RESUMO
RNA localization is highly regulated, with subcellular organization driving context-dependent cell physiology. Although proximity-based labelling 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. Here we develop α-alkoxy thioenol and chloroenol esters that function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a platform for spatial analysis of RNA: bioorthogonal acylating agents for proximity labelling 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 expand the scope of proximity labelling technologies and provide a powerful approach to interrogate the cellular organization of RNAs.