How to Make the CUTiest Sensor in Three Simple Steps for Computational Pedestrians

Genetically encoded FRET sensors for revealing local concentrations of second messengers in living cells have enormously contributed to our understanding of physiological and pathological processes. However, the development of sensors remains an intricate process. Using simulation techniques, we recently introduced a new architecture to measure intracellular concentrations of cAMP named CUTie, which works as a FRET tag for arbitrary targeting domains. Although our method showed quasi-quantitative predictive power in the design of cAMP and cGMP sensors, it remains intricate and requires specific computational skills. Here, we provide a simplified computer-aided protocol to design tailor-made CUTie sensors based on arbitrary cyclic nucleotide-binding domains. As a proof of concept, we applied this method to construct a new CUTie sensor with a significantly higher cAMP sensitivity (EC50 = 460 nM).This simple protocol, which integrates our previous experience, only requires free web servers and can be straightforwardly used to create cAMP sensors adapted to the physicochemical characteristics of known cyclic nucleotide-binding domains.