Improving FRET dynamic range with bright green and red fluorescent proteins
Open accessAbstract
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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Tau FRET sensors composed of tau P301S-Ruby2 and -Clover reporters aggregate and transmit FRET signals in the presence of seed-competent tau species.
“These reporter proteins aggregate and transmit FRET signals in the presence of seed-competent tau species (44, 45).”
From Modeling late-onset Alzheimer’s disease neuropathology via direct neuronal reprogramming (2024) · cites this paper as [45]
The Results sections on mRuby2 and RhoA responses show that Clover–mRuby2 functions as a FRET pair in other reporters. Our copy does not address tau P301S reporters, aggregation, or responses to seed-competent tau species; searches for those terms found no supporting passage. (LLM, conf=0.76)
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Self-citation
- Authors
- Lam, A. J., St-Pierre, F., Gong, Y., Marshall, J. D., Cranfill, P. J., Baird, M. A., McKeown, M. R., Wiedenmann, J., Davidson, M. W., Schnitzer, M. J., Tsien, R. Y., & Lin, M. Z.
- Affiliations
- Stanford University (US)
- Stanford Medicine (US)
- Howard Hughes Medical Institute (US)
- Florida State University (US)
- University of California San Diego (US)
- National Oceanography Centre (GB)
- Corresponding author
- Lin, Michael Z
- ORCID
- Venue
- Nature Methods
- Published
- 2012-09-09
- Volume
- 9
- Issue
- 10
- Pages
- 1005-1012
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC3461113
- OpenAlex
- W2059536603
- Cited by
- 732
- References
- 52
- Citations by year
- 2026: 29 · 2025: 32 · 2024: 45 · 2023: 36 · 2022: 48 · 2021: 63 · 2020: 62 · 2019: 63 · 2018: 51 · 2017: 58 · 2016: 65 · 2015: 72 · 2014: 51 · 2013: 50 · 2012: 6
- FWCI
- 24.79
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- ePrints Soton (University of Southampton) (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Cancer Institute (R01CA158448)
- National Institute of Neurological Disorders and Stroke (R01NS076860, R37NS027177, R01NS027177)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Physical Sciences › Materials Science › Materials Chemistry › Luminescence and Fluorescent Materials
- Keywords
- fluorescence resonance energy transfer, genetically encoded biosensors, photostability, photobleaching, photoconversion, neuronal action potentials, RhoA activation, growth cones
- MeSH terms
- Red Fluorescent Protein, Base Sequence, HeLa Cells, Humans, Luminescent Proteins, Molecular Sequence Data, Cyclic AMP-Dependent Protein Kinases, rhoA GTP-Binding Protein, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Calcium-Calmodulin-Dependent Protein Kinase Type 2, HEK293 Cells
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