Stabilization method of functional nucleic acid

Abstract

This invention is intended to enhance and improve the resistance of a single- or double-stranded nucleic acid fragment comprising a base sequence of a functional nucleic acid to degradation by nucleolytic enzymes in a simple and cost-effective manner. The single- or double-stranded nucleic acid fragment comprises, ligated to at least one end thereof, hairpin-shaped DNA comprising: (A) a nucleic acid region comprising 2 to 5 arbitrary nucleotides; (B) a nucleic acid region comprising a “gna” or “gnna” base sequence, wherein each “n” represents “g”, “t”, “a”, or “c”, a base analogue, or a modified base; and (C) a nucleic acid region comprising a base sequence complementary to the nucleic acid region (A), sequentially from the 5′ end toward the 3′ end.

1 . A nucleic acid comprising;
 a hairpin-shaped DNA comprising nucleic acid regions (A) to (C) below sequentially ligated from the 5′ end toward the 3′ end:
 (A) a first nucleic acid region comprising 2 to 5 arbitrary nucleotides; 
 (B) a second nucleic acid region comprising a “gna” or “gnna” base sequence, 
 wherein each “n” independently represents “g”, “t”, “a”, or “c”, a base analogue, or a modified base; and 
 (C) a third nucleic acid region comprising a base sequence complementary to the first nucleic acid region, 
   wherein the first nucleic acid region and the third nucleic acid region form a stem portion by base pairing with each other and the second nucleic acid region forms a loop portion, and a nucleic acid fragment (1) or (2) below:
 (1) a double-stranded nucleic acid fragment made by complete or partial base pairing; or 
 (2) a single-stranded nucleic acid fragment having at least one stem structure and at least one loop structure, 
   wherein at least one end of the nucleic acid fragment is ligated to the hairpin-shaped DNA.   
     
     
         2 . The nucleic acid according to  claim 1 , wherein the first nucleic acid region comprises “g” or “c” base. 
     
     
         3 . The nucleic acid according to  claim 1 , wherein the nucleic acid fragment is composed of DNA, RNA, and/or a derivative thereof. 
     
     
         4 . The nucleic acid according to  claim 3 , wherein, when the nucleic acid fragment is the double-stranded nucleic acid fragment (1), the double-stranded nucleic acid fragment comprises the siRNA base sequence. 
     
     
         5 . The nucleic acid according to  claim 4  comprising any of (1) to (4) below in which nucleic acid fragments and hairpin-shaped DNAs are sequentially ligated from the 5′ end toward the 3′ end:
 (1) a nucleic acid fragment comprising the base sequence of an siRNA sense strand, the hairpin-shaped DNA, a nucleic acid fragment comprising the base sequence of an siRNA antisense strand, and the hairpin-shaped DNA; 
 (2) the hairpin-shaped DNA defined in  claim 1 , a nucleic acid fragment comprising the base sequence of an siRNA sense strand, the hairpin-shaped DNA, and a nucleic acid fragment comprising the base sequence of an siRNA antisense strand; 
 (3) a nucleic acid fragment comprising the base sequence of an siRNA antisense strand, the hairpin-shaped DNA, a nucleic acid fragment comprising the base sequence of an siRNA sense strand, and the hairpin-shaped DNA; or 
 (4) the hairpin-shaped DNA defined in  claim 1 , a nucleic acid fragment comprising the base sequence of an siRNA antisense strand, the hairpin-shaped DNA, and a nucleic acid fragment comprising the base sequence of an siRNA sense strand. 
 
     
     
         6 . The nucleic acid according to  claim 4 , wherein the hairpin-shaped DNA is ligated to the ends according to any of (1) to (4) below:
 (1) the 5′ end and the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA sense strand;   (2) the 5′ end and the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA antisense strand;   (3) the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA sense strand and the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA antisense strand; and   (4) the 5′ end of a nucleic acid fragment comprising the base sequence of an siRNA sense strand and the 5′ end of a nucleic acid fragment comprising the base sequence of an siRNA antisense strand.   
     
     
         7 . The nucleic acid according to  claim 4 , wherein the hairpin-shaped DNA is ligated to the end according to any of (1) to (4) below:
 (1) the 5′ end of a nucleic acid fragment comprising the base sequence of an siRNA sense strand;   (2) the 5′ end of a nucleic acid fragment comprising the base sequence of an siRNA antisense strand;   (3) the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA sense strand; and   (4) the 3′ end of a nucleic acid fragment comprising the base sequence of an siRNA antisense strand.   
     
     
         8 . The nucleic acid according to  claim 4 , wherein each strand of the double-stranded nucleic acid fragment comprises 19 to 30 bases. 
     
     
         9 . The nucleic acid according to  claim 4 , wherein the double-stranded nucleic acid fragment has at least one mismatched region and/or bulge structure. 
     
     
         10 . The nucleic acid according to  claim 3 , wherein, when the nucleic acid fragment is the double-stranded nucleic acid fragment (1), the double-stranded nucleic acid fragment comprises a target molecule-binding region. 
     
