Nishihara Ryo, Hoshino Emi, Kakudate Yoshiki, Suzuki Koji, Kim Sung-Bae
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Kanagawa, Japan.
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Methods Mol Biol. 2021;2274:111-126. doi: 10.1007/978-1-0716-1258-3_11.
Coelenterazine (CTZ) is a common substrate to most marine luciferases and photoproteins. The present protocol introduces mammalian cell imaging with nine novel dye- and azide-conjugated CTZ analogues, which were synthesized by conjugating a series of fluorescent dyes or an azide group to the C-2 or C-6 position of CTZ backbone. The investigation on the optical properties revealed that azide-conjugated CTZs emit greatly selective bioluminescence (BL) to artificial luciferases (ALucs) and ca. 130 nm blue-shifted BL with Renilla luciferase variant 8.6 (RLuc8.6) in mammalian cells. The corresponding kinetic study explains that azide-conjugated CTZ exerts higher catalytic efficiency than CTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra and characteristic BRET spectra with artificial luciferase 16 (ALuc16). The present protocol shows that the minimal spectral overlap occurs among the pairs of [Furimazine/NanoLuc], [6-N-CTZ/ALuc26], [6-pi-OH-CTZ/RLuc8.6], and [6-N-CTZ/RLuc8.6] because of the substrate-driven luciferase specificity or color shifts, convincing a cross talk-free multiplex bioassay platform. The present protocol introduces a new toolbox to bioassays and multiplex molecular imaging platforms for mammalian cells.
腔肠素（CTZ）是大多数海洋荧光素酶和光蛋白的常见底物。本实验方案介绍了使用九种新型染料和叠氮化物共轭的CTZ类似物进行哺乳动物细胞成像，这些类似物是通过将一系列荧光染料或叠氮基团连接到CTZ主链的C-2或C-6位置合成的。对光学性质的研究表明，叠氮化物共轭的CTZ对人工荧光素酶（ALucs）发出极具选择性的生物发光（BL），并且在哺乳动物细胞中与海肾荧光素酶变体8.6（RLuc8.6）发出的BL发生约130 nm的蓝移。相应的动力学研究解释了叠氮化物共轭的CTZ比CTZ具有更高的催化效率。尼罗红共轭的CTZ与人工荧光素酶16（ALuc16）完全呈现红移的化学共振能量转移（CRET）光谱和特征性的生物发光共振能量转移（BRET）光谱。本实验方案表明，由于底物驱动的荧光素酶特异性或颜色变化，在[腔肠酰胺/纳米荧光素]、[6-N-CTZ/ALuc26]、[6-π-OH-CTZ/RLuc8.6]和[6-N-CTZ/RLuc8.6]对之间出现的光谱重叠最小，这证明了一个无串扰的多重生物测定平台。本实验方案为哺乳动物细胞的生物测定和多重分子成像平台引入了一个新的工具箱。
