Compounds and processes for single-pot attachment of a label to nucleic acid

Abstract

Compounds and methods are provided for a single-pot covalent attachment of a label to nucleic acids comprising forming a covalently attachable labeling reagent for alkylating the molecule. Then, combining the covalently attachable labeling reagent with a mixture containing the molecule, under conditions wherein the labeling reagent has reactivity with the molecule thereby forming a covalent bond.

We claim:  
     
         1 . A labeling compound for covalently attaching a label to a nucleic acid in a single-pot reaction consisting of an alkylating group comprising: mustards, three-membered rings and a label, wherein the compound has affinity for nucleic acid when alkylation reaction occurs.  
     
     
         2 . The compound of  claim 1  wherein the mustard is selected from the group consisting of nitrogen mustards, sulfur mustards and selenium mustards.  
     
     
         3 . The compound of  claim 1  wherein the three-membered ring is selected from the group consisting of aziridines, episulfides, epoxides, and cyclopropanes.  
     
     
         4 . The compound of  claim 1  wherein affinity for nucleic acid consists of positive charge.  
     
     
         5 . The compound of  claim 4  wherein the alkylating group is a tertiary mustard and the labeling reagent has a net charge greater than or equal to zero.  
     
     
         6 . The compound of  claim 1  wherein the label is selected from the group consisting of fluorescence-emitting molecules, hapten-containing molecules, proteins, and radioactive chemicals.  
     
     
         7 . The compound of  claim 1  wherein the label is selected from the group consisting of functional groups.  
     
     
         8 . The compound of  claim 1  wherein the label is separated from the alkylating group by a linker.  
     
     
         9 . The compound of  claim 8  wherein the linker is charge neutral.  
     
     
         10 . The compound of  claim 8  in which the linker is cationic.  
     
     
         11 . The compound of  claim 1  wherein the compound is selected from the group consisting of: the structures illustrated in FIG. 3, FIG. 6, FIG. 11A FIG. 11B, and FIG. 11C.  
     
     
         12 . The compound of  claim 1  wherein the nucleic acid consists of DNA.  
     
     
         13 . The compound of  claim 1  wherein the nucleic acid consists of RNA.  
     
     
         14 . The compound of  claim 13  wherein the nucleic acid consists of siRNA  
     
     
         15 . A labeling compound for covalently attaching a label to a nucleic acid having the structure comprising:  
       
         
           
           
               
               
           
         
       
       wherein A is a label selected from the group consisting of fluorescence-emitting compounds, radioactive compounds, haptens, immunogenic molecules, chemiluminescence-emitting compounds, proteins, and functional groups, wherein the net charge of the compound is greater than or equal to zero.  
     
     
         16 . The compound of  claim 15  wherein the compound contains an affinity group.  
     
     
         17 . The compound of  claim 15  wherein the compound contains a spacer.  
     
     
         18 . The compound of  claim 17  wherein the compound contains an affinity group.  
     
     
         19 . A process for forming a nucleic acid labeling reagent comprising: reacting an amine-reactive label with the amine of a compound selected from FIG. 2D and FIG. 6Ai.  
     
     
         20 . A kit comprising: 
 a) a receptacle containing a covalently attachable labeling reagent for alkylating nucleic acid in a single-pot reaction; and,    b) instructions for use.