Our approach involves labeling cellular RNA using chemically modified ribonucleoside analogs. We have developed a method to introduce these modified bases into RNA of living cells following short treatment with the modified nucleosides. The labels introduced into RNA are chemically derivatized using copper catalyzed click chemistry, which allows us to distinguish and physically separate newly synthesized RNA molecules (coding and non-coding) from mature RNA. We are using this approach to visualize sites of active transcription in living cells using fluorescence microscopy, and to determine the turnover rates of RNA molecules across the genome using quantitative PCR, microarray and cDNA sequencing. These approaches allow us to compare genomic regulatory patterns under a variety of stimuli and growth conditions, including, cytokine stimulation, oxidative stress and treatment with pharmacologically active small molecules.
