Single-stranded oligonucleotide

Abstract

To provide a single-stranded oligonucleotide that can control a target gene with high efficiency and can be easily produced.SOLUTION: There is provided a single-stranded oligonucleotide represented by the formula (I), wherein X is represented by Xa-Xb, Xa is coupled with Y, and Xb and Y hybridize. Xa is composed of 1 to 40 nucleotides, and contains at least one modified-nucleotide. Xb is composed of 4 to 40 nucleotides, and contains at least one modified-nucleotide. Y is composed of 4 to 40 nucleotides, and contains at least one ribonucleotide. Xz and Yz are composed of 5 to 40 nucleotides, and contain at least one modified-nucleotide. Nucleotide sequences X, Xz and Yz have an antisense sequence capable of hybridizing with a target RNA. Lx and Ly are composed of 0 to 20 nucleotides.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to single-stranded oligonucleotides.

Background Art

[0002] Antisense oligonucleotides (ASOs) are oligonucleotides complementary to mRNA, mRNA precursors, or ncRNAs (non-coding RNAs) such as ribosomal RNA, transfer RNA, and miRNA of target genes, and are single-stranded DNAs, RNAs, and / or their structural analogs consisting of about 8 to 30 bases. ASOs suppress the function of mRNA, mRNA precursors, or ncRNAs by forming double strands with the mRNA, mRNA precursors, or ncRNAs targeted by the antisense oligonucleotides.

[0003] However, since ASOs are easily degraded by nucleases in vivo and have low uptake efficiency into target cells, it is difficult to put them into practical use. In order to overcome these two major problems, studies on chemical modification of the oligonucleotide itself as an active ingredient and drug delivery systems (DDSs) for delivering oligonucleotides into target cells have been conducted for many years.

[0004] Examples of chemical modification of ASOs themselves include S-oligo (phosphorothioate) in which the phosphate moiety is modified, 2’,4’-BNA (bridged nucleic acid) / LNA (locked nucleic acid) in which the sugar moiety is modified (see Patent Documents 1 to 5), and the like.

[0005] Examples of DDSs include methods using carriers such as cationic liposomes and polymer micelles. Patent Document 6 describes an ASO in which a GalNAc (N-acetylgalactosamine) derivative, which is a sugar derivative having an interaction with an asialoglycoprotein receptor, is bound via a linker, and it is described that when the ASO is administered, the expression of the target gene in the liver is suppressed.

[0006] Patent Document 7 and Non-Patent Document 1 describe that by binding tocopherol (Toc) to a double-stranded oligonucleotide (HDO) containing an RNA oligonucleotide complementary to an ASO, in mice, compared with ASO, it was efficiently delivered and accumulated in the liver, and the expression of the target gene in the liver was suppressed. Patent Document 8 describes an ASO in which a GalNAc derivative is bound to an HDO via a linker, and it is described that when the antisense oligonucleotide is administered subcutaneously, the expression is suppressed more efficiently than the tocopherol (Toc) modified form.

[0007] Patent Document 9 describes that an oligonucleotide (HCDO) in which an ASO is bound to the RNA strand end of a double-stranded oligonucleotide unit of DNA and RNA suppresses the target RNA more efficiently than ASO.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

[0010] When applied to mammals including humans in a clinical setting as a pharmaceutical, a new nucleic acid pharmaceutical capable of efficiently controlling the expression of a target gene has been desired. In addition, when producing a double-stranded oligonucleotide (for example, the HDO, HCDO), a step of separately synthesizing an RNA strand complementary to the antisense strand and finally hybridizing these strands is required. Furthermore, when administering to animals or cells, it is necessary to suppress dissociation into single strands, and it is assumed that labor is also required for setting the handling conditions.

[0011] An object of the present invention is to provide a new oligonucleotide capable of efficiently controlling the expression of a target gene. Another object is to provide an oligonucleotide that can be produced more easily than a double-stranded oligonucleotide. [Means for Solving the Problems]

[0012] To achieve the above object, the present inventors have found that a single-stranded oligonucleotide in which an oligonucleotide chain (X chain) containing an antisense sequence and an oligonucleotide chain (Y chain) containing RNA are linked, the X chain consists of an Xa chain linked to the Y chain and an Xb chain not linked, and the Y chain and the Xb chain have a structure in which they are partially hybridized within the molecule, exhibits an antisense effect equal to or higher than that of a double-stranded oligonucleotide. In addition, since the single-stranded oligonucleotide is single-stranded, there is no hybridization step for forming a double-strand, and it can be efficiently produced. The present invention includes the following aspects.

[0013] 1. Formula (I): [Chemical formula] {Wherein, Y is a group derived from an oligonucleotide Y consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and containing at least one ribonucleotide, X is of the formula: [Chemical formula] (Wherein, Xb is a group derived from an oligonucleotide Xb consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide, Xa is a group derived from an oligonucleotide Xa consisting of 1 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide. Xa is bonded to the oligonucleotide Y and the oligonucleotide Xb at both ends thereof), and is a group derived from an oligonucleotide X consisting of 5 to 80 nucleotides, Xz is a group derived from an oligonucleotide Xz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide, Yz is a group derived from an oligonucleotide Yz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide, Lx is a group consisting of a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 0 to 20 nucleotides independently selected from the group consisting of oligonucleotides Lx, and binds to the Xb, Ly is a group consisting of a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 0 to 20 nucleotides independently selected from the group consisting of oligonucleotides Ly, m is 0 or 1, When m is 0, n is 0 or 1, When m is 1, n is 0, The oligonucleotide X has a nucleotide sequence X, the oligonucleotide Xa has a nucleotide sequence Xa, the oligonucleotide Xb has a nucleotide sequence Xb, the oligonucleotide Y has a nucleotide sequence Y, the oligonucleotide Xz has a nucleotide sequence Xz, the oligonucleotide Yz has a nucleotide sequence Yz, the oligonucleotide Lx has a nucleotide sequence Lx, and the oligonucleotide Ly has a nucleotide sequence Ly, The nucleotide sequence Xb is complementary to the nucleotide sequence Y, The nucleotide sequence X includes an antisense sequence that enables hybridization with the target RNA, When m is 1 and n is 0, The nucleotide sequence Xz includes an antisense sequence that enables hybridization with the target RNA, When m is 0 and n is 1, The nucleotide sequence Yz includes an antisense sequence that enables hybridization with the target RNA, When there are two or more of the antisense sequences, the target RNAs to which the respective antisense sequence portions hybridize may be the same or different} Is represented by a single-stranded oligonucleotide in which Xb and Y hybridize.

[0014] 2. The single-stranded oligonucleotide according to 1, wherein Xb is bound to Xa on the 3'-side and Y is bound to Xa on the 5'-side. 3. The single-stranded oligonucleotide according to 1, wherein Xb is bound to Xa on the 5'-side and Y is bound to Xa on the 3'-side. 4. The antisense sequence is, independently of each other, a sequence comprising at least 4 consecutive nucleotides recognized by RNaseH, or The single-stranded oligonucleotide according to any one of 1 to 3, which is a sequence comprising at least 1 sugar-modified nucleotide and not comprising 4 consecutive deoxyribonucleotides. 5. At least one antisense sequence is a sequence comprising at least 4 consecutive nucleotides recognized by RNaseH, and the antisense sequence portion is adjacent to and bound to the 5'-side and 3'-side of the sequence portion comprising at least 4 consecutive nucleotides recognized by the RNaseH and comprises sugar-modified nucleotides. The single-stranded oligonucleotide according to 4.

[0015] 6. The single-stranded oligonucleotide according to any one of 1 to 5, wherein the antisense sequence portion comprises a phosphorothioate bond. 7. The single-stranded oligonucleotide according to any one of 1 to 6, wherein the antisense sequence is a sequence consisting of 10 to 30 nucleotides comprising at least 1 deoxyribonucleotide. 8. The single-stranded oligonucleotide according to any one of 1 to 7, wherein the nucleotide sequence Y is a sequence comprising at least 4 consecutive nucleotides cleaved by RNaseH. 9. The single-stranded oligonucleotide according to any one of 1 to 8, wherein the oligonucleotide Y comprises 1 or more sugar-modified nucleotides on at least one of the 5'-side and 3'-side of the oligonucleotide Y. 10. The single-stranded oligonucleotide according to any one of 1 to 9, wherein m is 0 and n is 0.

[0016] 11. The single-stranded oligonucleotide according to any one of 1. to 9., wherein m is 0 and n is 1. 12. The single-stranded oligonucleotide according to 11., wherein the nucleotides contained in the oligonucleotide Ly are linked to each other by phosphodiester bonds. 13. The single-stranded oligonucleotide according to 11. or 12., wherein the oligonucleotide Ly is DNA or RNA. 14. The single-stranded oligonucleotide according to any one of 1. to 9., wherein m is 1 and n is 0. 15. The single-stranded oligonucleotide according to 14., wherein the nucleotides contained in the oligonucleotide Lx are linked to each other by phosphodiester bonds. 16. The single-stranded oligonucleotide according to any one of 14. or 15., wherein the oligonucleotide Lx is DNA or RNA. 17. The single-stranded oligonucleotide according to any one of 1. to 16., further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site. 18. The single-stranded oligonucleotide according to 17., wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof. 19. The single-stranded oligonucleotide according to 17. or 18., wherein the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol, and tocotrienol. 20. The single-stranded oligonucleotide according to 17. or 18., wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor.

[0017] 21. The single-stranded oligonucleotide according to 17. or 18., wherein the functional molecule is a peptide or protein selected from the group consisting of a ligand of a receptor and an antibody. 22. A pharmaceutical composition comprising the single-stranded oligonucleotide according to any one of 1. to 21. and a pharmaceutically acceptable carrier. 23. A method for controlling the function of a target RNA, comprising a step of contacting a single-stranded oligonucleotide according to any one of 1. to 21. with a cell. 24. A method for controlling the function of a target RNA in a mammal, comprising a step of administering a pharmaceutical composition containing a single-stranded oligonucleotide according to any one of 1. to 21. to the mammal. 25. A method for controlling the expression of a target gene, comprising a step of contacting a single-stranded oligonucleotide according to any one of 1. to 21. with a cell.

[0018] 26. A method for controlling the expression of a target gene in a mammal, comprising a step of administering a pharmaceutical composition containing a single-stranded oligonucleotide according to any one of 1. to 21. to the mammal. 27. A method for producing a single-stranded oligonucleotide according to any one of 1. to 21., comprising a step of extending a nucleotide chain at the 3'-end or 5'-end of an oligonucleotide containing at least one of X and Y.

Advantages of the Invention

[0019] According to the present invention, an oligonucleotide capable of controlling a target RNA with high efficiency can be provided.

[0020] The single-stranded oligonucleotide of the present invention can effectively control the expression of a target gene by its component antisense oligonucleotide and is useful as a nucleic acid pharmaceutical.

Brief Description of the Drawings

[0021]

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[0022] The terms used in this specification are used in the meanings commonly used in the art, unless otherwise specified. Each term used in this specification will be described below. In this specification, each term has the same meaning whether used alone or in combination with other terms, unless otherwise specified.

[0023] The "antisense effect" means that the function of a target RNA is controlled by hybridization between the target RNA selected corresponding to a target gene and, for example, an oligonucleotide having a sequence complementary to a partial sequence thereof. For example, when the target RNA is mRNA, it means that translation of the target RNA is inhibited by hybridization, a splicing function conversion effect such as exon skipping, and degradation of the target RNA by recognition of the hybridized portion. Examples of the oligonucleotide that causes the antisense effect include DNA and oligodeoxyribonucleotides, etc., but the oligonucleotide that causes the antisense effect is not limited to these, and may be RNA, oligoribonucleotides, or an oligonucleotide designed so that the antisense effect usually occurs.

[0024] "Target RNA" means mRNA, pre-mRNA or ncRNA, and includes mRNA transcribed from genomic DNA encoding a target gene, unmodified mRNA, pre-mRNA not subjected to splicing, ncRNA, and the like. The "target RNA" whose function is controlled by the antisense effect is not particularly limited, and examples thereof include RNA related to genes whose expression is enhanced in various diseases. The "target RNA" may be any RNA synthesized by DNA-dependent RNA polymerase, preferably mRNA or pre-mRNA. More preferably, it is mammalian mRNA or pre-mRNA, and still more preferably, human mRNA or pre-mRNA.

[0025] "Hybridize" means an act of forming a double strand between oligonucleotides containing complementary sequences or groups derived from oligonucleotides, and a phenomenon in which groups derived from oligonucleotides containing complementary sequences or oligonucleotides form a double strand.

[0026] "Complementary" means that two nucleobases can form a Watson-Crick type base pair (natural type base pair) or a non-Watson-Crick type base pair (Hoogsteen type base pair, etc.) via hydrogen bonds. Two oligonucleotides or groups derived from oligonucleotides can "hybridize" when their sequences are complementary. For two oligonucleotides or groups derived from oligonucleotides to hybridize, they do not need to be completely complementary, but the complementarity for two oligonucleotides or groups derived from oligonucleotides to hybridize is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, or 99% or more). The complementarity of sequences can be determined by using a computer program that automatically identifies partial sequences of oligonucleotides. For example, OligoAnalyzer is one such software provided by Integrated DNA Technologies. This program can also be used on the website. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two oligonucleotides or groups derived from oligonucleotides can hybridize. Also, those skilled in the art can easily design an antisense oligonucleotide complementary to a target RNA by using, for example, the BLAST program, etc., based on the information on the nucleotide sequence of the target RNA. For the BLAST program, reference can be made to Proceedings of the National Academy of Sciences of the United States of America (1990, 87 volumes, 2264 - 68 pages; 1993, 90 volumes, 5873 - 77 pages), and Journal of Molecular Biology (1990, 215 volumes, 403 pages), etc.

[0027] "Nucleotide" means a molecule that can be a constituent unit of a nucleic acid (oligonucleotide), and usually has a base as a constituent element. A nucleotide is composed of, for example, a sugar, a base, and a phosphate. Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, which will be described later.

[0028] "Oligonucleotide" means a molecule having a structure in which one or more of the above nucleotides are polymerized. When an "oligonucleotide" is composed of one nucleotide, that oligonucleotide can be referred to as a "nucleotide". The nucleotides contained in the "single-stranded oligonucleotide" molecule of the present invention are each independently linked to each other by a phosphodiester bond or a modified phosphodiester bond described later. The nucleotide at the 3'-end of the single-stranded oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3'-position, more preferably a hydroxy group, and usually has a hydroxy group. The nucleotide at the 5'-end of the single-stranded oligonucleotide molecule preferably has a hydroxy group or a phosphate group at its 5'-position, more preferably a hydroxy group, and usually has a hydroxy group.

[0029] "Group derived from an oligonucleotide" means a group obtained by removing a hydrogen atom, a hydroxy group, etc. from at least one of the hydroxy groups at the 3'-end and 5'-end of the oligonucleotide, and is indirectly covalently linked to another group (for example, a group derived from another oligonucleotide) by forming a phosphodiester bond or a modified phosphodiester bond. The hydroxy group at the 3'-end or 5'-end includes the hydroxy group of the phosphate group. For example, a group obtained by removing a hydrogen atom from the hydroxy group at the 3'-end of an oligonucleotide and a group obtained by removing a hydroxy group from the phosphate group at the 5'-end of another oligonucleotide form a phosphodiester bond or a modified phosphodiester bond.

[0030] "Nucleotide sequence" means the base sequence of the nucleotides constituting an oligonucleotide. The "nucleotide sequence portion" means a partial structure of a region having the nucleotide sequence among the oligonucleotide chains.

[0031] In this specification, whether the "nucleotide sequence" contains or does not contain a predetermined nucleotide or oligonucleotide chain, etc. has the same meaning as whether the corresponding "sequence portion containing a nucleotide" contains or does not contain the nucleotide or the oligonucleotide chain, etc.

[0032] The "sequence portion" means a partial structure of an oligonucleotide chain. For example, a sequence portion containing a nucleotide is a partial structure of a region containing the nucleotide among the oligonucleotide chains. That the nucleotide sequence is a sequence selected from predetermined nucleotides, a sequence in which predetermined nucleotides are continuous, etc. has the same meaning as that the corresponding nucleotide sequence portion is a sequence portion selected from the nucleotides, a sequence portion in which the nucleotides are continuous, etc., respectively.

[0033] "Deoxyribonucleotide" means a molecule in which the sugar is 2'-deoxyribose among the above-mentioned "nucleotides", a base is bonded to the 1'-position carbon atom of 2'-deoxyribose, and a phosphate group is present at the 3'-position or 5'-position. The deoxyribonucleotides in the present invention may be naturally occurring deoxyribonucleotides or deoxyribonucleotides in which the base portion or phosphodiester bond portion of naturally occurring deoxyribonucleotides is modified. Modifications of the base portion and modifications of the phosphodiester bond site may be applied in combination of multiple types to one deoxyribonucleotide. The modified deoxyribonucleotides are described, for example, in Journal of Medicinal Chemistry (2016, Vol. 59, No. 21, pp. 9645-9667), Medicinal Chemistry Communications (2014, Vol. 5, pp. 1454-1471), Future Medicinal Chemistry (2011, Vol. 3, No. 3, pp. 339-365), etc.

[0034] When the "deoxyribonucleotide" constitutes the single-stranded oligonucleotide molecule of the present invention, usually, the 3'-position of the deoxyribonucleotide is linked to other nucleotides or the like by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond), and the 5'-position of the deoxyribonucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond). The deoxyribonucleotide at the 3'-end of the single-stranded oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3'-position, and the 5'-position is as described above. The deoxyribonucleotide at the 5'-end of the single-stranded oligonucleotide molecule preferably has a hydroxy group or a phosphate group at its 5'-position, and the 3'-position is as described above.

[0035] "Oligodeoxyribonucleotide" means an oligonucleotide composed of the deoxyribonucleotides. The deoxyribonucleotides constituting the oligodeoxyribonucleotide may be the same or different from each other.

[0036] "DNA" means an oligonucleotide composed of natural deoxyribonucleotides. The natural deoxyribonucleotides constituting the DNA may be the same or different from each other.

