Highly Sensitive Chemigenetic FRET-Based Kinase Biosensors

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.