Fluorogenic "click" reaction for labeling and detection of DNA in proliferating cells

A thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), has been reported as a rapid labeling tool for direct measurement of cells in S-phase. The alkynyl group of EdU is a biologically inert group that undergoes an extremely selective reaction with azido-functionalized groups via Cu(I)-catalyzed alkyneazide cycloaddition (CuAAC or "click") reaction. Here we report the highly efficient reaction of the terminal alkynyl group of EdU with a pro-fluorogenic compound, 3-azido-7-hydroxycoumarin, to afford an intense fluorescent 1,2,3-triazole product, which occurs only after the CuAAC reaction. This new method eliminates concerns for residual fluorescence since the unreacted precursors are optically inactive. The procedure therefore does not require extensive wash steps to remove the unreacted fluorescent dyes in the sample, allowing for immediate quantification and visualization after the reaction. The advantage over currently available commercial products is its potential to streamline high-throughput applications and help minimize errors.