acid (e.g., locked or unlocked nucleic acids, glycol nucleic acids, etc.).

[0185] Each X 4 It can be either O or S independently;

[0186] Each X 5 It can be independently of O, S, NH or a bond;

[0187] Each R 9 H independently, or C with optional substitution 1﹣6 Alkyl, optionally substituted C 2﹣6 alkenyl, optionally substituted C 2﹣6 alkynyl, optionally substituted (C 1﹣9 Heterocyclic group)﹣C 1﹣6 -alkyl, optionally substituted (C 6﹣10 Aryl)﹣C 1﹣6 -alkyl, optionally substituted (C 3﹣8 (cycloalkyl)﹣C 1﹣6 -alkyl, –LinkA(–T) p Or the joined or fused parts;

[0188] Each LinkA is independently a multivalent connector (e.g., including -C(O)-N(H)-);

[0189] Each T is an independent auxiliary part;

[0190] R 10 It is a bond with the 3'-carbon atom of the nucleoside (x) in the chain;

[0191] R 11 It is a bond with the 5'-oxygen atom of the nucleoside (x+1) in the chain;

[0192] p is an integer from 1 to 6; and

[0193] t is an integer from 1 to 6.

[0194] In some aspects, the debase substitutions described herein are attached to the guiding strand of the polynucleotide molecule described herein. In certain aspects, the guiding strand described herein (e.g., within the seed region of the guiding strand) may include debase substitutions (e.g., internucleotide debase spacers of formula (III), where t is 1). In some aspects, debase substitutions (e.g., internucleotide debase spacers of formula (III), where t is 1) may be bonded to the 3' carbon atom of the second, third, fourth, or fifth nucleotide of the guiding strand described herein, starting from the 5' end. In some aspects, debase substitutions (e.g., internucleotide debase spacers of formula (III), where t is 1) may be bonded to the 3' carbon atom of the thirteenth, fourteenth, fifteenth, or sixteenth nucleotide of the guiding strand described herein, starting from the 5' end. In some aspects, debase substitutions are made on the fourth, fifth, sixth, seventh, eighth, and / or ninth nucleotides of the guiding strand described herein, starting from the 5' end.

[0195] In some respects, the polynucleotide molecules described herein contain one or more of the modifications described above. Therefore, in some respects, about 10% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 20% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 30% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 40% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 50% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 60% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 70% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 80% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, about 90% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the modifications described above. In other respects, 100% of the nucleotides from the polynucleotide molecules described herein are modified with one or more of the types of modifications described above.

[0196] In some aspects, the modifications of one or more types described herein occur at different locations within the polynucleotide molecule described herein. In some aspects, the modifications of one or more types described herein occur in seed regions within the polynucleotide molecule described herein. In some aspects, the modifications of one or more types described herein occur at the 3' end of the polynucleotide molecule described herein. In some aspects, the modifications of one or more types described herein occur at the 5' end of the polynucleotide molecule described herein. In some aspects, the modifications of one or more types described herein occur dispersedly within the polynucleotide molecule described herein. In some aspects, the modifications of one or more types described herein occur in clusters within the polynucleotide molecule described herein.

[0197] Guidance Chain

[0198] On the one hand, this paper describes specific modification motifs or patterns of double-stranded repressive polynucleotide molecules that include a transit strand and a guide strand.

[0199] In some aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at position 2 starting from the 5' end. In some aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at position 7 starting from the 5' end. In some aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at position 12 starting from the 5' end. In some aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at position 14 starting from the 5' end. In some aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at position 16 starting from the 5' end. In other aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at positions 2, 12, 14, 16, or a combination thereof starting from the 5' end. In other aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at positions 2, 7, 12, 14, 16, or a combination thereof starting from the 5' end. In other aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at at least three of positions 2, 12, 14, and 16 starting from the 5' end. In other aspects, the guiding strand comprises a nucleotide analog at one of positions 2-8 starting from the 5' end, and further comprises a 2'-fluorinated nucleotide at at least three of positions 2, 7, 12, 14, and 16 starting from the 5' end.

[0200] In some cases, the nucleotide analog is located at position 3 starting from the 5' end of the guide strand. In some cases, the nucleotide analog is located at position 3 starting from the 5' end of the guide strand, and the guide strand further comprises 2-thiouridine-3'-phosphate nucleotide. In some cases, the nucleotide analog is located at position 3 starting from the 5' end of the guide strand, and the guide strand further comprises 2'-O-methyl-2-thiouridine-3'-phosphate (u3). In some cases, the nucleotide analog is located at position 3 starting from the 5' end of the guide strand, and the guide strand further comprises at least one, at least two, at least three, or at least four 2'-F modified nucleotides. In some cases, the nucleotide analog is located at position 3 starting from the 5' end of the guide strand, and the guide strand further comprises at least one 2'-F modified nucleotide at positions 2, 7, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 3 of the guide strand starting from the 5' end, and the guide strand further contains 2'-F modified nucleotides at positions 2, 7, 12, 14, and 16 starting from the 5' end.

[0201] In some cases, the nucleotide analog is located at position 6, starting from the 5' end of the guide strand. In some cases, the nucleotide analog is located at position 6, starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least one of positions 2, 7, 12, 14, and 16, starting from the 5' end. In some cases, the nucleotide analog is located at position 6, starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least two of positions 2, 7, 12, 14, and 16, starting from the 5' end. In some cases, the nucleotide analog is located at position 6, starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least three of positions 2, 7, 12, 14, and 16, starting from the 5' end. In some cases, the nucleotide analog is located at position 6 of the guide strand starting from the 5' end, and the guide strand further includes 2'-fluorinated (2'-F) modified nucleotides at positions 2, 7, 12, 14 and 16 starting from the 5' end.

[0202] In some cases, the nucleotide analog is located at position 7 starting from the 5' end of the guide strand. In some cases, the nucleotide analog is located at position 7 starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least one of positions 2, 6, 8, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 7 starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least one of positions 2, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 7 starting from the 5' end of the guide strand, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least two of positions 2, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 7 of the guide strand starting from the 5' end, and the guide strand further comprises 2'-fluorinated (2'-F) modified nucleotides at at least three of positions 2, 12, 14, and 16 starting from the 5' end.

[0203] In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end. In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least one of positions 2, 7, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least two of positions 2, 7, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end, and the guide strand further comprises a 2'-fluorinated (2'-F) modified nucleotide at at least three of positions 2, 7, 12, 14, and 16 starting from the 5' end. In some cases, the nucleotide analog is located at position 8 of the guide strand starting from the 5' end, and the guide strand further includes 2'-fluorinated (2'-F) modified nucleotides at positions 2, 7, 12, 14 and 16 starting from the 5' end.

[0204] In some cases, the nucleotides of the guide strand include DNA or RNA. As described herein, in some cases, the DNA nucleotides include unmodified DNA, which includes: unmodified adenine nucleotide (A), unmodified guanine nucleotide (G), unmodified thymine nucleotide (T), or unmodified cytosine nucleotide (C). As described herein, in some cases, the RNA includes unmodified RNA, which includes: unmodified adenine nucleotide (A), unmodified guanine nucleotide (G), unmodified uracil nucleotide (U), or unmodified cytosine nucleotide (C).

[0205] In some cases, the nucleotide in the guiding strand includes DNA, RNA, nucleotide analogs, 2'-F modified nucleotides, or 2'-O-alkyl modified nucleotides. In some cases, 2'-O-alkyl modified nucleotides include 2'-O-methyl modified nucleotides. In some cases, the nucleotide in the guiding strand that is not a nucleotide analog or a 2'-F modified nucleotide is selected from DNA nucleotides, RNA nucleotides, and 2'-O-alkyl modified nucleotides. In some cases, the nucleotide in the guiding strand that is not a nucleotide analog or a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

[0206] In some cases, the guiding chain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains one, two, three, four, five, six, seven, or eight phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 phosphate-thioester modified nucleotide bonds. In some cases, the guiding chain contains 1 to 4, 2 to 4, or 3 to 4 phosphate-thioester modified nucleotide bonds.

[0207] In some cases, the guiding strand contains one phosphate-thioester modified nucleotide bond at the 5' end and one phosphate-thioester modified nucleotide bond at the 3' end. In some cases, the guiding strand contains two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guiding strand contains three phosphate-thioester modified nucleotide bonds at the 5' end and three phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guiding strand contains four phosphate-thioester modified nucleotide bonds at the 5' end and four phosphate-thioester modified nucleotide bonds at the 3' end.

[0208] In some cases, the guide chain contains nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-F, and / or nucleotide analogs described herein. In some cases, the guide strand contains a 5'-mFmmmXFmmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-fluorine (2'-F) modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-sulfur Ureidine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G) 2) 2',3'-Octocytidine-3'-phosphate (C2), 2',3'-Octocytidine-3'-phosphate (U2), 2'-Deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-Deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-fluoro (2'-F) modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0209] In some cases, the guide strand contains a 5'-mFmmmmXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and 2'-O-methyl-2-thiouridine. -3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) 2',3'-O-cytosine-3'-phosphate (C2), 2',3'-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0210] In some cases, the guide chain contains 5'- The sequence is mFmmmFXmmmmFmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (... u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2' ,3'-O-cytosine-3'-phosphate (C2), 2',3'-O-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-mFmmmFXmmmmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0211] In some cases, the guide chain contains 5'- The sequence is mFmmmmXFmmmFmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (... u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2' ,3'-O-cytosine-3'-phosphate (C2), 2',3'-O-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmmXFmmmmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0212] In some cases, the guide strand contains a 5'-mFmmmmFXmmmFmFmFmmmmmmm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and 2'-O-methyl-2-thiouridine. -3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) 2',3'-O-cytosine-3'-phosphate (C2), 2',3'-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmFXmmmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0213] In some cases, the guide chain contains 5'﹣ mFmmmmFmmmmXmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-... Thioururidine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3'-open-ring Cytidine-3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-mFmmmmFmmmmXmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0214] In some cases, the guide chain contains 5'﹣ mFXmmmFmmmmFmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-... Thioururidine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3'-open-ring Cytidine-3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-mFXmmmFmmmmFmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl L-threonine debasement. Nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3) and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0215] In some cases, the guide chain contains 5'﹣ mFmmmFXmmmmXmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-... Thioururidine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3'-open-ring Cytidine-3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-mFmmmFXmmmmXmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl L-threonine debasement. Nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3) and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0216] In some cases, the guide chain contains 5'- The sequence is mFmmmXFmmmmXmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate ( u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2' ,3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-mFmmmXFmmmmXmFmFmmmmmmm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-A). NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), and 2'-O-methylinosine-3'-phosphate (i).

[0217] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'﹣mFmmmmXmmmmYmFmFmmmmmmm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'﹣phosphate (T﹣T), acyclic L-threonine nucleic acid-adenine-3'﹣phosphate (T﹣A), acyclic N-acetyl L-threonine debased nucleic acid-3' phosphate (T﹣NAc), 1',2'﹣dideoxyribose. -3'-phosphate (dAB) and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and Y is selected from 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxythymidine-3'-phosphate (dT) and 2'-deoxy-2-thiothymidine-3'-phosphate (dT3).

[0218] In some cases, the guiding strand contains 2'-O-methyl modified nucleotides, 2'-F modified nucleotides, nucleotide analogs described herein, or phosphate-thioester nucleotide bonds. In some cases, the guiding strand contains one or more phosphate-thioester modified nucleotide bonds at the 5' end and one or more phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guiding strand contains two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end.

[0219] In some cases, the guide strand contains a 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O- Methyl-2-thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate Acid (G2), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0220] In some cases, the guide strand contains a 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O- Methyl-2-thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate Acid (G2), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0221] In some cases, the guide strand contains a 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O- Methyl-2-thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate Acid (G2), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0222] In some cases, the guide chain contains 5'- The sequence is msFsmmmFXmmmmFmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a thiophosphate nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) ), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a 5'-msFsmmmFXmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0223] In some cases, the guide chain contains 5'- The sequence is msFsmmmmFmmmmXmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) ), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-msFsmmmmFmmmmXmFmFmmmmmsmsm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl-L-... Threonine-3-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0224] In some cases, the nucleotide analog is located at at least one of positions 2-12 from the 5' end of the guiding strand, and the nucleotide analog comprises 2'-O-methyl-2-thiouridine-3'-phosphate (U3) or 2'-fluoro-2-thiouridine-3'-phosphate (U3f). In some cases, the nucleotide analog is located at position 12 at the 5' end of the guiding strand, and the nucleotide analog consists of 2'-fluoro-2-thiouridine-3'-phosphate (U3f). In some cases, the nucleotide analog is located at position 3 at the 5' end of the guiding strand, and the nucleotide analog comprises 2'-O-methyl-2-thiouridine-3'-phosphate (U3). In some cases, the nucleotide analog is located at position 3 at the 5' end of the guide strand, and the nucleotide analog consists of 2'-O-methyl-2-thiouridine-3'-phosphate (u3). In some cases, the guiding strand contains a nucleic acid sequence of 5'﹣mFmmmmFXmmmF'mFmFmmmmmmm﹣3', where m is a 2'﹣O﹣methyl modified nucleotide, F is a 2'﹣F modified nucleotide, X is selected from acyclic L﹣threonine nucleic acid﹣thymine﹣3'﹣phosphate (T﹣T), acyclic N﹣acetyl L﹣threonine debased nucleic acid﹣3' phosphate (T﹣NAc), 1',2'﹣dideoxyribose﹣3'﹣phosphate (dAB), and thymidine﹣ethylene glycol nucleic acid (GNA) S﹣ isomer (Tgn), and F' is 2'﹣fluoro﹣2﹣thiouridine﹣3'﹣phosphate (U3f). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmF'mFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and F' is 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0225] In some cases, the guide chain contains 5'- The sequence msFsXmmmFmmmmFmFmFmmmmmsmsm-3' is a nucleic acid sequence where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) The sequence contains 2',3'-octane cytidine-3'-phosphate (C2), 2',3'-octane uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFXmmmFmmmmFmFmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is 2'-O-methyl-2-thiouridine-3'-phosphate (u3). In some cases, the guiding strand contains a nucleic acid sequence of 5'﹣msFsXmmmFmmmmFmFmFmmmmmsmsm﹣3', where m is a 2'﹣O﹣methyl modified nucleotide, F is a 2'﹣F modified nucleotide, s is a thiophosphate nucleotide bond, and X is 2'﹣O﹣methyl﹣2﹣thiouridine﹣3'﹣phosphate (u3).

