Unnatural Amino Acid and Emerging Chemistry Approaches to Map RNA–Protein Interactions

Abstract RNA serves as both a genetic messenger and a functional non‐coding molecule, with its activity relying on interactions with diverse proteins. Characterizing RNA–protein interactions remains challenging, particularly for dynamic or low‐abundance complexes. Traditional crosslinking methods, such as UV‐254 nm irradiation and formaldehyde fixation, suffer from low efficiency, poor specificity, and broad reactivity, limiting their utility for high‐resolution interactome mapping. Recent advances in genetic code expansion (GCE) and unnatural amino acid (UAA) incorporation now enable chemoselective, site‐specific crosslinking chemistries that circumvent these limitations. This review highlights classes of crosslinkable UAAs, including benzophenone‐, diazirine‐, and aryl azide‐based moieties, which have been used for photo‐induced covalent capture of RNA–protein interactions. Next‐generation modalities, such as acetophenone derivatives, halogenated UAAs, bifunctional UAAs, acyl silanes, diaryl nitrones, and photoactivatable systems further expand the toolkit for photocrosslinking and bioorthogonal labelling. Beyond UV‐based approaches, latent bioreactive UAAs and proximity‐induced chemistries exploit electrophile–nucleophile substitution and masked acylating agents to enable covalent capture under physiological conditions. Together, these strategies provide unprecedented control over reactivity, temporal resolution and site specificity, advancing RNA‐targeted proteomics. Continued innovation in UAA‐based chemistry promise to transform how RNA–protein complexes are interrogated and manipulated with molecular precision.