SOLiD‐MaP: A Photoproximity Labeling Platform for Small Molecule Binding Site Mapping on RNA

RNA-targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand-RNA interactions have provided powerful foundations, yet many rely on direct cross-linking or covalent-capture chemistries whose performance depends on ligand-specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand-Directed manner for Mutational Profiling (SOLiD-MaP), a photochemical platform achieving the labeling resolution for small molecule binding site identification. Using the Mango-II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen-mediated RNA labeling and demonstrate target-selective labeling driven by ligand-localized photosensitization. We further show that labeling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next-generation sequencing readouts to infer ligand-proximal regions and unambiguously map binding sites. We further extended binding-region inference to a cellular context. SOLiD-MaP provides a new, orthogonal strategy for studying small molecule-RNA recognition and should support RNA-focused mechanism-of-action studies.