Rational Approach to Optimizing Conformation-Switching Aptamers for Biosensing Applications.

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensor Development

Background:

• Structure-switching aptamers (SSAs) are crucial for sensitive molecular detection but their de novo development is complex.
• Optimizing SSAs requires balancing binding affinity with target-dependent conformational changes for biosensor integration.

Purpose of the Study:

• To develop a rational optimization strategy for SSAs that improves binding affinity and conformational switching.
• To ensure aptamer optimization is compatible with real-world biosensor applications and relevant biofluids.
• To demonstrate proof-of-concept using a cortisol-binding aptamer for stress monitoring.

Main Methods:

• Utilized NMR and computational modeling to identify key structural features for optimizing conformational switching.
• Employed large-scale, microarray-based mutation analysis to map mutation-permissive regions and enhance binding affinity.
• Performed optimizations in relevant biofluids to facilitate direct biosensor integration.

Main Results:

• Successfully optimized SSA structure-switching and binding affinity through a rational design approach.
• Identified specific mutations that enhance aptamer performance in relevant biological conditions.
• Demonstrated the approach's efficacy with a cortisol-binding aptamer, showing potential for real-time stress monitoring.

Conclusions:

• The described rational approach significantly simplifies and improves SSA development for biosensing.
• Optimized SSAs, exemplified by the cortisol aptamer, can enable continuous monitoring of biomarkers for health assessment.
• This methodology is translatable to other aptamers, broadening its impact on biosensor technology.