Dissociation Constant (<i>K</i>_d) Measurement for Small‐Molecule Binding Aptamers: Homogeneous Assay Methods and Critical Evaluations

Since 1990, numerous aptamers have been isolated and discovered for use in various analytical, biomedical, and environmental applications. This trend continues to date. A critical step in the characterization of aptamer binding is to measure its binding affinity toward both target and non‐target molecules. Dissociation constant ( K d ) is the most commonly used value in characterizing aptamer binding. In this article, homogenous assays are reviewed for aptamers that can bind small‐molecule targets. The reviewed methods include label‐free methods, such as isothermal titration calorimetry, intrinsic fluorescence of target molecules, DNA staining dyes, and nuclease digestion assays, and labeled methods, such as the strand displacement reaction. Some methods are not recommended, such as those based on the aggregation of gold nanoparticles and the desorption of fluorophore‐labeled DNA from nanomaterials. The difference between the measured apparent K d and the true K d of aptamer binding is stressed. In addition, avoiding the titration regime and paying attention to the time required to reach equilibrium are discussed. Finally, it is important to include mutated non‐binding sequences as controls.