Single-stranded oligonucleotide

Abstract

Provided is a single-stranded oligonucleotide that is capable of controlling a target gene with high efficiency and can be easily produced. The single-stranded oligonucleotide is represented by the formula X-L-Y wherein X and Y hybridize by a first nucleotide sequence portion and a second nucleotide sequence portion. X is composed of 7 to 100 nucleotides, contains at least one modified nucleotide, and has a first nucleotide sequence that is capable of hybridizing with a second oligonucleotide and contains at least four contiguous nucleotides recognized by RNase H. Y is composed of 4 to 100 nucleotides, and has a second nucleotide sequence that is capable of hybridizing with a second oligonucleotide and contains at least one ribonucleotide. At least one of nucleotide sequence X and nucleotide sequence Y has an antisense sequence capable of hybridizing with a target RNA. L is a group derived from a third oligonucleotide that is degraded under physiological conditions.

1 . A single-stranded oligonucleotide represented by the formula:
   X-L-Y   wherein   X represents a group derived from a first oligonucleotide composed of 7 to 100 nucleotides that are independently selected from a deoxyribonucleotide, ribonucleotide and sugar-modified nucleotide and that contains at least one nucleotide of which at least one of a sugar moiety, base moiety and phosphate moiety has been modified,   Y represents a group derived from a second oligonucleotide composed of 4 to 100 nucleotides that are independently selected from a deoxyribonucleotide, ribonucleotide and sugar-modified nucleotide, and   L represents a group derived from a third oligonucleotide that respectively covalently bonds with the first oligonucleotide and the second oligonucleotide at both ends thereof and is degraded under physiological conditions; wherein,   the first oligonucleotide has a nucleotide sequence X, and the second oligonucleotide has a nucleotide sequence Y,   the nucleotide sequence X contains a first nucleotide sequence that is capable of hybridizing with at least a portion of the second oligonucleotide and contains at least four contiguous nucleotides recognized by RNase H,   the nucleotide sequence Y contains a second nucleotide sequence that is capable of hybridizing with at least a portion of the first oligonucleotide and contains at least one ribonucleotide,   the nucleotide sequence Y contains at least one antisense sequence capable of hybridizing with a target RNA, and   in the case of having two or more antisense sequences, the target RNA hybridized by each antisense sequence may be the same or different, wherein X and Y hybridize by the first nucleotide sequence portion and the second nucleotide sequence portion.   
     
     
         2 . The single-stranded oligonucleotide according to  claim 1 , wherein Y has the second nucleotide sequence portion between the antisense sequence portion and L. 
     
     
         3 . The single-stranded oligonucleotide according to  claim 1 , wherein X bonds to L on the 3′-side and Y bonds to L on the 5′-side. 
     
     
         4 . The single-stranded oligonucleotide according to  claim 1 , wherein X bonds to L on the 5′-side and Y bonds to L on the 3′-side. 
     
     
         5 . The single-stranded oligonucleotide according to  claim 1 , wherein nucleotides contained in the third oligonucleotide are mutually coupled through phosphodiester bonds. 
     
     
         6 . The single-stranded oligonucleotide according to  claim 1 , wherein the third oligonucleotide is DNA or RNA. 
     
     
         7 . The single-stranded oligonucleotide according to  claim 1 , wherein the first oligonucleotide contains a sugar-modified nucleotide bound adjacent to at least one of the 5′-side and 3′-side of the first nucleotide sequence portion. 
     
     
         8 . The single-stranded oligonucleotide according to  claim 1 , wherein the first oligonucleotide contains a sugar-modified nucleotide bound adjacent to the 5′-side and 3′-side of the first nucleotide sequence portion. 
     
     
         9 . The single-stranded oligonucleotide according to  claim 1 , wherein the first oligonucleotide contains a phosphorothioate bond. 
     
     
         10 . The single-stranded oligonucleotide according to  claim 1 , wherein the first nucleotide sequence is composed of 4 to 20 nucleotides including at least one deoxyribonucleotide. 
     
     
         11 . The single-stranded oligonucleotide according to  claim 1 , wherein the second nucleotide sequence is a sequence that contains at least four contiguous nucleotides cleaved by RNase H. 
     
     
         12 . The single-stranded oligonucleotide according to  claim 11 , wherein the second oligonucleotide contains a sugar-modified nucleotide bound adjacent to at least one of the 5′-side and 3′-side of the second nucleotide sequence portion. 
     
     
         13 . The single-stranded oligonucleotide according to  claim 11 , wherein at least one of the 5′-side and 3′-side of the second nucleotide sequence portion is coupled to an adjacent nucleotide through a phosphorothioate bond. 
     
     
         14 . The single-stranded oligonucleotide according to  claim 1 , wherein the antisense sequence contained by the nucleotide sequence Y is a sequence that contains at least four contiguous nucleotides recognized by RNase H when hybridizing with a target RNA. 
     
     
         15 . The single-stranded oligonucleotide according to  claim 1 , wherein the antisense sequence portion contained by the nucleotide sequence Y contains at least one sugar-modified nucleotide but does not contain an oligonucleotide strand composed of four contiguous deoxyribonucleotides. 
     
