Recommendations for the reduction of compound artifacts in time-resolved fluorescence resonance energy transfer assays.

ABSTRACT

Time-resolved (TR) fluorescence resonance energy transfer (FRET) is a widely accepted technology for high throughput screening (HTS), being able to detect and quantify the interactions of specific biomolecules in a homogeneous format. TR-FRET has several advantages for HTS applications that reduce assay artifacts such as compound interference. However, in some cases artifacts due to compound autofluorescence, color quenching, or signal stability are still observed. This report presents strategies addressing these issues by several means. One recommendation is the recording and visualization of differences in the donor/acceptor fluorescence, which allows the identification of compound artifacts. Another suggestion is to adjust the time delay, between excitation and recording of the fluorescence, in order to reduce compound interference. Furthermore, configuring the assay to allow the TR-FRET measurement to be taken at different time points, creating a reaction time course, allows background correction for each sample. Finally, the optimization of the FRET pair, to ensure assay signal stability under screening conditions, can improve the assay quality. This report presents examples of how these simple steps can be applied to enhance the quality of TR-FRET screening campaigns.