Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization.
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.
Hafner
Markus
M
Program Unit Chemical Biology & Medicinal Chemistry, LIMES Institute, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
Vianini
Elena
E
Albertoni
Barbara
B
Marchetti
Laura
L
Grüne
Imke
I
Gloeckner
Christian
C
Famulok
Michael
M
D052157:Aptamers, Nucleotide / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
D052157:Aptamers, Nucleotide; D020662:Guanine Nucleotide Exchange Factors