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Theinterconversionofguanosinetriphosphate(GTP)andguanosinediphosphate(GDP)isintegraltoawidevarietyofbiologicalcellularactivities~([1]).However,analyticalmethodswhichdirectlydetecttheratioofintracellularGTPandGDPconcentrationshavenotbeenavailable.Herein,wereportGNEPS,ageneticallyencodedfluorescentsensorthatenablesreal-timemonitoringoftheGTP:GDPratio,whichisafusionproteincomprisingaeukaryoticG-proteinandacircularlypermutedyellowfluorescentprotein.GNEPShasdistinctfluorescencespectrabetweenitsGTP-boundandGDP-boundstates.ItsapparentfluorescencesignalthereforedependsuponthecompetitivebindingofGTPandGDP.LivecellimagingexperimentsdemonstratedthatGNEPScanbeusedtomonitorspatiotemporalchangesintheintracellularGTP:GDPratioinvariouscelltypesandorganellesinresponsetometabolicperturbations~([2]).WeanticipatethatGNEPSwillbecomeavaluabletoolforunderstandingthemetabolicandregulatorycontributionsofguanosinenucleotides.