[0037] "Ribonucleotide" means a molecule among the above-mentioned "nucleotides" in which the sugar is ribose, a base is bonded to the carbon atom at the 1'-position of ribose, and a phosphate group is present at the 2'-position, 3'-position or 5'-position. The ribonucleotides in the present invention may be naturally occurring ribonucleotides or ribonucleotides in which the base portion or phosphodiester bond portion of the naturally occurring ribonucleotides is modified. Modifications of the base portion and modifications of the phosphodiester bond site may be applied in combination of multiple types to one ribonucleotide. The modified ribonucleotides are described, for example, in Journal of Medicinal Chemistry (2016, Vol. 59, No. 21, pp. 9645-9667), Medicinal Chemistry Communications (2014, Vol. 5, pp. 1454-1471), Future Medicinal Chemistry (2011, Vol. 3, No. 3, pp. 339-365), etc.

[0038] When the above-mentioned "ribonucleotide" constitutes the single-stranded oligonucleotide molecule of the present invention, typically, the 3'-position of the ribonucleotide is linked to another nucleotide by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond), and the 5'-position of the ribonucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond). The ribonucleotide at the 3'-end of the single-stranded oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at its 3'-position, and the 5'-position is as described above. The ribonucleotide at the 5'-end of the single-stranded oligonucleotide molecule has a hydroxy group or a phosphate group preferably at its 5'-position, and the 3'-position is as described above.

[0039] "Oligoribonucleotide" means an oligonucleotide composed of the above-mentioned ribonucleotides. The ribonucleotides constituting the oligoribonucleotide may be the same or different from each other.

[0040] "RNA" means an oligonucleotide composed of natural ribonucleotides. The natural ribonucleotides constituting RNA may be the same or different from each other.

[0041] "Sugar-modified nucleotide" means a nucleotide in which the sugar moiety of the deoxyribonucleotide or ribonucleotide is partially substituted by one or more substituents, or the entire sugar backbone is replaced by a sugar backbone different from ribose and 2'-deoxyribose (for example, a 5- to 6-membered sugar backbone such as hexitol or threose), or the entire sugar backbone or a ring portion of the sugar backbone is replaced by a 5- to 7-membered saturated or unsaturated ring (for example, cyclohexane, cyclohexene, morpholine, etc.) or a partial structure capable of forming a 5- to 7-membered ring by hydrogen bonding (for example, a peptide structure), or the ring of the sugar moiety is opened, and further, the opened portion is a modified nucleotide. The base portion of the "sugar-modified nucleotide" may be a naturally occurring base or a modified base. Also, the phosphodiester bond portion of the "sugar-modified nucleotide" may be a phosphodiester bond or a modified phosphodiester bond. Modifications of the base portion and the phosphodiester bond site may be combined in multiple types for one sugar-modified nucleotide. Modifications of the opened portion include, for example, halogenation, alkylation (for example, methylation, ethylization), hydroxylation, amination, and thiolation, etc., and also include demethylation, etc.

[0042] "Sugar-modified nucleotides" may be cross-linked nucleotides or non-cross-linked nucleotides. Examples of sugar-modified nucleotides include nucleotides and the like that are disclosed as being preferably used in the antisense method in, for example, Japanese Patent Application Laid-Open No. 10-304889, International Publication No. 2005 / 021570, Japanese Patent Application Laid-Open No. 10-195098, Japanese Patent Application Laid-Open No. 2002-521310, International Publication No. 2007 / 143315, International Publication No. 2008 / 043753, International Publication No. 2008 / 029619, and International Publication No. 2008 / 049085 (hereinafter, these documents are referred to as "documents related to the antisense method"). The said documents disclose nucleotides such as hexitol nucleotides (HNA), cyclohexene nucleotides (CeNA), peptide nucleic acids (PNA), glycol nucleic acids (GNA), threonucleotides (TNA), morpholino nucleic acids, tricyclo-DNA (tcDNA), 2'-O-methylated nucleotides, 2'-O-methoxyethyl (2'-MOE)-modified nucleotides, 2'-O-aminopropyl (2'-AP)-modified nucleotides, 2'-fluorinated nucleotides, 2'-F-arabinonucleotides (2'-F-ANA), cross-linked nucleotides (BNA (Bridged Nucleic Acid)), 2'-O-{(N-methylcarbamoyl)ethyl} (2'-MCE)-modified nucleotides, etc. Also, sugar-modified nucleotides are disclosed in Journal of Medicinal Chemistry (2016, 59, 21, 9645-9667), Medicinal Chemistry Communications (2014, 5, 1454-1471), Future Medicinal Chemistry (2011, 3, 3, 339-365), etc.

[0043] When the "sugar-modified nucleotide" constitutes the single-stranded oligonucleotide molecule of the present invention, for example, the 3'-position of the sugar-modified nucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond), and the 5'-position of the sugar-modified nucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, a phosphorothioate bond). The sugar-modified nucleotide at the 3'-end of the single-stranded oligonucleotide molecule of the present invention has, for example, a hydroxy group or a phosphate group preferably at its 3'-position, and the 5'-position is as described above. The sugar-modified nucleotide at the 5'-end of the single-stranded oligonucleotide molecule has, for example, a hydroxy group or a phosphate group preferably at its 5'-position, and the 3'-position is as described above.

[0044] The base moieties in deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides are preferably at least one selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U) and 5-methylcytosine (5-me-C).

[0045] Examples of modifications to the base moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include halogenation, methylation, ethylization, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, cyclobutylation, hydroxylation, amination, thiolation, and demethylation, etc. More specifically, 5-methylation, 5-fluorination, 5-bromination, 5-iodination, and N4-methylation of cytosine; 2-thiolation, 5-demethylation, 5-fluorination, 5-bromination, and 5-iodination of thymine; 2-thiolation, 5-fluorination, 5-bromination, and 5-iodination of uracil; N6-methylation and 8-bromination of adenine; N2-methylation and 8-bromination of guanine, etc. Also, examples of modifications to the base moiety in nucleotides are disclosed in Journal of Medicinal Chemistry-(2016, 59, 21, 9645-9667), Medicinal Chemistry Communications (2014, 5, 1454-1471), Future Medicinal Chemistry (2011, 3, 3, 339-365), etc., and these can be used for the base moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.

[0046] Examples of modifications of the phosphodiester bond moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include phosphorothioation, methylphosphonylation (including chiral-methylphosphonylation), methylthiophosphonylation, phosphorodithioation, phosphoramidation, phosphorodiamidation, phosphoramidothioation, and boranophosphate formation, among others. Further, examples of modifications of the phosphodiester bond moiety in nucleotides are disclosed in Journal of Medicinal Chemistry (2016, Vol. 59, No. 21, pp. 9645-9667), Medicinal Chemistry Communications (2014, Vol. 5, pp. 1454-1471), Future Medicinal Chemistry (2011, Vol. 3, No. 3, pp. 339-365), etc., and these can be used for the phosphodiester bond moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.

[0047] Examples of modifications in which the sugar moiety of a deoxyribonucleotide or ribonucleotide is partially substituted by one substituent include 2'-O-methylation, 2'-O-methoxyethyl (2'-MOE) modification, 2'-O-aminopropyl (2'-AP) modification, 2'-fluorination, and 2'-O-{(N-methylcarbamoyl)ethyl} (2'-MCE) modification, among others.

[0048] A "bridged nucleotide" is a sugar-modified nucleotide in which a bridging unit is substituted by two substitutions in the sugar moiety. For example, a nucleotide in which the 2'-position and the 4'-position are bridged can be mentioned.

[0049] As for the nucleotide in which the 2'-position and the 4'-position are bridged (2',4'-BNA), it may be a nucleotide having a sugar moiety in which the carbon atom at the 2'-position and the carbon atom at the 4'-position are bridged by two or more atoms. For example, C 2-6An alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently -O-, -NR 1 -(R 1 represents a hydrogen atom, C 1-6 alkyl group or halo C 1-6 alkyl group), and nucleotides having a sugar moiety crosslinked with -S-). Combining the above substitution and replacement, the group that crosslinks the 2'-position and 4'-position of 2',4'-BNA is -C(=O)-O-, -O-C(=O)-NR 1 -(R 1 represents a hydrogen atom, C 1-6 alkyl group or halo C 1-6 alkyl group), -C(=O)-NR 1 -(R 1 represents a hydrogen atom, C 1-6 alkyl group or halo C 1-6 alkyl group), -C(=S)-NR 1 -(R 1 represents a hydrogen atom, C 1-6 alkyl group or halo C 1-6 alkyl group), etc., and may contain a group represented by the like.

[0050] Examples of such BNA include Locked Nucleic Acid (registered trademark), also known as LNA, α-L-methyleneoxy(4'-CH 2 -O-2')BNA or β-D-methyleneoxy(4'-CH 2 -O-2')BNA, ethyleneoxy(4'-(CH 2 ) 2 -O-2')BNA, also known as ENA, β-D-thio(4'-CH 2 -S-2')BNA, aminooxy(4'-CH 2 -O-N(R 11 )-2')BNA (R 11 is H or CH 3 ), 2',4'-BNA NCAlso referred to as oxyamino (4’-CH 2 -N(R 12 )-O-2’)BNA (where R 12 is H or CH 3 ), 2’,4’-BNA COC , 3’-amino-2’,4’-BNA, 5’-methyl BNA, also referred to as cEt-BNA (4’-CH(CH 3 )-O-2’)BNA, also referred to as cMOE-BNA (4’-CH(CH 2 OCH 3 )-O-2’)BNA, amide-type BNA also referred to as AmNA (4’-C(=O)-N(R 13 )-2’)BNA (where R 13 is H or CH 3 ), and other BNAs known to those skilled in the art, etc.

[0051] "Nucleotide in which at least one of the sugar moiety, base moiety, and phosphate moiety is modified" means a deoxyribonucleotide in which at least one of the base moiety and phosphate moiety of a naturally occurring deoxyribonucleotide is modified, a ribonucleotide in which at least one of the base moiety and phosphate moiety of a naturally occurring ribonucleotide is modified, or a sugar-modified nucleotide.

[0052] "n-" means normal, "s-" means secondary, and "t-" means tertiary.

[0053] "Halogen atom" means a fluorine atom, chlorine atom, bromine atom, or iodine atom.

[0054] "C 1-6 alkyl group" means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, isopentyl group, neopentyl group, n-hexyl group, isohexyl group, etc.

[0055] "HaloC 1-6 alkyl group" means the above-mentioned "C 1-6It means a group in which a hydrogen atom at any position of the "alkyl group" is substituted with one or more of the said "halogen atoms".

[0056] "C 1-6 The "alkylene group" means a divalent group obtained by removing one hydrogen atom at an arbitrary position from a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Examples include a methylene group, an ethylene (ethanediyl) group, a propane-1,3-diyl group, a propane-2,2-diyl group, a 2,2-dimethyl-propane-1,3-diyl group, a hexane-1,6-diyl group, and a 3-methylbutane-1,2-diyl group, etc. "C 2-6 The "alkylene group" means 1-6 among the said "C 1-6 alkylene group", a linear or branched divalent group having 2 to 6 carbon atoms. Examples are the same as the said "C 1-6 alkylene group", except for the methylene group. "C 2-20 The "alkylene group" means a divalent group obtained by removing one hydrogen atom at an arbitrary position from a linear or branched saturated hydrocarbon group having 2 to 20 carbon atoms. Similarly, the "C 8-12 alkylene group" means a divalent group obtained by removing one hydrogen atom at an arbitrary position from a linear or branched saturated hydrocarbon group having 8 to 12 carbon atoms.

[0057] "C 2-20 The "alkenylene group" means a divalent group obtained by removing one hydrogen atom at an arbitrary position from a linear or branched unsaturated hydrocarbon group having 2 to 20 carbon atoms and containing at least one double bond.

[0058] The "oxo group" indicates a group (=O) in which an oxygen atom is substituted via a double bond. When the oxo group is substituted on a carbon atom, it forms a carbonyl group together with the said carbon atom.

[0059] The "thioxo group" means Sulfur a group (=S) in which an atom is substituted via a double bond. When the thioxo group is substituted on a carbon atom, it forms a thiocarbonyl group together with the said carbon atom.

[0060] Sugar-modified nucleotides are not limited to those exemplified herein. A number of sugar-modified nucleotides are known in the art, and for example, the sugar-modified nucleotides described in US Patent No. 8,299,039 to Tachas et al. (especially columns 17 to 22), or Journal of Medicinal Chemistry (2016, Vol. 59, 21, pp. 9645-9667), Medicinal Chemistry Communications (2014, Vol. 5, pp. 1454-1471), Future Medicinal Chemistry (2011, Vol. 3, No. 3, pp. 339-365), etc. can also be used as embodiments of the present invention.

[0061] A person skilled in the art can appropriately select and use sugar-modified nucleotides from among such sugar-modified nucleotides in consideration of viewpoints such as the antisense effect, the affinity for a partial sequence of the target RNA, and the resistance to nucleolytic enzymes.

[0062] "RNaseH" is generally known as a ribonuclease that recognizes a double-stranded structure in which DNA and RNA are hybridized and cleaves the RNA to produce single-stranded DNA. RNaseH can recognize not only a double-stranded structure in which DNA and RNA are hybridized, but also a double-stranded structure in which at least one of the base moiety, phosphodiester bond moiety, and sugar moiety of at least one of DNA and RNA is modified. For example, it can also recognize a double-stranded structure in which an oligodeoxyribonucleotide and an oligoribonucleotide are hybridized. Therefore, DNA can be recognized by RNaseH when hybridized with RNA. The same applies when at least one of the base moiety, phosphodiester bond moiety, and sugar moiety of at least one of DNA and RNA is modified. For example, typical examples include oligonucleotides in which the phosphodiester bond moiety of DNA is modified to phosphorothioate. RNA can be cleaved by RNaseH when hybridized with DNA. The same applies when at least one of the base moiety, phosphodiester bond moiety, and sugar moiety is modified in at least one of DNA and RNA. Examples of modifications of DNA and / or RNA that can be recognized by RNaseH are described, for example, in Nucleic Acids Research (2014, Vol. 42, No. 8, pp. 5378 - 5389), Bioorganic & Medicinal Chemistry Letters (2008, Vol. 18, pp. 2296 - 2300), Molecular Biosystems (2009, Vol. 5, pp. 838 - 843), Nucleic Acid Therapeutics (2015, Vol. 25, No. 5, pp. 266 - 274), The Journal of Biological Chemistry (2004, Vol. 279, No. 35, pp. 36317 - 36326), etc. The RNaseH used in the present invention is preferably mammalian RNaseH, more preferably human RNaseH, and particularly preferably human RNaseH1.

[0063] "At least four consecutive nucleotides recognized by RNaseH" includes four or more consecutive nucleotides and is not particularly limited as long as it is recognized by RNaseH. The consecutive nucleotides are preferably independently selected from deoxyribonucleotides and sugar-modified nucleotides, and more preferably independently selected from deoxyribonucleotides. These consecutive nucleotides may be the same or different from each other.

[0064] "At least four consecutive nucleotides cleaved by RNaseH" includes four consecutive nucleotides and is not particularly limited as long as it is cleaved by RNaseH, but includes at least one ribonucleotide. Further, it preferably includes an oligoribonucleotide, and more preferably includes RNA. The consecutive nucleotides are more preferably selected independently from ribonucleotides. Further, the consecutive nucleotides are more preferably linked to each other by phosphodiester bonds. These consecutive nucleotides may be the same or different from each other.

[0065] Next, the antisense sequence and the antisense sequence portion in the present invention will be described.

[0066] "Antisense sequence" means the base sequence of nucleotides constituting an oligonucleotide capable of hybridizing with a target RNA.

[0067] "Antisense sequence portion" means a partial structure of a region having the antisense sequence among an oligonucleotide chain.

[0068] In the present specification, whether the "antisense sequence" contains or does not contain a predetermined nucleotide or oligonucleotide chain, etc. has the same meaning as whether the corresponding "antisense sequence portion" contains or does not contain the nucleotide or the oligonucleotide chain, etc.

[0069] The antisense sequence portion does not need to hybridize with the entire target RNA, and it is sufficient to hybridize with at least a part of the target RNA. Usually, it hybridizes with at least a part of the target RNA. For example, an oligonucleotide having an antisense sequence complementary to a partial sequence of the target RNA (DNA, oligodeoxyribonucleotide, or an oligonucleotide designed to produce a normal antisense effect, etc.) hybridizes with at least a part of the target RNA, thereby controlling the expression of the target gene. Also, it is not necessary for the entire antisense sequence portion to hybridize, and a part may not hybridize, but it is preferable for the entire antisense sequence portion to hybridize.

[0070] The complementarity between the antisense sequence and the partial sequence of the target RNA is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). Although it is not necessary for their sequences to be completely complementary for the antisense sequence portion to hybridize with at least a part of the target RNA, it is even more preferable for them to be completely complementary.

[0071] The antisense sequence is preferably a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", or a sequence containing "at least 1 sugar-modified nucleotide and not containing 4 consecutive deoxyribonucleotides".

[0072] A person skilled in the art can easily determine a base sequence that conforms to an antisense sequence that "enables hybridization with the target RNA" by using a BLAST program or the like. The same applies to a nucleotide sequence that conforms to "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA".

[0073] "At least four consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" are usually 4 to 30 consecutive nucleotides, preferably 4 to 20 consecutive nucleotides, more preferably 5 to 16 consecutive nucleotides, still more preferably 6 to 12 consecutive nucleotides, and particularly preferably 8 to 10 consecutive nucleotides. The consecutive nucleotides are preferably independently selected from deoxyribonucleotides and sugar-modified nucleotides, more preferably independently selected from deoxyribonucleotides. Particularly preferably, they are 8 to 10 consecutive deoxyribonucleotides. These consecutive nucleotides may be the same or different from each other.

[0074] Also, from the viewpoint of excellent pharmacokinetics, it is preferable that at least one of these consecutive nucleotides is phosphorothioated. More preferably, at least one of the nucleotides at the 3'-end and 5'-end of these consecutive nucleotides is phosphorothioated, and even more preferably, both the nucleotides at the 3'-end and 5'-end are phosphorothioated. Still more preferably, 80% of the nucleotides of these consecutive nucleotides are phosphorothioated, and even more preferably, 90% of the nucleotides are phosphorothioated. Particularly preferably, all of these consecutive nucleotides are phosphorothioated.

