5-bromo-2'-deoxy-uridine labeled nucleotide triphosphates and nucleic acid probes and methods of making and using the same

Abstract

5-bromo-2′-deoxy-uridine (BrdU) labeled nucleotide triphosphates and nucleic acid probes are described herein. The BrdU labeled nucleotide triphosphates include a linker between the nucleotide triphosphate and the BrdU moiety. The linker can be cleavable or non-cleavable. The nucleotide triphosphates can be a ribonucleotide triphosphates, 2′-deoxyribonucleotide triphosphates or 2′,3′-dideoxyribonucleotide triphosphates. The nucleic acid probes can be used for in situ hybridization.

1 . A compound of the formula: 
       
         
           
           
               
               
           
         
       
       or a salt thereof, wherein “Nu” is a nucleotide triphosphate, a 2′-deoxynucleotide triphosphate or a 2′,3′-dideoxynucleotide triphosphate and wherein L is a linker. 
     
     
         2 . The compound of  claim 1 , wherein L has a length greater than a C 11  n-alkane carbon chain. 
     
     
         3 . The compound of  claim 1 , wherein “Nu” is uridine triphosphate, 2′-deoxyuridine triphosphate or 2′,3′-dideoxytriphosphate. 
     
     
         4 . The compound of  claim 1 , wherein the compound is of the formula: 
       
         
           
           
               
               
           
         
       
       or a salt thereof, wherein each X is independently H or OH and “L” is a linker. 
     
     
         5 . The compound of  claim 1 , wherein “L” is a linker having a chemical structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein n is an integer of from 6 to 12. 
     
     
         6 . A sodium salt of the compound of  claim 1 . 
     
     
         7 . The compound of  claim 1 , wherein the compound is of the formula: 
       
         
           
           
               
               
           
         
         or a salt thereof, wherein each X is independently H or OH. 
       
     
     
         8 . A sodium salt of the compound of  claim 7 . 
     
     
         9 . A polynucleotide represented by the formula: 
       
         
           
           
               
               
           
         
       
       wherein “Y” is a nucleoside, a nucleotide or a polynucleotide, “Z” is H, a nucleotide or a polynucleotide, “X” is H or OH, “B” is a nucleobase and “L” is a linker. 
     
     
         10 . The polynucleotide of  claim 9 , wherein the polynucleotide is represented by the formula: 
       
         
           
           
               
               
           
         
       
       wherein “Y” is a nucleoside, nucleotide or polynucleotide, “Z” is H, a nucleotide or a polynucleotide, “X” is H or OH and “L” is a linker. 
     
     
         11 . The polynucleotide of  claim 9 , wherein “L” is a linker having a chemical structure selected from the group consisting of: 
       
         
           
           
               
               
           
         
       
       wherein n is an integer of from 6 to 12. 
     
     
         12 . (canceled) 
     
     
         13 . A method for detecting the presence of a nucleic acid analyte in a tissue sample comprising:
 incubating the tissue sample with a composition comprising the polynucleotide of  claim 9 , wherein the polynucleotide binds to the nucleic acid analyte;   washing the sample;   contacting the sample with an anti-BrdU antibody conjugated to a detectable moiety or to an enzyme; and   detecting the detectable moiety or enzyme.   
     
     
         14 . The method of  claim 13 , further comprising fixing the sample prior to incubating the sample. 
     
     
         15 . The method of  claim 14 , wherein fixing comprises contacting the sample with an acid or a cross-linking agent. 
     
     
         16 . A composition comprising:
 adenosine triphosphate (ATP);   uridine triphosphate (UTP);   guanosine triphosphate (GTP);   cytidine triphosphate (CTP); and   a compound as set forth in  claim 1 , wherein “Nu” is a nucleotide triphosphate.   
     
     
         17 - 25 . (canceled)