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Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain
Spencer T Adams1, David M Mofford1, G S Kiran Kumar Reddy1
1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St, Worcester, MA, 01605, USA.
Angewandte Chemie (International Ed. in English)
|March 19, 2016
Summary
Engineered firefly luciferases can now distinguish between natural and synthetic substrates in live mouse brains. This breakthrough expands bioluminescence imaging capabilities for visualizing multiple biological events simultaneously.
Area of Science:
- Biochemistry
- Molecular Biology
- In vivo imaging
Background:
- Bioluminescence imaging enables visualization of biological processes in live animals.
- Current bioluminescence relies on limited natural luciferase-luciferin pairs, restricting its applications.
- The scope of bioluminescence imaging is constrained by the available enzyme-substrate combinations.
Purpose of the Study:
- To engineer firefly luciferase mutants capable of discriminating between natural and synthetic substrates.
- To demonstrate the utility of these engineered luciferases for in vivo imaging in mouse brains.
- To expand the toolkit for multiplexed bioluminescence imaging in live animals.
Main Methods:
- Utilized adeno-associated viral (AAV) vectors for expressing firefly luciferase mutants in mouse brains.
- Tested mutant luciferases for activity with natural d-luciferin and synthetic aminoluciferins (CycLuc1, CycLuc2).
- Assessed the performance of mutant luciferases with FAAH-sensitive luciferin amides.
Main Results:
- Developed firefly luciferase mutants that show reduced activity with d-luciferin.
- Demonstrated that these mutants exhibit bright luminescence with synthetic aminoluciferins CycLuc1 and CycLuc2.
- Successfully visualized gene expression and biological events in live mouse brains using engineered luciferases and synthetic substrates.
Conclusions:
- Mutant firefly luciferases can be engineered to selectively utilize synthetic substrates.
- This selectivity enables enhanced control and expanded applications for bioluminescence imaging in neuroscience and beyond.
- Further development of selective luciferases will facilitate simultaneous imaging of multiple biological events within the same live animal.