[0075] When the antisense sequence is a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", from the viewpoint of increasing the affinity for the partial sequence of the target RNA or the resistance to nucleases, it is preferable that 1 to 10 sugar-modified nucleotides are bound adjacent to at least one of the 3'-side and 5'-side of "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", more preferably 2 to 5 sugar-modified nucleotides are bound adjacent to at least one of the 3'-side and 5'-side, and even more preferably 2 to 3 sugar-modified nucleotides are bound adjacent to at least one of the 3'-side and 5'-side. Here, one or more deoxyribonucleotides and / or ribonucleotides may be included between the plurality of sugar-modified nucleotides on at least one of the 3'-side and 5'-side, but the plurality of sugar-modified nucleotides are preferably consecutive. Also, the one or more sugar-modified nucleotides are preferably bound adjacent to both the 3'-side and 5'-side of "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA". When a plurality of sugar-modified nucleotides are bound adjacent to at least one of the 3'-side and 5'-side of "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", "a plurality of sugar-modified nucleotides are bound adjacent" means that an oligonucleotide chain composed of the plurality of sugar-modified nucleotides and deoxyribonucleotides and ribonucleotides contained between the plurality of sugar-modified nucleotides are bound adjacent. When a plurality of sugar-modified nucleotides are bound adjacent to at least one of the 3'-side and 5'-side, each sugar-modified nucleotide may be the same or different.

[0076] The sugar-modified nucleotide moiety that binds adjacent to at least one of the 3'-side and 5'-side of the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" may or may not hybridize to the target RNA, but it is preferable from the same viewpoint as above that it hybridizes to the target RNA. When the antisense sequence portion contains the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA", one or more sugar-modified nucleotides are bound adjacent to the 3'-side and 5'-side, and when the sugar-modified nucleotide portion hybridizes to the target RNA, the one or more sugar-modified nucleotide portions are also part of the antisense sequence portion. That is, the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA", and one or more sugar-modified nucleotides that bind adjacent to the 3'-side and 5'-side constitute the antisense sequence portion. The antisense sequence portion is called a gapmer.

[0077] Also, from the viewpoint of excellent pharmacokinetics, at least one of the sugar-modified nucleotide moieties that bind adjacent to at least one of the 3'-side and 5'-side of the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" is preferably phosphorothioated, more preferably at least one of the sugar-modified nucleotide moieties adjacent to the 3'-side and at least one of the sugar-modified nucleotide moieties adjacent to the 5'-side are phosphorothioated, even more preferably 50% is phosphorothioated, even more preferably 80% is phosphorothioated. Also, it is preferable that all are phosphorothioated. When a plurality of sugar-modified nucleotides are adjacent to the 3'-side, the bond between the nucleotides is preferably phosphorothioated, and the same applies when a plurality of sugar-modified nucleotides are adjacent to the 5'-side.

[0078] The gapmer is preferably an oligonucleotide consisting of 1 to 10 sugar-modified nucleotides, an oligodeoxyribonucleotide consisting of 4 to 30 deoxyribonucleotides, and an oligonucleotide in which oligonucleotides consisting of 1 to 10 sugar-modified nucleotides are sequentially linked. More preferably, it is an oligonucleotide consisting of 2 to 5 sugar-modified nucleotides, an oligodeoxyribonucleotide consisting of 4 to 20 deoxyribonucleotides, and an oligonucleotide in which oligonucleotides consisting of 2 to 5 sugar-modified nucleotides are sequentially linked. Even more preferably, it is an oligonucleotide consisting of 2 or 3 sugar-modified nucleotides, an oligodeoxyribonucleotide consisting of 5 to 15 deoxyribonucleotides, and an oligonucleotide in which oligonucleotides consisting of 2 or 3 sugar-modified nucleotides are sequentially linked. Particularly preferably, it is an oligonucleotide consisting of 2 or 3 sugar-modified nucleotides, an oligodeoxyribonucleotide consisting of 8 to 12 deoxyribonucleotides, and an oligonucleotide in which oligonucleotides consisting of 2 or 3 sugar-modified nucleotides are sequentially linked. As another particularly preferred embodiment, it is an oligonucleotide consisting of 4 or 5 sugar-modified nucleotides, an oligodeoxyribonucleotide consisting of 8 to 12 deoxyribonucleotides, and an oligonucleotide in which oligonucleotides consisting of 4 or 5 sugar-modified nucleotides are sequentially linked.

[0079] When the antisense sequence is a sequence that "contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides", the antisense sequence portion may or may not contain ribonucleotides and may or may not contain deoxyribonucleotides, but it contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides. This antisense sequence portion is called a mixmer. The antisense sequence portion is preferably a partial structure of an oligonucleotide composed of nucleotides independently selected from deoxyribonucleotides and sugar-modified nucleotides, and the content rate of the sugar-modified nucleotides is, for example, 25% or more. From the viewpoint that the affinity for a partial sequence of the target RNA or the resistance to nucleases increases, the content rate of the sugar-modified nucleotides is preferably 30% or more, and more preferably 50% or more. From the same viewpoint, it is preferable that at least one of the nucleotides on the 3'-side and the 5'-side of this antisense sequence portion is a sugar-modified nucleotide, and it is more preferable that the nucleotides on the 3'-side and the 5'-side are sugar-modified nucleotides. As another aspect, the content rate of the sugar-modified nucleotides in the antisense sequence portion is preferably 100%.

[0080] It is more preferable that the antisense sequence portion that "contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides" does not contain three consecutive deoxyribonucleotides.

[0081] The antisense sequence portion (mixmer) that "contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides" is usually 4 to 30 consecutive nucleotides, preferably 8 to 25 consecutive nucleotides, and more preferably 10 to 20 consecutive nucleotides. These consecutive nucleotides may be the same or different from each other.

[0082] Also, from the viewpoint of excellent in vivo kinetics, among the nucleotides constituting the antisense sequence portion (mixmer) that "contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides", it is preferable that at least one nucleotide is phosphorothioated. More preferably, at least one of the nucleotides at the 3'-end and 5'-end of the antisense sequence portion is phosphorothioated. Among the bonds between the nucleotides included in the antisense sequence portion, it is further preferable that 80% are phosphorothioated, even more preferably 90% are phosphorothioated, and particularly preferably all are phosphorothioated.

[0083] The "sugar-modified nucleotide" contained in the antisense sequence portion may be a nucleotide with increased affinity for a partial sequence of the target RNA or a nucleotide with increased resistance to nucleolytic enzymes due to substitution or the like. Preferably, it is a 2'-O-methylated nucleotide, 2'-O-methoxyethyl (2'-MOE)-modified nucleotide, 2'-O-aminopropyl (2'-AP)-modified nucleotide, 2'-fluorinated nucleotide, 2'-F-arabinonucleotide (2'-F-ANA), bridged nucleotide (BNA (Bridged Nucleic Acid)) or 2'-O-methylcarbamoylethyl (2'-MCE)-modified nucleotide. More preferably, it is BNA or 2'-O-methylated nucleotide. Even more preferably, it is LNA containing a partial structure represented by the following formula (II), or 2'-O-methylated nucleotide. Particularly preferably, it is LNA. The "sugar-modified nucleotide" contained in the antisense sequence portion is particularly preferably a 2'-MOE-modified nucleotide and a 2'-MCE-modified nucleotide in addition to the above-mentioned bridged nucleotide.

[0084]

Chemical formula

[0085] In the formula, Base represents a base moiety, which is a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, and the purin-9-yl group and the 2-oxo-pyrimidin-1-yl group are either unmodified or modified. Here, the 2-oxo-pyrimidin-1-yl group is synonymous with the 2-oxo-1H-pyrimidin-1-yl group. Further, the purin-9-yl group and the 2-oxo-pyrimidin-1-yl group also include their respective tautomers.

[0086] The types, numbers, and positions of sugar-modified nucleotides, deoxyribonucleotides, and ribonucleotides in the antisense sequence portion can affect the antisense effect and the like exhibited by the single-stranded oligonucleotide disclosed herein. Since the types, numbers, and positions vary depending on the sequence of the target RNA and the like, it cannot be generally stated, but those skilled in the art can determine a preferred embodiment while referring to the descriptions in the literature regarding the antisense method. Further, the antisense effect of the single-stranded oligonucleotide after modification of the base portion, sugar portion, or phosphodiester bond portion is measured, and if the obtained measurement value is not significantly lower than that of the single-stranded oligonucleotide before modification (for example, if the measurement value of the single-stranded oligonucleotide after modification is 30% or more of the measurement value of the single-stranded oligonucleotide before modification), the modification can be evaluated as a preferred embodiment. The measurement of the antisense effect can be performed, for example, as shown in the examples described below, by introducing a test oligonucleotide into cells or the like, and using known techniques such as Northern blotting, quantitative PCR, Western blotting, etc. to appropriately measure the expression level of the target RNA controlled by the antisense effect exhibited by the test oligonucleotide, the expression level of cDNA related to the target RNA, the amount of protein related to the target RNA, and the like.

[0087] It is preferable that two nucleotides on at least one side of the 3'-side and the 5'-side of the antisense sequence portion "containing at least one sugar-modified nucleotide and not containing four consecutive deoxyribonucleotides" are sugar-modified nucleotides. The sugar-modified nucleotide is preferably a cross-linked nucleotide, and particularly preferably LNA. When the two nucleotides on the 3'-side of the antisense sequence portion are sugar-modified nucleotides, two or more of the three nucleotides on the 5'-side are sugar-modified nucleotides, and it is preferably linked in any of the following orders in sequence from the terminal side of the antisense sequence portion. When the two nucleotides on the 5'-side of the antisense sequence portion are sugar-modified nucleotides, two or more of the three nucleotides on the 3'-side are sugar-modified nucleotides, and it is preferably linked in any of the following orders in sequence from the terminal side of the antisense sequence portion. In these orders, the left side is the terminal side of the antisense sequence portion, and the right side is the inside of the antisense sequence portion. The sugar-modified nucleotide is preferably a cross-linked nucleotide, and particularly preferably LNA. Sugar-modified nucleotide - Sugar-modified nucleotide - Sugar-modified nucleotide Sugar-modified nucleotide - Sugar-modified nucleotide - Deoxyribonucleotide Sugar-modified nucleotide - Deoxyribonucleotide - Sugar-modified nucleotide

[0088] Next, the single-stranded oligonucleotide molecule of the present invention will be described. The single-stranded oligonucleotide of the present invention contains X and Y. Embodiments of the single-stranded oligonucleotide of the present invention include An embodiment that does not contain both Xz and Lx, and Yz and Ly (in the formula (I), m is 0 and n is 0), An embodiment that does not contain Xz and Lx but contains Yz and Ly (in the formula (I), m is 0 and n is 1), An embodiment that contains Xz and Lx but does not contain Yz and Ly (in the formula (I), m is 1 and n is 0) can be mentioned.

[0089] The following will describe Xa, Xb, X, Y, Xz, and Yz in the present invention. Although there are several embodiments in the present invention, first, the common points will be described.

[0090] Xa is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. It is a group consisting of 1 to 40 nucleotides independently selected from the group consisting of nucleotides, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base portion and the phosphate portion is modified. The oligonucleotide Xa contains at least one sugar-modified nucleotide. The oligonucleotide Xa has a nucleotide sequence Xa. Since Xa does not hybridize with Y, it is preferable that the nucleotide sequence Xa does not contain a sequence complementary to the nucleotide sequence Y.

[0091] The nucleotide sequence Xa is the base sequence of the nucleotides constituting the oligonucleotide Xa.

[0092] The number of nucleotides contained in Xa is from 1 to 40, preferably from 2 to 20, more preferably from 3 to 10, still more preferably from 4 to 8, even more preferably 4 or 5, and particularly preferably 5. The number of nucleotides contained in Xa is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure hybridized within the molecule, cost, synthesis yield, etc.

[0093] Xb is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. It is a group consisting of 4 to 40 nucleotides independently selected from the group, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base part and the phosphate part is modified. The oligonucleotide Xb contains at least one sugar-modified nucleotide. The oligonucleotide Xb has a nucleotide sequence Xb, and the nucleotide sequence Xb contains a sequence complementary to the nucleotide sequence Y.

[0094] The nucleotide sequence Xb is the base sequence of the nucleotides constituting the oligonucleotide Xb.

[0095] The number of nucleotides contained in Xb is 4 to 40, preferably 6 to 25, more preferably 8 to 16, still more preferably 9 to 13, and particularly preferably 9 to 11. The number of nucleotides contained in Xb is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure hybridized within the molecule, cost, synthesis yield, etc.

[0096] The oligonucleotide X is an oligonucleotide in which one end of the oligonucleotide Xa and one end of the oligonucleotide Xb are each covalently linked. The 5'-position of the nucleotide at the 5'-end of Xa and the 3'-position of the nucleotide at the 3'-end of Xb form a phosphodiester bond or a modified phosphodiester bond and are linked, or the 5'-position of the nucleotide at the 5'-end of Xb and the 3'-position of the nucleotide at the 3'-end of Xa form a phosphodiester bond or a modified phosphodiester bond and are linked.

[0097] X is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 5 to 80 nucleotides independently selected from the group, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base portion and the phosphate portion is modified. The oligonucleotide X contains at least two sugar-modified nucleotides. The oligonucleotide X has a nucleotide sequence X.

[0098] The nucleotide sequence X is the base sequence of the nucleotides constituting the oligonucleotide X. The nucleotide sequence X is synonymous with the nucleotide sequence (Xb-Xa).

[0099] The number of nucleotides contained in X is 5 to 80, preferably 8 to 45, more preferably 11 to 26, still more preferably 13 to 21, and particularly preferably 13 to 16. The number of nucleotides contained in X is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure hybridized within the molecule, cost, synthesis yield, and the like.

[0100] Y is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 4 to 40 nucleotides independently selected from the group, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base portion and the phosphate portion is modified. The oligonucleotide Y contains at least one ribonucleotide. The oligonucleotide Y has a nucleotide sequence Y, and the nucleotide sequence Y contains a sequence complementary to the nucleotide sequence Xb.

[0101] The nucleotide sequence Y is the base sequence of the nucleotides constituting the oligonucleotide Y.

[0102] The number of nucleotides contained in Y is from 4 to 40, preferably from 6 to 25, more preferably from 8 to 16, and particularly preferably from 10 to 13. The number of nucleotides contained in Y may be the same as or different from the number of nucleotides contained in Xb. The number of nucleotides contained in Y is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure hybridized within the molecule, cost, synthesis yield, etc. The difference between the number of nucleotides contained in Y and the number of nucleotides contained in Xb is preferably within 10, more preferably within 5, still more preferably within 4, even more preferably within 2, and particularly preferably 0.

[0103] Xz is a group derived from an oligonucleotide Xz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base moiety and the phosphate moiety is modified. The oligonucleotide Xz contains at least one sugar-modified nucleotide. The oligonucleotide Xz has a nucleotide sequence Xz.

[0104] The nucleotide sequence Xz is the base sequence of the nucleotides constituting the oligonucleotide Xz.

[0105] The number of nucleotides contained in Xz is from 5 to 40, preferably from 8 to 30, more preferably from 11 to 25, even more preferably from 12 to 21 bases, and particularly preferably from 13 to 14 bases. The number of nucleotides contained in Xz is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure in which X and Y hybridize within the molecule, cost, synthesis yield, etc.

[0106] Yz is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 5 to 40 nucleotides independently selected from the group consisting of oligonucleotides Yz consisting of nucleotides, and the deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides are each independently unmodified or at least one of the base portion and the phosphate portion is modified. The oligonucleotide Yz contains at least one sugar-modified nucleotide. The oligonucleotide Yz has a nucleotide sequence Yz.

[0107] The nucleotide sequence Yz is the base sequence of the nucleotides constituting the oligonucleotide Yz.

[0108] The number of nucleotides contained in Yz is 5 to 40, preferably 8 to 30, more preferably 11 to 25, even more preferably 12 to 21 bases, and particularly preferably 13 to 14 bases. The number of nucleotides contained in Yz is usually selected according to other factors such as the strength of the antisense effect on the target RNA, the stability of the structure in which X and Y hybridize within the molecule, cost, synthesis yield, and the like.

[0109] X and Y are linked in the order of Xb-Xa-Y. When Xb binds to Xa on the 3' side, Y binds to Xa on the 5' side. When Xb binds to Xa on the 5' side, Y binds to Xa on the 3' side.

[0110] Xa and Y are linked by a covalent bond, and the 5'-position of the nucleotide at the 5'-end of Xa and the 3'-position of the nucleotide at the 3'-end of Y form a phosphodiester bond or a modified phosphodiester bond to be linked, or the 5'-position of the nucleotide at the 5'-end of Y and the 3'-position of the nucleotide at the 3'-end of Xa form a phosphodiester bond or a modified phosphodiester bond to be linked. It is preferable that Xa and Y are linked by a phosphodiester bond.

[0111] Xa may or may not contain a sequence that is partially complementary within the group derived from the oligonucleotide of Xa.

[0112] Xb and Y hybridize within the molecule.

[0113] The nucleotide sequence Xb and the nucleotide sequence Y do not have to be completely complementary for Xb and Y to hybridize, but the complementarity is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). The nucleotide sequence Xb and the nucleotide sequence Y may be completely complementary.

[0114] It is not necessary for the whole of Y to hybridize to Xb which is a part of the antisense sequence portion, and a part of Y may not hybridize, but it is preferable for the whole to hybridize. When Y partially hybridizes with Xb which is a part of the antisense sequence portion, at least the terminal on the Xa side of Y preferably hybridizes with Xb. The number of nucleotides that hybridize partially is usually selected according to other factors such as the stability of the structure hybridized intermolecularly or intramolecularly, the strength of the antisense effect on the target RNA, cost, synthesis yield, etc.

[0115] The nucleotide sequence X contains an antisense sequence. Among the nucleotide sequence X, the proportion occupied by the antisense sequence is preferably 70% or more, more preferably 90% or more, and particularly preferably 100%. The antisense sequence contained in the nucleotide sequence X is a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA" or a sequence containing "at least 1 sugar-modified nucleotide and not containing 4 consecutive deoxyribonucleotides", and preferred embodiments are as described in the antisense sequence and the antisense sequence portion.

