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

The determination of k_on and k_off 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 k_on and k_off values as a function of their K_d values, a notable trend emerged. Across a range of K_d values spanning from 28 nM to 864 μM, the k_on value decreased from 2×10⁵ M^-1 s^-1 to 96 M^-1 s^-1, whereas the k_off value increased from 1.03×10^-3 s^-1 to 0.012 s^-1. Thus, both k_on and k_off contributed to the change of K_d in the same direction, although the range of k_on change was larger. Since experiments are often run at close to the K_d 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.