Dataset Information

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

ABSTRACT: Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

SUBMITTER: Yu H

PROVIDER: S-EPMC5934630 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Yu Haixiang H Yang Weijuan W Alkhamis Obtin O Canoura Juan J Yang Kyung-Ae KA Xiao Yi Y

Nucleic acids research 20180501 8

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dy ...[more]

PMID: 29361056

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Project description:Applications of synthetic biology spanning human health, industrial bioproduction, and ecosystem monitoring often require small molecule sensing capabilities, typically in the form of genetically encoded small molecule biosensors. Critical to the deployment of greater numbers of these systems are methods that support the rapid development of such biosensors against a broad range of small molecule targets. Here, we use a previously developed method for selection of RNA biosensors against unmodified small molecules (DRIVER) to perform a selection against a densely multiplexed mixture of small molecules, representative of those employed in high-throughput drug screening. Using a mixture of 5,120 target compounds randomly sampled from a large diversity drug screening library, we performed a 95-round selection and then analyzed the enriched RNA biosensor library using next generation sequencing (NGS). From our analysis, we identified RNA biosensors with at least 2-fold change in signal in the presence of at least 217 distinct target compounds with sensitivities down to 25 nM. Although many of these biosensors respond to multiple targets, clustering analysis indicated at least 150 different small-molecule sensing patterns. We also built a classifier that was able to predict whether the biosensors would respond to a new compound with an average precision of 0.82. Since the target compound library was designed to be representative of larger diversity compound libraries, we expect that the described approach can be used with similar compound libraries to identify aptamers against other small molecules with a similar success rate. The new RNA biosensors (or their component aptamers) described in this work can be further optimized and used in applications such as biosensing, gene control, or enzyme evolution. In addition, the data presented here provide an expanded compendium of new RNA aptamers compared to the 82 small molecule RNA aptamers published in the literature, allowing further bioinformatic analyses of the general classes of small molecules for which RNA aptamers can be found.

Dataset Information

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

Publications

In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality.

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