[0116] Xb is a part of the antisense sequence portion contained in X, hybridizes within the molecule, and Xa is a part of the antisense sequence portion contained in X and does not hybridize within the molecule. When the antisense sequence portion contained in X is a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA", it is preferable that a part of the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" is contained in Xb and hybridizes within the molecule, and a part is contained in Xa and does not hybridize within the molecule. As another aspect, it is preferable that the whole of the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" is contained in Xb and hybridizes. Among the "sugar moiety-modified nucleotide" sequence portions that are adjacent and bound to at least one of the 3'-side and 5'-side of the "at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA", the portion contained in Xb preferably hybridizes within the molecule, and the portion contained in Xa does not hybridize.

[0117] In the oligonucleotide X, an oligonucleotide chain consisting of 1 to 10 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar moiety-modified nucleotides may or may not be bound adjacent to the Xb side of the antisense sequence portion contained in the oligonucleotide X, but it is preferable not to bind. When binding, an oligonucleotide chain consisting of nucleotides independently selected from the group consisting of deoxyribonucleotides and ribonucleotides preferably binds adjacent to the end on the Xb side of the antisense sequence portion. When an oligonucleotide chain consisting of 1 to 10 nucleotides binds adjacent to the Xb side of the antisense sequence portion, it is preferable that the Xb side of the antisense sequence portion and the oligonucleotide chain are linked by a phosphodiester bond.

[0118] The nucleotide sequence Xz contains an antisense sequence. Among the nucleotide sequence Xz, the proportion occupied by the antisense sequence is preferably 70% or more, more preferably 90% or more, and particularly preferably 100%. The antisense sequence contained in the nucleotide sequence Xz is a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", or a sequence containing "at least one sugar-modified nucleotide and not containing 4 consecutive deoxyribonucleotides". Preferred embodiments and the like are as described in the antisense sequence and the antisense sequence portion.

[0119] The antisense sequence portion contained in Xz does not hybridize intramolecularly.

[0120] In the oligonucleotide Xz, an oligonucleotide chain consisting of 1 to 10 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides may or may not be bound adjacent to the end on the side that does not bind to Lx of the antisense sequence portion contained in the oligonucleotide Xz, but it is preferably not bound. When binding, an oligonucleotide chain consisting of nucleotides independently selected from the group consisting of deoxyribonucleotides and ribonucleotides preferably binds adjacent to the side of the antisense sequence portion contained in Xz that does not bind to Lx. When an oligonucleotide chain consisting of 1 to 10 nucleotides binds adjacent to the end on the side that does not bind to Lx of the antisense sequence portion, it is preferable that the antisense sequence portion and the oligonucleotide chain are linked by a phosphodiester bond.

[0121] The nucleotide sequence Yz contains an antisense sequence. Among the nucleotide sequence Yz, the proportion occupied by the antisense sequence is preferably 70% or more, more preferably 90% or more, and particularly preferably 100%. The antisense sequence contained in the nucleotide sequence Yz is a sequence containing "at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA", or a sequence containing "at least 1 sugar-modified nucleotide and not containing 4 consecutive deoxyribonucleotides". Preferred embodiments and the like are as described for the antisense sequence and the antisense sequence portion.

[0122] The antisense sequence portion contained in Yz does not hybridize intramolecularly.

[0123] In the oligonucleotide Yz, an oligonucleotide chain consisting of 1 to 10 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides may or may not be bound adjacent to the end on the side that does not bind to Ly of the antisense sequence portion contained in the oligonucleotide Yz, but it is preferably not bound. When binding, an oligonucleotide chain consisting of nucleotides independently selected from the group consisting of deoxyribonucleotides and ribonucleotides is preferably bound adjacent to the side that does not bind to Ly of the antisense sequence portion contained in Yz. When an oligonucleotide chain consisting of 1 to 10 nucleotides is bound adjacent to at least one of the 3'-side and 5'-side of the antisense sequence portion, it is preferable that the antisense sequence portion and the oligonucleotide chain are linked by a phosphodiester bond.

[0124] The types, numbers, and positions of the modified nucleotides, deoxyribonucleotides, and ribonucleotides in the oligonucleotide X may affect the antisense effect and the like exerted by the single-stranded oligonucleotide. Since the preferred embodiments vary depending on the types, sequences, etc. of the nucleotides to be modified, it cannot be generally stated. However, similar to the antisense sequence portion described above, it can be specified by measuring the antisense effect of the single-stranded oligonucleotide after modification. The same applies to Xz and Yz as to the oligonucleotide X.

[0125] When the oligonucleotides X and Xz hybridize to the same target RNA, their antisense sequences may be the same or different from each other. The oligonucleotides X and Xz may hybridize to different target RNAs, respectively.

[0126] When the oligonucleotides X and Yz hybridize to the same target RNA, their antisense sequences may be the same or different from each other. The oligonucleotides X and Yz may hybridize to different target RNAs, respectively.

[0127] The types, numbers, and positions of sugar moiety-modified nucleotides, deoxyribonucleotides, and ribonucleotides in Y may affect the antisense effect and the like exerted by the single-stranded oligonucleotide. Since the preferred embodiments vary depending on the types, sequences, etc. of the nucleotides to be modified, it cannot be generally stated. However, similar to the aforementioned antisense sequence portion, it can be specified by measuring the antisense effect of the modified oligonucleotide complex. From the viewpoint that Y is decomposed by nucleases such as RNaseH in specific cells, thereby generating an oligonucleotide containing the antisense sequence portion and being likely to exhibit an antisense effect, the nucleotide sequence Y preferably contains "at least 4 consecutive nucleotides cleaved by RNaseH" and preferably contains at least 1 ribonucleotide. The consecutive nucleotides are more preferably selected independently from ribonucleotides. Further, the consecutive nucleotides are more preferably linked to each other by phosphodiester bonds. These consecutive nucleotides may be the same or different from each other. Also, the nucleotide sequence Y preferably contains oligoribonucleotides and more preferably contains RNA. It is more preferable that the "at least 4 consecutive nucleotides cleaved by RNaseH" contain 4 to 25 consecutive nucleotides.

[0128] Next, each of the embodiments in the cases of [A] not containing both Xz and Lx, and Yz and Ly, [B] not containing Xz and Lx but containing Yz and Ly, and [C] containing Xz and Lx but not containing Yz and Ly will be described in order.

[0129] [A] In the case of not containing both Xz and Lx, and Yz and Ly (m = 0, n = 0)

[0130] The nucleotide sequence Y preferably contains at least 4 consecutive nucleotides that are cleaved by RNaseH, more preferably 4 to 25 consecutive nucleotides. These consecutive nucleotides may be the same or different from each other. Y preferably contains an oligoribonucleotide, more preferably contains RNA. Among the nucleotides on the 5'-side and 3'-side of the oligonucleotide Y, it is preferable that at least one of them is phosphorothioated. When Xb binds to Xa on the 3'-side and Y binds to Xa on the 5'-side, it is preferable that the 3'-side of the oligonucleotide Y is phosphorothioated. When Xb binds to Xa on the 5'-side and Y binds to Xa on the 3'-side, it is preferable that the 5'-side of the oligonucleotide Y is phosphorothioated. When Xb binds to Xa on the 3'-side and Y binds to Xa on the 5'-side, the 3'-side of the oligonucleotide Y preferably contains 1 to 10 sugar-modified nucleotides, more preferably 2 to 5 sugar-modified nucleotides, and even more preferably 2 or 3 sugar-modified nucleotides. When Xb binds to Xa on the 5'-side and Y binds to Xa on the 3'-side, the 5'-side of the oligonucleotide Y preferably contains 1 to 10 sugar-modified nucleotides, more preferably 2 to 5 sugar-modified nucleotides, and even more preferably 2 or 3 sugar-modified nucleotides. The plurality of sugar-modified nucleotides are preferably linked by phosphorothioate bonds. Here, a plurality of deoxyribonucleotides or ribonucleotides or both may be included between the plurality of sugar-modified nucleotides on at least one of the 3'-side and 5'-side, but the plurality of sugar-modified nucleotides are preferably consecutive. When at least one of the 3'-side and 5'-side of the oligonucleotide Y contains a plurality of sugar-modified nucleotides, each sugar-modified nucleotide may be the same or different from each other.

[0131] The sugar-modified nucleotides contained in at least one of the 3'-side and 5'-side of the oligonucleotide Y are preferably 2'-O-methylated nucleotides, 2'-MOE (2'-O-methoxyethyl)ated nucleotides, 2'-AP (2'-O-aminopropyl)ated nucleotides, 2'-fluorinated nucleotides, 2'-F-arabinonucleotides (2'-F-ANA), bridged nucleotides (BNA (Bridged Nucleic Acid)) or 2'-O-methylcarbamoylethylated nucleotides (MCE), more preferably BNA or 2'-O-methylated nucleotides, even more preferably LNA containing a partial structure represented by the following formula (II), or 2'-O-methylated nucleotides, and particularly preferably 2'-O-methylated nucleotides.

[0132] [Chemical formula]

[0133] In the formula, Base represents a base moiety and is a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, and the purin-9-yl group and the 2-oxo-pyrimidin-1-yl group are either unmodified or modified.

[0134] In another aspect, the nucleotides contained in Y are preferably independently selected from ribonucleotides. Also, the nucleotides contained in Y are preferably linked to each other by phosphodiester bonds.

[0135] [B] When not containing Xz and Lx and containing Yz and Ly (m = 0, n = 1)

[0136] The nucleotide sequence Y preferably contains at least 4 consecutive nucleotides that can be cleaved by RNaseH, and more preferably contains 4 to 25 consecutive nucleotides. These consecutive nucleotides may be the same or different from each other. The oligonucleotide Y preferably contains oligoribonucleotides, more preferably contains RNA, and particularly preferably is a group derived from RNA. The nucleotides contained in Y are preferably selected independently from ribonucleotides. The nucleotides contained in Y are preferably linked to each other by phosphodiester bonds.

[0137] [C] When including Xz and Lx and not including Yz and Ly (m = 1, n = 0)

[0138] A preferred embodiment of the nucleotide sequence Y is the same as the case of not including both [A] Xz and Lx, Yz and Ly described above.

[0139] Next, Lx, Ly, and the functional molecule will be described. The following is common to some of the above embodiments.

[0140] Lx is a group derived from an oligonucleotide Lx consisting of 0 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a linker that links the aforementioned Xb and Xz. Lx links the aforementioned Xb and Xz in the order of Xz-Lx-Xb. When m = 1 and the oligonucleotide Lx consists of 0 nucleotides, Xb and Xz are directly linked.

[0141] Ly is a group derived from an oligonucleotide Ly consisting of 0 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a linker that links the aforementioned Y and Yz. Ly links the aforementioned Y and Yz in the order of Y-Ly-Yz. When n is 1 and the oligonucleotide Ly consists of 0 nucleotides, Y and Yz are directly linked.

[0142] Lx and Xb are linked by a covalent bond. For example, it is preferable that an oxygen atom obtained by removing a hydrogen atom from the hydroxy group of the sugar moiety of the terminal nucleotide of Xb (including the partial structure replaced from the sugar backbone in the sugar moiety-modified nucleotide) is linked to the sugar moiety of the terminal nucleotide of Lx by a phosphodiester bond or a modified phosphodiester bond. Lx and Xz are linked by a covalent bond. For example, it is preferable that an oxygen atom obtained by removing a hydrogen atom from the hydroxy group of the sugar moiety of the terminal nucleotide of Xz (including the partial structure replaced from the sugar backbone in the sugar moiety-modified nucleotide) is linked to the sugar moiety of the terminal nucleotide of Lx by a phosphodiester bond or a modified phosphodiester bond. Similarly, it is preferable that Ly and Y are linked by the sugar moiety of the terminal nucleotide of Ly and the sugar moiety of the terminal nucleotide of Y, and it is preferable that Ly and Yz are linked by the sugar moiety of the terminal nucleotide of Ly and the sugar moiety of the terminal nucleotide of Yz. When Xb and Xz are directly linked, similarly, it is preferable that the sugar moiety of the terminal nucleotide of Xb and the sugar moiety of the terminal nucleotide of Xz are linked by a phosphodiester bond or a modified phosphodiester bond, and it is more preferable that they are linked by a phosphodiester bond. When Y and Yz are directly linked, similarly, it is preferable that the sugar moiety of the terminal nucleotide of Y and the sugar moiety of the terminal nucleotide of Yz are linked by a phosphodiester bond or a modified phosphodiester bond, and it is more preferable that they are linked by a phosphodiester bond. When the terminal nucleotide is a sugar moiety-modified nucleotide, the sugar moiety includes a partial structure replaced from the sugar backbone and the like.

[0143] When Xb binds to Xa on the 3'-side, Y binds to Xa on the 5'-side. Further, when m = 1, Xb binds to Lx on the 5'-side and Xz binds to Lx on the 3'-side. When Xb binds to Xa on the 3'-side, Y binds to Xa on the 5'-side, and further when n = 1, Y binds to Ly on the 3'-side and Yz binds to Ly on the 5'-side.

[0144] When Xb binds to Xa on the 5'-side, Y binds to Xa on the 3'-side. Further, when m = 1, Xb binds to Lx on the 3'-side and Xz binds to Lx on the 5'-side. When Xb binds to Xa on the 5'-side, Y binds to Xa on the 3'-side, and further when n = 1, Y binds to Ly on the 5'-side and Yz binds to Ly on the 3'-side.

[0145] It is desirable that Lx and Ly are decomposed more rapidly than the aforementioned antisense sequence portion.

[0146] The oligonucleotide Lx is preferably an oligonucleotide that is decomposed under physiological conditions.

[0147] The oligonucleotide Ly is preferably an oligonucleotide that is decomposed under physiological conditions.

[0148] Here, the "oligonucleotide that is decomposed under physiological conditions" may be an oligonucleotide that is decomposed by enzymes such as various DNases (deoxyribonucleases) and RNases under physiological conditions. The nucleotides constituting the oligonucleotide may or may not be chemically modified in part or in whole in the base, sugar, or phosphate bond.

[0149] The oligonucleotide Lx is preferably an oligonucleotide linked by phosphodiester bonds, more preferably an oligodeoxyribonucleotide or oligoribonucleotide, still more preferably DNA or RNA, and even more preferably RNA. The oligonucleotide Ly is the same as the oligonucleotide Lx.

[0150] Oligonucleotide Lx may or may not contain a partially complementary sequence within oligonucleotide Lx, but oligonucleotide Lx is preferably an oligonucleotide that does not contain a partially complementary sequence within oligonucleotide Lx. Examples of bases derived from such oligonucleotides include (N) linked by phosphodiester bonds k (where N is independently adenosine, uridine, cytidine, guanosine, 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, or 2'-deoxyguanosine, and k is an integer from 1 to 20 (number of repetitions)). Among them, k is preferably 1 to 12, more preferably 1 to 8, still more preferably 1 to 5, and even more preferably 1 to 3. Oligonucleotide Ly is the same as oligonucleotide Lx.

[0151] Functional molecules may be directly or indirectly bound to X (including Xa and Xb), Y, Xz, Yz, Lx, and Ly. In the case where neither [A] Xz and Lx nor Yz and Ly are included, the functional molecule is preferably bound to oligonucleotide Y. In the case where [B] Xz and Lx are not included and Yz and Ly are included, the functional molecule is preferably bound to oligonucleotide Xb or oligonucleotide Y. In the case where [C] Xz and Lx are included and Yz and Ly are not included, the functional molecule is preferably bound to oligonucleotide Y. The binding of the functional molecule to oligonucleotide Y or oligonucleotide Xb may be direct or indirect via other substances, but it is preferable that the functional molecule and oligonucleotide Y or oligonucleotide Xb are bound by a covalent bond, an ionic bond, or a hydrogen bond. From the viewpoint of high stability of the binding, it is more preferable that they are directly bound by a covalent bond or bound via a linker (linking group) by a covalent bond.

[0152] When the functional molecule binds to a single-stranded oligonucleotide by a covalent bond, it is preferred that the functional molecule is directly or indirectly bound to the 3'-end or 5'-end of the single-stranded oligonucleotide molecule. The bond between the linker or functional molecule and the terminal nucleotide of the single-stranded oligonucleotide molecule is selected according to the functional molecule. It is preferred that the linker or functional molecule and the terminal nucleotide of the single-stranded oligonucleotide molecule are linked by a phosphodiester bond or a modified phosphodiester bond, and more preferably linked by a phosphodiester bond. The linker or functional molecule may be directly linked to the oxygen atom at the 3'-position of the nucleotide at the 3'-end of the single-stranded oligonucleotide molecule or the oxygen atom at the 5'-position of the nucleotide at the 5'-end.

[0153] The structure of the "functional molecule" is not particularly limited, and a desired function is imparted to the single-stranded oligonucleotide by its binding. Examples of desired functions include a labeling function, a purification function, and a delivery function to a target site. Examples of molecules that impart a labeling function include compounds such as fluorescent proteins and luciferase. Examples of molecules that impart a purification function include compounds such as biotin, avidin, His-tag peptide, GST-tag peptide, and FLAG-tag peptide.

[0154] Also, from the viewpoint of delivering a single-stranded oligonucleotide to a target site (e.g., target cells) with high specificity and efficiency and very effectively controlling the expression of a target gene by the single-stranded oligonucleotide, it is preferred that a molecule having a function of delivering the single-stranded oligonucleotide to the target site is bound as the functional molecule. References such as European Journal of Pharmaceutical and Biopharmaceutics, Vol. 107, pp. 321-340 (2016), Advanced Drug Delivery Reviews, Vol. 104, pp. 78-92 (2016), Expert Opinion on Drug Delivery, Vol. 11, pp. 791-822 (2014), etc. can be referred to for molecules having such a delivery function.

[0155] As molecules that confer the function of delivering to the target RNA, for example, from the viewpoint of being able to deliver single-stranded oligonucleotides highly specifically and efficiently to the liver or the like, lipids and sugars can be mentioned. Such lipids include cholesterol; fatty acids; fat-soluble vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, vitamin K; intermediate metabolites such as acylcarnitine and acyl-CoA; glycolipids; glycerides; and their derivatives and the like. Among these, from the viewpoint of higher safety, cholesterol and vitamin E (tocopherols, tocotrienols) are preferred. Among them, tocopherols are more preferred, tocopherol is further preferred, and α-tocopherol is particularly preferred. Sugars include sugar derivatives that interact with asialoglycoprotein receptors.

