Developments of advanced NanoLuc-based bioluminescent systems for bioimaging and assays

Xiong, Ying, Biophysics - School of Medicine, University of Virginia
Ai, Huiwang, MD-MPHY Mole Phys & Biophysics, University of Virginia

Bioluminescence is an old yet fast advancing research area, where photons are
generated based on the oxidization of luciferins. The advent of NanoLuc has revolutionized
bioluminescence in imaging and bioanalytical assay because of its superior brightness,
miniaturized size and cofactor-free luminescence nature.
However, current NanoLuc system is still far from perfect. One of the most
prominent shortages is the blue-photon emission profile, hindering its performance in deep
tissue imaging. To improve it, in Chapter 2 we developed an amber-emitting luciferaseluciferin
pair QLuc-QTZ featuring significantly improved tissue penetration. Leveraging
its improved photophysical properties, we further developed an immuno-bioluminescence
imaging platform by utilizing QLuc as the nonradiative tracer in vivo.
Moreover, bioluminescence significantly outperforms fluorescence in macroscopic
imaging with high signal-to-noise ratio. Ca2+ and K+ are two important ions exerting
diversified functions in living organisms. Therefore, monitoring their spatial and temporal
dynamics in vivo is of great importance to deepen our understanding of their biological
functions. To pursue those arch goals, we successfully constructed a new bioluminescent
Ca2+ sensor featuring improved responses in optical window by exploiting a hybrid sensing
mechanism in Chapter 3. Instead of engineering luciferase we further developed the first
prototype of bioluminescent K+ sensor by engineering the luciferin in Chapter 4.
In summary, the studies described herein have successfully advanced the NanoLucbased
bioluminescence system and largely expanded the repository of bioluminescent tools
in biomedical research.

PHD (Doctor of Philosophy)
Bioluminescence, Biosensors, Protein engineering, Optical imaging, Coelenterazine synthesis
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