Kinetic ITC of DNA Aptamers Binding for Small Molecules and Implications for Binding Assays and Biosensors.

ABSTRACT

The determination of kon and koff values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data of various aptamers. Upon plotting their kon and koff values as a function of their Kd values, a notable trend emerged. Across a range of Kd values spanning from 28 nM to 864 µM, the kon value decreased from 2×105 M-1 s-1 to 96 M-1 s-1, whereas the koff value increased from 1.03×10-3 s-1 to 0.012 s-1. Thus, both kon and koff contributed to the change of Kd in the same direction, although the range of kon change was larger. Since experiments are often run at close to the Kd value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and offered insights into aptamer-based biosensors.