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Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization

Nature Protocols volume 3, pages 579–587 (2008)Cite this article

Abstract

Small molecule inhibitors of proteins are invaluable tools in research and as starting points for drug development. However, their screening can be tedious, as most screening methods have to be tailored to the corresponding drug target. Here, we describe a detailed protocol for a modular and generally applicable assay for the identification of small organic compounds that displace an aptamer complexed to its target protein. The method relies on fluorescence-labeled aptamers and the increase of fluorescence polarization upon their binding to the target protein. The assay has high Z′-factors, making it compatible with high-throughput screening. It allows easy automation, making fluorescence readout the time-limiting step. As aptamers can be generated for virtually any protein target, the assay allows identification of small molecule inhibitors for targets or individual protein domains for which no functional screen is available. We provide the step-by-step protocol to screen for antagonists of the cytohesin class of small guanosine exchange factors.

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Figure 1: Schematic representation of the ideal scenario probed by the aptamer-displacement screening assay by fluorescence polarization.
Figure 2: Workflow for the setup and execution of a fluorescence-polarization screen.
Figure 3: Nine possible modes of action for the compound in an aptamer-displacement assay system.
Figure 4: Screening setup to use M69 in a robust aptamer-displacement assay to screen for inhibitors of cytohesin (Cyh)1-Sec7.
Figure 5: Representative example of the screening results of a box of 88 compounds with the structures of three hits indicated.

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  1. Program Unit Chemical Biology & Medicinal Chemistry, LIMES Institute, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, Bonn, 53121, Germany

    Markus Hafner, Elena Vianini, Barbara Albertoni, Laura Marchetti, Imke Grüne, Christian Gloeckner & Michael Famulok

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Correspondence to Michael Famulok.

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Hafner, M., Vianini, E., Albertoni, B. et al. Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization. Nat Protoc 3, 579–587 (2008). https://doi.org/10.1038/nprot.2008.15

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