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

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Abstract
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.

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