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.

RNA 定位受到高度调控，亚细胞组织驱动环境依赖性细胞生理学。尽管使用高反应性自由基或卡宾的邻近标记技术为细胞内蛋白质组织的无偏映射提供了强大的方法，但无偏 RNA 映射的方法很少且相对不那么稳健。在这里，我们开发了 α-烷氧基硫烯醇酯和氯烯醇酯，它们在受控的酯暴露后充当有效的酰化剂。我们将这些探针与生物正交酯酶的亚细胞定位表达配对，建立一个用于 RNA 空间分析的平台：用于邻近标记和测序 (BAP-seq) 的生物正交酰化剂。我们证明，通过选择性地揭露感兴趣区域中的烯醇探针，我们可以绘制膜结合和无膜细胞器中的 RNA 分布图。控释酰化剂化学和相应的 BAP-seq 方法扩大了邻近标记技术的范围，并提供了一种强大的方法来研究 RNA 的细胞组织。