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In vitro selection of RNA aptamers against a composite small molecule-protein surface.

ABSTRACT: A particularly challenging problem in chemical biology entails developing systems for modulating the activity of RNA using small molecules. One promising new approach towards this problem exploits the phenomenon of 'surface borrowing,' in which the small molecule is presented to the RNA in complex with a protein, thereby expanding the overall surface area available for interaction with RNA. To extend the utility of surface borrowing to include potential applications in synthetic biology, we set out to create an 'orthogonal' RNA-targeting system, one in which all components are foreign to the cell. Here we report the identification of small RNA modules selected in vitro to bind a surface-engineered protein, but only when the two macromolecules are bound to a synthetic bifunctional small molecule.

SUBMITTER: Plummer KA

PROVIDER: S-EPMC1240114 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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In vitro selection of RNA aptamers against a composite small molecule-protein surface.

Plummer Kelly A KA Carothers James M JM Yoshimura Masahiro M Szostak Jack W JW Verdine Gregory L GL

Nucleic acids research 20050930 17

A particularly challenging problem in chemical biology entails developing systems for modulating the activity of RNA using small molecules. One promising new approach towards this problem exploits the phenomenon of 'surface borrowing,' in which the small molecule is presented to the RNA in complex with a protein, thereby expanding the overall surface area available for interaction with RNA. To extend the utility of surface borrowing to include potential applications in synthetic biology, we set ...[more]

PMID: 16199752

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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 selection of RNA aptamers against a composite small molecule-protein surface.

Publications

In vitro selection of RNA aptamers against a composite small molecule-protein surface.

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