A Dual-Readout F2 Assay That Combines Fluorescence Resonance Energy Transfer and Fluorescence Polarization for Monitoring Bimolecular Interactions

Abstract Forster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed “Dual-Readout F2 assay” with F2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 pr...