[0156] The "asialoglycoprotein receptor" exists on the surface of liver cells and has the function of recognizing the galactose residue of asialoglycoprotein and taking the molecule into the cell for degradation. The "sugar derivative that interacts with the asialoglycoprotein receptor" preferably has a structure similar to the galactose residue and is a compound that is taken into the cell by interaction with the asialoglycoprotein receptor, and examples include GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives. Also, from the viewpoint of being able to deliver the single-stranded oligonucleotide of the present invention highly specifically and efficiently to the brain, sugars (for example, glucose, sucrose, etc.) can be mentioned as the "functional molecule". Further, from the viewpoint of being able to deliver the single-stranded oligonucleotide highly specifically and efficiently to a specific organ by interacting with various proteins on the cell surface of each organ, receptor ligands, antibodies, peptides or proteins of their fragments can be mentioned as the "functional molecule".

[0157] The linker that binds a functional molecule to X (including Xa and Xb), Y, Xz, Yz, Lx, and Ly may be any linker that can stably bind the functional molecule and the oligonucleotide, as long as it can exert the function of the functional molecule as a single-stranded oligonucleotide molecule. Examples of the linker include a group derived from an oligonucleotide having 2 to 20 nucleotides, a group derived from a polypeptide having 2 to 20 amino acids, an alkylene group having 2 to 20 carbon atoms, and an alkenylene group having 2 to 20 carbon atoms. The group derived from an oligonucleotide having 2 to 20 nucleotides is a group obtained by removing a hydroxy group, a hydrogen atom, etc. from an oligonucleotide having 2 to 20 nucleotides. The group derived from a polypeptide having 2 to 20 amino acids is a group obtained by removing a hydroxy group, a hydrogen atom, an amino group, etc. from a polypeptide having 2 to 20 amino acids.

[0158] The linker is preferably a C 2-20 alkylene group or a C 2-20 alkenylene group (the methylene groups contained in the alkylene group and the alkenylene group are each independently unsubstituted or substituted with one or two substituents selected from the group consisting of a halogen atom, a hydroxy group, a protected hydroxy group, an oxo group, and a thioxo group. Further, the methylene groups of the alkylene group and the alkenylene group are each independently unsubstituted or replaced with -O-, -NR B -(R B is a hydrogen atom, a C 1-6 alkyl group, or a halo C 1-6 alkyl group), -S-, -S(=O)-, or -S(=O) 2 -). Here, by combining the above substitution and replacement, the linker is -C(=O)-O-, -O-C(=O)-NR 1 -(R 1 is a hydrogen atom, a C 1-6 alkyl group, or a halo C 1-6 alkyl group), -C(=O)-NR 1 -(R 1 is a hydrogen atom, a C 1-6 alkyl group, or a halo C1-6 (representing an alkyl group), -C(=S)-NR 1 -(R 1 is a hydrogen atom, C 1-6 alkyl group or halo C 1-6 (representing an alkyl group), -NR 1 -C(=O)-NR 1 -(R 1 are each independently a hydrogen atom, C 1-6 alkyl group or halo C 1-6 (representing an alkyl group), etc., and may contain a group represented by the like.

[0159] The linker is more preferably a C 2-20 alkylene group (the methylene groups of the alkylene group are each independently unsubstituted or substituted by -O-. The unsubstituted methylene groups are each independently unsubstituted or substituted by a hydroxy group or a protected hydroxy group), and still more preferably a C 8-12 alkylene group (the methylene groups of the alkylene group are each independently unsubstituted or substituted by -O-. The unsubstituted methylene groups are each independently unsubstituted or substituted by a hydroxy group), and particularly preferably a 1,8-octylene group. In another aspect, the linker is particularly preferably a group represented by the following formula (III).

[0160] [Chemical formula]

[0161] In the formula, one * represents the bonding position (atom constituting the nucleotide) with the group derived from the oligonucleotide, and the other * represents the bonding position (atom constituting the group derived from the functional molecule) with the group derived from the functional molecule.

[0162] In another aspect, the linker is more preferably a C 2-20An alkylene group (each methylene group of the alkylene group is independently unsubstituted or substituted by -O- or -NR B -(R B is a hydrogen atom or a C 1-6 alkyl group). The unsubstituted methylene groups are each independently unsubstituted or substituted by an oxo group), and more preferably, a group represented by the following formula: [Chemical formula] (wherein e is each independently an integer from 1 to 6), and particularly preferably, a group represented by the following formula: [Chemical formula] represented by the formula.

[0163] The protecting group of the "protected hydroxy group" is not particularly limited as long as it is stable when binding the functional molecule and the oligonucleotide. It is not particularly limited as a linker, and examples thereof include any protecting group described in Protective Groups in Organic Synthesis, 3rd Edition, published by John Wiley & Sons (1999), etc. Specifically, methyl group, benzyl group, p-methoxybenzyl group, tert-butyl group, methoxymethyl group, methoxyethyl group, 2-tetrahydropyranyl group, ethoxyethyl group, cyanoethyl group, cyanoethoxymethyl group, phenylcarbamoyl group, 1,1-dioxothiomorpholine-4-thiocarbamoyl group, acetyl group, pivaloyl group, benzoyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, [(triisopropylsilyl)oxy]methyl group (Tom group), 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl group (Cpep group), triphenylmethyl group (trityl group), monomethoxytrityl group, dimethoxytrityl group (DMTr group), trimethoxytrityl group, 9-phenylxanthen-9-yl group (Pixyl group), 9-(p-methoxyphenyl)xanthen-9-yl group (MOX group), etc. The protecting group of the "protected hydroxy group" is preferably a benzoyl group, trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, tert-butyldimethylsilyl group, triphenylmethyl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, 9-phenylxanthen-9-yl group or 9-(p-methoxyphenyl)xanthen-9-yl group, more preferably a monomethoxytrityl group, dimethoxytrityl group or trimethoxytrityl group, and even more preferably a dimethoxytrityl group.

[0164] Preferred single-stranded oligonucleotides for nucleic acid pharmaceuticals include those shown below. 1) Formula (I) [Chemical formula] {In the formula, Y is a group derived from an oligonucleotide Y consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and containing at least one ribonucleotide. X is a group represented by the formula: [Chemical formula] (In the formula, Xb is a group derived from an oligonucleotide Xb consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide. Xa is a group derived from an oligonucleotide Xa consisting of 1 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide. Xa is bonded to the oligonucleotide Y and the oligonucleotide Xb at both ends respectively), and is a group derived from an oligonucleotide X consisting of 5 to 80 nucleotides. Xz is a group derived from an oligonucleotide Xz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide. Yz is a group derived from an oligonucleotide Yz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and containing at least one sugar-modified nucleotide. Lx is a group derived from an oligonucleotide Lx consisting of 0 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. Ly is a group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, and is a group consisting of 0 to 20 nucleotides independently selected from the group consisting of nucleotides, and is a group consisting of nucleotides derived from the oligonucleotide Ly. m is 0 or 1, When m is 0, n is 0 or 1, When m is 1, n is 0, The oligonucleotide X has a nucleotide sequence X, the oligonucleotide Xa has a nucleotide sequence Xa, the oligonucleotide Xb has a nucleotide sequence Xb, the oligonucleotide Y has a nucleotide sequence Y, the oligonucleotide Xz has a nucleotide sequence Xz, the oligonucleotide Yz has a nucleotide sequence Yz, the oligonucleotide Lx has a nucleotide sequence Lx, and the oligonucleotide Ly has a nucleotide sequence Ly. The nucleotide sequence Xb is complementary to the nucleotide sequence Y. The nucleotide sequence X contains an antisense sequence that enables hybridization with the target RNA. When m is 1 and n is 0, The nucleotide sequence Xz contains an antisense sequence that enables hybridization with the target RNA. When m is 0 and n is 1, The nucleotide sequence Yz contains an antisense sequence that enables hybridization with the target RNA. When there are two or more of the antisense sequences, the target RNAs to which the respective antisense sequence portions hybridize may be the same or different. Is represented by, and is a single-stranded oligonucleotide in which Xb and Y hybridize.

[0165] 2) The single-stranded oligonucleotide according to 1), wherein Xb binds to Xa on the 3' side and Y binds to Xa on the 5' side. 3) The single-stranded oligonucleotide according to 1), wherein Xb binds to Xa on the 5' side and Y binds to Xa on the 3' side.

[0166] 4) The single-stranded oligonucleotide according to any one of 1) to 3), wherein the complementarity between the antisense sequence and the target RNA sequence is 70% or more. 5) The single-stranded oligonucleotide according to any one of 1) to 4), wherein the complementarity between the nucleotide sequence Xb and the nucleotide sequence Y is 70% or more. 6) Each nucleotide contained in the single-stranded oligonucleotide represented by the formula (I) is independently selected from the group consisting of a phosphodiester bond, a phosphorothioate bond, a methylphosphonate bond, a methylthiophosphonate bond, a phosphorodithioate bond, and a phosphoramidate bond, and is linked to each other by at least one kind, The single-stranded oligonucleotide according to any one of 1) to 5). 7) Each nucleotide contained in the single-stranded oligonucleotide represented by the formula (I) is independently selected from at least one kind of a phosphodiester bond and a phosphorothioate bond and is linked to each other, The single-stranded oligonucleotide according to any one of 1) to 6). 8) The single-stranded oligonucleotide according to any one of 1) to 7), wherein the antisense sequence portion contained in X contains a phosphorothioate bond. 9) The single-stranded oligonucleotide according to any one of 1) to 8), wherein the antisense sequence contained in the nucleotide sequence X is a sequence containing nucleotides linked to each other by a phosphorothioate bond. 10) The single-stranded oligonucleotide according to any one of 1) to 9), wherein the nucleotides contained in the oligonucleotide X are linked to each other by a phosphorothioate bond.

[0167] 11) The single-stranded oligonucleotide according to any one of 1) to 10), wherein at least one of the nucleotides on the 3'-side and 5'-side of the antisense sequence portion contained in the oligonucleotide X is a sugar-modified nucleotide. 12) The single-stranded oligonucleotide according to any one of 1) to 11), wherein the nucleotides on the 3'-side and 5'-side of the antisense sequence portion contained in the oligonucleotide X are sugar-modified nucleotides. 13) The single-stranded oligonucleotide according to any one of 1) to 12), wherein the antisense sequence contained in the nucleotide sequence X is a sequence consisting of 11 to 26 nucleotides independently selected from sugar-modified nucleotides and deoxyribonucleotides. 14) The single-stranded oligonucleotide according to any one of 1) to 13), wherein the antisense sequence contained in the nucleotide sequence X is a sequence consisting of 11 to 26 nucleotides containing at least one deoxyribonucleotide.

[0168] 15) The single-stranded oligonucleotide according to any one of 1) to 14), wherein the antisense sequence contained in the nucleotide sequence X is a sequence containing at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA. 16) The single-stranded oligonucleotide according to 15), wherein the antisense sequence portion contains sugar-modified nucleotides that are adjacently linked to the 5'-side and 3'-side of the "sequence portion containing at least 4 consecutive nucleotides recognized by RNaseH". 17) The single-stranded oligonucleotide according to any one of 15) or 16), wherein the "sequence containing at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA" is a sequence consisting of 4 to 20 nucleotides containing at least one deoxyribonucleotide.

[0169] 18) The single-stranded oligonucleotide according to any one of 1) to 14), wherein the antisense sequence portion contained in the nucleotide sequence X contains at least one sugar-modified nucleotide and does not contain 4 consecutive deoxyribonucleotides. 19) The single-stranded oligonucleotide according to any one of 1) to 13), wherein the antisense sequence contained in the nucleotide sequence X is a sequence consisting of 4 to 30 sugar-modified nucleotides.

[0170] 20) The single-stranded oligonucleotide according to any one of 1) to 19), wherein the nucleotide sequence Y is a sequence containing at least 4 consecutive nucleotides that are cleaved by RNaseH. 21) The single-stranded oligonucleotide according to any one of 1) to 20), wherein the nucleotide sequence Y is a sequence consisting of 6 to 25 ribonucleotides. 22) The single-stranded oligonucleotide according to any one of 1) to 21), wherein the oligonucleotide Y contains one or more sugar-modified nucleotides on at least one of the 5'-side and 3'-side of the oligonucleotide Y. 23) The single-stranded oligonucleotide according to any one of 1) to 22), wherein the oligonucleotide Y contains a phosphodiester bond. 24) The single-stranded oligonucleotide according to any one of 1) to 23), wherein at least one of the 5'-side and 3'-side of Y is linked to an adjacent nucleotide by a phosphodiester bond. 25) The single-stranded oligonucleotide according to any one of 1) to 24), wherein the oligonucleotide Xa consists of 3 to 10 nucleotides independently selected from the group consisting of deoxyribonucleotides and sugar-modified nucleotides, and the oligonucleotide Xb consists of 8 to 16 nucleotides independently selected from the group consisting of deoxyribonucleotides and sugar-modified nucleotides. 26) The single-stranded oligonucleotide according to 1) to 25), wherein m is 0 and n is 0. 27) The single-stranded oligonucleotide according to 26), wherein at least one of the nucleotides on the 5'-side and 3'-side of Y is phosphorothioated.

[0171] 28) The single-stranded oligonucleotide according to 1) to 25), wherein m is 1 and n is 0. 29) The single-stranded oligonucleotide according to 28), wherein the antisense sequence portion containing Xz contains phosphorothioate bonds. 30) The single-stranded oligonucleotide according to any one of 28) or 29), wherein the antisense sequence contained in the nucleotide sequence Xz is a sequence containing nucleotides linked to each other by phosphorothioate bonds. 31) The single-stranded oligonucleotide according to any one of 28) to 30), wherein the nucleotides contained in oligonucleotide Xz are linked to each other by phosphorothioate bonds.

[0172] 32) The single-stranded oligonucleotide according to any one of 28) to 31), wherein at least one of the nucleotides on the 3'-side and 5'-side of the antisense sequence portion contained in oligonucleotide Xz is a sugar-modified nucleotide. 33) The single-stranded oligonucleotide according to any one of 28) to 32), wherein the nucleotides on the 3'-side and 5'-side of the antisense sequence portion contained in oligonucleotide Xz are sugar-modified nucleotides. 34) The single-stranded oligonucleotide according to any one of 33) to 34), wherein the antisense sequence contained in the nucleotide sequence Xz is a sequence consisting of 11 to 26 nucleotides independently selected from sugar-modified nucleotides and deoxyribonucleotides. 35) The single-stranded oligonucleotide according to any one of 28) to 34), wherein the antisense sequence contained in the nucleotide sequence Xz is a sequence consisting of 11 to 26 nucleotides containing at least one deoxyribonucleotide.

[0173] 36) The single-stranded oligonucleotide according to any one of 28) to 35), wherein the antisense sequence contained in the nucleotide sequence Xz is a sequence containing at least 4 consecutive nucleotides recognized by RNaseH when hybridized to the target RNA. 37) The single-stranded oligonucleotide according to 36), wherein the antisense sequence portion contains sugar moiety-modified nucleotides that are adjacent and linked to the 5' side and the 3' side of the "sequence portion containing at least 4 consecutive nucleotides recognized by the RNaseH". 38) The single-stranded oligonucleotide according to any one of 36) or 37), wherein the "sequence containing at least 4 consecutive nucleotides recognized by the RNaseH when hybridized with the target RNA" is a sequence consisting of 4 to 20 nucleotides containing at least 1 deoxyribonucleotide.

[0174] 39) The single-stranded oligonucleotide according to any one of 28) to 35), wherein the antisense sequence portion contained in the nucleotide sequence Xz contains at least 1 sugar moiety-modified nucleotide and does not contain 4 consecutive deoxyribonucleotides. 40) The single-stranded oligonucleotide according to any one of 28) to 34), wherein the antisense sequence contained in the nucleotide sequence Xz is a sequence consisting of 4 to 30 sugar moiety-modified nucleotides. 41) The single-stranded oligonucleotide according to any one of 28) to 40), wherein the oligonucleotide Lx consists of 0 nucleotides, and Xb and Xz are linked by a phosphodiester bond. 42) The single-stranded oligonucleotide according to any one of 28) to 40), wherein Lx is a group derived from an oligonucleotide Lx consisting of 1 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar moiety-modified nucleotides. 43) The single-stranded oligonucleotide according to 42), wherein the oligonucleotide Lx contains a phosphodiester bond. 44) The single-stranded oligonucleotide according to any one of 42) or 43), wherein the nucleotides contained in the oligonucleotide Lx are linked to each other by a phosphodiester bond. 45) The single-stranded oligonucleotide according to any one of 42) to 44), wherein the oligonucleotide Lx consists of 1 to 8 nucleotides independently selected from the group consisting of deoxyribonucleotides and ribonucleotides. 46) The single-stranded oligonucleotide according to any one of 42) to 45), wherein the oligonucleotide Lx is an oligodeoxyribonucleotide or an oligoribonucleotide. 47) The single-stranded oligonucleotide according to any one of 42) to 46), wherein the oligonucleotide Lx is DNA or RNA. 48) The single-stranded oligonucleotide according to any one of 42) to 46), wherein the oligonucleotide Lx is RNA. 49) The single-stranded oligonucleotide according to any one of 28) to 48), wherein at least one of the nucleotides on the 5'-side and 3'-side of the oligonucleotide Y is phosphorothioated.

[0175] 50) The single-stranded oligonucleotide according to any one of 1) to 25), wherein m is 0 and n is 1. 51) The single-stranded oligonucleotide according to 50), wherein the antisense sequence portion contained in Yz contains phosphorothioate bonds. 52) The single-stranded oligonucleotide according to any one of 50) or 51), wherein the antisense sequence contained in the nucleotide sequence Yz is a sequence containing nucleotides linked to each other by phosphorothioate bonds. 53) The single-stranded oligonucleotide according to any one of 50) to 52), wherein the nucleotides contained in the oligonucleotide Yz are linked to each other by phosphorothioate bonds.

[0176] 54) The single-stranded oligonucleotide according to any one of 50) to 53), wherein at least one of the nucleotides on the 3'-side and 5'-side of the antisense sequence portion contained in the oligonucleotide Yz is a sugar-modified nucleotide. 55) The single-stranded oligonucleotide according to any one of 50) to 54), wherein the nucleotides on the 3'-side and 5'-side of the antisense sequence portion included in the oligonucleotide Yz are sugar-modified nucleotides. 56) The single-stranded oligonucleotide according to any one of 50) to 55), wherein the antisense sequence included in the nucleotide sequence Yz is a sequence consisting of 11 to 26 nucleotides independently selected from sugar-modified nucleotides and deoxyribonucleotides. 57) The single-stranded oligonucleotide according to any one of 50) to 56), wherein the antisense sequence included in the nucleotide sequence Yz is a sequence consisting of 11 to 26 nucleotides including at least one deoxyribonucleotide.

