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

 

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 (KD = 0.15 nM to 250 nM) to reduction of the on-rate (kON max = 3.8 x 108 M−1s−1), while the stability of the complex (kOFF) 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.

molecules I 2019

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