Biorthogonal masked acylating agents for proximity-dependent RNA labeling

RNA subcellular localization is highly regulated, with local enrichment driving geography- dependent cell physiology. While proximity-based labeling technologies that use highly reactive radicals or carbenes provide a powerful method for unbiased mapping of protein organization within a cell, methods for unbiased RNA mapping are scarce and comparably less robust. In this work, we develop novel α-alkoxy thioenol and chlorenol esters, which function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a novel method for spatial analysis of RNA: Bioorthogonal Acylating agents for Proximity labeling and sequencing (BAP-seq). We demonstrate that by selectively unmasking the enol probe in a locale of interest, we can map RNA distribution in membrane-bound and membrane-less organelles. The controlled-release acylating agent chemistry and corresponding BAP-seq method expands the scope of proximity labeling technologies and provides a powerful approach to interrogate the cellular organization of RNAs.