FRET Based High-Throughput Screening


Advanced molecular imaging tools combined with investigative tools like biochemical and cell based assays have thepotential to unravel complex molecular processes. These tools combined with high throughput screening can significantly impact diagnostics for cancer screening and accelerate drug discovery. These inventions are two new Fluorescence Resonance Energy Transfer (FRET) biosensor pairs, one composed of two new colors, mOrange2 and mCherry and the other composed of a modified high-sensitive ECFP/YPet pair that can significantly enhance the dynamic range of a variety of biosensors. The ECFP/YPet can detect signaling events with high spatio-temporal resolutions which makes it an ideal readout indicator for high throughput screening.

FRET technology and genetically encoded FRET biosensors are very useful in detecting active molecular events inlive cells with high temporal and spatial resolutions. FRET occurs when two flurophores are in proximity with the emission spectrum of the donoroverlapping with the excitation spectrum of the acceptor. To date, the most popular FRET pair is cyan and yellow fluorescent proteins (CFP and YFP).

The new FRET biosensor pairs mOrange2/mCherry are proteins with different colors and spectrally distinctive from the CFP/YFP pair. This invention opens up the possibility of lighting two diagnostic biomarkers in the same cell e.g., cancer cells, thus providing a double criterion high-fidelity assay to differentiate cancer vs. normal cells.

To provide proof-of-concept that mOrange2 and mCherry are suitable as novel FRET biosensors, they were operably linked to the protein recognition sequence of MMP-MT1. MMP-MT1 is an enzyme belonging to the matrix metalloproteinase family that has known roles in cancer metastasis. The basis of this assay is that when MT1-MMP is inactive, the mCherry and mOrange2 and positioned in proximity and favor a strong FRET between the two moieties. Indeed, in-vitro assays established that the mOrange/mCherry pair can serve as a reliable and sensitive indicator of the status of MT1-MMP activation and can potentially be applied to other biomarker assays.

The ECFP/YPet pair provides a high-sensitive biosensor for the visualization of molecular hierarchy at different subcellular locations inlive cells. In-vitro assays revealedthat the ECFP/YPet pair exhibits significantly enhanced dynamic range of the MT1-MMP biosensor compared to currently available CFP/YFP FRET pairs. When quantified, the ECFP/YPet pair showed a 570% change (% change in basal level upon stimulation of MT1-MMP) when compared to only 90-100% in existing FRET pairs. The ECFP/YPet pair has also been successfully applied to other classes of proteins, like kinases, that are important therapeutic targets in human cancer.

These and other genetically engineered biosensors can serve as a research tool to monitor different signaling cascades in live mammalian cells with high sensitivity. The developed MT1-MMP biosensor and potentially other protein biosensors can provide a powerful tool for the spatiotemporal imaging of protein functions in cancer development e.g. detection of circulating tumor cells (CTCs). Furthermore, these biosensors can serve as an excellent high-throughput reporting system forthe detection of cancer and the development of inhibitors for cancer therapeutics. 


The novel mOrange2/mCherry pair presents a method to simultaneously visualize two active signaling events in the same cell when combined with existing FRET pairsThe ECFP/YPet pair can serve as a high-sensitive biosensor with significantly enhanced dynamic ranges compared to existing CFP/YFP FRET pairs. The ECFP/Ypet pair can be operably linked to any protein recognition sequence to detect activity of that protein in live cells. The pair also includes a positively charged tag which allows 100% efficiency ofdetection. This invention demonstrates an ECFP/Ypet pair operably linked to MT1-MMP, a tumor metastasis biomarker, to accurately detect circulating tumor cells in blood samples.