[0226] In some cases, the guide chain contains 5'- The sequence is msFsmmmFXmmmmXmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) ), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding chain contains 5'-msFsmmmFXmmmmXmFmFmmmmmsmsm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a thiophosphate nucleotide internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0227] In some cases, the guide chain contains 5'- The sequence is msFsmmmXFmmmmXmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) ), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a 5'-msFsmmmXFmmmmXmFmFmmmmmsmsm-3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T) or acyclic L-threonine nucleic acid. -Adenine-3'-phosphate (T-A), acyclic N-acetyl-L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), and 2'-O-methylinosine-3'-phosphate (i).

[0228] In some respects, this paper describes a specific modification pattern in which the guiding strand comprises 5'﹣msFsmmmmFmmmmYmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, s represents a thiophosphate nucleotide bond, and Y is selected from 2'﹣O﹣methyl﹣2﹣thiouridine﹣3'﹣phosphate (u3), 2'﹣fluoro﹣2﹣thiouridine﹣3'﹣phosphate (U3f), 2'﹣deoxythymidine﹣3'﹣phosphate (dT), and 2'﹣deoxy﹣2﹣thiothymidine﹣3'﹣phosphate (dT3).

[0229] In some respects, this paper describes a specific modification pattern in which the guiding strand comprises 5'﹣msFsmmmIFmmmmYmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, s represents a thiophosphate nucleotide bond, I represents 2'﹣O﹣methylinosine﹣3'﹣phosphate (i), and Y is selected from 2'﹣O﹣methyl﹣2﹣thiouridine﹣3'﹣phosphate (u3), 2'﹣fluoro﹣2﹣thiouridine﹣3'﹣phosphate (U3f), 2'﹣deoxythymidine﹣3'﹣phosphate (dT), and 2'﹣deoxy﹣2﹣thiothymidine﹣3'﹣phosphate (dT3).

[0230] In some respects, this paper describes a specific modification pattern in which the guiding strand comprises 5'﹣msFsmmmmXmmmmmmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, s represents a phosphate thioester internucleotide bond, and X is selected from acyclic L﹣threonine nucleic acid﹣thymine﹣3'﹣phosphate (T﹣T), acyclic L﹣threonine nucleic acid﹣adenine﹣3'﹣phosphate (T﹣A), acyclic N﹣acetyl L﹣threonine debased nucleic acid﹣3' phosphate (T﹣NAc), 1',2'﹣dideoxyribose﹣3'﹣phosphate (dAB), and thymidine﹣ethylene glycol nucleic acid (GNA) S﹣ isomer (Tgn).

[0231] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'﹣msFsmmmmXmmmmYmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, s represents a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'﹣phosphate (T﹣T), acyclic L-threonine nucleic acid-adenine-3'﹣phosphate (T﹣A), acyclic N-acetyl L-threonine debaseted nucleic acid-3' phosphate (T﹣NAc), 1',2 Y is selected from '-dideoxyribose-3'-phosphate (dAB) and thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), and Y is selected from 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxythymidine-3'-phosphate (dT) and 2'-deoxy-2-thiothymidine-3'-phosphate (dT3).

[0232] In some cases, the guide chain contains 5'- The sequence is msFsmmmFXmmmmFmFmFmmmmmsmsm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a thiophosphate nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiourea Glycoside-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2) ), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i). In some respects, this paper describes a specific modification pattern in which the guiding strand comprises 5'-msFsmmmFXmmmmFmFmFmmmmmsmsm-3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a phosphate thioester internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).

[0233] In some cases, the guide chain contains 5'-msFsmmmmXFmmmFmFmFmmmmmsmsm The -3' nucleic acid sequence, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a thiophosphate nucleotide internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2 '-Fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3' -Cyclocytosine-3'-phosphate (C2), 2',3'-Cyclouridine-3'-phosphate (U2), 2'-Deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-Deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some respects, this paper describes a specific modification pattern in which the guiding strand comprises 5'﹣ msFsmmmmXFmmmFmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, s represents a phosphate thioester internucleotide bond, and X is selected from acyclic L﹣threonine nucleic acid﹣thymine﹣3'﹣phosphate (T﹣T), acyclic L﹣threonine nucleic acid﹣adenine﹣3'﹣phosphate (T﹣A), acyclic N﹣acetyl L﹣threonine debased nucleic acid﹣3' phosphate (T﹣NAc), 1',2'﹣dideoxyribose﹣3'﹣phosphate (dAB), and thymidine﹣ethylene glycol nucleic acid (GNA) S﹣ isomer (Tgn).

[0234] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'﹣msFsmmmmFmmmmFmFmFmmmmmsmsm ﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, and s represents a phosphate thioester nucleotide bond.

[0235] Passerby Chain

[0236] In some respects, this paper describes specific modification motifs for patterns of double-stranded repressive polynucleotide molecules comprising a guest strand and a guide strand. In other respects, this paper describes specific modification patterns for the guest strand.

[0237] In some cases, the guest strand contains one or more nucleotide analogs. In some cases, the guest strand contains one or more nucleotide analogs at positions opposite to the seed region of the guiding strand. In some cases, the guest strand contains one or more nucleotide analogs at positions 12-22 starting from the 5' end. In some cases, the guest strand contains one or more nucleotide analogs at positions 2-10 starting from the 3' end. In some cases, the guest strand contains one or more 2-amino-2'-O-methyladenosine-3'-phosphate (a1). In some cases, the guest strand contains 2-amino-2'-O-methyladenosine-3'-phosphate (a1) at positions 11, 12, 13, 14, or 15 starting from the 5' end. In some cases, the guest strand contains 2-amino-2'-O-methyladenosine-3'-phosphate (a1) at position 13 starting from the 5' end. In some cases, the transient chain contains 2-amino-2'-O-methyladenosine-3'-phosphate (a1) at position 14 starting from the 5' end. In some cases, the transient chain contains 2-amino-2'-O-methyladenosine-3'-phosphate (a1) at position 15 starting from the 5' end.

[0238] In some cases, the guest chain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight nucleotides modified with 2'-fluorine (2'-F). In some cases, the guest chain contains at least two, at least three, or at least four nucleotides modified with 2'-F. In some cases, the guest chain contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight nucleotides modified with 2'-fluorine (2'-F). In some cases, the guest chain contains one to eight, two to eight, three to eight, four to eight, five to eight, six to eight, or seven to eight nucleotides modified with 2'-fluorine (2'-F). In some cases, the guest chain contains one, two, three, four, five, six, seven, or eight nucleotides modified with 2'-fluorine (2'-F). In some cases, the guest chain contains one 2'-F modified nucleotide. In some cases, the guest chain contains two 2'-F modified nucleotides. In some cases, the guest chain contains three 2'-F modified nucleotides. In some cases, the guest chain contains four 2'-F modified nucleotides. In some cases, the guest chain contains five 2'-F modified nucleotides.

[0239] In some aspects, the guest chain contains a 2'-fluorinated nucleotide at position 7 starting from the 5' end. In some aspects, the guest chain contains a 2'-fluorinated nucleotide at position 9 starting from the 5' end. In some aspects, the guest chain contains a 2'-fluorinated nucleotide at position 11 starting from the 5' end. In some aspects, the guest chain contains a 2'-fluorinated nucleotide at at least one of positions 7, 9, and 11 starting from the 5' end. In some aspects, the guest chain contains a 2'-fluorinated nucleotide at positions 7, 9, 11, or a combination thereof starting from the 5' end. In some aspects, the guest chain contains a 2'-fluorinated nucleotide at positions 7, 9, and 11 starting from the 5' end.

[0240] In some cases, the nucleotides in the transit chain include DNA nucleotides or RNA nucleotides. As described herein, in some cases, the DNA nucleotides include unmodified DNA nucleotides, including: unmodified adenine nucleotides (A), unmodified guanine nucleotides (G), unmodified thymine nucleotides (T), or unmodified cytosine nucleotides (C). As described herein, in some cases, the RNA nucleotides include unmodified RNA nucleotides, including: unmodified adenine nucleotides (A), unmodified guanine nucleotides (G), unmodified uracil nucleotides (U), or unmodified cytosine nucleotides (C).

[0241] In some cases, the nucleotide in the guest chain includes DNA nucleotides, RNA nucleotides, nucleotide analogs, 2'-F modified nucleotides, or 2'-O-alkyl modified nucleotides. In some cases, 2'-O-alkyl modified nucleotides include 2'-O-methyl modified nucleotides. In some cases, the nucleotide in the guest chain that is not a nucleotide analog or a 2'-F modified nucleotide is selected from 2'-O-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-halogen modified nucleotides, DNA nucleotides, RNA nucleotides, ENA, BNA, LNA, and UNA. In some cases, the nucleotide in the guest chain that is not a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

[0242] In some cases, the guest chain contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains at least one phosphate-thioester modified nucleotide bond. In some cases, the guest chain contains at most one, at most two, at most three, at most four, at most five, at most six, at most seven, or at most eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains one, two, three, four, five, six, seven, or eight phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 6 to 8 phosphate-thioester modified nucleotide bonds. In some cases, the guest chain contains 1 to 4, 2 to 4, or 3 to 4 phosphate-thioester modified nucleotide bonds.

[0243] In some cases, the guest chain contains at least one, at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds at the 5' end. In some cases, the guest chain contains at least one phosphate-thioester modified nucleotide bond at the 5' end. In some cases, the guest chain contains at least one, at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guest chain contains at least one phosphate-thioester modified nucleotide bond at the 3' end.

[0244] In some cases, the guest chain contains one phosphate-thioester modified nucleotide bond at the 5' end. In some cases, the guest chain contains two phosphate-thioester modified nucleotide bonds at the 5' end. In some cases, the guest chain contains three phosphate-thioester modified nucleotide bonds at the 5' end. In some cases, the guest chain contains four phosphate-thioester modified nucleotide bonds at the 5' end.

[0245] In some cases, the guest chain contains one phosphate-thioester modified nucleotide bond at the 3' end. In some cases, the guest chain contains two phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guest chain contains three phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guest chain contains four phosphate-thioester modified nucleotide bonds at the 3' end.

[0246] In some cases, the guest chain contains one phosphate-thioester modified nucleotide bond at the 5' end and one phosphate-thioester modified nucleotide bond at the 3' end. In some cases, the guest chain contains two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guest chain contains three phosphate-thioester modified nucleotide bonds at the 5' end and three phosphate-thioester modified nucleotide bonds at the 3' end. In some cases, the guest chain contains four phosphate-thioester modified nucleotide bonds at the 5' end and four phosphate-thioester modified nucleotide bonds at the 3' end.

[0247] In some cases, the transient chain contains 5'﹣mmmmmmFmFmFmmmmmmmmmm﹣3', 5'﹣msmsmmmmFmFmFmmmmmmmm﹣3', 5'﹣mmmmmmFmFmFmmX'mmmmmmm﹣3', 5'﹣ msmsmmmmFmFmFmmX'mmmmmmm﹣3', 5'﹣ mmmmmmFmFmFmmmX'mmmmmm ﹣3', or 5'﹣ msmsmmmmFmFmFmmmX'mmmmmm ﹣3', where m is a 2'﹣O﹣methyl modified nucleotide, F is a 2'﹣F modified nucleotide, s is a phosphate thioester nucleotide bond, and X' is 2﹣amino﹣2'﹣O﹣methyladenosine﹣3'﹣phosphate (a1). In some respects, this paper describes a modified polynucleotide molecule comprising a guide strand and a guest strand, wherein the guest strand comprises 5'﹣mmmmmmFmFmFmmmmmmmmmm﹣3', 5'﹣msmsmmmmFmFmFmmmmmmmmmm﹣3', 5'﹣mmmmmmFmFmFmmX'mmmmmmm﹣3', 5'﹣ msmsmmmmFmFmFmmX'mmmmmmm﹣3', 5'﹣ mmmmmmFmFmFmmmX'mmmmmm ﹣3', or 5'﹣ msmsmmmmFmFmFmmmX'mmmmmm -3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide bond, and X' is 2-amino-2'-O-methyl adenosine-3'-phosphate (a1).

