Using Exonucleases for Aptamer Characterization, Engineering, and Sensing

selection to detect analytes with ultralow background and high sensitivity. Through this approach, we were able to detect analytes at nanomolar levels in biological samples, with the capacity for achieving multiplexed detection by using molecular beacons. Finally, we used exonucleases to develop a high throughput means of characterizing aptamer affinity and specificity for a variety of ligands. This approach has enabled more comprehensive analysis of aptamers by greatly increasing the number of aptamer candidates and aptamer-ligand pairs that can be tested in a single experiment. We have also demonstrated the success of this method as a means for identifying new mutant aptamers with augmented binding properties and for quantifying aptamer-target affinity. Our enzymatic technologies can greatly streamline the aptamer characterization and sensor development process, and with the adoption of robotics or liquid handling systems in the future, it should be possible to rapidly identify the most suitable aptamers for a particular application from hundreds to thousands of candidates.