The purpose of this project is to enhance the capabilities of a novel drug discovery platform that has already been brought to commercialization. Specifically, the focus is live cell fluorescence resonance energy transfer (FRET) assays monitored by changes in the donor?s fluorescence lifetime using our direct waveform recording (DWR) methodology, which affords exceptional speed and precision. Work to date has employed a green fluorescent protein (GFP) donor and a red fluorescent protein (RFP) acceptor, which involves excitation with a 473 nm microchip laser. It has long been our hope to switch the excitation to 532 nm, for which much better pulsed lasers are available. Among the benefits of shifting to longer excitation wavelength are 4X higher repetition rate (20 kHz vs. 5 kHz), 3X shorter pulse duration (0.75 ns vs. 2.5 ns), better pulse-to-pulse stability, better beam quality, and lower cost. Furthermore, the shift to longer wavelength reduces the occurrence of compound fluorescence, cellular autofluorescence, and light-induced cell damage. An attractive candidate FRET pair, just reported in the second half of 2016, employs an orange fluorescent protein (OFP) donor and a maroon fluorescent protein (MFP) acceptor. In addition to the above advantages It has a larger R0 and therefore greater FRET changes compared to the GFP-RFP pair. Preliminary work, using the human calcium pump as a model system, demonstrates that the change with the new FRET pair results in a nearly 2X increase in the signal change, and 2-3X reduction in the incidence of interference arising from compound fluorescence. The previous instrument, using 473 nm excitation for GFP, was developed during a Phase 1 and Phase 2 NIH STTR project and further refined with contract funding from a major pharma collaborator. The most recent form of such an instrument has two independent channels in which the fluorescence lifetime is monitored. One channel is near the peak of the donor emission. By strategically placing the wavelength of the second channel on the short wavelength end of the donor emission, even small amounts of compound fluorescence can be discerned and flagged. Additional strategies to further improve performance will be investigated in Phase 1, including optimization of cell density, focusing in well, evaluation of alternative digitizers and optimized sampling rate. With successful completion of the Phase 1 work, we 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 biosensors. New biosensors for a wide range of therapeutic targets will be developed and applied in Phase 2.
Project Terms: Adopted; Algorithms; Biological Assay; Biological Models; Biosensor; Ca(2+)-Transporting ATPase; Cell Density; cell injury; Cells; Cellular biology; Color; commercialization; Contracts; cost; Detection; drug discovery; Drug Screening; Economics; Evaluation; Fluorescence; Fluorescence Resonance Energy Transfer; Funding; Goals; Green Fluorescent Proteins; high throughput screening; Human; improved; Incidence; Industry; innovation; instrument; instrumentation; Investigation; Lasers; Lead; Letters; Libraries; Light; Measurement; Methodology; microchip; microscopic imaging; Molecular Biology; Monitor; novel therapeutics; Oranges; Performance; Phase; photonics; Physiologic pulse; Process; Proteins; Publications; red fluorescent protein; Reporting; Research; Rest; Sampling; SERCA2a; Signal Transduction; Small Business Technology Transfer Research; small molecule; Speed; Technology; Testing; therapeutic target; United States National Institutes of Health; Work;
