A red-shifted biosensor for intracellular detection of cAMP: the CUTieR the better

The concentration of cAMP changes inside the cell as part of different signaling pathways generating metabolic responses. Measuring the intracellular fluctuations in cAMP levels is key to understanding the mechanism of action of hormones, compounds, cell-pathogen interactions, and -signaling, among others. Based on coarse-grained simulations, we engineered a second generation of the CUTie cAMP sensor that uses the FRET pair Clover-mRuby2. This red-shifted (CUTieR) architecture overcomes the inconveniences of CFP and YFP emission overlapping spectra, making it suitable for high throughput analysis by flow cytometry (FC). Here we describe the generation and FC-based characterization of CUTieR expressed in two mammalian cell lines routinely used for research and biotechnological purposes.