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

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.