FRET-sensors have become important tools for intracellular imaging, but their dependence on external illumination presents some limitations, such as photobleaching and phototoxicity, which limit measurements over extended periods of time. Fluorescence measurements also suffer from autofluorescence and light scattering, which hampers in vivo imaging and measurements in strongly absorbing and scattering media such as blood. In principle, these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET). The recent development of brighter and more stable luciferases and the concomitant improvement in luciferase substrates have substantially decreased the sensitivity gap between fluorescence and bioluminescence. As a result, the application of BRET-sensors is no longer restricted to measurements on cell populations, but they can also be used for imaging of single living cells, and BRET has started to emerge as an attractive sensor format for point-of-care diagnostics. The aim of this chapter is to first provide a brief overview of the basic design principles for BRET-sensors. Next, important design considerations will be discussed in more detail by describing the development of three different classes of BRET-sensors, both from our own work and that of others. These examples are all based on the NanoLuc luciferase, a bright and very stable blue light-emitting luciferase developed by Promega that has quickly risen to prominence in the bioluminescence field.
Bibliografische notapart of a special issue:"Enzymes as Sensors"
Edited by Richard B. Thompson Carol A. Fierke