Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules
- 16 April 2020
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 92 (10), 6909-6917
- https://doi.org/10.1021/acs.analchem.9b05563
Abstract
Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically-labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, L-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte, is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.Keywords
Funding Information
- Division of Chemistry (CHE-1608949, CHE-1904424, CHE-1904885)
- Air Force Institute of Technology
- Division of Chemical, Bioengineering, Environmental, and Transport Systems (CBET-1818476)
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