Descrizione
5-ethynyl uridine (EU) is a commonly used molecular biology reagent for RNA synthesis studies. EU is readily taken up by living cells and phosphorylated through the pyrimidine salvage pathway. Generated 5-ethynyl uridine-5’-triphosphate is incorporated by RNA polymerases I, II, and III into de novo RNA instead of uridine, but not into DNA. EU-labeled nascent cellular RNA can be detected quickly and with high sensitivity via Click Chemistry following fluorescent visualization. Alkyne group attached at the 5-position of uridine in modified RNA reacts with dye or biotin azides via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Labeled RNA can be detected with different methods, e.g. fluorescent microscopy or flow cytometry, which allows estimating transcriptional levels in the cells. 5-ethynyl uridine has an advantage over its analog, 5-bromouridine, because azide-containing dyes are very small in size and exhibit better membrane permeability compared to antibodies used for the detection of 5-bromouridine. Thus, the Click Chemistry approach allows whole-mount staining of large samples like organs or tissue fragments.
