5-Ethynylcytidine as a new agent for detecting RNA synthesis in live cells by "click" chemistry.

Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimental technique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modified nucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfully synthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based on the biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Analogue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via "click" chemistry with fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC was efficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-ethynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and the rate of metabolism of EC was much faster than that of EU.