Enabling Fluorescence Lifetime Imaging Multiplexing Using UnaG through Its Modification with Canonical and...

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• Fluorogen-activating proteins are vital for microscopy and functional imaging, offering an oxygen-independent alternative to GFP-like proteins.
• Limitations in fluorophore selectivity and spectral channels hinder multicolor synthetic dye development.
• Poor cell permeability of synthetic dyes restricts their application in biological systems.

Purpose of the Study:

• To develop a palette of UnaG protein probes for fluorescence lifetime imaging microscopy (FLIM).
• To overcome limitations of synthetic dyes by utilizing bilirubin as a natural chromophore.
• To establish structure-lifetime relationships for UnaG-bilirubin complexes for in vivo applications.

Main Methods:

• Rational design and engineering of the UnaG protein.
• Utilizing point mutagenesis and noncanonical amino acid incorporation.
• Employing time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM).

Main Results:

• Generated UnaG variants with a wide range of fluorescence lifetimes (picoseconds to nanoseconds).
• Determined the limits of bilirubin lifetime variation based on protein structural changes.
• Demonstrated reliable distinction of minimal structural changes via in cellula lifetime analysis.

Conclusions:

• Engineered UnaG protein offers a versatile platform for multicolor FLIM probes.
• Bilirubin-based UnaG variants overcome limitations of traditional fluorescent proteins and synthetic dyes.
• Further modifications can optimize spectral and temporal characteristics for quantitative imaging.