The Hoechst Dye‐Binding RNA Aptamer Recognizes Its Ligand in a Highly Dynamic Fashion

RNA molecules can undergo dynamic processes that influence their overall function. The study presented here aims to characterize the ligand binding dynamics of the t ‐Bu Hoechst–RNA aptamer complex through a combination of nuclear magnetic resonance (NMR) spectroscopy and fluorescence techniques. Variable temperature NMR experiments revealed that the t ‐Bu Hoechst dye has two overlapping ligand binding sites. It moves between the two ligand binding sites in the aptamer on the slow to intermediate NMR exchange timescale. Fluorescence anisotropy experiments confirmed the presence of two fluorescence lifetimes. New variants of the RNA1 aptamer were designed and characterized by melting temperature studies, fluorescence emission, fluorescence titration, and one‐dimensional (1D) 1 H NMR titration experiments to identify the key ligand‐binding nucleotides. These studies showed that the nucleotides in Helix 2 adjacent to the bulge region have the largest impact on the aptamer's structure and ligand interactions, indicating that Helix 2 is the major binding site.