Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications

The utilization of structure-switching aptamers (SSAs) has enabled the development of novel sensing platforms for the sensitive and continuous detection of molecules. De novo development of SSAs, however, is complex and laborious. Here we describe a rational approach to SSA optimization that simultaneously improves aptamer binding affinity and introduces target-dependent conformation-switching for compatibility with real-world biosensor applications. Key structural features identified from NMR and computational modeling were used to optimize conformational switching in the presence of target, while large-scale, microarray-based mutation analysis was used to map regions of the aptamer permissive to mutation and identify combinations of mutations with stronger binding affinity. Optimizations were carried out in a relevant biofluid to ensure a seamless transition of the aptamer to a biosensing platform. Initial proof-of-concept for this approach is demonstrated with a cortisol binding aptamer but can easily be translated to other relevant aptamers. Cortisol is a hormone correlated with the stress response that has been associated with various medical conditions and is present at quantifiable levels in accessible biofluids. The ability to continuously track levels of stress in real-time via cortisol monitoring, which can be enabled by the aptamers reported here, is crucial for assessing human health and performance.