biostudies-literatureZeller MJNCRR NIH HHSNIAID NIH HHSNCI NIH HHSNational Institutes of HealthNIGMS NIH HHSNIH HHS438-448https://www.ebi.ac.uk/biostudies/studies/S-EPMC8938680biostudies-literatureUnknown17(2)RNA molecules can show high levels of cooperativity in their global folding and interactions with divalent ions. However, cooperativity at individual ligand-RNA interaction sites remains poorly understood. Here, we investigated the binding of thiamine and methylene diphosphonic acid (MDP, a soluble structural analogue of pyrophosphate) to the thiamine pyrophosphate riboswitch. These ligands each bind weakly at proximal subsites, with 10 μM and 1 mM affinities, respectively. The affinity of MDP moderately improves when thiamine or thiamine-like fragments are pre-bound to the RNA. Covalent linking of thiamine and MDP substantially increases riboswitch binding to a notable high affinity of 20 nM. Crystal structures and single-molecule correlated chemical probing revealed favorable induced fit effects upon binding of individual ligands and, unexpectedly, a substantial thermodynamically unfavorable RNA structural rearrangement upon binding of the linked thiamine-MDP ligand. Thus, linking of two ligands of modest affinity, accompanied by an unfavorable structural rearrangement, still yields a potent linked RNA-binding compound. Since complex ligands often bind riboswitches and other RNAs at proximal subsites, principles derived from this work inform and support fragment-linking strategies for identifying small molecules that interact with RNA specifically and with high affinity.ACS chemical biologySubsite Ligand Recognition and Cooperativity in the TPP Riboswitch: Implications for Fragment-Linking in RNA Ligand Discovery.PMC8938680P30 CA016086R01-EUREKA GM098662P30 GM124165R01 AI068462P30 GM133893R01 GM098662S10 OD021527R01 GM112940S10 RR029205Zeller MJSerganov AAube JNuthanakanti ALi KWeeks KMfalseSubsite Ligand Recognition and Cooperativity in the TPP Riboswitch: Implications for Fragment-Linking in RNA Ligand Discovery.RNA molecules can show high levels of cooperativity in their global folding and interactions with divalent ions. However, cooperativity at individual ligand-RNA interaction sites remains poorly understood. Here, we investigated the binding of thiamine and methylene diphosphonic acid (MDP, a soluble structural analogue of pyrophosphate) to the thiamine pyrophosphate riboswitch. These ligands each bind weakly at proximal subsites, with 10 μM and 1 mM affinities, respectively. The affinity of MDP moderately improves when thiamine or thiamine-like fragments are pre-bound to the RNA. Covalent linking of thiamine and MDP substantially increases riboswitch binding to a notable high affinity of 20 nM. Crystal structures and single-molecule correlated chemical probing revealed favorable induced fit effects upon binding of individual ligands and, unexpectedly, a substantial thermodynamically unfavorable RNA structural rearrangement upon binding of the linked thiamine-MDP ligand. Thus, linking of two ligands of modest affinity, accompanied by an unfavorable structural rearrangement, still yields a potent linked RNA-binding compound. Since complex ligands often bind riboswitches and other RNAs at proximal subsites, principles derived from this work inform and support fragment-linking strategies for identifying small molecules that interact with RNA specifically and with high affinity.2022-01-01T00:00:00Z2022 Feb2026-05-09T22:22:28.107Z2025-04-19T02:45:28.529ZS-EPMC89386803506069810.1021/acschembio.1c00880