Clickable APEX2 Probes for Enhanced RNA Proximity Labeling in Live Cells

Although APEX2-mediated proximity labeling has been extensively implemented for studying RNA subcellular localization in live cells, the biotin-phenoxyl radical used for labeling RNAs has a relatively low efficiency, which can limit its compatibility with other profiling methods. Herein, a set of phenol derivatives were designed as APEX2 probes through balancing reactivity, hydrophilicity, and lipophilicity. Among these derivatives, Ph_N₃ exhibited reliable labeling ability and enabled two biotinylation routes for downstream analysis. As a proof of concept, we used APEX2/Ph_N₃ labeling with high-throughput sequencing analysis to examine the transcriptomes in the mitochondrial matrix, demonstrating high sensitivity and specificity. To further expand the utility of Ph_N₃, we employed mechanistically orthogonal APEX2 and singlet oxygen (¹O₂)-mediated strategies for dual location labeling in live cells. Specifically, DRAQ5, a DNA-intercalating photosensitizer, was applied for nucleus-restricted ¹O₂ labeling. We validated the orthogonality of APEX2/Ph_N₃ and DRAQ5-¹O₂ at the imaging level, providing an attractive and feasible approach for future studies of RNA translocation in live cells.