Minimizing Artifacts in BRET and FRET Assays: Novel Controls for Enhanced RET Accuracy in Mammalian Systems

Resonance energy transfer (RET) techniques, such as fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), are powerful tools for probing protein-protein interactions, conformational changes, and post-translational modifications in live cells. These methods leverage the non-radiative transfer of energy from a donor molecule to an acceptor, offering real-time, highly sensitive insights into molecular dynamics within their native environment. However, RET assays are prone to concentration-dependent artifacts. At elevated concentrations, donor and acceptor molecules leading to non-specific energy transfer, which may produce misleading or false-positive signals. In contrast, low concentrations can reduce signal intensity, diminishing the sensitivity and accuracy of the measurements. To systematically address these limitations and refine the applicability of FRET and BRET in cellular studies, we designed and tested a variety of RET constructs. Specifically, mEGFP was used as a FRET donor in combination with the mCherry acceptor, and as a BRET acceptor in conjunction with the Nluc donor. These constructs were expressed both in the cytosol and at the plasma membrane, allowing for a comparative evaluation of RET efficiency in different cellular compartments. Our study demonstrates the critical need for precise control of donor and acceptor concentrations to mitigate non-specific energy transfer and ensure accurate detection of protein interactions. This work underscores the importance of optimizing experimental conditions for RET-based assays to enhance the reliability of FRET and BRET as investigative tools in live-cell protein interaction studies.