Fluorescent Protein Chromophore-Based Luciferins for Bioluminescence Imaging.

Area of Science:

• Biomedical Imaging
• Molecular Biology
• Biochemistry

Background:

• Bioluminescence imaging (BLI) is crucial for tracking cellular and molecular processes in vivo.
• Current BLI probes have limited tissue penetrance, restricting their use in deep tissue and preclinical models.
• Red-shifted probes are needed to enhance sensitivity and penetration depth in BLI.

Purpose of the Study:

• To develop novel red-shifted bioluminescent probes for improved deep tissue imaging.
• To engineer luciferases optimized for enhanced activity with the new probes.
• To expand the applications of bioluminescence imaging in preclinical research.

Main Methods:

• Synthesized two firefly luciferin analogues (FPLucs) based on fluorescent protein chromophores.
• Engineered luciferases using Rosetta-guided design and screening for optimal FPLuc processing.
• Evaluated probe performance in tissue mimics and assessed light output with engineered luciferases.

Main Results:

• Developed FPLucs emitting light >650 nm (peak emissions at 701 nm and 699 nm), suitable for tissue imaging.
• Engineered luciferases showed significantly improved light output with FPLucs compared to native firefly luciferase.
• Designer luciferase-FPLuc pairs demonstrated detectability in tissue mimics, indicating potential for in vivo applications.

Conclusions:

• Fluorophore-inspired luciferins, like FPLucs, offer a promising strategy for red-shifted bioluminescence.
• Engineered luciferase-FPLuc pairs enhance signal detection for deep tissue imaging.
• These advancements hold potential to broaden the scope and sensitivity of bioluminescence imaging.