A luminescent biosensor with increased dynamic range for intracellular cAMP.

The second messenger cAMP is a key mediator of signal transduction following activation of G-protein coupled receptors. Investigations on Gs-coupled receptors would benefit from a second messenger assay that allows continuous monitoring of kinetic changes in cAMP concentration over a broad dynamic range. To accomplish this, we have evolved a luminescent biosensor for cAMP to better encompass the physiological concentration ranges present in living cells. When compared to an immunoassay, the evolved biosensor construct was able to accurately track both the magnitude and kinetics of cAMP change using a far less labor intensive format. We demonstrate the utility of this construct to detect a broad range of receptor activity, together with showing suitability for use in high-throughput screening.