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 β-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 β-axial substituent. Using selective abasic substitution at this position, we quantified the energetic contributions associated with A20 in the native RNA core and with base displacement. Isothermal titration calorimetry and fluorescence measurements revealed that cryptic-site formation incurs a modest energetic penalty of ∼1.4 kcal mol ^-1 . Guided by this experimentally derived framework, computational conformational sampling recapitulated cryptic-site formation in the Cbl riboswitch and identified analogous cryptic sites in structurally unrelated RNAs from HIV-1 and HCV. These cryptic-site conformers were identified within low-energy conformational windows and exposed ligand-accessible surfaces through local base displacement. Finally, a ligand previously identified to target the env8 cryptic site bound both RNAs and yielded docking poses consistent with engagement of the newly exposed binding surfaces. Together, these results indicate that cryptic RNA binding sites can be both energetically accessible and chemically addressable, expanding the range of conformational states that may contribute to RNA ligandability.