[0248] Double-stranded polynucleic acid molecules

[0249] In some respects, this paper describes a specific modification pattern for a double-stranded polynucleotide molecule that includes a transit strand and a guide strand. In some cases, the guide strand contains the nucleic acid sequence mFmmmXFmmmmFmFmmmmmmm, and the guest strand contains mmmmmmFmFmFmmmmmmmm, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2' -O-methyl-2-thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate Phosphate (G2), 2',3'-octanecytidine-3'-phosphate (C2), 2',3'-octaneuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmmmmmmm-3', and the guest strand contains mmmmmmFmFmFmmmmmmmmmm, where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmX'mmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acids. -Thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thio Ureidine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3 X' is 2'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acids. -Thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thio Ureidine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3 X' is 2'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0250] In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer. Tgn, 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanine Glycoside-3'-phosphate (G2), 2',3'-octopycidine-3'-phosphate (C2), 2',3'-octopyuridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from... Cyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmX'mmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonyl groups. Thymine-3'-phosphate (T-T), acyclic L-threonine-adenosine-3'-phosphate (T-A), acyclic N-acetyl L-threonine-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl- 2-Thiouridine-3'-phosphate (U3), 2'-Fluoro-2-Thiouridine-3'-phosphate (U3f), 2-Amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-O-methyladenosine-3'-phosphate (A2), 2',3'-O-methyladenosine-3'-phosphate (G2), 2',3'-O-methyladenosine-3'-phosphate 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'. X'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0251] In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (T). gn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine- 3'-phosphate (G2), 2',3'-cytosine-3'-phosphate (C2), 2',3'-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guide strand contains a 5'-mFmmmmXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, and the pass strand contains 5'-msmsmmmmFmFmFmmX'mmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid- Adenine-3'-phosphate (T-A), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluorine U3-2-thiouridine-3'-phosphate (U3f), or 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2). 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).In some cases, the guide strand contains a 5'-mFmmmmXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, and the pass strand contains 5'-msmsmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenosine, and adenosine. Purine-3'-phosphate (T-A), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro -2-thiouridine-3'-phosphate (U3f), or 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2), 2 X' is 3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0252] In some cases, the guiding strand contains a 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GN). A) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'- Guanosine-3'-phosphate (G2), 2',3'-cytidine-3'-phosphate (C2), 2',3'-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and s is a phosphate thioester nucleotide bond. Furthermore, X is selected from acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guide strand contains a 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3' nucleic acid sequence, and the guest strand contains 5'-msmsmmmmFmFmFmmX'mmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid- Adenine-3'-phosphate (T-A), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluorine U3-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine-3'-phosphate (C2) 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonyl groups. Thymine-3'-phosphate (T-T), acyclic L-threonine-adenosine-3'-phosphate (T-A), acyclic N-acetyl L-threonine-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl- 2-Thiouridine-3'-phosphate (U3), 2'-Fluoro-2-Thiouridine-3'-phosphate (U3f), 2-Amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-O-methyladenosine-3'-phosphate (A2), 2',3'-O-methyladenosine-3'-phosphate (G2), 2',3'-O-methyladenosine-3'-phosphate 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0253] In some cases, the guide strand contains a 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3' nucleic acid sequence, and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (T). gn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine- 3'-phosphate (G2), 2',3'-cytosine-3'-phosphate (C2), 2',3'-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-mmmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmX'mmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleosides. Acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl-L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2 -Thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine- 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).In some cases, the guide strand contains a nucleic acid sequence of 5'-mFmmmmFXmmmmFmFmFmmmmmmm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleosides. Acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl-L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2 -Thiouridine-3'-phosphate (U3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'-open-cyclic guanosine-3'-phosphate (G2), 2',3'-open-cyclic cytidine- 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0254] In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the transit strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and s is a thiophosphorus... The ester-nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debaseted nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (G NA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2',3'- The compounds are guanosine-3'-phosphate (G2), cytidine-3'-phosphate (C2), uridine-3'-phosphate (U2), deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), thymidine-3'-phosphate (dT3), and methylinosine-3'-phosphate (i). In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the transit strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and s is a phosphate thioester nucleotide bond. X is selected from acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), and thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn).In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the transit strand contains 5'-msmsmmmmFmFmFmmX'mmmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonyl groups. Thymine-3'-phosphate (T-T), acyclic L-threonine-adenosine-3'-phosphate (T-A), acyclic N-acetyl L-threonine-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl- 2-Thiouridine-3'-phosphate (U3), 2'-Fluoro-2-Thiouridine-3'-phosphate (U3f), 2-Amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-O-methyladenosine-3'-phosphate (A2), 2',3'-O-methyladenosine-3'-phosphate (G2), 2',3'-O-methyladenosine-3'-phosphate 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).In some cases, the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the guest strand contains 5'-msmsmmmmFmFmFmmmX'mmmmmm-3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonyl groups. Thymine-3'-phosphate (T-T), acyclic L-threonine-adenosine-3'-phosphate (T-A), acyclic N-acetyl L-threonine-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl- 2-Thiouridine-3'-phosphate (U3), 2'-Fluoro-2-Thiouridine-3'-phosphate (U3f), 2-Amino-2'-O-methyladenosine-3'-phosphate (A1), C3-spacer (Pr), 2',3'-O-methyladenosine-3'-phosphate (A2), 2',3'-O-methyladenosine-3'-phosphate (G2), 2',3'-O-methyladenosine-3'-phosphate 3'-phosphate (C2), 2',3'-open-ring uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i), and X' is 2-amino-2'-O-methyladenosine-3'-phosphate (a1).

[0255] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'-msFsmmmmFmmmmYmFmFmmmmmsmsm-3' and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide internucleotide bond, and Y is selected from 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxythymidine-3'-phosphate (dT), and 2'-deoxy-2-thiothymidine-3'-phosphate (dT3).

[0256] In some cases, this paper describes a specific modification pattern in which the guiding strand contains a nucleic acid sequence of 5'-msFsmmmmFmmmmXmFmFmmmmmsmsm-3', and the transit strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm -3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a thiophosphate nucleotide internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluorine U3-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3'- The sequences include cytidine-3'-phosphate (C2), uridine-3'-phosphate (U2), deoxy-2-aminopurine nucleotide-3'-phosphate (dA3), thymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i). In some cases, the guide strand contains a 5'-msFsmmmmFmmmmXmFmFmmmmmsmsm-3' nucleic acid sequence, and the pass strand contains a 5'-msmsmmmmFmFmFmmmmmmmmm -3', where m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), and acyclic N-acetyl L-threonine. Alcohol-debasic nucleic acid -3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3) and 2'-fluoro-2-thiouridine-3'-phosphate (U3f).

[0257] In some respects, this paper describes a specific embellishment pattern, wherein the guiding chain contains 5'-msFsmmmIFmmmmYmFmFmmmmmsmsm-3', and the transient chain contains 5'-msmsmmmmFmFmFmmmmmmmmmm -3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide bond, I represents 2'-O-methylinosine-3'-phosphate (i), and Y is selected from 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxythymidine-3'-phosphate (dT), and 2'-deoxy-2-thiothymidine-3'-phosphate (dT3).

[0258] In some respects, this paper describes a specific embellishment pattern, wherein the guiding chain contains 5'-msFsmmmmXmmmmmmFmFmmmmmsmsm-3', and the transient chain contains 5'-msmsmmmmFmFmFmmmmmmmmmm -3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2' -Fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3' -Cyclocytosine-3'-phosphate (C2), 2',3'-Cyclouridine-3'-phosphate (U2), 2'-Deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-Deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0259] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'-msFsmmmmXmmmmYmFmFmmmmmsmsm-3' and the guest strand contains 5'-msmsmmmmFmFmFmmmmmmmmmm-3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide internucleotide bond, and Y is selected from 2'-O-methyl-2'-thiouridine-3'-phosphate (u3), 2'-fluoro-2'-thiouridine-3'-phosphate (U3f), 2'-deoxythymidine-3'-phosphate (dT), and 2'-deoxy-2'-phosphate. -Thiothymidine-3'-phosphate (dT3), and X is selected from acyclic L-threonine nucleic acid-thymidine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethyl GNA (Glycol Nucleic Acid) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-cyclic adenosine-3'-phosphate (A2), 2', 3'-O-guanosine-3'-phosphate (G2), 2',3'-O-cytidine-3'-phosphate (C2), 2',3'-O-uridine-3'-phosphate (U2), 2'-deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0260] In some respects, this paper describes a specific embellishment pattern, wherein the guiding chain contains 5'-msFsmmmFXmmmmFmFmFmmmmmsmsm-3', and the transient chain contains 5'-msmsmmmmFmFmFmmmmmmmmmm -3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2' -Fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3' -Cyclocytosine-3'-phosphate (C2), 2',3'-Cyclouridine-3'-phosphate (U2), 2'-Deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-Deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0261] In some respects, this paper describes a specific embellishment pattern, wherein the guiding chain contains 5'-msFsmmmmXFmmmFmFmFmmmmmsmsm-3', and the transient chain contains 5'-msmsmmmmFmFmFmmmmmmmmmm -3', where m represents a 2'-O-methyl modified nucleotide, F represents a 2'-F modified nucleotide, s represents a thiophosphate nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (T-T), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T-A), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-ethylene glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2' -Fluoro-2-thiouridine-3'-phosphate (U3f), 2-amino-2'-O-methyladenosine-3'-phosphate (a1), C3-spacer (Pr), 2',3'-open-ring adenosine-3'-phosphate (A2), 2',3'-open-ring guanosine-3'-phosphate (G2), 2',3' -Cyclocytosine-3'-phosphate (C2), 2',3'-Cyclouridine-3'-phosphate (U2), 2'-Deoxy-2-aminopurine nucleoside-3'-phosphate (dA3), 2'-Deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

[0262] In some respects, this paper describes a specific modification pattern in which the guiding strand contains 5'﹣msFsmmmmFmmmmFmFmFmmmmmsmsm﹣3' and the guest strand contains 5'﹣msmsmmmmFmFmFmmmmmmmmmm﹣3', where m represents a 2'﹣O﹣methyl modified nucleotide, F represents a 2'﹣F modified nucleotide, and s represents a phosphate thioester nucleotide bond.

[0263] Adhesion

[0264] Targeted portion

[0265] In some respects, the polynucleotide molecules described herein are coupled or conjugated to one or more targeting moieties to form polynucleotide-targeting moieties conjugate molecules. In some cases, the targeting moieties are selected based on their ability to selectively or preferably target the conjugate molecules described herein to a desired cell population, tissue, or organ. In some cases, the targeting moieties target cells, tissues, or organs that express a corresponding binding partner (e.g., a corresponding receptor or ligand) of the targeting moieties. For example, a polynucleotide molecule conjugated to N-acetylgalactosamine (GalNAc) can target hepatocytes expressing desialyl glycoprotein (ASGP-R). Any suitable GalNAc molecules known in the art to be used as targeting moieties are considered. Exemplary GalNAc molecules include tribranched GalNAc (e.g., L96). Another example of a targeting moiety is galactose. The targeting moieties can also be lipids, peptides, or small molecules.

[0266] The hybrid polynucleotide constructs disclosed herein may include a targeting portion (i.e., an intracellular targeting portion) that targets desired sites within the cell (e.g., the endoplasmic reticulum, Golgi apparatus, nucleus, or mitochondria). Non-limiting examples of intracellular targeting portions are provided in WO 2015 / 069932 and WO 2015 / 188197; the disclosures of the intracellular targeting portions in WO 2015 / 069932 and WO 2015 / 188197 are incorporated herein by reference.

[0267] Therefore, the polynucleotide molecules described herein may include one or more targeting moieties selected from intracellular targeting moieties, extracellular targeting moieties, and combinations thereof. Thus, including one or more targeting moieties (e.g., extracellular targeting moieties independently selected from folic acid, mannose, N-acetylgalactosamine, and prostate-specific membrane antigen) and one or more intracellular targeting moieties (e.g., moieties targeting the endoplasmic reticulum, Golgi apparatus, nucleus, or mitochondria) in the polynucleotide molecules described herein can facilitate the delivery of polynucleotides to specific sites within a specific cell population. In some aspects, the targeting moieties contain one or more mannose carbohydrates. The mannose targets the mannose receptor, a 175 kDa membrane-associated receptor expressed on hepatic sinusoidal cells and antigen-presenting cells (e.g., macrophages and dendritic cells). It is a highly efficient endocytic / recirculating receptor that binds to and internalizes mannosylated pathogens and proteins (Lennartz et al. J. Biol. Chem. 262:9942-9944, 1987; Taylor et al. J. Biol. Chem. 265:12156-62, 1990).

[0268] This article describes several targeting components. In some respects, the targeting components contain or specifically bind to proteins selected from the following: insulin, insulin-like growth factor receptor 1 (IGF1R), IGF2R, insulin-like growth factor (IGF; e.g., IGF 1 or 2), mesenchymal epithelial transforming factor receptor (c-met; also known as hepatocyte growth factor receptor (HGFR)), hepatocyte growth factor (HGF), epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), cytokinin, fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), platelet-derived growth factor (PDGF), vascular endothelial growth factor receptor (VEGFR), and tumor necrosis factor receptor. (TNFR), Tumor necrosis factor α (TNF-α), TNF-β, Folate receptor (FOLR), Folate, Transferrin, Transferrin receptor (TfR), Mesothelin, Fc receptor, c-kit receptor, c-kit, Integrins (e.g., α4 integrin or β-1 integrin), P-selectin, Sphingosine-1-phosphate receptor-1 (S1PR), Hyaluronic acid receptor, Leukocyte functional antigen-1 (LFA-1), CD4, CD11, CD18, CD20, CD25, CD27 CD52, CD70, CD80, CD85, CD95 (Fas receptor), CD106 (vascular cell adhesion molecule 1 (VCAM1)), CD166 (activated leukocyte adhesion molecule (ALCAM)), CD178 (Fas ligand), CD253 (TNF-associated apoptosis-inducing ligand (TRAIL)), ICOS ligand, CCR2, CXCR3, CCR5, CXCL12 (stromal cell-derived factor 1 (SDF-1)), interleukin-1 (IL-1) IL-1ra, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, CTLA-4, MART-1, gp100, MAGE-1, hepatic glycoprotein (Eph) receptor, mucosal addressin cell adhesion molecule 1 (MAdCAM-1), carcinoembryonic antigen (CEA), LewisY, MUC-1, epithelial cell adhesion molecule (EpCAM), cancer antigen 125 (CA125), prostate-specific membrane antigen (PSMA), TAG-72 antigen, and fragments thereof. In a further aspect, the targeting portion contains erythroblastoma leukemia virus oncogene homolog (ErbB) receptors (e.g., ErbB1 receptor; ErbB2 receptor; ErbB3 receptor; and ErbB4 receptor). In some aspects, the targeting portion contains one or more (e.g., 1 to 6) N-acetylgalactosamine (GalNAc). In some respects, the target portion contains one or more (e.g., 1 to 6) galactoses. In some respects, the target portion contains one or more (e.g., 1 to 6) mannoses.In other respects, the targeting portion contains a folic acid ligand. The folic acid ligand has the following structure:

[0269] .

