Fluorescent Chemical Compounds Having High Selectivity for Double Stranded DNA, and Methods for Their Use

Abstract

Chemical compounds having a high selectivity for double stranded DNA over RNA and single stranded DNA are disclosed. The chemical compounds are stains that become fluorescent upon illumination and interaction with double stranded DNA, but exhibit reduced or no fluorescence in the absence of double stranded DNA. The compounds can be used in a variety of biological applications to qualitatively or quantitatively assay DNA, even in the presence of RNA.

1 . A chemical compound having the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 12  is an alkyl group. 
       
     
     
         2 . The chemical compound of  claim 1 , wherein:
 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  are independently hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof.   
     
     
         3 . The chemical compound of  claim 1 , wherein at least one of R 1 , R 2 , R 3 , and R 4  comprises an aromatic group or an alkynyl group. 
     
     
         4 . The chemical compound of  claim 1 , wherein R 9  comprises an aromatic group, alkyl-aromatic, or an alkynyl group. 
     
     
         5 . The chemical compound of  claim 1 , wherein R 10  is an amine group. 
     
     
         6 . The chemical compound of  claim 1 , wherein:
 at least one of R 1 , R 2 , R 3 , and R 4  comprises an aromatic group or an alkynyl group; and   R 9  comprises an aromatic group, alkyl-aromatic group, or an alkynyl group.   
     
     
         7 . The chemical compound of  claim 1 , wherein:
 at least one of R 1 , R 2 , R 3 , and R 4  comprises an aromatic group or an alkynyl group;   R 9  comprises an aromatic group, alkyl-aromatic group, or an alkynyl group; and   R 10  comprises an amine group.   
     
     
         8 . The chemical compound of  claim 1 , wherein R 12  is a C 1 -C 8  alkyl group. 
     
     
         9 . The chemical compound of  claim 1 , wherein R 12  is a methyl group. 
     
     
         10 . The chemical compound of  claim 1 , further comprising one or more cations or anions. 
     
     
         11 . A kit comprising:
 DNA, RNA, or both DNA and RNA; and   a chemical compound having the structure:   
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 12  is an alkyl group. 
       
     
     
         12 . A method of detecting the presence or absence of double stranded DNA in a sample, the method comprising:
 providing a sample suspected of containing double stranded DNA;   contacting the sample with a chemical compound to prepare a test sample;   illuminating the test sample with energy; and   detecting emission of energy from the test sample;   wherein the chemical compound has the structure:   
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 12  is an alkyl group. 
       
     
     
         13 . The method of  claim 12 , further comprise calculating the concentration of double stranded DNA in the sample after the detecting step. 
     
     
         14 . A chemical compound having the structure: 
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 22  is an alkyl group. 
       
     
     
         15 . The chemical compound of  claim 14 , wherein:
 R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21  are independently hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof.   
     
     
         16 . The chemical compound of  claim 14 , wherein at least one of R 13 , R 14 , R 15 , and R 16  comprises an aromatic group or an alkynyl group. 
     
     
         17 . The chemical compound of  claim 14 , wherein R 19  comprises an aromatic group, alkyl-aromatic, or an alkynyl group. 
     
     
         18 . The chemical compound of  claim 14 , wherein R 20  is an amine group. 
     
     
         19 . The chemical compound of  claim 14 , wherein:
 at least one of R 13 , R 14 , R 15 , and R 16  comprises an aromatic group or an alkynyl group; and   R 19  comprises an aromatic group, alkyl-aromatic group, or an alkynyl group.   
     
     
         20 . The chemical compound of  claim 14 , wherein:
 at least one of R 13 , R 14 , R 15 , and R 16  comprises an aromatic group or an alkynyl group;   R 19  comprises an aromatic group, alkyl-aromatic group, or an alkynyl group; and   R 20  comprises an amine group.   
     
     
         21 . The chemical compound of  claim 14 , wherein R 22  is a C 1 -C 8  alkyl group. 
     
     
         22 . The chemical compound of  claim 14 , wherein R 22  is a methyl group. 
     
     
         23 . The chemical compound of  claim 14 , further comprising one or more cations or anions. 
     
     
         24 . A kit comprising:
 DNA, RNA, or both DNA and RNA; and   a chemical compound having the structure:   
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 22  is an alkyl group. 
       
     
     
         25 . A method of detecting the presence or absence of double stranded DNA in a sample, the method comprising:
 providing a sample suspected of containing double stranded DNA;   contacting the sample with a chemical compound to prepare a test sample;   illuminating the test sample with energy; and   detecting emission of energy from the test sample;   wherein the chemical compound has the structure:   
       
         
           
           
               
               
           
         
         wherein: 
         n is a non-negative integer; 
         X is oxygen or sulfur; 
         R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21  independently comprise hydrogen, a hydroxyl group, an alkoxy group, a thiol, a thioalkyl, a thioaryl, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an aromatic group, a primary amine group, a secondary amine group, a tertiary amine group, a reactive group, or combinations thereof; and 
         R 22  is an alkyl group. 
       
     
     
         26 . The method of  claim 25 , further comprise calculating the concentration of double stranded DNA in the sample after the detecting step.