Journal
NATURE METHODS
Volume 9, Issue 10, Pages 1005-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.2171
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Funding
- Burroughs Wellcome Fund
- Stanford University Bio-X Interdisciplinary Initiatives Project grant
- Siebel Foundation Scholarship
- Stanford CNC Program
- National Academy of Sciences Keck Futures Initiative
- National Science Foundation [1134416]
- US National Institutes of Health [R01NS076860, 4R37NS027177-23]
- Div Of Chem, Bioeng, Env, & Transp Sys
- Directorate For Engineering [1134416] Funding Source: National Science Foundation
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A variety of genetically encoded reporters use changes in fluorescence (or Forster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Forster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.
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