Red-shifted FRET biosensors for high-throughput fluorescence lifetime screening

Tory M. Schaaf, Ang Li, Benjamin D. Grant, Kurt Peterson, Samantha Yuen, Prachi Bawaskar, Evan Kleinboehl, Ji Li, David D. Thomas, Gregory D. Gillispie

Research output: Contribution to journalArticle

3 Scopus citations

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.

Original languageEnglish (US)
Article number99
JournalBiosensors
Volume8
Issue number4
DOIs
StatePublished - Oct 24 2018

Keywords

  • Biosensor
  • Drug screening
  • Fluorescence
  • Fluorescent proteins
  • High-throughput
  • Plate reader
  • SERCA
  • Small-molecule
  • Time-resolved FRET

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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. (2018). Red-shifted FRET biosensors for high-throughput fluorescence lifetime screening. Biosensors, 8(4), [99]. https://doi.org/10.3390/bios8040099