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.
