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 2×10 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.