     
         16 . The single-stranded oligonucleotide according to  claim 14 , wherein the nucleotide sequence X contains a sequence that is able to hybridize with at least a portion of the antisense sequence portion contained by the nucleotide sequence Y and contains at least four contiguous nucleotides cleaved by RNase H. 
     
     
         17 . The single-stranded oligonucleotide according to  claim 1 , wherein nucleotide sequence X contains the 5′-end or 3′-end. 
     
     
         18 . The single-stranded oligonucleotide according to  claim 1 , further containing a group represented by the formula:
   X′-L′-
   wherein   X is a group derived from a fourth oligonucleotide composed of 7 to 100 nucleotides that are independently selected from a deoxyribonucleotide, ribonucleotide and sugar-modified nucleotide and that contains at least one nucleotide of which at least one of the sugar moiety, base moiety and phosphate moiety has been modified, and   L′ represents a group derived from a fifth oligonucleotide that respectively covalently bonds with the first oligonucleotide and the fourth oligonucleotide at both ends thereof and is degraded under physiological conditions; wherein,   the fourth oligonucleotide has an antisense sequence capable of hybridizing with a target RNA.   
     
     
         19 . The single-stranded oligonucleotide according to  claim 18 , wherein
 X′ contains at least one sugar-modified nucleotide, and   the antisense sequence possessed by the fourth oligonucleotide contains at least four contiguous nucleotides recognized by RNase H when hybridizing with a target RNA.   
     
     
         20 . The single-stranded oligonucleotide according to  claim 18 , wherein the antisense sequence portion contained by the fourth oligonucleotide contains at least one sugar-modified nucleotide, but does not contain an oligonucleotide strand composed of four contiguous deoxyribonucleotides. 
     
     
         21 . The single-stranded oligonucleotide according to  claim 19 , wherein the antisense sequence portion contained by the fourth oligonucleotide hybridizes with at least a portion of the second oligonucleotide. 
     
     
         22 . The single-stranded oligonucleotide according to  claim 18 , wherein nucleotides contained in the fifth oligonucleotide are mutually coupled through phosphodiester bonds. 
     
     
         23 . The single-stranded oligonucleotide according to  claim 18 , wherein the fifth oligonucleotide is DNA or RNA. 
     
     
         24 . The single-stranded oligonucleotide according to  claim 1 , containing a sugar-modified nucleotide bound adjacent to at least one of the 5′-side and 3′-side of the antisense sequence portion. 
     
     
         25 . The single-stranded oligonucleotide according to  claim 1 , containing sugar-modified nucleotides bound adjacent to the 5′-side and 3′-side of the antisense sequence portion. 
     
     
         26 . The single-stranded oligonucleotide according to  claim 1 , wherein the antisense sequence is composed of 4 to 20 nucleotides including at least one deoxyribonucleotide. 
     
     
         27 . The single-stranded oligonucleotide according to  claim 1 , wherein the antisense sequence portion contains a phosphorothioate bond. 
     
     
         28 . The single-stranded oligonucleotide according to  claim 1 , wherein the sugar-modified nucleotide is at least one selected from the group consisting of hexitol nucleotides, cyclohexene nucleotides, peptide nucleic acids, glycol nucleic acids, threose nucleotides, morpholine nucleic acids, tricyclo-DNA, 2′-O-methyl nucleotides, 2′-O-methoxyethyl nucleotides, 2′-O-aminopropyl nucleotide, 2′-fluoronucleotide, 2′-F-arabinonucleotides, bridged nucleotides and 2′-O-methylcarbamoylethyl nucleotides. 
     
     
         29 . The single-stranded oligonucleotide according to  claim 1 , wherein nucleotide sequence Y contains the 5′-end or 3′-end. 
     
     
         30 . The single-stranded oligonucleotide according  claim 1 , further containing a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purifying function and a target site delivery function. 
     
     
         31 . The single-stranded oligonucleotide according to  claim 30 , wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins and derivatives thereof. 
     
     
         32 . The single-stranded oligonucleotide according to  claim 30 , wherein the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol and tocotrienol. 
     
     
         33 . The single-stranded oligonucleotide according to  claim 30 , wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor. 
     
     
         34 . The single-stranded oligonucleotide according to  claim 30 , wherein the functional molecule is a peptide or protein selected from the group consisting of receptor ligands and antibodies. 
     
     
         35 . A pharmaceutical composition containing the single-stranded oligonucleotide according to  claim 1  and a pharmacologically acceptable carrier. 
     
     
         36 . A method for controlling the function of a target RNA, comprising a step for contacting the single-stranded oligonucleotide according to  claim 1  with a cell. 
     
     
         37 . A method for controlling the function of a target RNA in a mammal, comprising a step for administering a pharmaceutical composition containing the single-stranded oligonucleotide according to  claim 1  to the mammal. 
     
     
         38 . A use of the single-stranded oligonucleotide according to  claim 1  for controlling the function of a target RNA in a mammal. 
     
     
         39 . A method for producing the single-stranded oligonucleotide according to  claim 1 , comprising a step for elongating the nucleotide strand at the 3′-end or 5′-end of an oligonucleotide containing at least one of X, L, and Y.