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. To investigate, we constructed and probed “generic” structured RNA libraries using homogeneous loop sequences. We employed 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 4000-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 data to refine structure analysis. 12 samples are analyzed, including replicates for each conditions: (1) A-case RNA library treated with 200mM NAIN3 (duplicate); (2) A-case RNA library treated with 25mM 1M7 (duplicate); (3) A-case RNA library treated DMSO (duplicate, as control); (4) U-case RNA library treated with 200mM NAIN3 (duplicate); (5) U-case RNA library treated with 25mM 1M7 (duplicate); (6) U-case RNA library treated DMSO (duplicate, as control).