A Red Fluorescent Genetically Encoded Biosensor for the Visualization of ATP in Live Cells

Adenosine triphosphate (ATP) serves as the universal energy currency in cellular metabolism. However, real-time analysis of ATP dynamics in prokaryotes remains a challenge due to significant intracellular pH fluctuations and high background interference. To address this, we developed IGAS, a novel genetically encoded biosensor engineered by integrating a binding protein derived from Bacillus subtilis PS3 with the acid-resistant fluorescent protein cpmCherry and miRFP670nano3. Characterization revealed that IGAS exhibits a robust 2.8-fold dynamic range, high selectivity for ATP, and remarkable pH stability. When expressed in E. coli, IGAS enabled real-time monitoring of intracellular ATP fluctuations throughout the bacterial growth cycle, demonstrating high consistency with standard luciferase assays. Furthermore, guided by molecular dynamics (MD) simulations, we identified key residues to engineer IGAS variants with tunable affinities. These sensors were successfully applied to diverse cellular environments, ranging from cytoplasmic targeting to mammalian cell surface display. Collectively, our results demonstrate the excellent reversibility and versatility of IGAS, establishing it as a powerful tool for dynamic ATP detection in complex biological systems.