[0270] Some targeting moieties may include bufotin, gastrin, gastrin-releasing peptide, tumor growth factor (TGF) (e.g., TGF-α or TGF-β), or vaccinia virus growth factor (VVGF). Non-peptide targeting moieties may also be used and may include, for example, steroids, carbohydrates, vitamins, and lectins. Some targeting moieties may include peptides such as somatostatin or somatostatin analogs (e.g., growth inhibitory peptide or lanreotide), bufotin, or antibodies or their antigen-binding fragments. Antibodies may be of any known class or subclass, such as IgG, IgA, IgM, IgD, or IgE. Typically, antibodies belonging to the IgG class are preferred. According to techniques known in the art, antibodies may be derived from any species. However, antibodies are typically human, mouse, or rabbit-derived. Furthermore, antibodies may be polyclonal or monoclonal, but are typically monoclonal. Human or chimeric (e.g., humanized) antibodies may be used for targeting moieties. Targeting moieties may include antigen-binding fragments of the antibody. Such antibody fragments may include, for example, Fab', F(ab')2, Fv or Fab fragments, single-domain antibodies, ScFv or other antigen-binding fragments. Fc fragments may also be used for targeting. These antibody fragments may be prepared, for example, by digestion with proteases, such as by pepsin or papain digestion, reductive alkylation, or recombinant techniques. The materials and methods used to prepare antibody fragments are well known to those skilled in the art. See, for example, Parham, J. Immunology, 131:2895, 1983; Lamoyi et al., J. Immunological Methods, 56:235, 1983.

[0271] Other peptides used as the targeting helper moiety in the polynucleotide molecules described herein may be selected from KiSS peptides and analogues, bony fish tension peptide II peptides and analogues, GnRH Peptides I and II and analogues, depreotide, vapreotide, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), RGD-containing peptides, melanocyte-stimulating hormone (MSH) peptides, neurotensin, calcitonin, glutathione, YIGSR (leukocyte-stimulating peptide, e.g., P483H, which contains the heparin-binding region of platelet factor-4 (PF-4) and a lysine-rich sequence), atrial natriuretic peptide (ANP), β-amyloid peptide, delta-opioid antagonists (such as ITIPP (psi)), annexin-V, endothelin, leukotriene B4 (LTB4), chemokines (e.g., N-formyl-methionyl-leucyl-phenylalanine-lysine (fMLFK), GP) IIb / IIIa receptor antagonists (e.g., DMP444), human neutrophil elastase inhibitors (EPI-HNE-2 and EPI-HNE-4), plasmin inhibitors, antimicrobial peptides, apticides (P280 and P274), platelet-reactive protein receptors (including analogs such as TP-1300), bitistatin, pituitary adenylate cyclase type I receptor (PAC1), fibrin α-chains, peptides derived from phage display libraries, and their conserved alternatives.

[0272] One or more (e.g., 1 to 6) target portions can be connected via –LinkA – to MOIETY or X2 in formula (V', V'', V''', V'''', V''''', V''''').

[0273] In some cases, the targeting portion includes a desialyl glycoprotein receptor targeting portion. In some cases, the desialyl glycoprotein receptor targeting portion comprises N-acetylgalactosamine (GalNAc) or galactose. In some aspects, the targeting portion includes one or more (e.g., 1 to 6 or 1 to 3) desialyl glycoprotein receptor ligands (e.g., GalNAc). In some aspects, the desialyl glycoprotein receptor ligand (e.g., GalNAc) is attached to -LinkA- via an anomeric carbon (e.g., where the anomeric carbon is a carbon atom in an acetal or sesamyl acetal). In some aspects, the desialyl glycoprotein receptor ligand (e.g., GalNAc) comprises an anomeric carbon bonded to a trivalent, tetravalent, pentavalent, or hexavalent linker, wherein the anomeric carbon is part of a sesamyl acetal group. Compared to hybrid polynucleotide constructs with desialyl glycoprotein receptor ligands (e.g., GalNAc) attached to the linker via methamine acetal, hybrid polynucleotide constructs with better efficacy in gene silencing can be produced.

[0274] In some cases, the targeting moiety is conjugated via a linker. In some aspects, formula (V) shows a linker and three desialyl glycoprotein receptor targeting moieties, each containing GalNAc. In some cases, the conjugates described herein contain only one desialyl glycoprotein receptor targeting moiety, and therefore the conjugate comprises the structure of formula (V) with any two targeting moieties removed. In some cases, the conjugates described herein contain only two desialyl glycoprotein receptor targeting moieties, and therefore the conjugates described herein comprise the structure of formula (V) with any one targeting moieties removed.

[0275]

[0276] (V),

[0277] One of Y1 and Y2 is a nucleotide, or both Y1 and Y2 are nucleotides, and Y1 and Y2 are consecutive or adjacent nucleotides from the polynucleotide molecule described herein.

[0278] In some aspects, the connector and targeting portion described herein are coupled to the 3' end of the transit chain (e.g., as shown in formulas (V', V'''', V''''', V''''')). In some aspects, the connector and targeting portion described herein are coupled to the 5' end of the transit chain (e.g., as shown in formula (V'') or (V''')). In some aspects, the connector and targeting portion described herein are coupled to the 3' end of the guide chain (e.g., as shown in formulas (V'), (V''''), (V'''''), (V''''')). In some aspects, the connector and targeting portion described herein are coupled to the 5' end of the guide chain (e.g., as shown in formula (V'') or (V''')).

[0279] (V'), wherein Z in formula (V') corresponds to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F or -O-methoxyethyl), and R in formula (V') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0280] (V''),

[0281] In formula (V''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (V'') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0282] (V'''),

[0283] In formula (V'''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (V''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0284] (V''''),

[0285] In formula (V''''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (V'''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0286] (V'''''),

[0287] In formula (V'''''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (V''''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0288]

[0289] (V''''''),

[0290] In formula (V''''''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (V'''''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0291] In some cases, the 3' end of the transit strand (or sense strand) of a polynucleotide molecule (e.g., transit strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formulas (V), (V'), (V''''), (V'''''), (V''''')). In some cases, the 5' end of the transit strand (or sense strand) of a polynucleotide molecule (e.g., transit strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formulas (V), (V''), or (V''')). In some cases, the nucleic acid in the transit strand (or sense strand) (not at the 5' or 3' end) (e.g., transit strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formula (V)). In some cases, the 3' end of the guide strand (or antisense strand) of the polynucleotide molecule (e.g., guide strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formulas (V), (V'), (V''''), (V'''''), (V''''')). In some cases, the 5' end of the guide strand (or antisense strand) of the polynucleotide molecule (e.g., guide strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formula (V), (V''), or (V''')). In other cases, the nucleic acid within the guide strand (or antisense strand) of the polynucleotide molecule (not at the 5' or 3' end) (e.g., guide strand sequences from Tables 1, 3, 5, 7, 9, 11, 13, 15, or 17) is conjugated to X2-GalNAc (see formula (V)).

[0292] In some cases, one or more endosome escape portions (e.g., 1 to 6 or 1 to 3) may be attached as auxiliary portions to the polynucleotide constructs or hybrid polynucleotide constructs disclosed herein. Exemplary endosome escape portions include chemotherapeutic agents described herein (e.g., quinolones such as chloroquine); fused lipids (e.g., dioleoylphosphatidylethanolamine (DOPE)); and polymers such as polyethyleneimine (PEI); poly(β-amino esters); polypeptides such as polyarginine (e.g., octaarginine) and polylysine (e.g., octalysine); proton sponges, viral capsids, and peptide transduction domains. For example, fusion peptides can be derived from the M2 protein of influenza A virus; peptide analogs of influenza virus hemagglutinin; the HEF protein of influenza C virus; transmembrane glycoproteins of filamentous viruses; transmembrane glycoproteins of rabies virus; transmembrane glycoproteins (G) of vesicular stomatitis virus; fusion proteins of Sendai virus; transmembrane glycoproteins of Semliki forest virus; fusion proteins of human respiratory syncytial virus (RSV); fusion proteins of measles virus; fusion proteins of Newcastle disease virus; fusion proteins of sheep exmyelinating virus; fusion proteins of murine leukemia virus; fusion proteins of HTL virus; and fusion proteins of simultaneous immunodeficiency virus (SIV). Other components that can be used to facilitate endosome escape are described in Dominska et al., Journal of Cell Science, 123(8):1183-1189, 2010. For example, specific examples of endosome escape portions are provided in WO 2015 / 188197, including portions suitable for conjugation to polynucleotide constructs for hybridization disclosed herein; the disclosure of these endosome escape portions is incorporated herein by reference.

[0293] As described herein, one or more internal escape portions (e.g., 1 to 6 or 1 to 3) can be attached to MOIETY or X2 in formula (V', V'', V''', V'''', V''''' or V'''''') via -LinkA.

[0294] One or more cell-penetrating peptides (CPPs) (e.g., 1 to 6 or 1 to 3) may be attached as helper moieties to the polynucleotide constructs or hybrid polynucleotide constructs disclosed herein. As disclosed herein, CPPs can be reversibly linked to the hybrid polynucleotides via disulfide bonds. Thus, upon delivery to a cell, CPPs can be cleaved intracellularly, for example by intracellular enzymes (e.g., protein disulfide isomerases, thioredoxins, or thioesterases), thereby releasing the polynucleotide.

[0295] CPPs are known in the art (e.g., TAT or Arg8) (Snyder and Dowdy, 2005, Expert Opin. Drug Deliv. 2, 43-51). For example, specific examples of CPPs including portions suitable for conjugation to polynucleotide constructs disclosed herein are provided in WO 2015 / 188197; the disclosure of these CPPs is incorporated herein by reference.

[0296] CPPs are positively charged peptides that facilitate the delivery of biological cargo into cells. The cationic charge of CPPs is believed to be crucial to their function. Furthermore, the transduction of these proteins appears to be independent of cell type, and they can be efficiently transduced in virtually all cultured cells without apparent toxicity (Nagahara et al., Nat. Med. 4:1449-52, 1998). Besides full-length proteins, CPP has been successfully used to induce intracellular responses to DNA (Abu-Amer, ibid.), antisense polynucleotides (Astriab-Fisher et al., Pharm. Res, 19:744-54, 2002), small molecules (Polyakov et al., Bioconjug. Chem. 11:762-71, 2000), and even inorganic 40 nm iron particles (Dodd et al., J. Immunol. Methods 256:89-105, 2001; Wunderbaldinger et al., Bioconjug. Chem. 13:264-8, 2002; Lewin et al., Nat. Biotechnol. 18:410-4, 2000; Josephson et al., Bioconjug. Chem. 10:186-91). The intake of (1999) indicates that particle size has considerable flexibility in the process.

[0297] In one embodiment, the CPP that can be used in the methods and compositions described herein comprises a peptide characterized by significant α-helicality. Transfection has been found to be optimized when the CPP exhibits significant α-helicality. In another embodiment, the CPP comprises a sequence containing basic amino acid residues substantially aligned along at least one face of the peptide. The CPP described herein can be a natural or synthetic peptide.

[0298] As described herein, one or more cell-penetrating peptides (e.g., 1 to 6 or 1 to 3) can be attached to MOIETY or X2 in the formula (V', V'', V''', V'''', V''''', V''''') via -LinkA-.

[0299] The polynucleotide constructs and hybrid polynucleotide constructs disclosed herein may also include covalently attached auxiliary moieties based on neutral polymers. Neutral polymers include poly(C1-6 epoxy), such as poly(ethylene glycol) and poly(propylene glycol), and copolymers thereof, such as diblock and triblock copolymers. Other examples of polymers include esterified poly(acrylic acid), esterified poly(glutamic acid), esterified poly(aspartic acid), poly(vinyl alcohol), poly(ethylene-co-vinyl alcohol), poly(N-vinylpyrrolidone), poly(ethyl oxazoline), poly(alkyl acrylate), poly(acrylamide), poly(N-alkylacrylamide), poly(N-acryloylmorpholine), poly(lactic acid), poly(glycolic acid), poly(dioxanone), poly(caprolactone), styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyurethane, N-isopropylacrylamide polymer, and poly(N,N-dialkylacrylamide). Exemplary polymer auxiliary portions may have molecular weights of less than 100, 300, 500, 1000, or 5000 Da (e.g., greater than 100 Da). Other polymers are known in the art.

[0300] As described herein, one or more polymers (e.g., 1 to 6 or 1 to 3) can be attached to MOIETY or X2 in the formula (V', V'', V''', V'''', V''''', V''''') via -LinkA-.

[0301] splicing joint

[0302] In some respects, the polynucleotide molecules described herein comprise a guide strand or transit strand bonded to at least one group of formula (I).

