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

Abstract 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 2×10 5 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.