Developments of advanced NanoLuc-based bioluminescent systems for bioimaging and assays 134 views

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

English

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2022-09-19