Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

Abstract

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 minutes. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded GFP donor and RFP acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate-reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells

“…Because FLT measurements are susceptible to interference from fluorescence of the test compound itself, we also acquired fluorescence emission spectra by scanning the same 384-well plates using a recently developed spectral plate reader 32 (Fig. 4c ) and applied a spectral similarity index to flag fluorescent compounds 36 , 37 . In total, 21 compounds were identified as Hits, which altered FRET from SERCA2a to PLB in at least 2 replicates after filtering out compounds with interfering fluorescence (false positives).…”

“…This instrument uses a unique direct waveform recording technology that enables high-throughput FLT detection at high precision 69 . We have previously demonstrated the performance of this plate reader with known fluorescence standards, as well as with a FRET-based HTS method that targets SERCA 22 , 36 , 37 .…”

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays

“…We acquired both FLT waveforms and fluorescence spectra, as previously described 19,33,34 . As previously observed 19 , Hit effects were greatest following a 2-hour incubation with the library compounds ( Supplementary Fig.…”

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

“…Schaaf et al used a highly sophisticated spectral unmixing plate reader to measure intramolecular CFP-YFP FRET from an ER-targeted calcium sensor as a follow-up 17 to a screen involving FRET between green fluorescent protein (GFP) and red fluorescent protein (RFP) that measured activity-associated conformational changes in SERCA ATPases. 18 Honarnejad et al used a confocal imaging plate reader to measure intracellular calcium using a CFP-YFP FRET reporter construct as part of a screen for compounds that can inhibit amyloid fibril formation. 19 As conventional fluorescent plate readers are commonly available, and intramolecular FRET between CFP and YFP serves as the basis of a multitude of different reporter constructs, 20 we thought that it might be useful to define the expression level and FRET ratio change needed to measure FRET reliably in a fluorescent plate reader, as this might encourage more widespread adoption of intramolecular CFP-YFP FRET sensors for screening.…”