Journal
NATURE BIOTECHNOLOGY
Volume 23, Issue 3, Pages 355-360Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt1066
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Funding
- NIBIB NIH HHS [EB-000205] Funding Source: Medline
- NIGMS NIH HHS [F32 GM111018] Funding Source: Medline
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Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence(1). FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations(2), binding interactions(3) and catalytic activity(4), but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.
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