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A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example
Open accessAbstract
We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors.
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Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., & Tsien, R. Y. (2004) · Nature Biotechnology · doi:10.1038/nbt1037 · Cited by 4,619
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Improving FRET dynamic range with bright green and red fluorescent proteins
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., et al. (2012) · Nature Methods · doi:10.1038/nmeth.2171 · Cited by 732
Raffaello, A., De Stefani, D., Sabbadin, D., Teardo, E., Merli, G., Picard, A., Checchetto, V., Moro, S., Szabò, I., & Rizzuto, R. (2013) · The EMBO Journal · doi:10.1038/emboj.2013.157 · Cited by 490
Klarenbeek, J., Goedhart, J., van Batenburg, A., Groenewald, D., & Jalink, K. (2015) · PLOS ONE · doi:10.1371/journal.pone.0122513 · Cited by 311
Self-citationSingle Cell Optical Imaging and Spectroscopy
Stender, A. S., Marchuk, K., Liu, C., Sander, S., Meyer, M. W., Smith, E. A., Neupane, B., Wang, G., Li, J., Cheng, J.-X., et al. (2013) · Chemical Reviews · doi:10.1021/cr300336e · Cited by 292
Metadata
- Authors
- van der Krogt, G. N. M., Ogink, J., Ponsioen, B., & Jalink, K.
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2008-04-02
- Volume
- 3
- Issue
- 4
- Pages
- e1916
- ISSN
- 1932-6203 (ISSN-L)
- OpenAlex
- W2079245879
- Cited by
- 174
- References
- 1
- FWCI
- 6.06
- Type
- journal article
- Language
- en
- Open access
- gold
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- van der Krogt, G. N. M., Ogink, J., Ponsioen, B., & Jalink, K. (2008). A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example. PLoS ONE. https://doi.org/10.1371/journal.pone.0001916
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