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Biosensors 2019, 9(1), 33; https://doi.org/10.3390/bios9010033

In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics

Institute of Pharmacology and Toxicology, University of Zurich, 8057 Zurich, Switzerland
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Received: 1 December 2018 / Revised: 12 February 2019 / Accepted: 19 February 2019 / Published: 28 February 2019
(This article belongs to the Special Issue FRET-Based Biosensors)
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Abstract

In previous studies, we encountered substantial problems using the CFP_YFP Förster resonance energy transfer (FRET) pair to analyze protein proximity in the endoplasmic reticulum of live cells. Bleed-through of the donor emission into the FRET channel and overlap of the FRET emission wavelength with highly variable cellular autofluorescence significantly compromised the sensitivity of our analyses. Here, we propose mCerulean3 and mRuby3 as a new FRET pair to potentially overcome these problems. Fusion of the two partners with a trypsin-cleavable linker allowed the direct comparison of the FRET signal characteristics of the associated partners with those of the completely dissociated partners. We compared our new FRET pair with the canonical CFP_YFP and the more recent mClover3_mRuby3 pairs and found that, despite a lower total FRET signal intensity, the novel pair had a significantly better signal to noise ratio due to lower donor emission bleed-through. This and the fact that the mRuby3 emission spectrum did not overlap with that of common cellular autofluorescence renders the mCerulean3_mRuby3 FRET pair a promising alternative to the common CFP_YFP FRET pair for the interaction analysis of membrane proteins in living cells. View Full-Text
Keywords: 3D fluorescence spectra; FAMPIR; fluorescent protein; excitation 3D fluorescence spectra; FAMPIR; fluorescent protein; excitation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Erismann-Ebner, K.; Marowsky, A.; Arand, M. In-Vitro Characterization of mCerulean3_mRuby3 as a Novel FRET Pair with Favorable Bleed-Through Characteristics. Biosensors 2019, 9, 33.

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