[0177] 58) The single-stranded oligonucleotide according to any one of 50) to 57), wherein the antisense sequence included in the nucleotide sequence Yz is a sequence including at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA. 59) The single-stranded oligonucleotide according to 58), wherein the antisense sequence portion includes sugar-modified nucleotides that are adjacently bound to the 5'-side and 3'-side of the "sequence portion including at least 4 consecutive nucleotides recognized by RNaseH". 60) The single-stranded oligonucleotide according to any one of 58) or 59), wherein the "sequence including at least 4 consecutive nucleotides recognized by RNaseH when hybridized with the target RNA" is a sequence consisting of 4 to 20 nucleotides including at least one deoxyribonucleotide.

[0178] 61) The single-stranded oligonucleotide according to any one of 50) to 57), wherein the antisense sequence portion included in the nucleotide sequence Yz includes at least one sugar-modified nucleotide and does not include 4 consecutive deoxyribonucleotides. 62) The single-stranded oligonucleotide according to any one of 50) to 56), wherein the antisense sequence contained in the nucleotide sequence Yz is a sequence consisting of 4 to 30 sugar-modified nucleotides. 63) The single-stranded oligonucleotide according to any one of 50) to 62), wherein the oligonucleotide Ly consists of 0 nucleotides, and Y and Yz are linked by a phosphodiester bond. 64) The single-stranded oligonucleotide according to any one of 50) to 62), wherein Ly is a group derived from an oligonucleotide Ly consisting of 1 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. 65) The single-stranded oligonucleotide according to 64), wherein the oligonucleotide Ly contains a phosphodiester bond. 66) The single-stranded oligonucleotide according to 64) or 65), wherein the nucleotides contained in the oligonucleotide Ly are linked to each other by a phosphodiester bond. 67) The single-stranded oligonucleotide according to any one of 64) to 66), wherein the oligonucleotide Ly consists of 1 to 8 nucleotides independently selected from the group consisting of deoxyribonucleotides and ribonucleotides. 68) The single-stranded oligonucleotide according to any one of 64) to 67), wherein the oligonucleotide Ly is an oligodeoxyribonucleotide or an oligoribonucleotide. 69) The single-stranded oligonucleotide according to any one of 64) to 68), wherein the oligonucleotide Ly is DNA or RNA. 70) The single-stranded oligonucleotide according to any one of 64) to 69), wherein the oligonucleotide Ly is RNA.

[0179] 71) The single-stranded oligonucleotide according to any one of 1) to 70), wherein the sugar-modified nucleotides are each independently a 2'-O-methylated nucleotide, a 2'-O-methoxyethylated nucleotide, a 2'-O-aminopropylated nucleotide, a 2'-fluorinated nucleotide, a 2'-F-arabinonucleotide, a cross-linked nucleotide, or a 2'-O-methylcarbamoylethylated nucleotide. 72) The single-stranded oligonucleotide according to any one of 1) to 71), wherein the sugar-modified nucleotides are each independently a 2'-O-methylated nucleotide or an LNA.

[0180] 73) The single-stranded oligonucleotide according to any one of 1) to 72), further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target RNA. 74) The single-stranded oligonucleotide according to 73), wherein the group derived from the functional molecule is directly or indirectly bonded to the nucleotide at the 5'-end of the single-stranded oligonucleotide represented by formula (I). 75) The single-stranded oligonucleotide according to 73), wherein the group derived from the functional molecule is directly or indirectly bonded to the nucleotide at the 3'-end of the single-stranded oligonucleotide represented by formula (I).

[0181] 76) The group derived from the functional molecule is a C 2-20 alkylene group or a C 2-20 alkenylene group (the methylene groups contained in the alkylene group and the alkenylene group are each independently unsubstituted or substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a protected hydroxy group, an oxo group, and a thioxo group. Also, the methylene groups of the alkylene group and the alkenylene group are each independently not replaced or -O-, -NR B -(R B is a hydrogen atom, a C 1-6 alkyl group, or a halo C 1-6 alkyl group), -S-, -S(=O)-, or -S(=O)2 - which is replaced by -), or directly linked by a covalent bond, to a single-stranded oligonucleotide represented by formula (I), the single-stranded oligonucleotide according to any one of 73) to 75). 77) C to which a group derived from the functional molecule is linked 2-20 an alkylene group or C 2-20 an alkenylene group and a nucleotide at the 5'-end or 3'-end of the single-stranded oligonucleotide represented by formula (I) are linked by a phosphodiester bond or a modified phosphodiester bond, the single-stranded oligonucleotide according to any one of 73) to 75). 78) C to which a group derived from the functional molecule is linked 2-20 an alkylene group or C 2-20 an alkenylene group and a nucleotide at the 5'-end or 3'-end of the single-stranded oligonucleotide represented by formula (I) are linked by a phosphodiester bond, the single-stranded oligonucleotide according to any one of 73) to 75). 79) The functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof, the single-stranded oligonucleotide according to any one of 73) to 78).

[0182] 80) The functional molecule is a lipid selected from the group consisting of cholesterol, fatty acids, fat-soluble vitamins, glycolipids, and glycerides, the single-stranded oligonucleotide according to any one of 73) to 79). 81) The functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol, and tocotrienol, the single-stranded oligonucleotide according to any one of 73) to 80). 82) The functional molecule is tocopherol, and the hydroxy group of tocopherol is C 2-20A single-stranded oligonucleotide according to any one of 73) to 75), which is bound to a nucleotide at the 5'-end or 3'-end of a single-stranded oligonucleotide represented by formula (I) via an alkylene group (in the alkylene group, each methylene group is independently unsubstituted or substituted by -O-. Each unsubstituted methylene group is independently unsubstituted or substituted by a hydroxy group). 83) The hydroxy group of tocopherol is represented by the following formula (III)

Chemical formula

[0183] B-1) Formula:

Chemical formula

[0184] B-2) The formula

Chemical formula

[0185] B-5) Xa 2 is a group derived from the oligonucleotide Xa 2 consisting of two or three deoxyribonucleotides, the single-stranded oligonucleotide according to any one of B-1) to B-4).

[0186] B-6) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, and 2'-O-methylcarbamoylethylated nucleotide, the single-stranded oligonucleotide according to any one of B-1) to B-5). B-7) Xb1 and Xa 2 The single-stranded oligonucleotide according to any one of B-1) to B-6), wherein the sugar-modified nucleotide contained in 2 is LNA. B-8) Y 1 The single-stranded oligonucleotide according to any one of B-1) to B-7), wherein the sugar-modified nucleotide contained in 1 is a 2'-O-methylated nucleotide.

[0187] B-9) Xb 1 , Xb 2 , Xa 1 , Xa 2 and Y 1 The nucleotides contained in 1 are linked to each other by phosphorothioate bonds, and the nucleotides contained in Y 2 2 are linked to each other by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-1) to B-8), represented by B-10) Xb 1 and Xb 2 , Xb 2 and Xa 1 , Xa 1 and Xa 2 and Y 2 and Y 1 The respective terminal nucleotides of 2 and Y 2 2 are linked to each other by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-1) to B-9), wherein the respective terminal nucleotides of Xa

[0188] In the above B-1) to B-10), the oligonucleotide Xb is represented by Xb 1 -Xb 2 , the oligonucleotide Xa is represented by Xa 2 -Xa 1 , and the oligonucleotide Y is represented by Y 2 -Y 1 1 .

[0189] B-11) Formula

Chemical formula

[0190] B-12) The formula [Chemical formula] (In the formula, Xb 1 is a group derived from an oligonucleotide Xb consisting of 4 or 5 sugar-modified nucleotides 1 ; Xb 2 is a group derived from an oligonucleotide Xb consisting of 8 to 10 deoxyribonucleotides 2 ; Xa consists of 4 or 5 sugar-modified nucleotides, Y 2 is a group derived from an oligonucleotide Y consisting of 8 to 10 ribonucleotides 2 ; Y 1 is a group derived from an oligonucleotide Y consisting of 4 or 5 sugar-modified nucleotides 1 ; B is C 2-20 alkylene group or C 2-20An alkenylene group (the methylene groups contained in the alkylene group and the alkenylene group are each independently unsubstituted or substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a protected hydroxy group, an oxo group, and a thioxo group. Further, the methylene groups of the alkylene group and the alkenylene group are each independently unsubstituted or replaced by -O-, -NR B -(R B is a hydrogen atom, C 1-6 alkyl group or halo C 1-6 alkyl group. ), -S-, -S(O)- or -S(O) 2 - is replaced by. ), and A is a group derived from a functional molecule. ) represented by the single-stranded oligonucleotide according to 73). B-13) B is C 2-20 an alkylene group (the methylene groups of the alkylene group are each independently unsubstituted or replaced by -O-. The unsubstituted methylene groups are each independently unsubstituted or substituted by a hydroxy group. ), and A is a group derived from tocopherol, the single-stranded oligonucleotide according to B-12). B-14) B is linked to the terminal nucleotide of Y 1 by a phosphodiester bond, the single-stranded oligonucleotide according to B-12) or B-13).

[0191] B-15) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, and 2'-O-methylcarbamoylethylated nucleotide, the single-stranded oligonucleotide according to any one of B-11) to B-14). B-16) Xb 1 and the sugar-modified nucleotides contained in Xa are each independently LNA or 2'-O-methoxyethylated nucleotide, the single-stranded oligonucleotide according to any one of B-11) to B-15). B-17) Y 1The single-stranded oligonucleotide according to any one of B-11) to B-16), wherein the sugar-modified nucleotide contained therein is a 2'-O-methylated nucleotide.

[0192] B-18) Xb 1 , Xb 2 , Xa and Y 1 The nucleotides contained in are linked to each other by phosphorothioate bonds, and Y 2 The nucleotides contained in are linked to each other by phosphodiester bonds. ) The single-stranded oligonucleotide according to any one of B-11) to B-17). B-19) Xb 1 and Xb 2 , Xb 2 and Xa and Y 2 and Y 1 The respective terminal nucleotides of are linked by phosphorothioate bonds, and the respective terminal nucleotides of Xa and Y 2 are linked by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-11) to B-18).

[0193] In the above B-11) to B-19), the oligonucleotide Xb is Xb 1 -Xb 2 represented by, and the oligonucleotide Y is Y 2 -Y 1 represented by.

[0194] B-20) Formula

Chemical formula

[0195] B-21) Formula

Chemical formula

[0196] B-24) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, and 2'-O-methylcarbamoylethylated nucleotide, the single-stranded oligonucleotide according to any one of B-20) to B-23). B-25) Xb 1 , Xb 3 and the sugar-modified nucleotides contained in Xa are each independently LNA or 2'-O-methoxyethylated nucleotide, the single-stranded oligonucleotide according to any one of B-20) to B-24). B-26) Y1 The single-stranded oligonucleotide according to any one of B-20) to B-25), wherein the sugar moiety-modified nucleotide contained therein is a 2'-O-methylated nucleotide.

[0197] B-27) Xb 1 , Xb 2 , Xb 3 , Xa and Y 1 The nucleotides contained in are linked to each other by phosphorothioate bonds, and Y 2 The nucleotides contained in are linked to each other by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-20) to B-26), represented by). B-28) Xb 1 and Xb 2 , Xb 2 and Xb 3 , Xb 3 and Xa and Y 2 and Y 1 The respective terminal nucleotides of are linked by phosphorothioate bonds, and the respective terminal nucleotides of Xa and Y 2 are linked by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-20) to B-27).

[0198] In the above B-20) to B-28), the oligonucleotide Xb is Xb 1 -Xb 2 -Xb 3 represented by, and the oligonucleotide Y is Y 2 -Y 1 represented by.

[0199] B-29) Formula

Chemical formula

[0200] B-32) Xa 2is an oligonucleotide Xa consisting of 2 or 3 deoxyribonucleotides 2 is a single-stranded oligonucleotide according to any one of B-29) to B-31), which is a group derived from

[0201] B-33) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotides, 2'-O-methoxyethylated nucleotides, and 2'-O-methylcarbamoylethylated nucleotides, and are single-stranded oligonucleotides according to any one of B-29) to B-32). B-34) Xb 1 and Xa 2 The sugar-modified nucleotides contained in are LNA, 2'-O-methoxyethylated nucleotides, or 2'-O-methylcarbamoylethylated nucleotides, and are single-stranded oligonucleotides according to any one of B-29) to B-33).

[0202] B-35) Xb 1 , Xb 2 , Xa 1 and Xa 2 The nucleotides contained in are linked to each other by phosphorothioate bonds, and Y 0 The nucleotides contained in are linked to each other by phosphodiester bonds. The single-stranded oligonucleotide according to any one of B-29) to B-34) represented by B-36) Xb 1 and Xb 2 , Xb 2 and Xa 1 and Xa 1 and Xa 2 The terminal nucleotides of each are linked by phosphorothioate bonds, and Xa 2 and Y 0 The terminal nucleotides of each are linked by phosphodiester bonds, and are single-stranded oligonucleotides according to any one of B-29) to B-35).

[0203] In the above B-29) to B-36), the oligonucleotide Xb is Xb 1-Xb 2 represented by, and the oligonucleotide Xa is Xa 2 -Xa 1 represented by, and the oligonucleotide Y is Y 0 represented by.

[0204] B-37) The formula

Chemical formula

[0205] B-40) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, and 2'-O-methylcarbamoylethylated nucleotide, the single-stranded oligonucleotide described in any one of B-37) to B-39). B-41) Xb 1 The sugar-modified nucleotides contained in Xb and Xa are each independently LNA, 2'-O-methoxyethylated nucleotide, or 2'-O-methylcarbamoylethylated nucleotide, the single-stranded oligonucleotide described in any one of B-37) to B-40).

[0206] B-42) Xb 1 , Xb 2 and the nucleotides contained in Xa are linked to each other by phosphorothioate bonds, and the nucleotides contained in Y 0 are linked to each other by phosphodiester bonds. The single-stranded oligonucleotide described in any one of B-37) to B-41). B-43) Xb 1 and Xb 2 and the terminal nucleotides of Xb 2 and Xa are linked by phosphorothioate bonds, and the terminal nucleotides of Xa and Y 0 are linked by phosphodiester bonds. The single-stranded oligonucleotide described in any one of B-37) to B-42).

[0207] In the above B-37) to B-43), the oligonucleotide Xb is Xb 1 -Xb 2 represented by, and the oligonucleotide Y is Y 0 represented by.

[0208] B-44) Formula

Chemical formula

[0209] B-47) The sugar-modified nucleotides are each independently selected from the group consisting of LNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, and 2'-O-methylcarbamoylethylated nucleotide. The single-stranded oligonucleotide according to any one of B-44) to B-46). B-48) Xb 1 , Xb 3 and the sugar-modified nucleotides contained in Xa are each independently LNA, 2'-O-methoxyethylated nucleotide or 2'-O-methylcarbamoylethylated nucleotide. The single-stranded oligonucleotide according to any one of B-44) to B-47). B-49) Xb 1 , Xb 3 and the sugar-modified nucleotides contained in Xa are 2'-O-methylcarbamoylethylated nucleotides. The single-stranded oligonucleotide according to any one of B-44) to B-47).

[0210] B-50) Xb 1 , Xb 2 , Xb 3 , and the nucleotides contained in Xa are linked to each other by phosphorothioate bonds, and Y 0The nucleotides contained therein are linked to each other by phosphodiester bonds. A single-stranded oligonucleotide according to any one of B-44) to B-48), represented by B-51) Xb 1 and Xb 2 , Xb 2 and Xb 3 and Xb 3 The respective terminal nucleotides of Xa are linked by phosphorothioate bonds, and the respective terminal nucleotides of Xa and Y 0 are linked by phosphodiester bonds. A single-stranded oligonucleotide according to any one of B-44) to B-50).

[0211] In the above B-44) to B-51), the oligonucleotide Xb is represented by Xb 1 -Xb 2 -Xb 3 and the oligonucleotide Y is represented by Y 0 as represented.

[0212] B-52) A single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-51), wherein the base moiety in deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides is at least one selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U) and 5-methylcytosine (5-me-C).

[0213] D-1) A medicament containing, as an active ingredient, a single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52).

[0214] Xb 1 -Xb 2 -Xa 2 -Xa 1 The portion represented by is an antisense sequence portion, and the portion represented by Xb 1 -Xb 2 and Y 2 -Y 1The conceptual diagram of the single-stranded oligonucleotide described in B-1), in which the part represented by hybridizes intramolecularly, is shown in Figure 1. In the single-stranded oligonucleotide shown in Figure 1, Xb consisting of 2 or 3 sugar-modified nucleotides 1 and Xb consisting of 6 to 8 deoxyribonucleotides 2 and Xa consisting of 1 to 3 deoxyribonucleotides 2 and Xa consisting of 2 or 3 sugar-modified nucleotides 1 and Y consisting of 6 to 8 ribonucleotides 2 and Y consisting of 2 or 3 sugar-modified nucleotides 1 are linked in this order. The binding direction from Xb 1 to Y 1 may be either in the 5' to 3' direction or in the 3' to 5' direction. In Figure 1, Xb 2 and Y 2 form a double strand. Xb 1 and Y 1 form a double strand.

[0215] Xb 1 -Xb 2 -Xa 2 -Xa 1 The part represented by is the antisense sequence part, and the part represented by Xb 1 -Xb 2 and the part represented by Y 2 -Y 1 hybridize intramolecularly. The conceptual diagram of the single-stranded oligonucleotide described in B-2) is shown in Figure 2. In the single-stranded oligonucleotide shown in Figure 2, Xb consisting of 2 or 3 sugar-modified nucleotides 1 and Xb consisting of 6 to 8 deoxyribonucleotides 2 and Xa consisting of 1 to 3 deoxyribonucleotides 2 and Xa consisting of 2 or 3 sugar-modified nucleotides 1 and Y consisting of 6 to 8 ribonucleotides 2 and Y consisting of 2 or 3 sugar-modified nucleotides 1 and C 2-20B, which is an alkylene group or the like, and A, which is a group derived from a functional molecule, are bonded in this order. Xb 1 to Y 1 The bonding direction from Xb 2 to Y 2 may be in the 5' to 3' direction or the 3' to 5' direction. In Figure 2, Xb 1 and Y 1 form a double strand. Xb

[0216] Xb 1 -Xb 2 -Xa represents an antisense sequence portion, and the portion represented by Xb 1 -Xb 2 and the portion represented by Y 2 -Y 1 hybridize within the molecule. A conceptual diagram of the single-stranded oligonucleotide described in B-11) is shown in Figure 3. In the single-stranded oligonucleotide shown in Figure 3, Xb 1 consisting of 4 or 5 sugar-modified nucleotides, Xb 2 consisting of 8 to 10 deoxyribonucleotides, Xa consisting of 4 or 5 sugar-modified nucleotides, Y 2 consisting of 8 to 10 ribonucleotides, and Y 1 consisting of 4 or 5 sugar-modified nucleotides are bonded in this order. Xb 1 to Y 1 The bonding direction may be in the 5' to 3' direction or the 3' to 5' direction. In Figure 3, Xb 2 and Y 2 form a double strand. Xb 1 and Y 1 form a double strand.

