Coupling RNA annealing and strand displacement: a FRET-based microplate reader assay for RNA chaperone activity.
Proteins with RNA chaperone activity help RNAs to obtain their native conformations, and many of them are active in the two basic reactions-RNA annealing and strand displacement. Therefore, we developed a time-saving in vitro assay that detects protein-facilitated annealing and strand displacement of fluorophore-labeled oligoribonucleotides in a microplate reader The two reactions are followed byfluorescence resonance energy transfer (FRET) in real-time, and the effect of the proteins on the reaction constants can be quantified. The high-throughput property of the fluorescence microplate reader the kinetic characterization, and the material-saving aspect of this assay enables a fast and convenient classification of proteins according to their RNA chaperone activity in annealing and strand displacement.
Rajkowitsch
Lukas
L
Max F. Perutz [corrected] Laboratories, University of Vienna, Vienna, Austria.
Schroeder
Renée
R
D001665:Binding Sites; D001681:Biological Assay / Q000379:methods; D003199:Computer Systems; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018832:Molecular Chaperones; D016601:RNA-Binding Proteins; D012313:RNA