Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries

Longwei Liu, Praopim Limsakul, Xianhui Meng, Yan Huang, Reed E. S. Harrison, Tse-Shun Huang, Yiwen Shi, Yiyan Yu, Krit Charupanit, Sheng Zhong, Shaoying Lu, Jin Zhang, Shu Chien, Jie Sun () and Yingxiao Wang ()
Additional contact information
Longwei Liu: University of California
Praopim Limsakul: University of California
Xianhui Meng: Zhejiang University School of Medicine
Yan Huang: Hunan University
Reed E. S. Harrison: University of California
Tse-Shun Huang: University of California
Yiwen Shi: University of California
Yiyan Yu: University of California
Krit Charupanit: Faculty of Medicine, Prince of Songkla University
Sheng Zhong: University of California
Shaoying Lu: University of California
Jin Zhang: University of California
Shu Chien: University of California
Jie Sun: Zhejiang University School of Medicine
Yingxiao Wang: University of California

Nature Communications, 2021, vol. 12, issue 1, 1-16

Abstract: Abstract The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.

Date: 2021
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DOI: 10.1038/s41467-021-25323-x

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