Cryptic RNA binding sites are energetically accessible and chemically addressable

Cryptic binding sites generated by local conformational dynamics have become an important concept in protein-targeted ligand discovery, yet their energetic accessibility and relevance to RNA recognition remain less well understood. Here, we use the env8 cobalamin (Cbl) riboswitch as a model system to investigate the energetic consequences of cryptic-site formation through base displacement. Structural analysis revealed that binding of {beta}-axial substituted Cbl derivatives displaces a conserved adenosine (A20) from the RNA core, exposing a previously hidden binding site that is subsequently occupied by the {beta}-axial substituent. Using selective abasic substitution at this position, we q