[0303]

[0304] (I)

[0305] Or its salt, or its stereoisomer,

[0306] in

[0307] Each X 1 It can be either O or S independently;

[0308] Each X 2 It can be independently of O, S, NH or a bond;

[0309] MOIETY is an optional substitution of C. 2﹣10 Alkane - tetramethyl or -M group 1 –M 2 –M 3– where each M 1 and each M 3 Independently for C that is absent or arbitrarily substituted 1﹣6 Alkylene, and M 2 C is an optional substitute 3﹣9 Heterocyclic tetramers, optionally substituted C 6﹣10 Aromatic - tetramethyl or optionally substituted C 3﹣8 Cycloalkanes -tetramethyl;

[0310] Each R 1 and each R 2 H independently, or C with optional substitution 1﹣16 Alkyl, optionally substituted C 2﹣16 Heteroalkyl, conjugated moiety, or –LinkA (–T) p As long as at least one R 1 Or at least one R 2 It is a join part or –LinkA(–T) p That's all;

[0311] Each R 3 H independently, or C with optional substitution 1﹣16 Alkyl, optionally substituted C 2﹣16 Heteroalkyl, optionally substituted C 2﹣16 alkenyl, optionally substituted C 2﹣16 alkynyl, optionally substituted (C 1﹣9 Heterocyclic group)﹣C 1﹣6 -alkyl, optionally substituted (C 6﹣10 Aryl)﹣C 1﹣6 -alkyl, optionally substituted (C 3﹣8 cycloalkyl)﹣C 1﹣6 -alkyl, conjugated, or –LinkA (–T) p ;

[0312] R 4 H, or C with optional substitution 1﹣6 Alkyl group, –LinkA (–T) p Or –Sol;

[0313] Each LinkA is independently a multivalent connector (e.g., including at least one multivalent connector of -C(O)-N(H)- (e.g., including at least one multivalent connector of -C(O)-N(H)- bonded to T)).

[0314] Each T is an independent auxiliary part;

[0315] Sol is a solid support;

[0316] m is an integer from 1 to 6;

[0317] Each n is independently 0 or 1;

[0318] Each p is an independent integer from 1 to 6; and

[0319] q is an integer from 0 to 3.

[0320] At least one group of formula (I) may be bonded to the 5' end, 3' end, inter-nucleotide phosphate, inter-nucleotide thiophosphate, or inter-nucleotide dithiophosphate of the polynucleotide. When at least one group of formula (I) is bonded to an inter-nucleotide phosphate, inter-nucleotide thiophosphate, or inter-nucleotide dithiophosphate, q is 0. The polynucleotide construct contains no more than one Sol.

[0321] The –LinkA– group may comprise 0 to 3 polyvalent monomers (e.g., optionally substituted C1-6 alkane-triyl, optionally substituted C1-6 alkane-tetrayl, or trivalent nitrogen atom) and one or more divalent monomers (e.g., 1 to 40), wherein each divalent monomer is independently an optionally substituted C1-6 alkylene; optionally substituted C2-6 alkenyl; optionally substituted C2-6 ynynyl; optionally substituted C3-8 cycloalkylene; optionally substituted C3-8 cycloalkenyl; optionally substituted C6-14 arylene; optionally substituted C1-9 heteroarylene having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted C1-9 heterocyclic group having 1 to 4 heteroatoms selected from N, O, and S; imino; optionally substituted N; O; or S(O)m, wherein m is 0, 1, or 2. In some respects, each monomer is independently an optionally substituted C1-6 alkylene; an optionally substituted C3-8 cycloalkylene; an optionally substituted C3-8 cycloalkenylene; an optionally substituted C6-14 arylene; an optionally substituted C1-9 heteroarylene having 1 to 4 heteroatoms selected from N, O and S; an optionally substituted C1-9 heterocyclic group having 1 to 4 heteroatoms selected from N, O and S; an imino; an optionally substituted N; O; or S(O)m, wherein m is 0, 1 or 2 (e.g., m is 2). In some respects, each monomer is independently a optionally substituted C1-6 alkylene; optionally substituted C3-8 cycloalkylene; optionally substituted C3-8 cycloalkenyl; optionally substituted C6-14 arylene; optionally substituted C1-9 heteroarylene having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted C1-9 heterocyclic group having 1 to 4 heteroatoms selected from N, O, and S; optionally substituted N; O; or S(O)m, where m is 0, 1, or 2 (e.g., m is 2). The non-biologically reversible linker connecting the auxiliary moiety to the conjugated moiety or its reaction product may comprise 2 to 500 (e.g., 2 to 300 or 2 to 200) such monomers. The -LinkA- group may include poly(epoxide) (e.g., polyethylene oxide, polypropylene oxide, poly(trimethylene oxide), polybutane oxide, poly(tetramethylene oxide), and their diblock or triblock copolymers). In some aspects, non-biological reversible linkers include polyethylene oxide (e.g., poly(ethylene oxide) with a molecular weight less than 1 kDa).

[0322] The –LinkA(–T)p group in formula (I) can be prepared by the process described in the following section. In some cases, –LinkA(–T)p is of formula (II):

[0323] –Q 1 –Q 2 ([–Q 3 –Q 4 –Q 5 ] s –Q 6–T) p ,

[0324] (II)

[0325] in

[0326] Each s is an integer from 0 to 20 (e.g., 0 to 10), where repeating units may be the same or different;

[0327] Q 1 It is a splice joint (e.g., [–Q) 3 –Q 4 –Q 5 ] s –Q C –, where Q C C is an optional substitute 2﹣12 Heteroalkylene (e.g., heteroalkylene containing –C(O)–N(H)–, –N(H)–C(O)–, –S(O)2–N(H)– or –N(H)–S(O)2–), optionally substituted C 1﹣12 Thioyl heterocyclic groups (e.g., , , , or ), optional substitution of C 1﹣12 Heterocyclic groups (e.g., 1,2,3-triazole-1,4-diyl or...) ), cyclobutane-3-ene-1,2-dione-3,4-diyl or pyridine-2-ylhydrazone);

[0328] If p is 1, then Q 2 It is a linear group (e.g., [–Q) 3 –Q 4 –Q 5 ] s –), or if p is an integer from 2 to 6, then Q 2 It is a branched group (e.g., [–Q) 3 –Q 4 –Q 5 ] s –Q 7 ([–Q 3 –Q 4 –Q 5 ] s –(Q 7 ) p1 ) p2 (where P1 is 0 or 1, and p2 is 0, 1, 2, or 3).

[0329] Each Q 3 and each Q 6Independently, -CO-, -NH-, -O-, -S-, -SO2-, -OC(O)-,

[0330] –COO–, –NHC(O)–, –C(O)NH–, –CH2–, –CH2NH–, –NHCH2–, –CH2O– or –OCH2–;

[0331] Each Q 4 Independently, C is non-existent or arbitrarily substituted. 1﹣12 Alkylene, optionally substituted C 2﹣12 alkenyl, optionally substituted C 2﹣12 Ethyne group, optionally substituted C 2﹣12 Heteroalkylene, optionally substituted C 6﹣10 aryl, optionally substituted C 1﹣9 heteroaryl or optionally substituted C 1﹣9 Sub-heterocyclic groups;

[0332] Each Q 5 Independently, -CO-, -NH-, -O-, -S-, -SO2-, -CH2-, -C(O)O-, -OC(O)-,

[0333] –C(O)NH–, –NH–C(O)–, –NH–CH(R a –C(O)– or –C(O)–CH(R) a )–NH–;

[0334] Each Q 7 C is independently optional substitution 1﹣6 Alkyl-trimethyl, optional substituted C 1﹣6 Alkyl-tetramethyl, optionally substituted C 2﹣6 Heteroalkanes - trimethyl or optionally substituted C 2﹣6 Heteroalkanes - tetramethyl; and

[0335] Each R a It is independently an H or amino acid side chain;

[0336] As long as Q 3 Q 4 and Q 5 The presence of at least one of them is sufficient.

[0337] In some respects, each Q 4 Independently, C is non-existent or arbitrarily substituted. 1﹣12 Alkylene, optionally substituted C 2﹣12 alkenyl, optionally substituted C 2﹣12 Ethyne group, optionally substituted C 2﹣12 Heteroalkylene or optionally substituted C 1﹣9Sub-heterocyclic group. In some respects, s is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0338] Therefore, in equation (II), if each p1 is 0, then LinkA may include a single branch point, or if at least one p1 is 1, then LinkA may include multiple branch points.

[0339] In equation (II), Q 1 It can be –O–Q L –Q C –, where Q L C is an optional substitute 2﹣12 Heteroalkylene, optionally substituted C 1﹣12 Alkylene or – (optionally substituted C 1﹣6 Alkylene) – (optionally substituted C 6﹣10 (Aspartic) –. In some respects, Q L C is an optional substitute 2﹣12 Heteroalkylene or optionally substituted C 1﹣12 Alkylene. In formula (II), Q C It could be:

[0340] , , or .

[0341] In equation (II), Q 2 It can be the formula [–Q] 3 –Q 4 –Q 5 ] s – linear groups, where Q 3 Q 4 and Q 5 As defined for equation (II). Alternatively, Q 2 It can be a branched group [–Q] 3 –Q 4 –Q 5 ] s –Q 7 ([–Q 3 –Q 4 –Q 5 ] s –(Q 7 ) p1 ) p2 , where each Q 7 C is independently optional substitution 1﹣6 Alkyl-trimethyl, optional substituted C 1﹣6 Alkyl-tetramethyl, optionally substituted C2﹣6 Heteroalkanes - trimethyl or optionally substituted C 2﹣6 Heteroalkanes - tetramethyl;

[0342] in

[0343] p1 is 0 or 1;

[0344] p2 is 0, 1, 2, or 3;

[0345] in,

[0346] When p1 is 0, LinkA is a trivalent or tetravalent connector, and...

[0347] When p1 is 1, LinkA is a tetravalent, pentavalent, or hexavalent connector.

[0348] In some respects, p1 is 0.

[0349] In some respects, Q 7 yes:

[0350] , , or .

[0351] This document and WO 2015 / 188197 describe compounds that can be used to prepare the group –LinkA(-T)p of formula (I). Non-limiting examples of –LinkA include:

[0352] , , ,

[0353] (i) (ii) (iii)

[0354] ,

[0355] (iv)

[0356] ,

[0357] (v)

[0358] ,

[0359] (vi)

[0360] ,

[0361] (vii)

[0362] , ,

[0363] (viii) (ix)

[0364] ,

[0365] (x)

[0366] ,

[0367] (xi)

[0368] ,

[0369] (xii)

[0370] ,

[0371] (xiii)

[0372] ,as well as

[0373] (xiv)

[0374]

[0375] (xv)

[0376] in

[0377] R 18 It is the key with MOIETY.

[0378] Each R 19 It is a key independent of the auxiliary part.

[0379] Each m5 is an integer from 1 to 20.

[0380] Each m6 is an integer from 1 to 10 independently.

[0381] m7 is an integer from 1 to 6, and

[0382] Each X 6 It can be either O or S on its own.

[0383] In equation (II), when the splice joint is of equation [–Q] 3 –Q 4 –Q 5 ] s –Q C –when, –Q 2 ([–Q 3 –Q 4 –Q 5 ] s –Q 6 –T) p It could be:

[0384] , , ,

[0385] (xvi) (xvii) (xviii)

[0386] ,

[0387] (xix)

[0388] ,

[0389] (xx)

[0390] ,

[0391] (xxi)

[0392] ,

[0393] (xxii)

[0394] ,

[0395] (xxiii)

[0396] ,or

[0397] (xxiv)

[0398]

[0399] (xxv)

[0400] in

[0401] R 20 Is with Q 1 China Q C The key,

[0402] Each R 19 It is a key independent of the auxiliary part.

[0403] Each m5 is an integer from 1 to 20.

[0404] Each m6 is an integer from 1 to 10 independently.

[0405] m7 is an integer from 1 to 6, and

[0406] Each X 6 It can be either O or S on its own.

[0407] In some respects, the joint described herein is cuttable. In other respects, the joint described herein is not cuttable.

[0408] In some respects, the polynucleotide molecules described herein comprise a guide strand or transit strand bonded to at least one group of formula (IV).

[0409]

[0410] (IV)

[0411] At least one of Y1 or Y2 is a nucleotide in a polynucleotide molecule.

[0412] In some cases, the adapter is contained in form (IV). In some cases, the adapter of the last nucleotide at the 3' end of the transit strand of the multinucleic acid molecule and the desialylated glycoprotein receptor targeting portion are shown in (V'), (V''''), (V'''''), or (V''''') as described herein.

[0413] In some cases, Y1 is the last nucleotide at the 3' end or the first nucleotide at the 5' end of one strand of a polynucleotide molecule. In some cases, Y1 is the last nucleotide at the 3' end or the first nucleotide at the 5' end of a transit strand of a polynucleotide molecule. In some cases, Y1 is the last nucleotide at the 3' end or the first nucleotide at the 5' end of a transit strand of a polynucleotide molecule, and Y2 is a 3-hydroxy-propoxy group. In some cases, Y2 is the first nucleotide at the 5' end or the last nucleotide at the 3' end of one strand of a polynucleotide molecule. In some cases, Y2 is the first nucleotide at the 5' end or the last nucleotide at the 3' end of a transit strand of a polynucleotide molecule. In some cases, Y2 is the first nucleotide at the 5' end or the last nucleotide at the 3' end of a transit strand of a polynucleotide molecule, and Y1 is a 3-hydroxy-propoxy group. In other cases, Y1 and Y2 are two consecutive nucleotides in one strand of a polynucleotide molecule.

[0414] In some aspects, the target portion described herein is concatenated with the 3' end of the transit chain (e.g., formula (IV')). In some aspects, the target portion described herein is concatenated with the 5' end of the transit chain (e.g., formula (IV'') or (IV''')). In some aspects, the target portion described herein is concatenated with the 3' end of the guide chain (e.g., formula (IV')). In some aspects, the target portion described herein is concatenated with the 5' end of the guide chain (e.g., formula (IV'') or (IV''')).

