Generation of a FRET-based sensor for real time imaging of NADPH in living cells

The aim of this project was to generate a group of fluorescence resonance energy transfer (FRET) biosensors for the detection of the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) in live cells. Taking into consideration the relevant role of NADPH in several metabolic pathways and the lack of reliable methods to assess NADPH levels, the development of new tools that would allow us to dissect the role of this essential cofactor at the subcellular level is of great importance and wide applicability.

As planned in the proposal, we constructed several variations of the sensor using different fluorophores and linkers. They were all successfully expressed in our cell system. Unfortunately, none of them were found to be specifically reacting with NADPH, most likely due to inadequate spatial conformation of the active fluorophores.