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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.
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DNA Aptamer–Cyanine Complexes as Generic Colorimetric Small‐Molecule Sensors
Abstract Aptamers are promising biorecognition elements for sensors. However, aptamer‐based assays often lack the requisite levels of sensitivity and/or selectivity because they typically employ structure‐switching aptamers with attenuated affinity and/or utilize reporters that require aptamer labeling or which are susceptible to false positives. Dye‐displacement assays offer a label‐free, sensitive means for overcoming these issues, wherein target binding liberates a dye that is complexed with the aptamer, producing an optical readout. However, broad utilization of these assays has been limited. Here, we demonstrate a rational approach to develop colorimetric cyanine dye‐displacement assays that can be broadly applied to DNA aptamers regardless of their structure, sequence, affinity, or the physicochemical properties of their targets. Our approach should accelerate the development of mix‐and‐measure assays that could be applied for diverse analytical applications.
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- Award ID(s):
- 2135005
- PAR ID:
- 10361563
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 61
- Issue:
- 3
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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