High-Throughput Aptamer Characterization via Real-Time Nuclease Digestion

selection has become more straightforward, it remains challenging to accurately characterize aptamer binding in an accurate and high-throughput manner. Here, we present a next-generation version of our previously reported first-generation exonuclease digestion assays for screening aptamer-ligand interactions in a parallel and label-free manner. Our new real-time exonuclease (RT-Exo) assay delivers throughput exceeding that of the previous-generation method by orders of magnitude, with considerably less labor and resource consumption. The RT-Exo assay uses a fluorogenic aptamer as a signal reporter to monitor exonucleolytic digestion of aptamers in real time. Once samples are prepared and loaded into a microplate, a reader can autonomously monitor the digestion process, generating hundreds of data points per sample. Our fluorogenic reporter functions under many buffer and salt conditions, and we have validated the performance of RT-Exo with multiple aptamers of diverse structures binding to targets of varying physicochemical properties and divergent binding affinities. Finally, we showcase the utility of RT-Exo by screening mutants as part of structure-activity studies and its impressive throughput by profiling aptamer binding to 120 fentanyl analogs in a single run with just one experimentalist. With the greatly increased throughput, precision, and simplicity of the RT-Exo assay, the aptamer characterization process should become far more streamlined and efficient.