Fluorescence-Activating and Absorption-Shifting Tags for Advanced Imaging and Biosensing

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Fluorescent labels and biosensors are crucial tools in biological and medical research for real-time imaging.
• Chemogenetic reporters, combining synthetic fluorophores with genetic tags, offer an alternative to purely genetically encoded fluorescent proteins.
• Fluorescence-activating and absorption-shifting tags (FASTs) are engineered proteins that bind and stabilize fluorophores, enhancing fluorescence.

Purpose of the Study:

• To present the expanding toolbox of FASTs and their applications in live-cell imaging and biosensing.
• To highlight the engineering of FASTs for diverse spectral properties, fluorogen selectivity, and biosensor development.
• To provide an overview of current advancements and future directions in FAST technology.

Main Methods:

• Directed protein evolution of the photoactive yellow protein (PYP) to create FAST variants.
• Engineering of hydroxybenzylidene rhodanine chromophores for enhanced fluorescence stabilization within the FAST protein cavity.
• Molecular engineering and circular permutation to develop FAST variants for specific applications like cell-surface labeling and analyte-specific biosensing.

Main Results:

• FASTs bind and stabilize fluorophores, overcoming non-radiative decay and enabling high-contrast imaging without washing steps.
• Development of FAST variants with tunable spectral properties and membrane-impermeant fluorogens for selective cell-surface labeling.
• Creation of circular-permutated and split FAST variants for advanced biosensing and imaging of dynamic protein-protein interactions.

Conclusions:

• FASTs represent a powerful chemogenetic platform for advanced live-cell imaging and biosensing.
• The modularity and adaptability of FASTs allow for diverse applications in biological research.
• Continued engineering of FASTs promises further innovations in visualizing cellular processes.