Long-Wavelength FRET Pairs for Fluorescence Lifetime HTS Assays in Living Cells

Project Summary The purpose of this project is to enhance the capabilities of a novel drug discovery platform that has already been brought to commercializationSpecificallythe focus is live cell fluorescence resonance energy transferFRETassays monitored by changes in the donor s fluorescence lifetime using our direct waveform recordingDWRmethodologywhich affords exceptional speed and precisionWork to date has employed a green fluorescent proteinGFPdonor and a red fluorescent proteinRFPacceptorwhich involves excitation with anm microchip laserIt has long been our hope to switch the excitation tonmfor which much better pulsed lasers are availableAmong the benefits of shifting to longer excitation wavelength areX higher repetition ratekHz vskHzX shorter pulse durationns vsnsbetter pulse to pulse stabilitybetter beam qualityand lower costFurthermorethe shift to longer wavelength reduces the occurrence of compound fluorescencecellular autofluorescenceand light induced cell damageAn attractive candidate FRET pairjust reported in the second half ofemploys an orange fluorescent proteinOFPdonor and a maroon fluorescent proteinMFPacceptorIn addition to the above advantages It has a larger Rand therefore greater FRET changes compared to the GFP RFP pairPreliminary workusing the human calcium pump as a model systemdemonstrates that the change with the new FRET pair results in a nearlyX increase in the signal changeandX reduction in the incidence of interference arising from compound fluorescenceThe previous instrumentusingnm excitation for GFPwas developed during a Phaseand PhaseNIH STTR project and further refined with contract funding from a major pharma collaboratorThe most recent form of such an instrument has two independent channels in which the fluorescence lifetime is monitoredOne channel is near the peak of the donor emissionBy strategically placing the wavelength of the second channel on the short wavelength end of the donor emissioneven small amounts of compound fluorescence can be discerned and flaggedAdditional strategies to further improve performance will be investigated in Phaseincluding optimization of cell densityfocusing in wellevaluation of alternative digitizers and optimized sampling rateWith successful completion of the Phaseworkwe believe the new FRET pair will quickly replace the existing GFP RFP pair as the one of choice for high throughput screening using live cell biosensorsNew biosensors for a wide range of therapeutic targets will be developed and applied in PhaseProject Narrativesentences maxThis research seeks a dramatic improvement in the reliability of high throughput drug screening in living cellsby shifting fluorescence detection to longer wavelengthsThis involves retooling of cell and molecular biologyas well as instrumentationThe goal is to improve the speedprecisionand economic efficiency of this critical early phase of drug discovery