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Biosensors 2018, 8(4), 99; https://doi.org/10.3390/bios8040099

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

1
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA
2
Fluorescence Innovations Inc., Minneapolis, MN 55455, USA
3
Photonic Pharma LLC, Minneapolis, MN 55410, USA
*
Authors to whom correspondence should be addressed.
Received: 20 September 2018 / Revised: 16 October 2018 / Accepted: 19 October 2018 / Published: 24 October 2018
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Abstract

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets. View Full-Text
Keywords: time-resolved FRET; biosensor; drug screening; SERCA; small-molecule; high-throughput; plate reader; fluorescence; fluorescent proteins time-resolved FRET; biosensor; drug screening; SERCA; small-molecule; high-throughput; plate reader; fluorescence; fluorescent proteins
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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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Schaaf, T.M.; Li, A.; Grant, B.D.; Peterson, K.; Yuen, S.; Bawaskar, P.; Kleinboehl, E.; Li, J.; Thomas, D.D.; Gillispie, G.D. Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening. Biosensors 2018, 8, 99.

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