Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide

Straightforward methods for detecting adenosine-to-inosine (A-to-I) RNA editing are key to a better understanding of its regulation, function, and connection with disease. We address this need by developing a novel reagent, N-(4-ethynylphenyl)acrylamide (EPhAA), and illustrating its ability to selectively label inosine in RNA. EPhAA is synthesized in a single step, reacts rapidly with inosine, and is "click"-compatible, enabling flexible attachment of fluorescent probes at editing sites. We first validate EPhAA reactivity and selectivity for inosine in both ribonucleosides and RNA substrates, and then apply our approach to directly monitor in vitro A-to-I RNA editing activity using recombinant ADAR enzymes. This method improves upon existing inosine chemical-labeling techniques and provides a cost-effective, rapid, and non-radioactive approach for detecting inosine formation in RNA. We envision this method will improve the study of A-to-I editing and enable better characterization of RNA modification patterns in different settings.