<p><b>ABSTRACT </b></p>
<p>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 an 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 – 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.</p>
Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation
