Two Novel Red-FRET ERK Biosensors in the 670-720nm Range.

DeCuzzi, N. L.; Hu, J. Y.; Xu, F.; Rodriguez, B.; Pargett, M.; Albeck, J. G.

doi:10.1101/2024.11.30.626109

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bioRxiv · 10.1101/2024.11.30.626109

Two Novel Red-FRET ERK Biosensors in the 670-720nm Range.

DeCuzzi, N. L.·Hu, J. Y.·Xu, F.·Rodriguez, B.·Pargett, M.·Albeck, J. G.

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Abstract

Cell fate decisions are regulated by intricate signaling networks, with Extracellular signal-Regulated Kinase (ERK) being a central regulator. However, ERK is rarely the only signaling pathway involved, creating a need to study multiple signaling pathways simultaneously at the single-cell level. Many existing fluorescent biosensors for ERK and other pathways have significant spectral overlap, limiting their ability to be multiplexed. To address this limitation, we developed two novel red-FRET ERK biosensors, REKAR67 and REKAR76, which operate in the 670-720 nm range using miRFP670nano3 and miRFP720. REKAR67 and REKAR76 differ in fluorophore position, which impacts biosensor characteristics; REKAR67 displayed a higher dynamic range but greater signal variance than REKAR76. Mixed populations of REKAR67 or REKAR76 displayed similar Signal-to-Noise ratio (SNR), but in clonal cell populations, REKAR76 had a significantly higher SNR. Overall, our red-FRET ERK biosensors were highly consistent with existing ERK FRET biosensors and in reporting ERK activity and are spectrally compatible with CFP/YFP FRET and cpGFP -based biosensors. Both REKAR biosensors expand the available methods for measuring single-cell ERK activity.

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DeCuzzi, N. L., Hu, J. Y., Xu, F., Rodriguez, B., Pargett, M., Albeck, J. G.. 2024-12-02. Two Novel Red-FRET ERK Biosensors in the 670-720nm Range.. https://doi.org/10.1101/2024.11.30.626109

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