ATPLyzer—An Advanced Ratiometric Multi‐Color Biosensor for Long‐Term Monitoring of ATP Dynamics

ABSTRACT

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. Multi‐color ATPLyzer variants with green‐orange (GO) and green‐apple (GA) consist of cpGFP‐based green reporter and Large Stokes Shift reference FPs (LSSmOrange or LSSmApple). Additionally, ATPLyzer affinity variants display different dissociation constants (K _D ’s) in the millimolar and micromolar range in vitro and exhibit high specificity for ATP over ADP. Further non‐binding variants were created serving as controls in in vivo applications. 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 tunable tool for minimally invasive, time‐resolved monitoring of intracellular ATP dynamics.