Acylation probing of "generic" RNA libraries reveals critical influence of loop constraints on reactivity.

The reactivity of RNA 2'-OH acylation is broadly useful both in probing structure and in preparing conjugates. To date, this reactivity has been analyzed in limited sets of biological RNA sequences, leaving open questions of how reactivity varies inherently without regard to sequence in structured contexts. We constructed and probed "generic" structured RNA libraries using homogeneous loop sequences, employing deep sequencing to carry out a systematic survey of reactivity. We find a wide range of RNA reactivities among single-stranded sequences, with nearest neighbors playing substantial roles. Remarkably, certain small loops are found to be far more reactive on average (up to 4,000-fold) than single-stranded RNAs, due to conformational constraints that enhance reactivity. Among loops, we observe large variations in reactivity based on size, type, and position. The results lend insights into RNA designs for achieving high-efficiency local conjugation and provide new opportunities to refine structure analysis.