[0415] (IV'),

[0416] In formula (IV'), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (IV') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0417] (IV'')

[0418] In formula (IV''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (IV'') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0419] (IV'''),

[0420] In formula (IV'''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (IV''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0421] (IV''''),

[0422] In formula (IV''''), Z is a part corresponding to one of the sugar modifications described herein (e.g., -H, -OH, -O-methyl, -F, or -O-methoxyethyl), and R in formula (IV'''') is adenine, uracil, guanine, cytosine, thymine, debasement, etc.

[0423] In some aspects, a linker conjugated to one or more target portions, as shown in formula (IV'') or (IV'''), is added to the first nucleotide at the 5' end. In some aspects, a linker conjugated to one or more GalNAc, as shown in formula (V'') or (V'''), is added to the first nucleotide at the 5' end. In some aspects, the modification pattern comprises one or more phosphate thioester-modified nucleotide internucleotide bonds. In some aspects, the modification pattern is shown in formula (VII). In some aspects, 5' end modifications known in the art are applied to one or more inverted nucleotides.

[0424] Pharmaceutical Composition

[0425] The delivery of the polynucleotide molecules described herein can be achieved by contacting cells with the construct using a variety of methods. In certain respects, the polynucleotide molecules described herein are formulated with a variety of excipients, mediators, and carriers, as described more fully elsewhere herein.

[0426] The pharmaceutical compositions described herein can be prepared into forms suitable for administration to a subject, including the hybridized polynucleotide constructs disclosed herein, using carriers, excipients, and mediators. Commonly used excipients include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycol and solvents (such as sterile water), alcohols, glycerol, and polyols. Intravenous mediators include fluids and nutritional supplements. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. Other pharmaceutically acceptable mediators include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, etc., as described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippencott Williams & Wilkins (2005) and The United States Pharmacopeia: The National Formulary (USP 36NF31), published in 2013. The pH and precise concentration of the various components in the pharmaceutical composition are adjusted according to conventional techniques in the art. See Goodman and Gilman, The Pharmacological Basis for Therapeutics.

[0427] The pharmaceutical compositions described herein can be administered topically or systemically. The therapeutically effective dose can vary depending on a variety of factors, such as the degree of infection in the subject, the individual's age, sex, and weight. Dosing regimens can be adjusted to provide the optimal therapeutic response. For example, several fractionated doses can be administered daily, or the dose can be reduced proportionally according to the urgency of the treatment situation.

[0428] Pharmaceutical compositions can be administered in convenient ways, such as by injection (e.g., subcutaneous, intravenous, intraoral, etc.), oral administration, ocular application, inhalation, topical application, or rectal administration. Depending on the route of administration, the pharmaceutical composition may be coated with a material to protect it from enzymes, acids, and other natural conditions that could inactivate it. Pharmaceutical compositions can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent microbial growth.

[0429] Suitable injectable pharmaceutical compositions include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. The compositions are typically sterile and fluid, to the extent that they are easy to inject. The compositions are typically stable under the conditions of production and storage, and preserved against contamination by microorganisms such as bacteria and fungi. The medium can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size (in the case of dispersions), and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents are used in the compositions, such as sugars, polyols such as mannitol and sorbitol, or sodium chloride. Extended absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0430] A sterile injectable solution can be prepared by incorporating the desired amount of the pharmaceutical composition with one or a combination of the ingredients listed above into a suitable solvent as needed, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the pharmaceutical composition into a sterile medium containing a basic dispersion medium and the other desired ingredients listed above.

[0431] For ease of administration and dosage uniformity, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, unit dosage form refers to a physically discrete unit suitable as a unit dose for the target of treatment; each unit containing a predetermined amount of the pharmaceutical composition is calculated to combine with a desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form are related to the characteristics of the pharmaceutical composition and the specific therapeutic effect to be achieved. For convenient and effective administration, an effective amount of the main pharmaceutical composition is compounded with a suitable pharmaceutically acceptable carrier in acceptable unit dosage forms. In the case of compositions containing supplemental active ingredients, the dosage is determined by referring to the usual dosage and method of administration of the ingredient.

[0432] Pharmaceutical compositions can be administered orally, for example, in enteric-coated unit dosage forms, such as in a carrier. Pharmaceutical compositions and other ingredients can also be encapsulated in hard or soft-shell gelatin capsules or compressed into tablets. For oral therapeutic use, the pharmaceutical composition can be mixed with excipients and used in ingestible tablets, lozenges, capsules, pills, sheets, etc. Such compositions and formulations may contain at least 1% by weight of the active compound. Of course, the percentage of the composition and formulation can vary and can conveniently be between about 5% to about 80% by weight per unit. Tablets, lozenges, pills, capsules, etc., may also contain: binders such as gum arabic, gum arabic, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint, wintergreen oil, or cherry flavoring. When the dosage unit form is a capsule, it may also contain a liquid carrier in addition to the materials of the types described above. Various other materials may exist as coatings or to otherwise alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrups or elixirs may contain agents, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange. Any material used to prepare any dosage unit form may have pharmaceutically acceptable purity and be substantially non-toxic at the amount used. Furthermore, pharmaceutical compositions may be incorporated into sustained-release formulations and formulations.

[0433] The pharmaceutical compositions described herein may comprise one or more permeation enhancers that promote the bioavailability of the polynucleotide molecules described herein. WO 2000 / 67798, Muranishi, 1990, Crit. Rev. Ther. DrugCarrier Systems, 7, 1, Lee et al., 1991, Crit. Rev. Ther. Drug Carrier Systems, 8, 91 are incorporated herein by reference in their entirety. In some aspects, the permeation enhancer is enteric. In some aspects, the permeation enhancer is transdermal. In some aspects, the permeation enhancer facilitates crossing the blood-brain barrier. In some aspects, the permeation enhancer improves permeability in oral, nasal, buccal, pulmonary, vaginal, or corneal delivery models. In some aspects, the permeation enhancer is a fatty acid or a derivative thereof. In some aspects, the permeation enhancer is a surfactant or a derivative thereof. In some aspects, the permeation enhancer is a bile salt or a derivative thereof. In some aspects, the permeation enhancer is a chelating agent or a derivative thereof. In some aspects, the permeation enhancer is a non-chelating non-surfactant or a derivative thereof. In some aspects, the permeation enhancer is an ester or a derivative thereof. In some respects, the penetration enhancer is an ether or a derivative thereof. In other respects, the penetration enhancer is arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, decanoic acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, didecanoate, tridecanoate, monooleate, dilaurin, 1-monodecanoate, 1-dodecylazine-heptane-2-one, acylcarnitine, acylcholine, or their monoglycerides, diglycerides, or pharmaceutically acceptable salts. In one specific respect, the penetration enhancer is sodium decanoate (C10). In some applications, the penetration enhancer is chenodeoxycholic acid (CDCA), ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glutocholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidic acid, or sodium saccharide dihydrofusidic acid. In other applications, the penetration enhancer is polyoxyethylene-9-lauryl ether or polyoxyethylene-20-cetyl ether.

[0434] For the polynucleotide molecules described herein, suitable pharmaceutically acceptable salts include (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, and calcium, and polyamines such as spermine and spermidine; (ii) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and (iii) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid.

[0435] Although the hybrid polynucleotide constructs described herein may not require the use of excipients for delivery to target cells, the use of excipients can be advantageous in some respects. Therefore, for delivery to target cells, the hybrid polynucleotide molecules described herein can nonvalently bind excipients to form complexes. Excipients can be used to alter post-delivery biodistribution, enhance uptake, increase the half-life or stability of the chains in the hybrid polynucleotide constructs (e.g., improve nuclease resistance), and / or increase targeting to specific cell or tissue types.

[0436] Exemplary excipients include condensing agents (e.g., agents capable of attracting or binding nucleic acids through ionic or electrostatic interactions); fusion agents (e.g., agents capable of fusing with and / or transporting across cell membranes); proteins targeting specific cell or tissue types (e.g., thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactant protein A, or any other protein); lipids; lipopolysaccharides; lipid micelles or liposomes (e.g., formed from phospholipids such as phosphatidylcholine, fatty acids, glycolipids, ceramides, glycerides, cholesterol, or any combination thereof); and nanoparticles (e.g., silica, lipids, carbohydrates, or...). Other pharmaceutically acceptable polymer nanoparticles); polyplexes formed from cationic polymers and anionic agents (e.g., CROs), wherein exemplary cationic polymers include polyamines (e.g., polylysine, polyarginine, polyamidoamine, and polyethyleneimine); cholesterol; dendritic polymers (e.g., polyamidoamine (PAMAM) dendritic polymers); serum proteins (e.g., human serum albumin (HSA) or low-density lipoprotein (LDL)); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid). Lipids; synthetic polymers (e.g., polylysine (PLL), polyethyleneimine, poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-hydroxyacetic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, pseudopeptide-polyamine, pseudopeptide polyamine, or polyamine); cationic moieties (e.g., cationic lipids, cationic porphyrins, quaternary ammonium chlorides of polyamines). Salts or α-helical peptides; polyvalent sugars (e.g., polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose); vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, folic acid, vitamin B12, riboflavin, biotin, or pyridoxal); cofactors; or drugs that disrupt the cytoskeleton to increase uptake (e.g., paclitaxel, vincristine, vinblastine, pinocembrin, nocodazole, japlakinolide, red sea sponge A, phalloidin, swinholide A, indanocine, or myoservin).

[0437] Other therapeutic agents as described herein can be combined with the polynucleic acid molecules described herein and incorporated into the pharmaceutical compositions described herein.

[0438] method

[0439] In some aspects, this document describes a method for regulating the expression of a target gene in a subject, comprising: administering to the subject a polynucleotide molecule, a polynucleotide conjugate, or a pharmaceutical composition as described herein, thereby regulating the expression of a target gene in the subject. In some aspects, this document describes a method for regulating the expression of a target RNA in a subject, comprising: administering to the subject a polynucleotide molecule, a polynucleotide conjugate, or a pharmaceutical composition as described herein, thereby regulating the expression of a target RNA in the subject.

[0440] In some aspects, this document describes a method for regulating the mRNA or protein expression levels of a target gene in a cell, comprising: exposing the cell to an inhibitory polynucleotide molecule described herein, thereby regulating the mRNA or protein expression levels of the target gene in the cell. In some aspects, this document describes a method for regulating the mRNA or protein expression levels of a target gene in a desired subject, comprising: administering to the subject an inhibitory polynucleotide molecule described herein or a pharmaceutical composition described herein, thereby regulating the mRNA or protein expression levels of the target gene in the subject.

[0441] In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 10% or at least 10%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 20% or at least 20%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 30% or at least 30%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 40% or at least 40%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 50% or at least 50%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 60% or at least 60%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 70% or at least 70%. In some aspects, compared with a negative control, the method described herein reduces the expression of the target gene in the subjects by about 80% or at least 80%. In some respects, the methods described herein reduce the expression of target genes in subjects by approximately 90% or at least 90% compared to negative controls. In some respects, the methods described herein reduce the expression of target genes in subjects by approximately 100% compared to negative controls.

[0442] In some aspects, the methods described herein achieve an IC50 value of approximately 5 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 10 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 15 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 20 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 25 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 30 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 35 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 40 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 45 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 50 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 55 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 60 nM. In some aspects, the methods described herein achieve an IC50 value of approximately 65 nM. In some aspects, the method described herein achieves an IC50 value of approximately 70 nM. In some aspects, the method described herein achieves an IC50 value of approximately 75 nM. In some aspects, the method described herein achieves an IC50 value of approximately 80 nM. In some aspects, the method described herein achieves an IC50 value of approximately 85 nM. In some aspects, the method described herein achieves an IC50 value of approximately 90 nM. In some aspects, the method described herein achieves an IC50 value of approximately 95 nM. In some aspects, the method described herein achieves an IC50 value of approximately 100 nM.

[0443] In one aspect, this document provides a method for suppressing off-target effects caused by the guide strand of an inhibitory polynucleotide molecule in a cell, comprising introducing the inhibitory polynucleotide molecule described herein into the cell. In another aspect, this document provides a method for suppressing off-target effects caused by the guide strand of an inhibitory polynucleotide molecule in a subject, comprising administering the inhibitory polynucleotide molecule described herein to the subject. In some cases, this document provides a method for improving the stability and efficacy of the guide strand of an inhibitory polynucleotide molecule in a cell, comprising introducing the inhibitory polynucleotide molecule described herein into the cell. In some cases, this document provides a method for improving the stability and efficacy of the guide strand of an inhibitory polynucleotide molecule in a subject, comprising administering the inhibitory polynucleotide molecule described herein to the subject.

