RE-SELEX: restriction enzyme-based evolution of structure-switching aptamer biosensors

• Authors: Aimee A Sanford, Emory University; Alexandra E Rangel, University of Utah, College Of Science; Trevor A Feagin, University of Utah, College Of Science; Robert G Lowery, BellBrook Labs, LLC; Hector S Argueta-Gonzalez, Emory University; Jennifer Heemstra, Emory University
• Journal Title: Chemical Science
• Volume: Volume 12, Number 35
• Publisher: The Royal Society of Chemistry | 2021-09-21, Pages 11692-11702
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1039/d1sc02715h
• Subjects: Chemistry, General
• Keywords: biosensors

Abstract

Aptamers are widely employed as recognition elements in small molecule biosensors due to their ability to recognize small molecule targets with high affinity and selectivity. Structure-switching aptamers are particularly promising for biosensing applications because target-induced conformational change can be directly linked to a functional output. However, traditional evolution methods do not select for the significant conformational change needed to create structure-switching biosensors. Modified selection methods have been described to select for structure-switching architectures, but these remain limited by the need for immobilization. Herein we describe the first homogenous, structure-switching aptamer selection that directly reports on biosensor capacity for the target. We exploit the activity of restriction enzymes to isolate aptamer candidates that undergo target-induced displacement of a short complementary strand. As an initial demonstration of the utility of this approach, we performed selection against kanamycin A. Four enriched candidate sequences were successfully characterized as structure-switching biosensors for detection of kanamycin A. Optimization of biosensor conditions afforded facile detection of kanamycin A (90 μM to 10 mM) with high selectivity over three other aminoglycosides. This research demonstrates a general method to directly select for structure-switching biosensors and can be applied to a broad range of small-molecule targets.