Labelling Strategies for the Sensitive Detection of Analytes

Abstract

The present invention relates to methods and reagents for detecting analytes, e.g. nucleic acids. The new methods and reagents allow a simple and sensitive detection even in complex biological samples.

1 : A reagent kit for detecting an analyte in a sample comprising:
 (a) a functionalized compound comprising
 (i) at least one Click functional group selected from alkyne and 
 azide groups which is a first reaction partner for a Click reaction said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring, and 
 (ii) a nucleic acid or nucleic acid analogue building block, wherein the functional group is attached to a nucleobase, 
   (b) a second reaction partner for said Click reaction comprising the complementary azide or alkyne group, wherein the second reaction partner further comprises a marker group or a marker precursor group, and   (c) optionally a marker forming reagent capable of converting marker precursor groups to marker groups.   
     
     
         2 : The reagent kit of  claim 1 , wherein the at least one Click functional group is an alkyne group. 
     
     
         3 : The reagent kit of  claim 1 , wherein the at least one Click functional group is attached to position 5 or 6 of a pyrimidine nucleobase or to position 7 or 8 of a purine nucleobase. 
     
     
         4 : The reagent kit of  claim 1 , wherein the second reaction partner comprises a fluorescence marker group. 
     
     
         5 : The reagent kit of  claim 1 , wherein the functional group is attached to the compound via a spacer. 
     
     
         6 : The reagent kit of  claim 5 , wherein the spacer is a flexible spacer or an at least partially rigid spacer. 
     
     
         7 : The reagent kit of  claim 6 , wherein the spacer comprises at least one group selected from alkene groups, alkyne groups, cyclic groups, particularly aromatic and heteroaromatic groups, and combinations thereof. 
     
     
         8 : The reagent kit of  claim 1 , wherein the nucleobase is selected from naturally occurring and non-naturally occurring purine and pyrimidine bases. 
     
     
         9 : The reagent kit of  claim 8 , wherein the nucleobase is selected from the group consisting of adenine, 7-deazaadenine, guanine, 7-deazaguanine, cytosine, thymidine, uracil, inosine and xanthine. 
     
     
         10 : The reagent kit of  claim 1 , wherein the functionalized compound is a building block for the chemical or enzymatic synthesis of nucleic acids or nucleic acid analogues or a precursor thereof, wherein enzymatic synthesis occurs by DNA polymerase or RNA polymerase. 
     
     
         11 : The reagent kit of  claim 1 , wherein the functionalized compound is a nucleoside triphosphate, particularly a ribose, 2′-deoxyribose or 2′,3′-dideoxyribose nucleoside triphosphate or a nucleoside phosphoramidite, H-phosphonate or phosphotriester. 
     
     
         12 : The reagent kit of  claim 1 , wherein the compound is a building block for the synthesis of peptide nucleic acids, morpholino nucleic acids or locked nucleic acids. 
     
     
         13 : The reagent kit of  claim 1 , wherein the functional group is attached to the nucleobase via a spacer having a chain length of up to 10 atoms. 
     
     
         14 : The reagent kit of  claim 13 , wherein the chain length is of up to 3 atoms. 
     
     
         15 : The reagent kit of  claim 1 , wherein the second reaction partner comprises an azide group and wherein the marker group is a fluorescence marker group.