Small‐Molecule Benzo‐Phenoselenazine Derivatives for Multi‐Subcellular Biomolecule Profiling

Abstract Elucidating the subcellular localization of RNAs and proteins is fundamental to understanding their biological functions. Genetically encoded proteins/enzymes provide an attractive approach to target many proteins of interest, but are limited to specific cell lines. Although small‐molecule‐based methods have been explored, a comprehensive system for profiling multiple locations in living cells, comparable to fusion‐protein techniques, is yet to be established. In this study, we introduce a novel proximity labeling strategy employing a suite of small molecules derived from benzo‐phenoselenazine (e.g., selenium‐containing Nile Blue [SeNB]), which achieves proximity labeling through singlet oxygen generation upon near‐infrared light activation in the presence of propargylamine. These SeNB compounds allow for selective labeling of RNAs and proteins within living cells, exhibiting a distinct preference for organelle membranes, which are systematically investigated via in vitro , computational, and in cellulo examinations. Our findings highlight the capabilities of SeNB derivatives as wash‐free and genetics‐free approaches to illuminate the subcellular localization of biological molecules with deep penetration and high spatial resolution. Moreover, SeNB derivatives are capable of elucidating inter‐organelle interactions at the molecular level, as evidenced by proteomic and transcriptomic analyses, thus holding significant potential for advancing our understanding of cellular processes related to disease progression and therapeutic development.