Entropy Driving the Mg²⁺-Induced Folding of TPP Riboswitch RNA

Mg²⁺ is well known to facilitate the structural folding of RNA. However, the thermodynamic and dynamic roles of Mg²⁺ in RNA folding remain elusive. Here, we exploit single-molecule fluorescence resonance energy transfer (smFRET) and isothermal titration calorimetry (ITC) to study the mechanism of Mg²⁺ in facilitating the folding of thiamine pyrophosphate (TPP) riboswitch RNA. The results of smFRET identify that the presence of Mg²⁺ compacts the RNA and enlarges the conformational dispersity among individual RNA molecules, resulting in a large gain of entropy. The compact yet flexible conformations triggered by Mg²⁺ may help the riboswitch recognize its specific ligand and further fold. This is supported by the ITC experiments, in which the Mg²⁺-induced RNA folding is driven by entropy (ΔS) instead of enthalpy (ΔH). Our results complement the understanding of the Mg²⁺-induced RNA folding. The strategy developed in this work can be used to model other RNAs' folding under different conditions.