Comparison of Aptamer Signaling Mechanisms Reveals Disparities in Sensor Response and Strategies to Eliminate False Signals.

Abstract

Aptamers are nucleic acid-based affinity reagents that have been incorporated into a variety of molecular sensor formats. However, many aptamer sensors exhibit insufficient sensitivity and specificity for real-world applications, and although considerable effort has been dedicated to improving sensitivity, sensor specificity has remained largely neglected and understudied. In this work, we have developed a series of sensors using aptamers for the small-molecule drugs flunixin, fentanyl, and furanyl fentanyl and compare their performance─in particular, focusing on their specificity. Contrary to expectations, we observe that sensors using the same aptamer operating under the same physicochemical conditions produce divergent responses to interferents depending on their signal transduction mechanism. For instance, aptamer beacon sensors are susceptible to false-positives from interferents that weakly associate with DNA, while strand-displacement sensors suffer from false-negatives due to interferent-associated signal suppression when both the target and interferent are present. Biophysical analyses suggest that these effects arise from aptamer-interferent interactions that are either nonspecific or induce aptamer conformational changes that are distinct from those induced by true target-binding events. We also demonstrate strategies for improving the sensitivity and specificity of aptamer sensors with the development of a "hybrid beacon," wherein the incorporation of a complementary DNA competitor into an aptamer beacon selectively hinders interferent─but not target─binding and signaling, while simultaneously overcoming signal suppression by interferents. Our results highlight the need for systematic and thorough testing of aptamer sensor response and new aptamer selection methods that optimize specificity more effectively than traditional counter-SELEX.