Papadopoulos, A.; Kaiser, C. F.; Schlumpberger, P.; Esser, J.; Reiners, J.; Gertzen, C. G. W.; Grossmann, G.; Smits, S.

Adenosine triphosphate (ATP) is a central molecule in cellular metabolism, serving as the primary energy currency that links catabolic and anabolic pathways. Monitoring intracellular ATP in vivo is essential for understanding the dynamics of metabolic states, as well as intracellular functions and intercellular interactions in health and disease. We report the design and application of ATPLyzer, a series of genetically encoded, ratiometric biosensors for the monitoring of ATP levels in living cells. The matryoshka design consists of an ATP-binding cassette linked to a circularly permutated GFP coupled with an internal large stokes shift reference fluorophore, allowing for single-wavelength excitation and ratiometric output. This design overcomes limitations of conventional biosensors, reliance on dual excitation wavelengths, and susceptibility to photobleaching. Multi-colour ATPLyzer variants with different dissociation constants were characterized in vitro, exhibiting high specificity for ATP over ADP. Monitoring ATP in Escherichia coli confirmed in vivo utility and revealed growth-phase and carbon-supply-dependent ATP dynamics. The ATPLyzer biosensor offers a robust and tuneable tool for minimally invasive, time-resolved monitoring of intracellular ATP dynamics.