Isothermal titration calorimetry (ITC)

All isothermal titration calorimetry (ITC) measurements were performed using a MicroCal iTC₂₀₀ instrument (Malvern Pananalytical, UK) at 25°C. Samples of unlabeled RNAs and all ligands were prepared in 25 mM potassium phosphate buffer (pH 6.2) containing 50 mM potassium chloride and 2 mM magnesium actetate. The ligand (400–4000 μM) was injected into a solution of 40–100 μM RNA. After an initial delay of 120 s, the first injection of 0.2 μl was followed by 19 serial injections of 2 μl, separated by intervals of 180 s using a reference power of 11 μcal⁻¹ and a stirring speed of 750 rpm with high feedback mode. The titration of the ligand into the buffer solution resulted in negligible evolution of heat. Three independent titrations for env9b-derived RNAs and two independent titrations for Pde-1-1 or SK209-52 RNAs were performed for each ligand and the reported K_D values are the average from these titrations. The thermograms were processed using Origin 7.0 (OriginLab) assuming a one-site binding model. The first injection was excluded from the analysis. An overview of all binding parameters derived from the ITC measurements is given in Supplementary Table S1.