Abstract The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA?protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5? untranslated regions of SARS-CoV-2 RNA.

Summary Spatially resolved in situ tagging of the cell of interest is crucial for in-depth mechanistic dissection of multicellular architectures or processes. With continuing interest in bioorthogonal photocatalytic decaging chemistry, we herein report the extracellular-targeted photocatalytic decaging system (CAT-Ex) for spatially resolved cell tagging and surface proteome profiling under living conditions. An antibody-conjugated photocatalysis system was established and extensively validated, enabling photocatalytic decaging of biotin precursors and proximal quinone methide probes on target cells. Visible-light-controlled selective cell tagging in cell mixture as well as in primary cells from tumor xenografts were demonstrated. Spatially resolved membrane proteome profiling was further achieved by coupling quinone methide decaging chemistry with CAT-Ex, revealing a potential microdomain protein cluster surrounding the endogenous HER2 receptor. Finally, we expanded our strategy to photocatalytic prodrug decaging for selective tumor cell killing, establishing CAT-Ex as a general platform for diverse photo-controlled molecular manipulations on targeted cells with spatial-temporal precision.