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

Abstract

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.

Comprehensive Analytical Comparison of Strategies Used for Small Molecule Aptamer Evaluation

“…Consistent with previous reports, these methods displayed improved precision and improved accuracy compared to previously-reported values 12,16 . However, FP requires intrinsic fluorescence of the target or target labelling and thus is not scalable to all small molecule targets.…”

Studying small molecule–aptamer interactions using MicroScale Thermophoresis (MST)

“…However, main issues of the current state of the art methods are low sensitivity, high sample consumption, immobilization of one of the partners, mass transport limitations on surfaces or buffer restrictions. Besides truly label free calorimetric approaches, which measure the enthalpy of the binding event (isothermal titration calorimetry, ITC) [4][5][6], SPR (Surface Plasmon Resonance) based assays with immobilized molecules are performed [7,8]. Other methods are isocratic elution [9], equilibrium filtration [10,11] and in-line probing [12][13][14].…”

In Vitro Reversible Translation Control Using γPNA Probes

“…35 This method should be useful for studying the kinetics of strand displacement for any other natural or synthetic nucleic acid. With growing interest in nucleic acid−based computing, SPR provides an attractive platform on which to study hybridization reactions in a label-free, automated, mediumthroughput manner.…”