[0217] Xb 1 -Xb 2 -Xa represents an antisense sequence portion, and the portion represented by Xb 1 -Xb 2 and the portion represented by Y 2 -Y 1The conceptual diagram of the single-stranded oligonucleotide described in B-12), in which the part represented by hybridizes intramolecularly, is shown in Figure 4. In the single-stranded oligonucleotide shown in Figure 4, Xb consists of 4 or 5 sugar-modified nucleotides 1 and Xb consists of 8 to 10 deoxyribonucleotides 2 and Xa consists of 4 or 5 sugar-modified nucleotides and Y consists of 8 to 10 ribonucleotides 2 and Y consists of 4 or 5 sugar-modified nucleotides 1 and C 2-20 B, which is an alkylene group or the like, and A, which is a group derived from a functional molecule, are bonded in this order. The bonding direction from Xb 1 to Y 1 may be either in the 5'-to-3' direction or in the 3'-to-5' direction. In Figure 4, Xb 2 and Y 2 form a double strand. Xb 1 and Y 1 form a double strand.

[0218] Xb 1 -Xb 2 -Xb 3 -Xa represents the antisense sequence part, and the part represented by Xb 1 -Xb 2 -Xb 3 and the part represented by Y 2 -Y 1 hybridize intramolecularly. The conceptual diagram of the single-stranded oligonucleotide described in B-20) is shown in Figure 5. In the single-stranded oligonucleotide shown in Figure 5, Xb consists of 4 to 6 sugar-modified nucleotides 1 and Xb consists of 8 to 10 deoxyribonucleotides 2 and Xb consists of 1 or 2 sugar-modified nucleotides 3 and Xa consists of 3 or 4 sugar-modified nucleotides and Y consists of 9 to 12 ribonucleotides 2 and Y consists of 4 to 6 sugar-modified nucleotides 1 are bonded in this order. The bonding direction from Xb 1 to Y 1The binding direction to [it] may be either in the 5'-to-3' direction or in the 3'-to-5' direction. In Figure 5, Xb 2 and Y 2 form a double strand. Xb 1 and Y 1 form a double strand.

[0219] Xb 1 -Xb 2 -Xb 3 -Xa represents an antisense sequence portion, and the portion represented by Xb 1 -Xb 2 -Xb 3 and the portion represented by Y 2 -Y 1 hybridize intramolecularly. The conceptual diagram of the single-stranded oligonucleotide described in B-21) is shown in Figure 6. In the single-stranded oligonucleotide shown in Figure 6, Xb 1 consisting of 4 to 6 sugar-modified nucleotides, Xb 2 consisting of 8 to 10 deoxyribonucleotides, Xb 3 consisting of 1 or 2 sugar-modified nucleotides, Xa consisting of 3 or 4 sugar-modified nucleotides, Y 2 consisting of 9 to 12 ribonucleotides, Y 1 consisting of 4 to 6 sugar-modified nucleotides, C 2-20 B which is an alkylene group or the like, and A which is a group derived from a functional molecule are bonded in this order. The binding direction from Xb 1 to Y 1 may be either in the 5'-to-3' direction or in the 3'-to-5' direction. In Figure 6, Xb 2 and Y 2 form a double strand. Xb 1 and Y 1 form a double strand.

[0220] Xb 1 -Xb 2 -Xa 2 -Xa 1 represents an antisense sequence portion, and the portion represented by Xb 1 -Xb 2 and Y 0The conceptual diagram of the single-stranded oligonucleotide described in B-29), in which the part represented by hybridizes intramolecularly, is shown in Fig. 7. In the single-stranded oligonucleotide shown in Fig. 7, Xb consists of 2 or 3 sugar-modified nucleotides 1 and Xb consists of 6 to 8 deoxyribonucleotides 2 and Xa consists of 1 to 3 deoxyribonucleotides 2 and Xa consists of 2 or 3 sugar-modified nucleotides 1 and Y consists of 8 to 11 ribonucleotides 0 and C 2-20 An alkylene group or the like, B, and a group derived from a functional molecule, A, are bonded in this order. The bonding direction from Xb 1 to Y 0 may be either in the 5' to 3' direction or in the 3' to 5' direction. In Fig. 7, Xb 2 and Y 0 form a double strand. Xb 1 and Y 0 form a double strand.

[0221] Xb 1 -Xb 2 -Xa represents an antisense sequence part, and the part represented by Xb 1 -Xb 2 and the part represented by Y 0 hybridize intramolecularly. The conceptual diagram of the single-stranded oligonucleotide described in B-37) is shown in Fig. 8. In the single-stranded oligonucleotide shown in Fig. 8, Xb consists of 4 or 5 sugar-modified nucleotides 1 and Xb consists of 8 to 10 deoxyribonucleotides 2 and Xa consists of 4 or 5 sugar-modified nucleotides, and Y consists of 12 to 15 ribonucleotides 0 and C 2-20 An alkylene group or the like, B, and a group derived from a functional molecule, A, are bonded in this order. The bonding direction from Xb 1 to Y 0 may be either in the 5' to 3' direction or in the 3' to 5' direction. In Fig. 8, Xb 2 and Y 0forms a double-strand with Xb 1 and Y 0 forms a double-strand with

[0222] Xb 1 -Xb 2 -Xb 3 The part represented by -Xa is an antisense sequence part, and Xb 1 -Xb 2 -Xb 3 The part represented by and Y 0 The part represented by hybridize intramolecularly. The conceptual diagram of the single-stranded oligonucleotide described in B-44) is shown in Fig. 9. In the single-stranded oligonucleotide shown in Fig. 9, Xb consisting of 4 to 6 sugar-modified nucleotides 1 and Xb consisting of 8 to 10 deoxyribonucleotides 2 and Xb consisting of 1 or 2 sugar-modified nucleotides 3 and Xa consisting of 3 or 4 sugar-modified nucleotides and Y consisting of 13 to 18 ribonucleotides 0 and C 2-20 B which is an alkylene group or the like and A which is a group derived from a functional molecule are bonded in this order. Xb 1 From Xb to Y 0 The bonding direction may be from the 5' to 3' direction or from the 3' to 5' direction. In Fig. 9, Xb 2 and Y 0 form a double-strand. Xb 1 and Y 0 form a double-strand with

[0223] Preferred methods of using the single-stranded oligonucleotide of the present invention include those shown below. E-1) A method for controlling the function of a target RNA, comprising the step of contacting a cell with the single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52). E-2) A method for controlling the function of a target RNA in a mammal, comprising the step of administering to the mammal a pharmaceutical composition containing the single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52). The method according to E-2), wherein the mammal is a human. The method according to E-2) or E-3), wherein the administration route is enteral. The method according to E-2) or E-3), wherein the administration route is parenteral. Use of a single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52) for controlling the function of a target RNA in a mammal. Use of a single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52) for manufacturing a medicament for controlling the function of a target RNA in a mammal. The use according to E-6) or E-7), wherein the mammal is a human.

[0224] The control of the function of the target RNA in the present invention means that the antisense sequence portion covers a part of the target RNA by hybridization, resulting in inhibition of translation or regulation or conversion of splicing functions such as exon skipping, or degradation of the target RNA that may occur by recognition of the hybridized portion of the antisense sequence portion and a part of the target RNA, thereby suppressing the function of the target RNA.

[0225] A method for controlling the expression of a target gene, comprising the step of contacting a cell with a single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52). A method for controlling the expression of a target gene in a mammal, comprising the step of administering to the mammal a pharmaceutical composition comprising a single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52). The method according to E-10), wherein the mammal is a human. The method according to E-10) or E-11), wherein the administration route is enteral. The method according to E-10) or E-11), wherein the administration route is parenteral. Use of the single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52) for controlling the expression of a target gene in a mammal. E-15) Use of the single-stranded oligonucleotide according to any one of 1) to 85) and B-1) to B-52) for producing a medicament for controlling the expression of a target gene in a mammal. E-16) The use according to E-14) or E-15), wherein the mammal is a human.

[0226] As described above, the preferred embodiments of the single-stranded oligonucleotide have been explained. However, the single-stranded oligonucleotide of the present invention is not limited to the above embodiments. The single-stranded oligonucleotide includes, for example, those that exist via their tautomerism and geometric isomerism, regardless of whether they are in the ring or outside the ring, and also includes those that exist as a mixture thereof or a mixture of each isomer. Further, when an asymmetric center exists, or when an asymmetric center is generated as a result of isomerization, those that exist as each optical isomer and a mixture in any ratio are also included. In the case of a compound having two or more asymmetric centers, diastereomers due to each optical isomer also exist. The present invention includes those containing all these types in any ratio.

[0227] The present invention also includes pharmaceutically acceptable salts of the single-stranded oligonucleotide represented by formula (I). The single-stranded oligonucleotide represented by formula (I) can be converted into a pharmaceutically acceptable salt as necessary, or can be liberated from the generated salt. Examples of pharmaceutically acceptable salts of the single-stranded oligonucleotide represented by formula (I) include salts with alkali metals (such as lithium, sodium, potassium), alkaline earth metals (such as magnesium, calcium), ammonium, organic bases (such as triethylamine, trimethylamine), amino acids (such as glycine, lysine, glutamic acid), inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid), or organic acids (such as acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid). In particular, a partial structure represented by -P(=O)(OH)- is converted into an anionic partial structure represented by -P(=O)(O - )-, and may form a salt with an alkali metal (such as lithium, sodium, potassium, etc.), an alkaline earth metal (such as magnesium, calcium, etc.) or ammonium. Further, a partial structure represented by -P(=O)(SH)- that forms a phosphorothioate bond is converted into an anionic partial structure represented by -P(=O)(S - )-, and similarly, may form a salt with an alkali metal, an alkaline earth metal or ammonium.

[0228] The present invention also includes prodrugs of single-stranded oligonucleotides represented by formula (I). A prodrug is a derivative of a pharmaceutical compound having a group that can be decomposed chemically or metabolically, and is a compound that is decomposed by solvolysis or in vivo under physiological conditions and is induced into a pharmacologically active pharmaceutical compound. Methods for selecting and manufacturing appropriate prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam 1985). In the case of the present invention, when having a hydroxyl group, prodrugs such as acyloxy derivatives produced by reacting the compound with an appropriate acyl halide, an appropriate acid anhydride or an appropriate alkyloxycarbonyl halide compound are exemplified. Particularly preferred structures as prodrugs include -O-COC 2 H 5 , -O-CO(t-Bu), -O-COC 15 H 31 , -O-CO(m-CO 2 Na-Ph), -O-COCH 2 CH 2 CO 2 Na-OCOCH(NH 2 )CH 3 , -O-COCH 2 N(CH 3 ) 2 or -O-CH 2 OC(=O)CH3 Examples include the following. When the single-stranded oligonucleotide forming the present invention has an amino group, a prodrug produced by reacting a compound having an amino group with a suitable acid halide, a suitable mixed acid anhydride, or a suitable alkyloxycarbonyl halide compound is exemplified. Particularly preferred structures as prodrugs include -NH-CO(CH 2 ) 20 OCH 3 , -NH-COCH(NH 2 )CH 3 , -NH-CH 2 OC(=O)CH 3 and the like.

[0229] The single-stranded oligonucleotide represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof can exist in any crystalline form and as any hydrate depending on the production conditions, and these crystalline forms, hydrates, and mixtures thereof are also included in the scope of the present invention. It may also exist as a solvate containing an organic solvent such as acetone, ethanol, 1-propanol, 2-propanol, etc., and all of these forms are included in the scope of the present invention.

[0230] The single-stranded oligonucleotide can be prepared by appropriately selecting a method known to those skilled in the art. For example, those skilled in the art can design the nucleotide sequence of the single-stranded oligonucleotide based on the information of the nucleotide sequence of the target RNA and synthesize it using a commercially available nucleic acid automatic synthesizer (manufactured by Applied Biosystems, Beckman, GeneDesign, etc.). It can also be synthesized by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzyme. That is, the method for producing a single-stranded oligonucleotide according to the present embodiment can include a step of extending the nucleotide chain at the 3'-end or 5'-end of an oligonucleotide containing at least one of X, Y, Xz, Yz, Lx, and Ly (among others, an oligonucleotide containing at least one of X and Y).

[0231] Many methods for binding a functional molecule to the oligonucleotide are well known in the art, and reference can be made to, for example, European Journal of Pharmaceutical and Biopharmaceutics, Vol. 107, pp. 321-340 (2016), Advanced Drug Delivery Reviews, Vol. 104, pp. 78-92 (2016), Expert Opinion on Drug Delivery, Vol. 11, pp. 791-822 (2014), etc. For example, after binding a functional molecule and a linker by a known method, it can be induced into an amidite form by an amidite reagent or into an H-phosphonate form by an H-phosphonate reagent, and then bound to an oligonucleotide.

[0232] A single-stranded oligonucleotide can be prepared by purifying the obtained oligonucleotide by reverse-phase column chromatography or the like. Then, the prepared single-stranded oligonucleotide is mixed in an appropriate buffer, denatured at 90-98 °C for several minutes (for example, 5 minutes), and then hybridized at 30-70 °C for 1-8 hours, whereby a single-stranded oligonucleotide hybridized within the molecule can be prepared. The step of hybridizing within the molecule may be omitted in some cases.

[0233] The single-stranded oligonucleotide can effectively control the expression of a target gene. Therefore, the present invention can provide a composition for controlling the expression of a target gene, for example, by an antisense effect, containing the single-stranded oligonucleotide as an active ingredient. In particular, the single-stranded oligonucleotide can obtain a high medicinal effect by administration at a low concentration, and pharmaceutical compositions for treating, preventing, and improving diseases associated with enhanced expression of a target gene, such as metabolic diseases, tumors, and infectious diseases, can also be provided in some embodiments.

[0234] A composition containing a single-stranded oligonucleotide can be formulated by known pharmaceutical methods. For example, it can be used enterally (such as orally) or parenterally as capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, troches, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, topical applications, ointments, plasters, poultices, transdermal absorption preparations, lotions, inhalants, aerosols, injections, suppositories, etc.

[0235] In these formulations, it can be appropriately combined with a pharmacologically or foodstuff-acceptable carrier, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetening agent, thickening agent, taste and odor correcting agent, solubilizing agent or other additives, etc.

[0236] The dosage form of the composition containing a single-stranded oligonucleotide is not particularly limited, but includes enteral (such as oral) or parenteral administration. More preferably, it includes intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intracerebroventricular administration, nasal administration, intravitreal administration, etc. and administration by infusion.

[0237] The diseases that can be treated, prevented, or improved by nucleic acid drugs using single-stranded oligonucleotides are not particularly limited. Examples include diseases caused by gene expression, such as metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, and rare genetic diseases. More specifically, hypercholesterolemia, hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama type congenital muscular dystrophy, Ullrich type congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, integrin deficiency, Walker-Warburg syndrome, etc.), Becker type muscular dystrophy, limb-girdle muscular dystrophy, Miyoshi type muscular dystrophy, facioscapulohumeral muscular dystrophy, etc.), Huntington's disease, Alzheimer's disease, transthyretin type amyloidosis, familial amyloid cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type 2 diabetes, chronic kidney disease, RS virus infection, Ebola hemorrhagic fever, Marburg fever, HIV, influenza, hepatitis B, hepatitis C, liver cirrhosis, chronic heart failure, myocardial fibrosis, atrial fibrillation, prostate cancer, melanoma, breast cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, cholangiocarcinoma, cervical cancer, liver cancer, lung cancer, leukemia, non-Hodgkin lymphoma, atopic dermatitis, glaucoma, age-related macular degeneration, etc. Depending on the type of the disease, the gene causing the disease can be set as the target gene, and further, according to the sequence of the target gene, the expression control sequence (for example, an antisense sequence) can be appropriately set.

[0238] In addition to primates such as humans, diseases of various other mammals can be treated, prevented, or improved by a composition containing single-stranded oligonucleotides. For example, but not limited thereto, diseases of mammalian species including cows, sheep, goats, horses, dogs, cats, guinea pigs, or other bovine, ovine, equine, canine, feline, rodent species such as mice can be treated. Also, the composition containing single-stranded oligonucleotides can be applied to other species such as birds (for example, chickens).

[0239] When administering or ingesting a composition containing a single-stranded oligonucleotide to an animal including a human, the dosage or intake amount is appropriately selected according to the age, weight, symptoms, health condition, type of the composition (such as pharmaceuticals, food and drink, etc.) of the subject, etc., but the dosage or intake amount is preferably 0.0001 mg / kg / day to 100 mg / kg / day in terms of single-stranded oligonucleotide conversion.

[0240] The single-stranded oligonucleotide can very effectively control the expression of a target gene. Therefore, it is possible to provide a method of administering a single-stranded oligonucleotide to an animal including a human and controlling the expression of the target gene by an antisense effect. Further, it is also possible to provide a method for treating, preventing, and improving various diseases accompanied by enhanced expression of a target gene, etc., including administering a composition containing a single-stranded oligonucleotide to an animal including a human.

Examples

[0241] Hereinafter, the present invention will be described more specifically based on Examples and Comparative Examples, but the embodiments are not limited to the following Examples. In the following Examples and FIGS. 10, 12, and 14, "Example" means an example, "Comparative" means a comparative example, and "control" means a control.

