Monitoring the Dynamic Regulation of the Mitochondrial GTP‐to‐GDP Ratio with a Genetically Encoded Fluorescent Biosensor

The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is known to be integral to a wide variety of biological cellular activities, yet to date there are no analytical methods available to directly detect the ratio of intracellular GTP to GDP. Herein, we report GRISerHR, a genetically encoded fluorescent biosensor to monitor the GTP : GDP ratio in multiple cell types and in various organelles under metabolic perturbation. Additionally, we characterized the differential mitochondrial GTP : GDP ratios resulting from genetic modulation of two isoforms of a tricarboxylic acid (TCA) cycle enzyme (succinyl-CoA synthetase; SCS-ATP and SCS-GTP) and of a phosphoenolpyruvate (PEP) cycle enzyme (PEPCK-M). Thus, our GRISerHR sensor achieves spatiotemporally precise detection of dynamic changes in the endogenous GTP : GDP ratio in living cells and can help deepen our understanding about the energy metabolic contributions of guanosine nucleotides in biology.