posted on 2022-12-21, 18:36authored byLu Xiao, Linglan Fang, Sayantan Chatterjee, Eric T. Kool
RNA
2′-OH acylation is widely used both for mapping structure
and for conjugating RNA, generally relying on selective reactions
with unpaired nucleotides over paired ones. Common reagents for this
acylation have been chiefly restricted to two similar aryl scaffolds,
leaving open the question of how more broadly varied reagent structure
might affect selectivity. Here, we prepared a set of 10 structurally
diverse acylimidazole reagents and employed deep sequencing to profile
their reactivity and selectivity in an RNA library of systematically
varied structure. We show that structure-directed reactivity profiles
vary significantly with the reagent scaffold, and we document new
acylating agents that have altered selectivity profiles, including
reagents that show elevated selectivity within loops, as well as compounds
with reduced off-target reactivity in loop closing base pairs. Interestingly,
we also show that the simplest reagent (acetylimidazole) is cell permeable
and is small enough to map RNA structure in the presence of protein
contacts that block other reagents. Finally, we describe reagents
that show elevated selectivity within small loops, with applications
in site-selective labeling. The results provide new tools for improved
conjugation and mapping of RNA.