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

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.

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

“…Our approach to developing this drug discovery pipeline has been consistent to previous efforts with other biosensor systems (e.g. tau and TNFR1, amongst others) 84,[90][91][92][103][104][105] . At the core of any fluorescent assay is the potential of artifacts being introduced into the model system through the labeling of protein with synthetic fluorophores or fusion to large fluorescent XFPs.…”

“…Another concern with HTS campaigns is the physiological relevance of the cellular platform being deployed. The choice of HEK293 cells for our FRET HTS primary assay implementation is strongly rooted in the thorough vetting of this cell lines performance in multiple HTS campaigns that interrogated a wide range of protein targets 84,[90][91][92][103][104][105] . We acknowledge that not using neuronal cell lines in a primary HTS platform targeting neurodegeneration imparts a disconnect between the physiological and epigenetic link between cell type and disease environment.…”

“…To generate the aSyn FRET constructs aSyn cDNA was cloned into a GFP-linker-RFP plasmid (linker contains 32 amino acids, GFP-32AA-RFP) that was previously characterized. 106 . QuikChange mutagenesis (Agilent Technologies, Santa Clara, CA) was performed to monomerize the GFP via A206K mutation and to produce the donor GFP-aSyn construct.…”

“…Cell cultures were maintained in an incubator with 5% CO2 (Forma Series II Water Jacket CO2 Incubator, Thermo Scientific) at 37℃. The intermolecular (oligomer) and intra-molecular (monomer-conformation) aSyn FRET biosensors were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 (Invitrogen) with GFP-aSyn and aSyn-RFP (1:8 DNA plasmid concentration ratio) or GFP-aSyn-RFP plasmid, respectively The effectiveness of HEK293 cells transfected with FRET constructs as a HTS platform has been demonstrated in our previous work 84, [90][91][92][103][104][105] .…”

A novel small molecule screening platform for disrupting toxic tau oligomers in cells

“…Stable cell lines expressing GFP-tau-RFP or tau-GFP with the largest population of expressing cells were selected by fluorescence microscopy. The GFPlinker-RFP (linker contains 32 amino acids, GFP-32AA-RFP) control stable cell line was generated as described previously 68 . The control cells expressing only free soluble fluorophores (GFP or RFP only) were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 with plasmids containing GFP or RFP DNAs at the same plasmid concentration as the intermolecular tau FRET biosensor.…”