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Improving FRET dynamic range with bright green and red fluorescent proteins


A variety of genetically encoded reporters use changes in fluorescence (or Förster) 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 Förster 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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Figure 1: Assessment of FRET reporter performance using Förster equations and emission spectra.
Figure 2: Clover-mRuby2 and ECFP-Venus responses in the CaMKIIα reporter Camuiα.
Figure 3: Clover-mRuby2 FRET in voltage sensing.
Figure 4: Reporting of fast local RhoA activation in neurons with Raichu-RhoA-CR.

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We thank Y. Hayashi (RIKEN Brain Science Institute) for the Camuiα plasmid, J. Zhang (John Hopkins Medicine) for the AKAR2 plasmid, M. Matsuda (Kyoto University) for the Raichu-RhoA plasmid and P. Ramasamy (Stanford University) for the pcDNA3.1/Puro-CAG plasmid. We thank N. Desai for help with cloning of the voltage sensors, members of the Lin laboratory for helpful discussion, and M.E. Greenberg (Harvard Medical School) for advice and resources during axon guidance experiments. This work was supported by the Burroughs Wellcome Fund (M.Z.L.), a Stanford University Bio-X Interdisciplinary Initiatives Project grant (M.Z.L. and M.J.S.), a Siebel Foundation Scholarship (A.J.L.), the Stanford CNC Program (Y.G., J.D.M. and M.J.S.), the National Academy of Sciences Keck Futures Initiative (Y.G., J.D.M. and M.J.S.), National Science Foundation grant 1134416 (M.Z.L.) and US National Institutes of Health grants R01NS076860 (M.Z.L.) and 4R37NS027177-23 (R.Y.T.). M.Z.L. is a Rita Allen Foundation Scholar.

Author information




M.Z.L. conceived the study. A.J.L., F.S.-P., M.Z.L., Y.G. and J.D.M. designed and performed FRET experiments and analyzed data. M.R.M. and M.Z.L. created and characterized fluorescent protein variants. M.A.B. and M.W.D. created and characterized fluorescent protein targeting fusions. P.J.C. and M.W.D. performed live-cell photobleaching experiments. J.W. provided unique reagents. M.J.S. and R.Y.T. provided ideas and advice. A.J.L., F.S.-P., and M.Z.L. wrote the manuscript.

Corresponding author

Correspondence to Michael Z Lin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–16 (PDF 12717 kb)

Complete time-lapse imaging series of the experiment in Fig. 4e demonstrates reporting of PKA by AKAR2-CR under continuous illumination.

Forskolin was added to 50 μM at time 0 to activate PKA in HEK293 cells expressing AKAR2-CR. Cells were continuously illuminated by 450–470 nm light from a 150-W xenon arc lamp passed through a 10% neutral-density filter, and emission filters were cycled between Clover and mRuby2 wavelengths as quickly as possible. Ratiometric images are shown, in which blue denotes a baseline-normalized mRuby2/Clover emission ratio of 0.8 and red an emission ratio of 1.6. (AVI 3248 kb)

Time-lapse imaging of RhoA activity during ephrin-A–induced growth-cone retraction in an embryonic cortical neuron.

Neurons expressing Raichu-RhoA-CR were treated at 1 d in vitro with 5 μg ml−1 preclustered ephrin-A5 at time 0 and images taken every 2 min. Ratiometric images are shown, in which blue denotes a baseline-normalized mRuby2/Clover emission ratio of 0.9 and red an emission ratio of 1.8. Asterisks mark locations of the growth cone showing transient ephrin-A–induced RhoA activity. (MOV 605 kb)

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Lam, A., St-Pierre, F., Gong, Y. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat Methods 9, 1005–1012 (2012).

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