De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy.

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• De novo protein design offers potential for creating novel molecular tools.
• Existing designed fluorophore binders often fall short of the demands for high-resolution fluorescence microscopy.

Purpose of the Study:

• To design de novo Rhodamine Binder (Rhobin) tags with enhanced properties for advanced cellular imaging.
• To create a versatile tool for live and fixed cell imaging across various subcellular targets.

Main Methods:

• De novo protein design of Rhodamine Binder (Rhobin) tags.
• Characterization of tag properties including size, brightness, and hyperstability.
• Application in live and fixed cell imaging in mammalian cells and extremophiles.

Main Results:

• Successful design of Rhobin tags combining ideal size, brightness, and hyperstability.
• Demonstrated utility in live and fixed cell imaging of diverse subcellular targets.
• Enabled live super-resolution STED and PAINT microscopy with reduced photobleaching.
• Showcased applicability in the extremophile Sulfolobus acidocaldarius at 75°C.

Conclusions:

• Rhobin tags represent a significant advancement in protein design for fluorescence microscopy.
• These tags offer unprecedented capabilities for live cell imaging, including super-resolution techniques.
• Rhobin provides a foundation for developing a new generation of fluorescent tags and biosensors for challenging biological systems.