Composition for measuring the binding affinity between nucleic acid and test substance, and use thereof

Abstract

In one embodiment of the present invention, a composition is disclosed for measuring a binding affinity between a nucleic acid and a test substance, which contains an organic fluorescent substance capable of binding to an RNA and which emits fluorescence having an intensity greater while the organic fluorescent substance is liberated from an RNA than while the organic fluorescent substance is bound to an RNA. This enables a highly accurate and easy measurement of a binding affinity between a test substance and a nucleic acid, and allows various substances to be examined as a test substance.

1 . A composition for measuring a binding affinity between a nucleic acid and a test substance, comprising an organic fluorescent substance which is capable of binding to an RNA and which emits fluorescence having an intensity greater while the organic fluorescent substance is liberated from the RNA than while the organic fluorescent substance is bound to the RNA. 
     
     
         2 . The composition as set forth in  claim 1 , comprising a compound represented by the following General Formula (1): 
       
         
           
           
               
               
           
         
         where each of R 1 , R 2 , R 3 , and R 4  is independently a hydrogen atom, a hydroxyl group, a halogen atom, or a C1 to C8 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom. 
       
     
     
         3 . The composition as set forth in  claim 1 , wherein the compound is represented by the following General Formula (2): 
       
         
           
           
               
               
           
         
         where R 5  is a hydrogen atom, a hydroxyl group, a halogen atom, or a C2 to C8 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom. 
       
     
     
         4 . The composition as set forth in  claim 1 , wherein the compound is represented by the following Structural Formula (3): 
       
         
           
           
               
               
           
         
       
     
     
         5 . The composition as set forth in  claim 1 , wherein the compound is represented by the following General Formula (12): 
       
         
           
           
               
               
           
         
         where n is 3, 4, or 5. 
       
     
     
         6 . The composition as set forth in  claim 1 , wherein the compound is represented by the following Structural Formula (13): 
       
         
           
           
               
               
           
         
       
     
     
         7 . The composition as set forth in  claim 1 , wherein the compound is represented by the following General Formula (14): 
       
         
           
           
               
               
           
         
         where n is 1 or 2; and each of R 10  and R 11  is independently an alkyl group or a carboxyl group. 
       
     
     
         8 . The composition as set forth in  claim 1  wherein the compound is represented by the following General Formula (16): 
       
         
           
           
               
               
           
         
         where each of R 12 , R 13 , R 14 , and R 15  is independently a hydrogen atom, a hydroxyl group, or a C1 to C5 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom. 
       
     
     
         9 . The composition as set forth in  claim 8 , wherein the compound is represented by the following General Formula (17): 
       
         
           
           
               
               
           
         
         where each of R 16  and R 18  is independently a hydroxyl group or a C1 to C5 alkoxyl group which may be substituted with an oxygen atom and/or a nitrogen atom at one or more carbons; X 1  is an oxygen atom, a nitrogen atom, or a sulfur atom; R 17  is a C1 to C5 alkylene group, one or more hydrogen atoms of which may be substituted with one or more functional groups selected from the group consisting of a hydroxyl group, an amino group, and an alkyl group. 
       
     
     
         10 . The composition as set forth in  claim 8 , wherein the compound is represented by the following General Formula (18): 
       
         
           
           
               
               
           
         
         where each of R 19  and R 21  is independently a hydroxyl group or a C1 to C5 alkoxyl group which may be substituted with an oxygen atom and/or a nitrogen atom at one or more carbons; each of X 2  and X 3  is independently an oxygen atom, a nitrogen atom, or a sulfur atom; each of R 20  and R 22  is independently a C1 to C5 alkylene group, one or more hydrogen carbons of which may be substituted with one or more functional groups selected from the group consisting of a hydroxyl group, an amino group, and an alkyl group. 
       
     
     
         11 . A kit for measuring a binding affinity between a nucleic acid and a test substance, comprising an organic fluorescent substance which is capable of binding to an RNA and which emits fluorescence having an intensity greater while the organic fluorescent substance is liberated from the RNA than while the organic fluorescent substance is bound to the RNA. 
     
     
         12 . The kit as set forth in  claim 11 , comprising:
 a compound represented by the following General Formula (1):   
       
         
           
           
               
               
           
         
       
       where each of R 1 , R 2 , R 3 , and R 4  is independently a hydrogen atom, a hydroxyl group, a halogen atom, or a C1 to C8 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom; and/or
 a compound represented by the following General Formula (16): 
 
       
         
           
           
               
               
           
         
         where each of R 12 , R 13 , R 14 , and R 15  is independently a hydrogen atom, a hydroxyl group, or a C1 to C5 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom. 
       
     
     
         13 . A method for measuring a binding affinity between a nucleic acid and a test substance, comprising:
 a first measuring step for measuring fluorescence emitted in response to irradiation of light onto a first solution obtained by mixing a nucleic acid with an organic fluorescent substance which is capable of binding to an RNA and which emits fluorescence having an intensity greater while the organic fluorescent substance is liberated from the RNA than while the organic fluorescent substance is bound to the RNA;   a second measuring step for measuring fluorescence emitted in response to irradiation of light onto a second solution obtained by further mixing the first solution with a test substance; and   a comparing step for comparing (i) the fluorescence measured in the first measuring step with (ii) the fluorescence measured in the second measuring step.   
     
     
         14 . The method as set forth in  claim 13 , wherein:
 the first measuring step measures the fluorescence emitted in response to irradiation of light onto the first solution obtained by mixing a nucleic acid with   a compound represented by the following General Formula (1):   
       
         
           
           
               
               
           
         
         where each of R 1 , R 2 , R 3 , and R 4  is independently a hydrogen atom, a hydroxyl group, a halogen atom, or a C1 to C8 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom; and/or a compound represented by the following General Formula (16): 
       
       
         
           
           
               
               
           
         
         where each of R 12 , R 13 , R 14 , and R 15  is independently a hydrogen atom, a hydroxyl group, or a C1 to C5 organic group which may contain one or more atoms selected from the group consisting of a hydrogen atom, a nitrogen atom, an oxygen atom, a sulfur atom, and a halogen atom; 
         the second measuring step measures the fluorescence emitted in response to irradiation of light onto the second solution obtained by further mixing the first solution with a test substance; and
 the comparing step compares (i) the fluorescence measured in the first measuring step with (ii) the fluorescence measured in the second measuring step.