Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo

The NanoLuc luciferase (NLuc) and its furimazine (FRZ) substrate have revolutionized bioluminescence (BL) assays and imaging. However, the use of the NLuc-FRZ luciferase- luciferin pair for mammalian tissue imaging is hindered by the low tissue penetration of the emitting blue photons. Here, we present the development of an NLuc mutant, QLuc, which catalyzes the oxidation of a synthetic QTZ luciferin for bright and red-shifted emission peaking at -585 nm. Compared to other small single domain NLuc mutants, this amber-light-emitting luciferase exhibited improved performance for imaging deep-tissue targets in live mice. Leveraging this novel bioluminescent reporter, we further pursued in vivo immunobioluminescence imaging (immunoBLI), which used a fusion protein of a single-chain variable antibody fragment (scFv) and QLuc for molecular imaging of tumor-associated antigens in a xenograft mouse model. As one of the most red-shifted NLuc variants, we expect QLuc to find broad applications in noninvasive mammalian imaging. Moreover, the immunoBLI method complements immunofluorescence imaging and immuno-positron emission tomography (immunoPET), serving as a convenient and nonradioactive molecular imaging tool for animal models in basic and preclinical research.

Automated summary

The NanoLuc luciferase and its furimazine substrate have greatly improved bioluminescence assays and imaging. However, their use for mammalian tissue imaging is limited by the low tissue penetration of blue photons. In this study, a mutant NLuc called QLuc was developed, which catalyzes the oxidation of a synthetic QTZ luciferin to produce bright and red-shifted emission. This red-shifted luciferase showed improved performance for imaging deep-tissue targets in live mice. Using QLuc, the researchers also demonstrated the use of immunoBLI for molecular imaging of tumor-associated antigens in a xenograft mouse model. QLuc is expected to have broad applications in noninvasive mammalian imaging, and immunoBLI serves as a convenient and nonradioactive molecular imaging tool for animal models in basic and preclinical research.