[0444] Example

[0445] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein. For all sequences presented herein, the oligonucleotide structure representation is written from left to right (5' to 3'). Unless otherwise specified, monomer codes present in the oligonucleotide code are linked by a 5'-3' phosphodiester bond (followed by a 3' internucleotide bond when written from left to right). The abbreviations of the nucleotide monomers used in the oligonucleotide structure representation are as follows: "A" represents adenosine-3'-phosphate; "a" represents 2'-O-methyladenosine-3'-phosphate; "Af" represents 2'-fluoroadenosine-3'-phosphate; "dA" represents 2'-deoxyadenosine-3'-phosphate; "dA3" represents 2'-deoxy-2-aminopurine nucleotide-3'-phosphate. Acids; "a1" represents 2-amino-2'-O-methyladenosine-3'-phosphate; "A2" represents 2',3'-open-cyclic adenosine-3'-phosphate; "C" represents cytidine-3'-phosphate; "c" represents 2'-O-methylcytidine-3'-phosphate; "Cf" represents 2'-fluorocytidine- 3'-phosphate; "dC" represents 2'-deoxycytidine-3'-phosphate; "C2" represents 2',3'-open-cytidine-3'-phosphate; "G" represents guanosine-3'-phosphate; "g" represents 2'-O-methylguanosine-3'-phosphate; "Gf" represents 2'-fluoroguanosine-3'-phosphate; "dG" represents 2'-deoxyguanosine-3'-phosphate; "G2" represents 2',3'-open-guanosine-3'-phosphate; "i" represents 2'-O-methylinosine-3'-phosphate; "U" represents uridine-3'-phosphate; "u" represents 2'-O-methyluridine-3'-phosphate. "Uf" represents 2'-fluorouridine-3'-phosphate; "u3" represents 2'-O-methyl-2-thiouridine-3'-phosphate; "U3f" represents 2'-fluoro-2-thiouridine-3'-phosphate; "dU" represents 2'-deoxyuridine-3'-phosphate; "U2" represents 2 ',3'-open-ring uridine-3'-phosphate; "T" represents 5-methyluridine-3'-phosphate; "t" represents 2'-O-methyl-5-methyluridine-3'-phosphate; "Tf" represents 2'-fluoro-5-methyluridine-3'-phosphate; "dT" represents thymidine-3'-phosphate; “dT3” represents 2’-deoxy-2’-thiothymidine-3’-phosphate; “s” represents 3’-thiophosphate ester; “(Pr)” represents C3-spacer; “(T-T)” represents acyclic L-threonine nucleic acid-thymidine-3’-phosphate; “(T-A)” represents acyclic L-threonine nucleic acid-adenine-3’-phosphate; “(T-NAc)” represents acyclic N-acetyl L-threonine debased nucleic acid-3’-phosphate; “(dAB)” represents 1’,2’-dideoxyribose-3’-phosphate; and “(Tgn)” represents thymidine-ethylene glycol nucleic acid (GNA) S-isomer.

[0446] Example 1 – Testing AGT siRNA in transgenic mice

[0447] The sequences of the evaluated siRNAs are specified in Table 1, with the 3' end of each transit strand (or sense strand) conjugated to GalNAc via X2 (see Equation (V')). Seed region modifications of the parental sequence SRS-001711 were evaluated. Transgenic AGT mice (HuAoGen mice, B6.Cg-Tg(hAGT)2041Sig / J, Jackson Laboratory) were assigned to treatment groups (n=5, 3 males, 2 females). AGT siRNA was administered as a single subcutaneous injection of 3 mg / kg to each treatment group on day 0. Blood samples were collected before administration (days -7 and 0) and on days 7, 14, 21, 28, and 35. Blood was processed into plasma, and the levels of circulating AGT protein in all plasma samples were analyzed using an AGT ELISA assay (IBL America, #27412). Results for each individual were calculated as the percentage of circulating AGT protein remaining relative to the pre-drug mean (days -7, day 0), and group means are listed in Table 2. Furthermore, hepatic hAGT mRNA was measured on day 35 using specific primers via qPCR (normalized to mouse ACTB). Figure 1 As shown, expression relative to the saline control group is expressed as mean ± standard deviation. In summary, these results indicate that these seed region-modified siRNAs effectively reduce human AGT mRNA in the liver and AGT protein in plasma. For example, SRS-002055, SRS-002060, and SRS-002061 reduced plasma and hepatic AGT mRNA to levels similar to those of SRS-001711.

[0448] Example 2 – Testing AGT siRNA in transgenic mice

[0449] The sequences of the evaluated siRNAs are specified in Table 3, where the 3' end of each guest strand (or sense strand) is conjugated to GalNAc via X2 (see formula (V'''')). Seed region modifications of the parental sequences SRS-002085, SRS-002088, and SRS-002093 were evaluated. Transgenic AGT mice (HuAoGen mice, B6.Cg-Tg(hAGT)2041Sig / J, Jackson Laboratory) were assigned to treatment groups (n=4 or 5, 1–2 males, 2–3 females). A single subcutaneous injection of 3 mg / kg AGT siRNA was administered to each treatment group on day 0. Blood samples were collected before administration (days -7 and 0) and on day 28. Blood was processed into plasma, and circulating AGT protein levels in all plasma samples were analyzed using an AGT ELISA assay (IBL America, #27412). The results for each individual on day 28 were calculated as the percentage of circulating AGT protein remaining relative to the pre-drug mean (days -7, 0), and group means are listed in Table 4. Additionally, hepatic hAGT mRNA was measured on day 35 using specific primers via qPCR (normalized to mouse ACTB). Figure 2 As shown, expression relative to the saline control group is expressed as mean ± standard deviation. These results indicate that the seed region modifications evaluated against the parental sequence of SRS-002093 are well tolerated. For example, SRS-002094 and SRS-002095 reduced plasma AGT by -83% and -80%, respectively, on day 28. Furthermore, SRS-002094 and SRS-002095 reduced hepatic AGT mRNA by -69% and -74%, respectively, on day 35. These reductions were similar to improvements when compared to the parental sequence of SRS-002093. The results demonstrate that these seed region-modified siRNAs can reduce AGT mRNA expression in the liver and AGT protein in plasma.

[0450] Example 3 – Testing AGT siRNA in transgenic mice

[0451] The sequences of the evaluated siRNAs are specified in Table 5, where the 3' end of each transient (or sense) strand is conjugated to GalNAc via X2 (see Formula (V')). Transgenic AGT mice (HuAoGen mice, B6.Cg﹣Tg(hAGT)2041Sig / J, Jackson Laboratory) were assigned to treatment groups (n=4, 2–3 males, 1–2 females). A single subcutaneous injection of 2 mg / kg AGT siRNA was administered to each treatment group on day 0. Blood samples were collected before administration (days –7, 0) and on days 7, 14, 21, 28, 35, and 42. Blood was processed into plasma, and the levels of circulating AGT protein in all plasma samples were analyzed using an AGT ELISA assay (IBL America, #27412). Results for each individual were calculated as the percentage of circulating AGT protein remaining relative to the pre-drug mean (days -7, day 0), and group means are listed in Table 6. Furthermore, hepatic hAGT mRNA was measured on day 42 using specific primers via qPCR (normalized to mouse ACTB). Figure 3 As shown, expression relative to the saline control group is expressed as mean ± standard deviation. In summary, these results indicate that these siRNAs effectively reduce human AGT mRNA in the liver and reduce AGT protein in plasma.

[0452] Example 4 – Testing AGT siRNA in non-human primates

[0453] The sequences of the siRNAs used in the non-human primate studies are specified in Table 7, where the 3' end of each guest strand (or sense strand) is conjugated to GalNAc via X2 (see Formula (V')). Four male cynomolgus monkeys were assigned to each treatment group. A single subcutaneous injection of 2 mg / kg AGT siRNA (as shown in Table 7) was administered to each treatment group on day 1. Blood samples were collected before administration (days -14, -7, and 1), and on days 8, 15, 22, 29, and 36. Blood was processed into serum, and the levels of circulating AGT protein in all serum samples were analyzed using the AGT ELISA assay (IBL America, #27412). Figure 4 As shown in Table 8, serum AGT levels decreased after administration of AGT siRNA. Results for each individual were calculated as a percentage change in circulating AGT protein relative to the time point prior to day 1 dosing, and group values ​​are expressed as mean ± standard deviation.

[0454] Example 5 – Testing ANGPTL3 siRNA in transgenic mice

[0455] The selected siRNAs or PBS shown in Table 9 were administered as a single subcutaneous dose to transgenic ANGPTL3 mice (n=5 per group), which were commercially available from Shanghai Model Organisms Center, Inc. (C57BL / 6-Angptl3). em2 (hANGPTL3)Smoc (Catalogue No. NM-HU-210036). The 3' end of the transient / sense strand of each siRNA is conjugated to a three-branched GalNAc moiety (X2-GalNAc in formula (V')). The selected siRNA was administered at a single subcutaneous dose of 1 mg / kg on day 0. Serum hANGPTL3 protein levels were measured by ELISA (R&D Systems, Catalogue No. DANL30) on days -4 (before administration) and on days 7, 14, 21, 28, and 35. For each individual, the percentage change in serum hANGPTL3 relative to the mean of the PBS group was calculated at each time point. The mean percentage change in serum hANGPTL3 protein levels relative to the PBS group and the standard error are reported in Table 10. The results are plotted and shown in Figure 5 middle.

[0456] Example 6 – Testing selected Lp(a) siRNA in cynomolgus monkeys

[0457] Eight Lp(a) siRNAs were tested in cynomolgus monkeys: SRS-000018, SRS-001955, SRS-001958, SRS-000016, SRS-002041, SRS-002042, SRS-002064, and SRS-002065, the sequences of which are specified in Table 11. The 3' end of the transient (or sense) strand of each siRNA is conjugated to GalNAc via X2 (see formula (V')). The Lp(a) siRNAs have sequences that cross-react with the cynomolgus monkey LPA gene. Each Lp(a) siRNA or saline was administered as a single subcutaneous injection of 1 mg / kg to male cynomolgus monkeys (each treatment group / siRNA, n=4). Animals were fasted overnight before blood samples were collected on days -15, -8, and 1 (before administration) and on days 4, 8, 11, 15, 22, 29, 36, 43, and 50. Additional blood samples were collected on days 57, 64, 71, 78, 85, 99, 113, 127, 141, and 155 for the groups treated with SRS-000018, SRS-00016, SRS-002064, and SRS-002065. Circulating Lp(a) protein levels in all serum samples were analyzed using an Lp(a) ELISA assay (Abcam, catalog number ab212165). Results for each individual animal are expressed as a percentage change in circulating Lp(a) protein relative to the pre-administration (day 1) time point. Mean percentage change and standard deviation for each treatment group are plotted on [data missing]. Figure 6 The results are shown in Table 12. The results indicated that serum Lp(a) protein levels decreased after administration of Lp(a) siRNA.

[0458] Example 7: Testing selected Lp(a) siRNA in cynomolgus monkeys

[0459] Eight Lp(a) siRNAs were tested in cynomolgus monkeys: SRS-000018, SRS-001955, SRS-001958, SRS-000016, SRS-002041, SRS-002042, SRS-002064, and SRS-002065, the sequences of which are specified in Table 13. The 3' end of the transient strand (or sense strand) of each siRNA is conjugated to GalNAc via X2 (see formula (V')). The Lp(a) siRNAs have sequences that cross-react with the cynomolgus monkey LPA gene. Each Lp(a) siRNA was administered as a single subcutaneous injection of 0.5 mg / kg to male cynomolgus monkeys (each treatment group / siRNA, n=4). Animals were fasted overnight before blood samples were collected on days -15, -8, and 1 (before administration) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 99, and 113. Circulating Lp(a) protein levels in all serum samples were analyzed using an Lp(a) ELISA assay (Abcam, catalog number ab212165). Results for each individual animal are expressed as a percentage change in circulating Lp(a) protein relative to the pre-administration (day 1) time point. Mean percentage change and standard deviation for each treatment group are plotted on [data missing]. Figure 7 The results are shown in Table 14. The results indicated that serum Lp(a) protein levels decreased after administration of Lp(a) siRNA.

[0460] Example 8: Testing selected Lp(a) siRNA in cynomolgus monkeys

[0461] Three Lp(a) siRNAs were tested in cynomolgus monkeys: SRS-002064, SRS-002378, and SRS-002379, whose sequences are specified in Table 15. The 3' end of the transient (or sense) strand of each siRNA is conjugated to GalNAc via X2 (see formula (V')). The Lp(a) siRNAs have sequences that cross-react with the cynomolgus monkey LPA gene. Each Lp(a) siRNA was administered as a single subcutaneous injection of 0.5 mg / kg to male cynomolgus monkeys (each treatment group / siRNA, n=4). Animals were fasted overnight before blood samples were collected on days -15, -8, and 1 (before administration) and on days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, and 78. Lp(a) circulating protein levels in all serum samples were analyzed using an Lp(a) ELISA assay (Abcam, catalog number ab212165). Results for each individual animal are expressed as a percentage change in circulating Lp(a) protein relative to the pre-drug administration time point (day 1). Mean percentage change and standard deviation for each treatment group are plotted on [data missing]. Figure 8The results are shown in Table 16. The results indicated that serum Lp(a) levels decreased after administration of the modified Lp(a) siRNA.

[0462] Example 9: Testing selected ApoC3 siRNA in APOC3 transgenic mice

[0463] The siRNAs listed in Table 17 were administered subcutaneously to APOC3 transgenic mice (Jackson Lab, B6; CBA-Tg(APOC3)3707 / Bres / J, strain number 006907) at a dose level of 0.5 mg / kg. The 3' end of the transaminase chain was conjugated to GalNAc via an X2 linker (see formula (V')). Plasma was collected from all groups on days -7, 0 (before administration), and on days 7, 14, 21, 28, and 35. Plasma hAPOC3 protein levels were measured using an ELISA assay (Abcam, catalog number: ab154131). For normalization, the APOC3 level for each animal at each time point was divided by the animal's pre-administration (day 0) level. Then, for each individual at a specific time point, the pre-administration normalized value was normalized to the mean pre-administration normalized value of the mediator control group. Mean results and standard deviations are listed in Table 18 and plotted on [the graph / chart]. Figure 9 At a dose of 0.5 mg / kg, most seed region-modified siRNAs still resulted in reduced hAPOC3 protein expression.

