Probing adenosine-to-inosine editing reactions using RNA-containing nucleoside analogs

Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect these changes have on the rate of enzyme-catalyzed deamination reveals features of the editing reaction and guides the design of inhibitors. We describe the synthesis of select nucleoside analog phosphoramidites and their incorporation into RNAs that mimic known editing sites by solid phase synthesis, and analyze the interaction of these synthetic RNAs with ADARs using deamination kinetics and quantitative gel mobility shift assays.