Methods and compositions for labeling nucleic acids

Abstract

The present invention relates to methods for the labeling of nucleic acid polymers in vitro and in vivo. In particular, the methods include a [ 3+2 ] cycloaddition between a nucleotide analogue incorporated into a nucleic acid polymer and a reagent attached to a label. Such methods do not require fixation and denaturation and therefore can be applied to the labeling of nucleic acid polymers in living cells and in organisms. Also provided are methods for measuring cellular proliferation. In these methods, the amount of label incorporated into the DNA is measured as an indication of cellular proliferation. The methods of the invention can be used in a wide variety of applications including clinical diagnosis of diseases and disorders in which cellular proliferation is involved, toxicity assays, and as a tool for the study of chromosomes' ultrastructures.

1 . A method of labeling a nucleic acid polymer, comprising steps of:
 providing a nucleic acid polymer comprising at least one nucleotide analogue that comprises a first reactive unsaturated group; and   contacting the nucleic acid polymer with a reagent comprising a second reactive unsaturated group attached to a label, such that a [3+2] cycloaddition occurs between the first and second unsaturated groups.   
     
     
         2 . The method of  claim 1 , wherein the first reactive unsaturated group comprises a 1,3-dipole and the second reactive unsaturated group comprises a dipolarophile or wherein the first reactive unsaturated group comprises a dipolarophile and the second reactive unsaturated group comprises a 1,3-dipole. 
     
     
         3 . The method of  claim 2 , wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl bond. 
     
     
         4 . The method of  claim 1 , wherein the label is directly detectable, or wherein the label comprises a fluorescent agent. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the label is indirectly detectable, or wherein the label comprises a hapten. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid polymer is inside a cell or in an organism. 
     
     
         9 . The method of  claim 8 , wherein the at least one nucleotide analogue is incorporated into the nucleic acid polymer during DNA replication. 
     
     
         10 . The method of  claim 1 , wherein the step of contacting is performed under aqueous conditions in presence of Cu(I). 
     
     
         11 . The method of  claim 8 , wherein the step of contacting is performed under aqueous conditions in absence of Cu(I) and the reagent further comprises a Cu chelating moiety. 
     
     
         12 - 40 . (canceled) 
     
     
         41 . A nucleic acid polymer comprising at least one nucleotide analogue attached to a label, wherein said nucleic acid polymer is prepared by the method of  claim 1 . 
     
     
         42 . The nucleic acid polymer of  claim 41 , wherein the at least one nucleotide analogue comprises a cycloadduct resulting from a [3+2] cycloaddition between an ethynyl group and an azide group. 
     
     
         43 . The nucleic acid polymer of  claim 41 , wherein the label is directly detectable, or wherein the label comprises a fluorescent agent. 
     
     
         44 . (canceled) 
     
     
         45 . The nucleic acid polymer of  claim 41 , wherein the label is indirectly detectable, or wherein the label comprises a hapten. 
     
     
         46 - 52 . (canceled) 
     
     
         53 . A cell comprising a nucleic acid polymer according to  claim 42 . 
     
     
         54 . A kit for labeling a nucleic acid polymer comprising:
 at least one nucleoside analogue that comprises a first reactive unsaturated group; and   a reagent comprising a second reactive unsaturated group attached to a label.   
     
     
         55 . The kit of  claim 54 , wherein the first reactive unsaturated group comprises a 1,3-dipole, the second reactive unsaturated group comprises a dipolarophile and the first and second reactive unsaturated groups can react via [3+2] cycloaddition, or wherein the first reactive unsaturated group comprises a dipolarophile, the second reactive unsaturated group comprises a1,3-dipole, and the first and second reactive unsaturated groups can react via [3+2] cycloaddition. 
     
     
         56 . (canceled) 
     
     
         57 . The kit of  claim 55 , wherein the 1,3-dipole comprises an azide group and the dipolarophile comprises an ethynyl group. 
     
     
         58 . The kit of  claim 54 , wherein the label is directly detectable, or wherein the label comprises a fluorescent agent. 
     
     
         59 . (canceled) 
     
     
         60 . The kit of  claim 54 , wherein the label is indirectly detectable, or wherein the label comprises a hapten. 
     
     
         61 . (canceled) 
     
     
         62 . The kit of  claim 54 , further comprising an aqueous medium and Cu(I). 
     
     
         63 - 104 . (canceled)