A DNA-based biosensor assay for the kinetic characterization of ion-dependent aptamer folding and protein binding

Therapeutic and diagnostic aptamers are nucleic acid oligomers designed to bind tightly and specifically to their target. Here, we present a switchSENSE®-based aptasensor for the detailed kinetic characterization of aptamer folding and aptamer–analyte interaction, employing the thrombin-binding aptamer (TBA) as model system. We observed antiparallel G-quadruplex formation induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer (K⁺ > NH₄⁺ >> Na⁺ > Li⁺). Intriguingly, we traced strong ion-dependent differences in the affinity of thrombin for TBA (K_D = 0.15 nM to 250 nM) to reduction of the on-rate (k_ON max = 3.8 x 10⁸ M⁻¹s⁻¹), while the stability of the complex (k_OFF) remained stable in all conditions. Because both folding and target interaction can be resolved directly, switchSENSE® is a useful analytical tool for in-depth characterization of aptamer–ion and aptamer–protein interactions.