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bioRxiv · 10.64898/2026.02.06.702976

Sensitive and Selective Next-Generation FRET-based PKA Biosensors

Zhang, J.-F.·Lin, W.·Huang, L.·Kim, S. H.·He, X.·Frei, M. S.·Zhong, H.·Mehta, S.·Zhang, J.
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Abstract

The cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway regulates diverse cellular processes through precise spatiotemporal control across subcellular compartments. Forster resonance energy transfer (FRET)-based A-kinase activity reporters (AKARs) have enabled live-cell visualization of PKA activity, but their limited dynamic range constrains detection of subtle or compartment-specific signaling events. Here, we present a suite of sixth-generation cyan/yellow FRET-based PKA sensors (the AKAR6 series) with substantially enhanced sensitivity and improved selectivity. Systematic optimization of the FRET donor-acceptor pair and sensor backbone yields superior performance versus previous best-in-class FRET-based AKARs, enabling robust detection of subtle PKA activity changes across diverse experimental modalities, including flow cytometry, fluorescence lifetime-based FRET, and two-photon imaging in brain slices. We further leverage kinome atlas data to engineer a variant with improved selectivity for more accurate visualization of nuclear PKA activity. Using the AKAR6 toolkit, we showed that in contrast to strong GPCR-induced PKA activities across all tested compartments in PC12 cells, growth factors stimulated a significant PKA activity at the trans-Golgi network but no detectable activity at cis-Golgi, signifying highly compartmentalized PKA signaling at the sub-organelle level. Furthermore, NGF and EGF induced sustained and transient PKA activity, respectively, across various intracellular compartments, including the nucleus, suggesting that growth factor-specific temporal controls are maintained across subcellular compartments. Together, the AKAR6 toolkit provides a sensitive, selective, and versatile platform for dissecting compartmentalized PKA signaling across cells and tissues.

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Zhang, J.-F., Lin, W., Huang, L., Kim, S. H., He, X., Frei, M. S., Zhong, H., Mehta, S., Zhang, J.. 2026-02-09. Sensitive and Selective Next-Generation FRET-based PKA Biosensors. https://doi.org/10.64898/2026.02.06.702976

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