Kinetic and Equilibrium Binding Characterization of Aptamers to Small Molecules using a Label-Free, Sensitive, and Scalable Platform

[Image: see text] Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer–target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.