Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, United States.
Center for Membrane and Cell Physiology, School of Medicine, University of Virginia, Charlottesville, Virginia 22908, United States.
J Am Chem Soc. 2022 Aug 10;144(31):14101-14111. doi: 10.1021/jacs.2c02320. Epub 2022 Aug 1.
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 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.
纳米萤光素酶（NLuc）及其荧光素（FRZ）底物彻底改变了生物发光（BL）检测和成像。然而，由于发射的蓝光光子在组织中的穿透深度较低，NLuc-FRZ 荧光素酶-荧光素对哺乳动物组织成像的应用受到了限制。在这里，我们介绍了一种 NLuc 突变体 QLuc 的开发，它可以催化合成的 QTZ 荧光素的氧化，产生明亮且红移的发射，峰值在约 585nm。与其他小型单结构域 NLuc 突变体相比，这种琥珀光发射荧光素酶在活体小鼠深层组织目标成像方面表现出了更好的性能。利用这种新型生物发光报告物，我们进一步进行了免疫生物发光成像（immunoBLI），该方法使用单链可变抗体片段（scFv）和 QLuc 的融合蛋白，对异种移植小鼠模型中的肿瘤相关抗原进行分子成像。作为最红移的 NLuc 变体之一，我们预计 QLuc 将在非侵入性哺乳动物成像中得到广泛应用。此外，immunoBLI 方法补充了免疫荧光成像和免疫正电子发射断层扫描（immunoPET），作为基础和临床前研究中动物模型的一种方便且无放射性的分子成像工具。
