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Design of an ultrabright biosensor for dynamic imaging of kinase activity in cells

Li, X.; Tan, S. K.; Chung, C.-I.; Hatstat, K. A.; Zhao, Q.; Luo, J.; DeGrado, W. F.; shu, X.

2025-08-16 bioengineering
10.1101/2025.08.13.669976 bioRxiv
Show abstract

Protein kinases regulate almost every major signaling pathway. Visualizing spatiotemporal dynamics of kinase activity is thus essential to understand cell signaling. Here we report a de novo-designed activity reporter of kinase, dubbed NOVARK, which contains a single polypeptide chain with multiple modular motifs that act as specific kinase substrates and reporters. NOVARK undergoes phosphorylation-induced higher order-assembly, which are detectable as ultrabright GFP droplets with a greater dynamic range than existing Forster resonance energy transfer-based kinase reporters. We designed versions of NOVARK that rapidly and reversibly report intracellular activity of protein kinase A, C, and ERK following stimulation/inhibition by upstream GPCR agonists. Our work provides a generalizable platform that enables the design of ultrabright biosensors for illuminating dynamic architecture of kinase signaling.

Published in Proceedings of the National Academy of Sciences (predicted rank #2) · training set

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