A Rapid, Label-Free, and Scalable Method for Characterizing Binding Properties of Small Molecule Aptamers

Small molecule-binding aptamers have emerged as versatile sensing and targeting elements for diagnostic, therapeutic, and gene-regulatory applications. Approximately 60 small molecule-binding RNA aptamers have been selected in vitro; however, limited characterization and functional validation have hindered their broader use in synthetic biology applications. For example, the majority of in vivo engineered RNA-based gene regulatory devices exclusively incorporate the theophylline aptamer as the sensing component. One of the challenges associated with developing and implementing new small molecule-binding aptamers is the ability to rapidly and reliably validate their binding properties. Here we describe a rapid and sensitive method for characterizing kinetic and binding affinity properties of small molecule aptamers based on the Biacore surface plasmon resonance biosensor platform. We validated the SPR-based aptamer characterization strategy by comparing the SPR-measured binding properties of previously studied aptamers and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. This panel included both natural and in vitro selected aptamers, providing insight regarding differences in their binding properties. Our system is label-free, scalable, and supports the analysis of different aptamer–target pairs and binding conditions with the same platform. Thus, the described SPR-based aptamer characterization method provides a versatile and powerful tool to facilitate the rapid validation of RNA aptamer sensors, supporting their broader application in engineering RNA devices and multicomponent systems.