[0242] (Example 1, Comparative Example 1) The oligonucleotides described in Table 1 were prepared using a nucleic acid automatic synthesizer nS-8II (manufactured by Gene Design Inc.). The target gene is mouse scavenger receptor class B member 1 (SRB1). In the sequence listing in Table 1, “(L)” means LNA, “(M)” means 2′-O-methylated nucleotide, small alphabet letters mean deoxyribonucleotides, capital alphabet letters (excluding the alphabet letters with the above-mentioned (L) and (M)) mean ribonucleotides, “^” means phosphorothioate bond, and “5” means that the base of the nucleotide is 5-methylcytosine. “Toc-TEG-” means that a part obtained by removing a hydrogen atom from the hydroxy group of tocopherol represented by the following formula (IV) is bonded via a group represented by the following formula (III-2) [Chemical formula] (In the formula, * represents the bonding position with the oligonucleotide Y, and ** represents the bonding position with tocopherol.), which means that a part obtained by removing a hydrogen atom from the hydroxy group of tocopherol represented by the following formula (IV) is bonded. [Chemical formula]

[0243] [Table 1]

[0244] The intramolecular hybridization in Example 1 and the intermolecular hybridization of two oligonucleotides in Comparative Example 1 were carried out by heating at 95° C. for 5 minutes and then leaving at a constant temperature of 37° C. for 1 hour. The confirmation of hybridization was confirmed by non-denaturing polyacrylamide gel electrophoresis.

[0245] [Evaluation Example 1] C57BL / 6J mice (male, 5 weeks old, Charles River Laboratories Japan, Inc.) were intravenously administered with Example 1 and Comparative Example 1 dissolved in physiological saline (Otsuka Seishoku Chuo, Otsuka Pharmaceutical Factory, Inc.) at a dose of 40 nmol / kg in terms of the amount of antisense oligonucleotide per mouse. As a control, only physiological saline (Otsuka Seishoku Chuo, Otsuka Pharmaceutical Factory, Inc.) was administered. Three days after the administration, blood was collected from the orbital venous plexus, and then liver tissue was collected under isoflurane anesthesia. RNA extraction from the liver was performed using the RNeasy Mini Kit (Qiagen) according to the recommended protocol of Qiagen. cDNA was obtained from the total RNA using PrimeScript RT Master Mix (Takara Bio Inc.). Real-time PCR was performed using the obtained cDNA and TaqMan (registered trademark) Gene Expression ID (Applied Biosystems) on a 7500 Real-Time PCR System (Applied Biosystems) to quantify the mRNA amount of SRB1. In real-time PCR, the mRNA amount of Cyclophilin, a housekeeping gene, was also quantified simultaneously, and the mRNA amount of SRB1 relative to the mRNA amount of Cyclophilin was evaluated as the expression level of SRB1. The results are shown in Fig. 10. The primers used were TaqMan Gene Expression Assay (Applied Biosystems), and the Assay IDs were as follows: For mouse SRB1 quantification: Mm00450234_m1 For mouse cyclophilin quantification: Mm0234230_g1

[0246] As is clear from Fig. 10, it was confirmed that the single-stranded oligonucleotide (Example 1) according to the present invention exhibited an equivalent antisense effect compared to HDO (Comparative Example 1).

[0247] [Evaluation Example 2] The results of non-denaturing polyacrylamide gel electrophoresis before and after the intramolecular hybridization treatment in Example 1 are shown in FIG. 11. As the single-stranded DNA size marker, a single-stranded DNA size marker for electrophoresis manufactured by Gene Design was used. This contains single-stranded DNAs with 15, 20, 30, 40, 50, 60, and 80 nucleotides. As the double-stranded RNA size marker, a double-stranded RNA size marker for electrophoresis manufactured by Gene Design was used. This contains double-stranded RNAs with 17, 21, 25, and 29 base pairs. In FIG. 11, "Lane No." represents the lane number in the electrophoresis test, "Example No." represents the example number, "before" represents before the above hybridization treatment, "after" represents after the above hybridization treatment, "ss-DNA size marker" represents the single-stranded DNA size marker, "ds-RNA size marker" represents the double-stranded RNA size marker, "mer" represents the number of bases, and "bp" represents the number of base pairs.

[0248] As is clear from FIG. 11, it was confirmed that the single-stranded oligonucleotide according to the present invention takes on the structure of intramolecular hybridization without going through a special hybridization step or by simple heating and cooling operations.

[0249] (Example 2, Comparative Example 2) The oligonucleotides described in Table 2 were prepared using a nucleic acid automatic synthesizer nS-8II (manufactured by Gene Design). The target gene is human Phosphatase and Tensin Homolog Deleted from Chromosome 10 (PTEN). In the sequence listing in Table 2, "(m)" means a 2'-O-methoxyethylated nucleotide, "5" means that the base of the nucleotide is 5-methylcytosine, "5(x)" means that the base of the deoxyribonucleotide is 5-methylcytosine, and the other sequence listings are the same as in Table 1. [Table 2]

[0250] In Examples 2 and Comparative Example 3, the intramolecular hybridization was carried out by heating at 95°C for 5 minutes and then leaving it at a constant temperature of 37°C for 1 hour. The confirmation of hybridization was confirmed by non-denaturing polyacrylamide gel electrophoresis.

[0251] [Evaluation Example 3] Cells of the human liver cancer-derived cell line HuH-7 were seeded in a 96-well plate at 3000 cells / well and cultured at 37°C with 5% CO 2 for 24 hours. Each oligonucleotide in Table 2 was added to each well using Lipofectamine (registered trademark) RNAiMax (manufactured by Thermo Fisher Scientific) so that its final concentration was 1 nM (transfection). After 4 hours, the medium was changed, and after another 20 hours, the cells were collected, and total RNA was extracted from the cells using an RNeasy mini kit (manufactured by QIAGEN). cDNA was obtained from the total RNA using PrimeScript RT Master Mix (manufactured by Takara Bio Inc.). Real-time PCR was performed using the obtained cDNA and TaqMan (registered trademark) Gene Expression ID (manufactured by Applied Biosystems) with a 7500 Real-Time PCR System (manufactured by Applied Biosystems) to quantify the mRNA amount of PTEN. In real-time PCR, the mRNA amount of the housekeeping gene GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was also quantified simultaneously, and the mRNA amount of PTEN relative to the mRNA amount of GAPDH was evaluated as the expression level of PTEN, respectively. Cells that did not undergo the transfection operation were used as a control. The results are shown in Figure 12. The primers used were TaqMan Gene Expression Assay (manufactured by Applied Biosystems), and the Assay ID was as follows: For human PTEN quantification: Hs02621230 For human GAPDH quantification: Hs99999905_m1

[0252] As is apparent from FIG. 12, it was confirmed that the single-stranded oligonucleotide according to the present invention (Example 2) exhibits a higher antisense effect as compared with the ASO (Comparative Example 2). Further, it was confirmed that the single-stranded oligonucleotide according to the present invention (Example 2) exhibits an antisense effect equal to or higher than that of the HDO (Comparative Example 3) linked with an oligonucleotide.

[0253] [Evaluation Example 4] The results of non-denaturing polyacrylamide gel electrophoresis before and after the intramolecular hybridization treatment in Example 2 are shown in FIG. 13. As the single-stranded DNA size marker, a single-stranded DNA size marker for electrophoresis manufactured by GeneDesign was used. As the double-stranded RNA size marker, a double-stranded RNA size marker for electrophoresis manufactured by GeneDesign was used. The abbreviations and the like in FIG. 13 are the same as those in FIG. 11.

[0254] As is apparent from FIG. 13, it was confirmed that the single-stranded oligonucleotide according to the present invention takes on the structure of intramolecular hybridization without undergoing a special hybridization process or by simple heating and cooling operations.

[0255] (Example 3, Comparative Examples 4 and 5) The oligonucleotides described in Table 3 were prepared using a nucleic acid automatic synthesizer nS-8II (manufactured by GeneDesign). The target gene is mouse scavenger receptor class B member 1 (SRB1). In the sequence listing in Table 3, “(V)” means 2′-O-methylcarbamoylethylated nucleotide, and the other sequence listings are the same as those in Tables 1 and 2.

Table 3

[0256] In Example 3 and Comparative Example 5, intramolecular hybridization was performed by heating at 95°C for 5 minutes and then leaving it at a constant temperature of 37°C for 1 hour. The confirmation of hybridization was carried out by non-denaturing polyacrylamide gel electrophoresis.

[0257] [Evaluation Example 5] The same evaluation method as in Evaluation Example 1 was used. Each oligonucleotide of Example 3 and Comparative Example 5 in Table 3 was intravenously administered at a dose of 0.7 μmol / kg per mouse in terms of the amount of antisense oligonucleotide, and the oligonucleotide of Comparative Example 4 was intravenously administered at a dose of 1.4 μmol / kg per mouse in terms of the amount of antisense oligonucleotide. As a control, only physiological saline (Otsuka Seishoku Chuzhu, Otsuka Pharmaceutical Factory Co., Ltd.) was administered. Three days after administration, the amount of mRNA of SRB1 relative to the amount of mRNA of Cyclophilin in the liver tissue was evaluated as the expression level of SRB1. The results are shown in Fig. 14.

[0258] As is clear from Fig. 14, it was confirmed that the single-stranded oligonucleotide according to the present invention (Example 3) exhibits a higher antisense effect compared to the ASO (Comparative Example 4). In addition, it was confirmed that the single-stranded oligonucleotide according to the present invention (Example 3) exhibits an antisense effect equal to or higher than that of the HDO (Comparative Example 5) linked with an oligonucleotide.

[0259] [Evaluation Example 6] The results of non-denaturing polyacrylamide gel electrophoresis before and after the intramolecular hybridization treatment in Example 3 are shown in Fig. 15. As a size marker for single-stranded DNA, a single-stranded DNA size marker for electrophoresis manufactured by Gene Design was used. As a size marker for double-stranded RNA, a double-stranded RNA size marker for electrophoresis manufactured by Gene Design was used. The abbreviations in Fig. 15 are the same as those in Fig. 11.

[0260] As is clear from Fig. 15, it was confirmed that the single-stranded oligonucleotide according to the present invention takes an intramolecular hybridization structure without undergoing a special hybridization process or by simple heating and cooling operations.

Industrial Applicability

[0261] By using the single-stranded oligonucleotide of the present invention, it becomes possible to deliver an antisense nucleic acid to a specific organ (cell) with high specificity and efficiency, and to effectively control the function of the target RNA and / or effectively suppress the expression of the target gene by the nucleic acid. In addition, various molecules such as lipids (e.g., tocopherol, cholesterol), sugars (e.g., glucose, sucrose), proteins, peptides, antibodies, etc. can be applied as functional molecules for delivering to a specific organ to the single-stranded oligonucleotide of the present invention. Therefore, the single-stranded oligonucleotide of the present invention can target various organs, tissues, and cells. Furthermore, even if the single-stranded oligonucleotide of the present invention is modified to confer resistance to RNase etc., its antisense effect does not decrease, so it can be used even in the form of enteral administration. Therefore, the single-stranded oligonucleotide of the present invention is excellent in that high drug efficacy can be obtained by administration at a low concentration and side effects can be reduced by suppressing the distribution in organs other than the target of the antisense nucleic acid. Thus, it is useful as a pharmaceutical composition etc. for treating and preventing diseases associated with enhanced function of target RNA and / or enhanced expression of target genes, such as metabolic diseases, tumors, infectious diseases, etc.

Claims

1. Formula (I) 【Chemical 1】 {wherein Y is a group derived from an oligonucleotide Y consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides and containing at least one ribonucleotide, X is a formula 【Chemical Formula 2】 (wherein Xb is a group derived from an oligonucleotide Xb consisting of 4 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides and containing at least one sugar-modified nucleotide, Xa is a group derived from an oligonucleotide Xa consisting of 1 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides and containing at least one sugar-modified nucleotide, and Xa binds to the oligonucleotide Y and the oligonucleotide Xb at both ends thereof), and is a group derived from an oligonucleotide X consisting of 5 to 80 nucleotides, Xz is a group derived from an oligonucleotide Xz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides and containing at least one sugar-modified nucleotide, Yz is a group derived from an oligonucleotide Yz consisting of 5 to 40 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides and containing at least one sugar-modified nucleotide, Lx is a group derived from an oligonucleotide Lx consisting of 0 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and binds to the Xb, Ly is a group derived from an oligonucleotide Ly consisting of 0 to 20 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, m is 0 or 1, when m is 0, n is 0 or 1, when m is 1, n is 0, The oligonucleotide X has a nucleotide sequence X, the oligonucleotide Xa has a nucleotide sequence Xa, the oligonucleotide Xb has a nucleotide sequence Xb, the oligonucleotide Y has a nucleotide sequence Y, the oligonucleotide Xz has a nucleotide sequence Xz, the oligonucleotide Yz has a nucleotide sequence Yz, the oligonucleotide Lx has a nucleotide sequence Lx, and the oligonucleotide Ly has a nucleotide sequence Ly. The nucleotide sequence Xb is complementary to the nucleotide sequence Y. The nucleotide sequence X contains an antisense sequence that enables hybridization with the target RNA. Xa is a part of the antisense sequence portion contained in X, and Xb is a part of the antisense sequence portion contained in X. When m is 1 and n is 0, the nucleotide sequence Xz contains an antisense sequence that enables hybridization with the target RNA. When m is 0 and n is 1, the nucleotide sequence Yz contains an antisense sequence that enables hybridization with the target RNA. When having two or more of the antisense sequences, the target RNAs with which the respective antisense sequence portions hybridize may be the same or different. At least one antisense sequence consists of nucleotides independently selected from deoxyribonucleotides and sugar-modified nucleotides, contains at least one sugar-modified nucleotide, does not contain four consecutive deoxyribonucleotides, and the content rate of the sugar-modified nucleotides is 25% or more. A single-stranded oligonucleotide represented by the formula, in which Xb and Y hybridize.

2. The single-stranded oligonucleotide according to claim 1, wherein Xb binds to Xa on the 3'-side and Y binds to Xa on the 5'-side.

3. The single-stranded oligonucleotide according to claim 1, wherein Xb binds to Xa on the 5'-side and Y binds to Xa on the 3'-side.

4. The antisense sequence independently contains a sequence of at least four consecutive nucleotides recognized by RNaseH, or The single-stranded oligonucleotide according to any one of claims 1 to 3, which contains at least one sugar-modified nucleotide and does not contain four consecutive deoxyribonucleotides.

5. At least one antisense sequence is a sequence containing at least 4 consecutive nucleotides recognized by RNaseH, and the antisense sequence portion is adjacent to the 5' side and the 3' side of the sequence portion containing at least 4 consecutive nucleotides recognized by the RNaseH and contains sugar moiety-modified nucleotides linked thereto. The single-stranded oligonucleotide according to claim 4.

6. The single-stranded oligonucleotide according to any one of claims 1 to 5, wherein the antisense sequence portion contains a phosphorothioate bond.

7. The single-stranded oligonucleotide according to any one of claims 1 to 6, wherein the antisense sequence is a sequence consisting of 10 to 30 nucleotides containing at least 1 deoxyribonucleotide.

8. The single-stranded oligonucleotide according to any one of claims 1 to 7, wherein the nucleotide sequence Y is a sequence containing at least 4 consecutive nucleotides cleaved by RNaseH.

9. The single-stranded oligonucleotide according to any one of claims 1 to 8, wherein the oligonucleotide Y contains one or more sugar moiety-modified nucleotides on at least one of the 5' side and the 3' side of the oligonucleotide Y.

10. The single-stranded oligonucleotide according to any one of claims 1 to 9, wherein m is 0 and n is 0.

11. The single-stranded oligonucleotide according to any one of claims 1 to 9, wherein m is 0 and n is 1.

12. The single-stranded oligonucleotide according to claim 11, wherein the nucleotides contained in the oligonucleotide Ly are linked to each other by phosphodiester bonds.

13. The single-stranded oligonucleotide according to claim 11 or 12, wherein the oligonucleotide Ly is DNA or RNA.

14. The single-stranded oligonucleotide according to any one of claims 1 to 9, wherein m is 1 and n is 0.

15. The single-stranded oligonucleotide according to claim 14, wherein the nucleotides contained in the oligonucleotide Lx are linked to each other by phosphodiester bonds.

16. The single-stranded oligonucleotide according to any one of claims 14 or 15, wherein the oligonucleotide Lx is DNA or RNA.

17. The single-stranded oligonucleotide according to any one of claims 1 to 16, wherein the sugar-modified nucleotide is at least one selected from the group consisting of hexitol nucleotide, cyclohexene nucleotide, peptide nucleic acid, glycol nucleic acid, threonucleotide, morpholino nucleic acid, tricyclo-DNA, 2'-O-methylated nucleotide, 2'-O-methoxyethylated nucleotide, 2'-O-aminopropylated nucleotide, 2'-fluorinated nucleotide, 2'-F-arabinonucleotide, cross-linked nucleotide, and 2'-O-methylcarbamoylethylated nucleotide.

18. The single-stranded oligonucleotide according to any one of claims 1 to 17, further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site.

19. The single-stranded oligonucleotide according to claim 18, wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof.

20. The single-stranded oligonucleotide according to claim 18 or 19, wherein the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol, and tocotrienol.

21. The single-stranded oligonucleotide according to claim 18 or 19, wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor.

22. The single-stranded oligonucleotide according to claim 18 or 19, wherein the functional molecule is a peptide or protein selected from the group consisting of a ligand of a receptor and an antibody.

23. A pharmaceutical composition comprising the single-stranded oligonucleotide according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

24. A method for controlling the function of a target RNA, comprising the step of contacting the single-stranded oligonucleotide according to any one of claims 1 to 22 with a cell (excluding human cells).

25. A method for controlling the function of a target RNA in a mammal (excluding humans), comprising the step of administering a pharmaceutical composition comprising the single-stranded oligonucleotide according to any one of claims 1 to 22 to the mammal.

26. A method for controlling the expression of a target gene, comprising the step of contacting the single-stranded oligonucleotide according to any one of claims 1 to 22 with a cell (excluding human cells).

27. A method for controlling the expression of a target gene in a mammal (excluding humans), comprising the step of administering to the mammal a pharmaceutical composition comprising the single-stranded oligonucleotide according to any one of claims 1 to 22.

28. A method for producing the single-stranded oligonucleotide according to any one of claims 1 to 22, comprising the step of extending a nucleotide chain at the 3'-end or 5'-end of an oligonucleotide comprising at least one of X and Y.

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