Abstract
Therapeutic and diagnostic nucleic acid aptamers are designed to bind tightly and specifically to their target. The combination of structural and kinetic analyses of aptamer interactions has gained increasing importance. Here, we present a fluorescence-based switchSENSE aptasensor for the detailed kinetic characterization of aptamer–analyte interaction and aptamer folding, employing the thrombin-binding aptamer (TBA) as a model system. Thrombin-binding aptamer folding into a G-quadruplex and its binding to thrombin strongly depend on the type and concentration of ions present in solution. We observed conformational changes induced by cations in real-time and determined the folding and unfolding kinetics of the aptamer. The aptamer’s affinity for K+ was found to be more than one order of magnitude higher than for other cations (K+ > NH4+ >> Na+ > Li+). The aptamer’s affinity to its protein target thrombin in the presence of different cations followed the same trend but differed by more than three orders of magnitude (KD = 0.15 nM to 250 nM). While the stability (kOFF) of the thrombin–TBA complex was similar in all conditions, the cation type strongly influenced the association rate (kON). These results demonstrated that protein–aptamer binding is intrinsically related to the correct aptamer fold and, hence, to the presence of stabilizing ions. Because fast binding kinetics with on-rates exceeding 108 M−1s−1 can be quantified, and folding-related phenomena can be directly resolved, switchSENSE is a useful analytical tool for in-depth characterization of aptamer–ion and aptamer–protein interactions.
Highlights
Aptamers are single-stranded oligonucleotides with ideally high-binding affinity and specificity for their targets [1,2]
We showed the dependence of thrombin association on the type of cation in solution and resolved on-rates of thrombin to thrombin-binding aptamer (TBA) up to the 108 M−1s−1 range in best conditions, which contain cations that facilitate thrombin binding by pre-folding of the G-quadruplex
We demonstrate how the switchSENSE technology can resolve aptamer folding and unfolding upon interaction with small molecules, such as ions, and provide rates of target kinetics
Summary
Aptamers are single-stranded oligonucleotides with ideally high-binding affinity and specificity for their targets [1,2]. Due to the presence of some similarities and its many advantages, aptamers are often compared to antibodies, with similar hopes for diagnostic and therapeutic applications [6]. Both can be designed to bind their targets with very high affinities. The observable trend in the field of antibodies to develop bispecific or trispecific antibodies with two or more binding sites combined, applies to aptamers as well [7]. During the very reproducible production it is easy to include site-specific chemical modifications or labels in aptamers
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