Department of Chemistry, Connecticut College, New London, CT 06320, USA.
Department of Chemistry "G. Ciamician", University of Bologna, 40126 Bologna, Italy.
Int J Mol Sci. 2022 Feb 23;23(5):2451. doi: 10.3390/ijms23052451.
Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.
荧光素酶催化发光反应，使其底物（荧光素）、分子氧，并且通常还有其他辅助因子产生彩虹色。这些生物发光（BL）系统为基础研究和医学应用提供了各种各样的可能性。由于基于 BL 的非侵入性动物成像（BLI）应用的重要性，特别是在支持癌症研究方面，新的 BL 系统通过工程化改造甲虫荧光素酶（Luc）变体和合成底物组合得以开发，以产生红色到近红外（nIR）光，从而提高成像灵敏度和分辨率。为了激发 BLI 研究的应用并推进用于 BLI 的改进试剂的发展，我们系统地比较了七种具有 LH 的甲虫 Luc 和九种底物的光谱和 BL 特性，其中包括两种新的含喹啉环的类似物。这些在体外和转染有 基因的活 HEK293T 细胞中进行的实验结果使我们能够鉴定出与以前报道的相比具有改进性质的 Luc/类似物组合，并提供可能与体内成像应用相关的活细胞 BL 数据。此外，我们发现了具有强候选酶/底物对的体外生物标志物应用，这些应用需要具有最小可见光成分的 nIR 光源。值得注意的是，我们的一种新底物与以前开发的 Luc 变体配对，被证明是体外 nIR 的极好来源，并且可能是提高 BLI 分辨率的有用 BL 系统。
