Parallel Screening for Rapid Identification of Orthogonal Bioluminescent Tools

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Bioluminescence imaging (BLI) using luciferase enzymes and luciferin substrates is vital for biological research, particularly in rodent models.
• A key limitation of BLI is the scarcity of distinguishable probes, restricting simultaneous multicolor imaging.
• Developing orthogonal probe sets is crucial for advancing multiplexed biological tracking.

Purpose of the Study:

• To establish a rapid and scalable method for identifying novel substrate-selective luciferases.
• To generate a library of mutually orthogonal bioluminescence probes for multicomponent imaging.
• To validate the utility of newly identified orthogonal probe pairs in biological systems.

Main Methods:

• A parallel screening strategy involving luciferin analogues and panels of mutant luciferase enzymes.
• Computational analysis (in silico) of screening data to identify orthogonal enzyme-substrate pairs.
• In vitro validation of selected orthogonal pairs, followed by in vivo testing in cell and animal models.

Main Results:

• Screened 159 mutant enzymes against 12 luciferins, revealing thousands of potentially orthogonal pairs.
• Validated over 100 orthogonal pairs in vitro, demonstrating sufficient selectivity for biological applications.
• Successfully applied three validated orthogonal probe sets in cell and animal models, proving in vivo efficacy.

Conclusions:

• The developed parallel screening approach is generalizable and scalable for discovering orthogonal bioluminescence probes.
• This method significantly streamlines the search for larger collections of probes, advancing multiplexed BLI.
• The findings pave the way for more sophisticated multicolor imaging in biological and biomedical research.