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Highly Sensitive Chemigenetic FRET-Based Kinase Biosensors.

Xinchang He, Daojia R Zhou, Yu Huan

Biorxiv : the Preprint Server for Biology

|February 23, 2026

Summary

This summary is machine-generated.

Chemigenetic biosensors overcome limitations of traditional FRET sensors, offering high sensitivity and dynamic range for studying cellular signaling. These advanced tools enable precise, live-cell imaging of biochemical activities in complex biological systems.

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Area of Science:

Cellular Biology
Biochemistry
Molecular Imaging

Background:

Fluorescent protein biosensors enable live-cell measurement of biochemical activities.
Förster Resonance Energy Transfer (FRET)-based biosensors have limitations in dynamic range and spectral properties.
Chemigenetic approaches offer a solution by combining synthetic fluorophores with self-labeling protein tags.

Purpose of the Study:

To develop highly sensitive, chemigenetic FRET-based biosensors with red-shifted emission and improved dynamic range.
To demonstrate the versatility of this platform for various biomolecules, including kinases, GTPases, and second messengers.
To enable robust multiplexed imaging and visualization of signaling networks in cells and tissues.

Main Methods:

Pairing fluorescent protein donors with HaloTag acceptors conjugated to far-red fluorophores.
Developing and validating chemigenetic biosensors for multiple kinase targets, small GTPases, and second messengers.
Utilizing two-photon fluorescence lifetime imaging for visualization in acute brain slices.

Main Results:

A suite of highly sensitive chemigenetic FRET biosensors with red-shifted emission and unprecedented dynamic range was created.
The platform demonstrated generalizability across various targets, maintaining high sensitivity.
Robust multiplexed activity imaging of signaling networks and clear visualization of kinase activity in brain slices were achieved.

Conclusions:

Chemigenetic biosensors provide a powerful toolkit for studying signaling networks with enhanced sensitivity and spectral flexibility.
These tools overcome previous limitations, enabling quantitative mapping of signaling events in cells and tissues.
The developed sensor toolkit illuminates spatiotemporal regulation of signaling networks.