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

Ying Xiong, Yiyu Zhang, Zefan Li, Md Shamim Reza, Xinyu Li, Xiaodong Tian, View ORCID ProfileHuiwang Ai
doi: https://doi.org/10.1101/2022.05.23.493143
Ying Xiong
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Yiyu Zhang
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Zefan Li
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Md Shamim Reza
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Xinyu Li
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Xiaodong Tian
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
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Huiwang Ai
1Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
2Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, VA, 22908, USA
3The UVA Cancer Center, University of Virginia, Charlottesville, VA, 22908, USA
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  • ORCID record for Huiwang Ai
  • For correspondence: huiwang.ai@virginia.edu
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ABSTRACT

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. This amber-light-emitting luciferase-luciferin pair 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 imaging in mammals. 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.

Competing Interest Statement

HA and YX are listed as inventors of a patent or a patent application covering some luciferase and luciferin variants described in this work.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 24, 2022.
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An Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging in Vivo
Ying Xiong, Yiyu Zhang, Zefan Li, Md Shamim Reza, Xinyu Li, Xiaodong Tian, Huiwang Ai
bioRxiv 2022.05.23.493143; doi: https://doi.org/10.1101/2022.05.23.493143
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An Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging in Vivo
Ying Xiong, Yiyu Zhang, Zefan Li, Md Shamim Reza, Xinyu Li, Xiaodong Tian, Huiwang Ai
bioRxiv 2022.05.23.493143; doi: https://doi.org/10.1101/2022.05.23.493143

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