     
         11 . The nucleic acid according to  claim 10 , wherein nucleic acid fragments and hairpin-shaped DNAs are sequentially ligated from the 5′ end toward the 3′ end in (1) or (2) below:
 (1) a nucleic acid fragment constituting the double-stranded nucleic acid fragment, the hairpin-shaped DNA, the other nucleic acid fragment constituting the double-stranded nucleic acid fragment, and the hairpin-shaped DNA; or 
 (2) the hairpin-shaped DNA, a nucleic acid fragment constituting the double-stranded nucleic acid fragment, the hairpin-shaped DNA, and the other nucleic acid fragment constituting the double-stranded nucleic acid fragment. 
 
     
     
         12 . The nucleic acid according to  claim 10 , wherein the nucleic acid fragments and hairpin-shaped DNAs below are sequentially ligated from the 5′ end toward the 3′ end and both ends are ligated to each other:
 a nucleic acid fragment constituting the double-stranded nucleic acid fragment, the hairpin-shaped DNA, the other nucleic acid fragment constituting the double-stranded nucleic acid fragment, and the hairpin-shaped DNA. 
 
     
     
         13 . The nucleic acid according to  claim 12 , which has a nick in a nucleic acid fragment region constituting the double-stranded nucleic acid fragment. 
     
     
         14 . The nucleic acid according to  claim 12 , wherein each of the nucleic acid fragment regions constituting the double-stranded nucleic acid fragment contains a nick, and the nick is not paired with the other nick. 
     
     
         15 . The nucleic acid according to  claim 10 , wherein the hairpin-shaped DNA is ligated to
 the 5′ end and the 3′ end of any one of the nucleic acid fragment constituting a double-stranded nucleic acid fragment or   the 5′ or 3′ end of each of the nucleic acid fragment constituting a double-stranded nucleic acid fragment.   
     
     
         16 . The nucleic acid according to  claim 10 , wherein the target molecule is a transcription regulator. 
     
     
         17 . The nucleic acid according to  claim 3 , wherein, when the nucleic acid fragment is the single-stranded nucleic acid fragment (2), the single-stranded nucleic acid fragment comprises a base sequence of a functional nucleic acid. 
     
     
         18 . The nucleic acid according to  claim 17 , wherein at least one stem structure formed via intramolecular annealing of the single-stranded nucleic acid fragment has a mismatched region or bulge structure. 
     
     
         19 . The nucleic acid according to  claim 17 , wherein the functional nucleic acid is selected from the group consisting of a single-stranded miRNA precursor, shRNA, a nucleic acid aptamer, a ribozyme (including deoxyribozyme), a molecular beacon or riboswitch, and a U1 adaptor. 
     
     
         20 . The nucleic acid according to  claim 17 , wherein the hairpin-shaped DNA is ligated to either the 5′ or 3′ end of the single-stranded nucleic acid fragment. 
     
     
         21 . A pharmaceutical composition comprising, as an active ingredient, the nucleic acid according to  claim 1 . 
     
     
         22 . The pharmaceutical composition according to  claim 21 , which comprises a pharmaceutically acceptable carrier. 
     
     
         23 . A method for enhancing resistance of a nucleic acid fragment to degradation by a nucleolytic enzyme
 by ligating hairpin-shaped DNA comprising the nucleic acid regions (A) to (C) below sequentially ligated from the 5′ end toward the 3′ end:   (A) a first nucleic acid region comprising 2 to 5 arbitrary nucleotides;   (B) a second nucleic acid region comprising a “gna” or “gnna” base sequence, wherein each “n” independently represents “g”, “t”, “a”, or “c”, a base analogue, or a modified base; and   (C) a third nucleic acid region comprising a base sequence complementary to the first nucleic acid region,   wherein the first nucleic acid region and the third nucleic acid region form a stem portion by base pairing with each other and the second nucleic acid region forms a loop portion,   to at least one end of the nucleic acid fragment (1) or (2) below:   (1) a double-stranded nucleic acid fragment made by the complete or partial base pairing; or   (2) a single-stranded nucleic acid fragment having at least one stem structure and at least one loop structure.   
     
     
         24 . The method according to  claim 23 , wherein the first nucleic acid region comprises “g” or “c” base. 
     
     
         25 . The method according to  claim 23 , wherein the nucleic acid fragment is composed of DNA, RNA, and/or a derivative thereof. 
     
     
         26 . The method according to  claim 25 , wherein, when the nucleic acid fragment is the double-stranded nucleic acid fragment (1), the double-stranded nucleic acid fragment comprises the siRNA base sequence. 
     
     
         27 . The method according to  claim 25 , wherein, when the nucleic acid fragment is the double-stranded nucleic acid fragment (1), the double-stranded nucleic acid fragment comprises a target molecule-binding region. 
     
     
         28 . The method according to  claim 25 , wherein, when the nucleic acid fragment is the single-stranded nucleic acid fragment (2), the single-stranded nucleic acid fragment comprises a functional nucleic acid. 
     
     
         29 . The method according to  claim 28 , wherein the functional nucleic acid is selected from the group consisting of shRNA, pri-miRNA, pre-miRNA, a nucleic acid aptamer, a ribozyme (including deoxyribozyme), a molecular beacon or riboswitch, and a U1 adaptor.