[0464] While preferred aspects of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Many variations, alterations, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the aspects of this disclosure described herein may be employed in the practice of this disclosure. The appended claims are intended to define the scope of this disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0465] Table 1. Sequences used in transgenic mouse research

[0466]

[0467] Table 2. Percentage of residual plasma AGT relative to the pre-drug mean

[0468]

[0469] Table 3. Sequences used in transgenic mouse research

[0470]

[0471]

[0472] Table 4. Percentage of residual plasma AGT relative to the pre-drug mean

[0473]

[0474] Table 5. Sequences used in transgenic mouse research

[0475]

[0476]

[0477] Table 6. Percentage of residual plasma AGT relative to the pre-drug mean

[0478]

[0479] Table 7. Sequences used in non-human primate research

[0480]

[0481]

[0482] Table 8. Percentage change in serum AGT relative to pre-dose levels on day 1.

[0483]

[0484] Table 9. Sequences used in transgenic mouse research

[0485]

[0486]

[0487] Table 10. Changes in the percentage of serum ANGPTL3 relative to the mediator

[0488]

[0489] Table 10 (continued)

[0490]

[0491] Table 11. Modified Lp(a) siRNA sequences used in in vivo studies

[0492]

[0493]

[0494] Table 12. Percentage change in serum LP(a) relative to pre-dose levels on day 1.

[0495]

[0496]

[0497] Table 13. Modified Lp(a) siRNA sequences used in in vivo studies

[0498]

[0499]

[0500] Table 14. Percentage change in serum LP(a) relative to pre-dose levels on day 1.

[0501]

[0502] Table 15. Modified Lp(a) siRNA sequences used in in vivo studies

[0503]

[0504] Table 16. Percentage change in serum LP(a) relative to pre-dose levels on day 1.

[0505]

[0506] Table 17. Modified ApoC3 siRNA sequences used in in vivo studies in transgenic mice

[0507]

[0508]

[0509] Table 18. Normalized APOC3 protein expression relative to pre-drug administration and control.

[0510]

[0511]

Claims

1. A repressive polynucleotide molecule comprising a guest strand (sense strand) and a guide strand (antisense strand), wherein the guide strand comprises a nucleotide analog selected from: acyclic L-threonine nucleic acid-thymidine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

2. A repressive polynucleotide molecule comprising a transient strand (sense strand) and a guiding strand (antisense strand), wherein the guiding strand comprises the following formula: 5’- (A) n1 -(B) n2 -(C) n3 -(D) n4 -(E) n5 -3’ Wherein A includes one or more nucleotides selected from the following: 2'-O-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-halogen modified nucleotides, DNA, RNA, ENA, BNA, LNA, UNA, and n1 is 1-7; B contains one or more nucleotide analogs selected from the following: acyclic L-threonine nucleic acid-thymidine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i), and n2 is 1-5; C is selected from DNA, RNA, 2'-O-alkyl modified nucleotides and 2'-halogen modified nucleotides and is composed of unmodified or identically modified nucleotides, and n3 is 0-6; D is selected from nucleotides modified with 2'-O-alkyl and nucleotides modified with 2'-halogen, including nucleotides that are modified differently from their adjacent nucleotides, and n4 is 4-8; and E is selected from nucleotides modified with 2'-O-alkyl, nucleotides modified with 2'-alkoxy, nucleotides modified with 2'-halogen, DNA, RNA, ENA, BNA, LNA, and UNA, and n5 is 3-10.

3. The inhibitory polynucleotide molecule as described in claim 1 or claim 2, wherein the inhibitory polynucleotide molecule is siRNA.

4. The inhibitory polynucleotide molecule according to any one of claims 1-3, wherein the nucleotide analog is located at the seed region (positions 2-8) of the guide strand starting from the 5' end.

5. The inhibitory polynucleotide molecule according to any one of claims 1-4, wherein the nucleotide analog is located at any one of positions 4-8 of the guide strand starting from the 5' end.

6. The inhibitory polynucleotide molecule according to any one of claims 1-5, wherein the nucleotide analog is located at any one of positions 5-8 of the guide strand starting from the 5' end.

7. The inhibitory polynucleotide molecule according to any one of claims 1-6, wherein the nucleotide analog is located at any one of positions 6-8 of the guide strand starting from the 5' end.

8. The inhibitory polynucleotide molecule according to any one of claims 1-7, wherein the nucleotide analog is located at position 6 of the guide strand starting from the 5' end.

9. The inhibitory polynucleotide molecule of any one of claims 1-8, wherein the nucleotide analog is located at position 6 of the guiding strand starting from the 5' end, and the guiding strand comprises 2'-F modified nucleotides at at least three of positions 2, 7, 12, 14, and 16 starting from the 5' end.

10. The inhibitory polynucleotide molecule of any one of claims 1-9, wherein the nucleotide analog is located at position 6 of the guide strand starting from the 5' end, and the guide strand comprises 2'-F modified nucleotides at positions 2, 7, 12, 14 and 16 starting from the 5' end.

11. The inhibitory polynucleotide molecule according to any one of claims 1-7, wherein the nucleotide analog is located at position 7 of the guide strand starting from the 5' end.

12. The inhibitory polynucleotide molecule according to any one of claims 1-7 and 11, wherein the nucleotide analog is located at position 7 of the guide strand starting from the 5' end, and the guide strand comprises 2'-F modified nucleotides at at least three of positions 2, 12, 14 and 16 starting from the 5' end.

13. The inhibitory polynucleotide molecule of any one of claims 1-7 and 11-12, wherein the nucleotide analog is located at position 7 of the guide strand starting from the 5' end, and the guide strand comprises 2'-F modified nucleotides at positions 2, 12, 14 and 16 starting from the 5' end.

14. The inhibitory polynucleotide molecule of any one of claims 1-7, wherein the nucleotide analog is located at position 8 of the guide strand starting from the 5' end.

15. The inhibitory polynucleotide molecule of any one of claims 1-7 and 14, wherein the nucleotide analog is located at position 8 of the guiding strand starting from the 5' end, and the guiding strand comprises 2'-F modified nucleotides at at least three of positions 2, 7, 12, 14 and 16 starting from the 5' end.

16. The inhibitory polynucleotide molecule of any one of claims 1-7 and 14-15, wherein the nucleotide analog is located at position 8 of the guiding strand starting from the 5' end, and the guiding strand comprises 2'-F modified nucleotides at positions 2, 7, 12, 14 and 16 starting from the 5' end.

17. The inhibitory polynucleotide molecule of any one of claims 1-16, wherein the nucleotide of the guide strand that is not a nucleotide analog or a 2'-F modified nucleotide is selected from DNA, RNA and 2'-O-alkyl modified nucleotides.

18. The inhibitory polynucleotide molecule of any one of claims 1-17, wherein the nucleotide of the guiding strand that is not the nucleotide analog or a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

19. The inhibitory polynucleotide molecule of any one of claims 1-18, wherein the guiding strand comprises at least two, at least three, or at least four phosphate-thioester modified nucleotide bonds.

20. The inhibitory polynucleotide molecule according to any one of claims 1-19, wherein the guiding strand comprises two phosphate-thioester modified nucleotide bonds at the 5' end and two phosphate-thioester modified nucleotide bonds at the 3' end.

21. The inhibitory polynucleotide molecule according to any one of claims 1-20, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmFmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), and acyclic N-ethylhexylene-methyl ... Acyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

22. The inhibitory polynucleotide molecule according to any one of claims 1-21, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (T... A) Acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

23. The inhibitory polynucleotide molecule according to any one of claims 1-20, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), and acyclic N-ethylhexyl ... Acyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

24. The inhibitory polynucleotide molecule according to any one of claims 1-20 or 23, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA) and acyclic L-threonine nucleic acid-thymine-3'-phosphate. (TT), acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i).

25. The inhibitory polynucleotide molecule according to any one of claims 1-20, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmmFXmmmFmFmFmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), and acyclic N-ethylhexylene-methyl ... Acyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

26. The inhibitory polynucleotide molecule according to any one of claims 1-20 or 25, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide bond, and X is selected from acyclic L-threonine nucleic acid-adenine-3'-phosphate (TA) and acyclic L-threonine nucleic acid-thymine-3'-phosphate. (TT), acyclic N-acetyl-L-threonol debased nucleic acid-3' phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3) and 2'-O-methylinosine-3'-phosphate (i).

27. The inhibitory polynucleotide molecule of any one of claims 1-26, wherein the transient chain comprises at least two, at least three, or at least four 2'-F modified nucleotides.

28. The inhibitory polynucleotide molecule of any one of claims 1-27, wherein the transient chain comprises three 2'-F modified nucleotides.

29. The inhibitory polynucleotide molecule of any one of claims 1-28, wherein the transient chain comprises a 2'-F modified nucleotide at at least one of positions 7, 9, and 11 starting from the 5' end.

30. The inhibitory polynucleotide molecule of any one of claims 1-29, wherein the transient chain comprises 2'-F modified nucleotides at positions 7, 9, and 11 starting from the 5' end.

31. The inhibitory polynucleotide molecule according to any one of claims 1-30, wherein the nucleotide in the transit chain that is not a 2'-F modified nucleotide is any one of a 2'-O-alkyl modified nucleotide, a 2'-alkoxy modified nucleotide, a 2'-halogen modified nucleotide, DNA, RNA, ENA, BNA, LNA, and UNA.

32. The inhibitory polynucleotide molecule according to any one of claims 1-31, wherein the nucleotide in the passchain that is not a 2'-F modified nucleotide is a 2'-O-methyl modified nucleotide.

33. The inhibitory polynucleotide molecule according to any one of claims 1-32, wherein the passchain comprises at least one phosphate thioester modified nucleotide inter-bond.

34. The inhibitory polynucleotide molecule according to any one of claims 1-33, wherein the transient chain comprises at least one phosphate-thioester modified nucleotide internucleotide bond at the 5' end.

35. The inhibitory polynucleotide molecule of any one of claims 1-34, wherein the transient chain comprises two phosphate-thioester modified nucleotide inter-bonds at the 5' end.

36. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmXFmmmmFmFmFmmmmmmm-3', and the guest strand comprises 5'-mmmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl-modified nucleotide, F is a 2'-F-modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP), and acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP). Purine-3'-phosphate (TA), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

37. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmXFmmmmFmFmFmmmmmsmsm-3', and the guest strand comprises 5'-msmsmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleoside, and thymine-3'-phosphate (TT). Acid-adenine-3'-phosphate (TA), acyclic N-acetyl-L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

38. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmmXmmmmFmFmFmmmmmmm-3', and the guest strand comprises 5'-mmmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl-modified nucleotide, F is a 2'-F-modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP), and acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP). Purine-3'-phosphate (TA), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

39. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmmXmmmmFmFmFmmmmmsmsm-3', and the guest strand comprises 5'-msmsmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), and acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT). Cyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

40. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-mFmmmmFXmmmFmFmFmmmmmmm-3', and the guest strand comprises 5'-mmmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl-modified nucleotide, F is a 2'-F-modified nucleotide, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP), and acyclic L-threonine nucleic acid-adenosine-3'-phosphate (ATP). Purine-3'-phosphate (TA), acyclic N-acetyl-L-threonol debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

41. The inhibitory polynucleotide molecule according to any one of claims 1-35, wherein the guiding strand comprises a nucleic acid sequence of 5'-msFsmmmmFXmmmFmFmFmmmmmsmsm-3', and the guest strand comprises 5'-msmsmmmmFmFmFmmmmmmmmmm-3', wherein m is a 2'-O-methyl modified nucleotide, F is a 2'-F modified nucleotide, s is a phosphate thioester nucleotide internucleotide bond, and X is selected from acyclic L-threonine nucleic acid-thymine-3'-phosphate (TT), ... Cyclic L-threonine nucleic acid-adenine-3'-phosphate (TA), acyclic N-acetyl L-threonine debased nucleic acid-3'-phosphate (T-NAc), 1',2'-dideoxyribose-3'-phosphate (dAB), thymidine-glycol nucleic acid (GNA) S-isomer (Tgn), 2'-O-methyl-2-thiouridine-3'-phosphate (u3), 2'-fluoro-2-thiouridine-3'-phosphate (U3f), 2'-deoxy-2-thiothymidine-3'-phosphate (dT3), and 2'-O-methylinosine-3'-phosphate (i).

42. The inhibitory polynucleotide molecule according to any one of claims 1-41, wherein the transit chain is conjugated to the target portion.

43. The inhibitory polynucleotide molecule of claim 42, wherein the targeting portion is a desialyl glycoprotein receptor targeting portion.

44. The polynucleotide conjugate of claim 43, wherein the desialylate glycoprotein receptor targeting portion comprises N-acetylgalactosamine (GalNAc) or galactose.

45. The inhibitory polynucleotide molecule of claim 43 or 44, wherein the targeting portion is coupled via a linker.

46. ​​The inhibitory polynucleotide molecule of claim 45, wherein the adapter comprises the following formula (IV). At least one of Y1 and Y2 is a nucleotide in the polynucleic acid molecule.

47. The polynucleotide conjugate of claim 46, wherein the linker of the last nucleotide at the 3' end of the transient chain of the polynucleotide molecule and the desialylate glycoprotein receptor targeting portion are as follows: （V’）、 (V'''') (V''''') or (V''''''), where Z in formula (V'), (V''''), (V'''') or (V''''') is -H, -OH, -O-methyl, -F or -O-methoxyethyl, and R in formula (V'), (V''''), (V'''') or (V''''') is adenine, uracil, guanine, cytosine, thymine, debasement or other.

48. A method for regulating the mRNA or protein expression level of a target gene in a cell or a desired object, comprising: The inhibitory polynucleotide molecule as described in any one of claims 1-47 is contacted with the cell or administered to the object to regulate the mRNA expression or protein expression level of the target gene in the object.

49. A method for inhibiting off-target effects caused by the guide strand of an inhibitory polynucleotide molecule in a cell, comprising introducing the inhibitory polynucleotide molecule as described in any one of claims 1-47 into the cell.

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