High throughput screening of biosensors directly in mammalian cells enables live cell imaging and drug screening

by Longwei Liu

Fluorescent biosensors based on fluorescence resonance energy transfer (FRET), a microscope imaging technology that uses fluorescent color changes to measure active molecular actions, have revolutionized biomedical science by enabling the direct measurement of signaling activities in living cells.

However, scientists face a big challenge when developing FRET biosensors—they are largely developed by trial and error, making it cumbersome for scientists to identify high-performance FRET biosensors. Now, bioengineers at the University of California San Diego developed a technology that can identify such biosensors with ease.

The technology, called FRET-Seq platform, is the first to accomplish this feat. It couples FRET signals to next-generation sequencing techniques that are capable of screening large-scale libraries directly in mammalian cells. The FRET readings from single cells expressing the biosensors are then used to screen and sort cells into different groups. The sorted cells then get analyzed by next-generation sequencing, which helps scientists to identify the biosensor sequences.

The UC San Diego team, led by postdoctoral researcher Longwei Liu and former Ph.D. student Praopim Limsakul from the lab of bioengineering professor Peter Yingxiao Wang, detailed their work in a paper published Aug 19 in Nature Communications.

FRET-Seq also uses a new self-activating FRET (saFRET) design, in which a kinase domain is linked to the conventional biosensor and causes the activation. This design can overcome difficulties in mammalian-cell library screening caused by the heterogenic kinase activities from individual cells. Counter-sorting strategy associated with this design further improves both sensitivity and specificity of biosensors during the screening process.

The biosensors developed through this platform have better sensitivity when applied in live-cell imaging, which allows applications evaluating immune T cell functions and screening drugs. In fact, ZAP70 is a critical kinase involved in many diseases, including autoimmunity, organ transplant rejection, graft-versus-host disease, or B cell CLL. Using the ZAP70 biosensor designed in this work, Liu and colleagues have screened a kinase inhibitor library and identified several inhibitors, including FDA-approved cancer drugs, that can be repurposed to inhibit ZAP70 activity and hence, related autoimmune diseases.

Looking into the future, the team is extending this FRET-seq technology as a general platform for the development of other high-performance and ultrasensitive biosensors for single cell imaging. The team is also integrating the high content screening platforms equipped with fully automated cellular imaging apparatus and analysis algorithms to screen large-scale compound libraries for drug discovery.

Other contributors of this work include: Yan Huang, Reed E. S. Harrison, Tse-Shun Huang, Yiwen Shi, Yiyan Yu, Krit Charupanit, Sheng Zhong, Shaoying Lu, Jin Zhang, and Shu Chien as well as the team of Xianhui Meng and Jie Sun from Zhejiang University.