Reagents and methods for direct labeling of nucleotides

Abstract

The present invention provides systems and methods for production of activatable diazo-derivatives for use in labeling nucleotides. Labeling nucleotides is accomplished by contacting a stable hydrazide derivative of a detectable moiety with an activating polymer reagent which is used to directly label the nucleotide sample. Labeling occurs on the phosphate backbone of the nucleotide which does not perturb hybridization of the labeled nucleotide with its anti-sense strand. Since the method involves direct labeling, all types of nucleotides can be labeled without prior amplification or alteration.

1 - 20 . (canceled) 
     
     
         21 . A reaction mixture for directly labeling and isolating a nucleophilic analyte comprising:
 a. a protected labeling compound;   b. a polymer-based activating reagent having a polymer-bound moiety which generates a reactive labeling compound when contacted with said protected labeling compound, wherein said reactive labeling compound has a different density and from said activating reagent;   c. an analyte having a nucleophilic group capable of reacting with said reactive labeling compound to form a self-separated, labeled nucleophilic analyte;   wherein the protected labeling compound comprises the structure:   
       
         
           
           
               
               
           
         
       
       wherein R is selected from the group consisting of hydrogen, alkyl, aryl, fused aryl, —NH 2 , CH 2 -halogen, —CN, ═O, −S—S—R 1 , —OH, —OR 1 , —NHR 1 , —CF 3 , —NO 2 , —CN, —COOR 2 , —OP(OR 3 ) 3 , N(R 4 )R 5  and —OCO—R 2 ; 
       wherein R′ is selected from the group consisting of hydrogen, alkyl and aryl; 
       wherein R″ is selected from the group consisting of an alkylsulfonyl, arylsulfonyl, substituted alkylsulfonyl and a substituted arylsulfonyl; 
       wherein X is selected from the group consisting of alkyl, substituted alkyl, —CH2, O-esters, N-amides, sulfonamide, urea, thiourea, thioether, maleimidothioether, and chlorotriazinylamino; 
       wherein R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and are selected from the group consisting of hydrogen, alkyls and substituted alkyls; 
       wherein L is a linking group comprising one or more polyatomic groups selected from the group consisting of V and Q; wherein V comprises from one to eight monoatomic groups which may be the same or different; wherein Q comprises an optional spacer moiety having from one to eight atoms; and 
       wherein D comprises a detectable moiety which is distinct from R 1 -R 5  and R″ 
       wherein said activating reagent comprises the structure:
   R-L-O − Alk + : 
 
       wherein 
       R is a polymeric support containing an alkyl or aryl group, 
       L is a linking arm, 
       and Alk+ is an alkali metal salt selected from the group consisting of Li+, Na+, K+, and Cs+. 
     
     
         22 . The reaction mixture of  claim 21 , wherein said nucleophile is selected from the group consisting of a mono-, di- or triphosphate of adenosine, thymine, cytosine, guanosine, uridine, or a purinosine and pyrimidinosine. 
     
     
         23 . The reaction mixture of  claim 22 , wherein said nucleophile is a constituent of a DNA or RNA oligonucleotide or polynucleotide. 
     
     
         24 . The reaction mixture of  claim 21 , wherein said detectable moiety is selected from the group consisting of a dye, a fluorochrome, an enzyme, a chemiluminescent compound, biotin, digoxigenin, avidin, streptavidin, an antibody and a lectin. 
     
     
         25 . The reaction mixture of  claim 21 , wherein said linking group comprises less than twenty carbon atoms. 
     
     
         26 . The reaction mixture of  claim 25 , wherein the carbon atoms of the linking group (L) are non-branched. 
     
     
         27 . The reaction mixture of  claim 21 , wherein said linking group comprises four carbon atoms and one heteroatom. 
     
     
         28 . The reaction mixture of  claim 27 , wherein the heteroatom is selected from the group consisting of oxygen and nitrogen. 
     
     
         29 . The reaction mixture of  claim 21 , wherein said linking group is a polyethyleneglycol. 
     
     
         30 . The reaction mixture of  claim 21 , wherein said linking group is selected from the group consisting of an oligolysine and a polylysine. 
     
     
         31 . The reaction mixture of  claim 21 , wherein the nucleophilic group of the analyte is selected from the group consisting of a phosphate group, a phosphoester, a carboxyl group and a thiolate group. 
     
     
         32 . The reaction mixture of  claim 21 , wherein the activating reagent has the general structure: 
       
         
           
           
               
               
           
         
       
     
     
         33 . The reaction mixture of  claim 21 , wherein said labeled analyte is hybridized to a microarray. 
     
     
         34 . The reaction mixture of  claim 21 , wherein the protected labeling compound has the structure: 
       
         
           
           
               
               
           
         
       
     
     
         35 . A method for labeling and isolating a nucleophilic analyte in a single reaction mixture comprising:
 a. in a single reaction mixture contacting a protected labeling compound with a polymer-based activating reagent having a different density from said protected labeling compound and in a reaction mixture to generate a reactive labeling compound; and   b. contacting said reaction mixture with a nucleophilic analyte having a nucleophilic group capable of reacting with said reactive labeling compound to form a labelled nucleophilic analyte that is self-separated from the activating reagent by density;   wherein the protected labeling compound comprises the structure:   
       
         
           
           
               
               
           
         
       
       where R is selected from the group consisting of hydrogen, alkyl, aryl, fused aryl, NH 2 , CH 2 -halogen, —CN, ═O, —S—S—R 1 , —OH, —OR 1 , —NHR 1 , —CF 3 , —NO 2 , —CN, —COOR 2 , —OP(OR 3 ) 3 , N(R 4 )R 5  and —OCO—R 2 ; 
       where R′ is selected from the group consisting of hydrogen, alkyl and aryl; 
       where R″ is selected from the group consisting of an alkylsulfonyl, arylsulfonyl, substituted alkylsulfonyl and a substituted arylsulfonyl; 
       where X is selected from the group consisting of alkyl, substituted alkyl, —CH2, O-esters, N-amides, sulfonamide, urea, thiourea, thioether, maleimidothioether, and chlorotriazinylamino; 
       where R 1 , R 2 , R 3 , R 4  and R 5  are the same or different and are selected from the group consisting of hydrogen, alkyls and substituted alkyls; 
       where L is a linking group comprising one or more polyatomic groups selected from the group consisting of V and Q; wherein V comprises from one to eight monoatomic groups which may be the same or different; wherein Q comprises an optional spacer moiety having from one to eight atoms; 
       where D comprises a detectable moiety which is distinct from R 1 -R 5  and R″; and 
       where said activating reagent comprises the structure: 
       R-L-O − Alk + : where R is a polymeric support for an alkyl or aryl group, L is a linking arm, and Alk+ is an alkali metal salt selected from the group consisting of Li+, Na+, K+, and Cs+. 
     
     
         36 . The method of  claim 35  where the activating reagent has the general structure: 
       
         
           
           
               
               
           
         
       
     
     
         37 . The method of  claim 35  wherein the protected labeling compound has the structure: