Bioluminescence, a phenomenon of production and emission of light as the result of a biochemical reaction, has broad applications in biology, biotechnology, and biomedical sciences. Bioluminescent reporters used in laboratories are mostly derivatives of two major luciferase families: ATP-dependent insect luciferases and ATP-independent marine luciferases. Despite that ATP-dependent luciferase-luciferin pairs, such as firefly luciferase (FLuc)-D-luciferin, have been widely usedfor in vivo bioluminescence imaging (BLI), they consume ATP for photon production and this metabolic disruption issue cannot be addressed by simply improving these reporters. Moreover, any bioluminescent biosensors derived from them are intrinsically ATP-dependent. On the other hand, ATP-independent marine luciferase-luciferin pairs, such as NanoLuc-furimazine (FRZ), have found broad applications in vitro, but they are far from optimal for in vivo BLI due to low photon penetrationdepth in tissue caused by their blue emission, poor substrate solubility and stability, and/or low substrate permeability through the blood-brain barrier (BBB). By integrating synthetic/medicinal chemistry, protein engineering, and in vivo validation, we are adressing the aforementioned caveats of ATP-independent bioluminescent reporters. This project will derive ATP-independent, bioluminescent imaging modalities with greatly enhanced biocompatibility, robustness, and in vitro and in vivo sensitivity. These new tools will enable and streamline a large array of applications in live cells, tissues, and animals. Moreover, because of their ATP-independency, these new reporters will be best suited for the development of various bioluminescent biosensors.
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