Azide- and Dye-Conjugated Coelenterazine Analogues for Imaging Mammalian Cells

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-N₃-CTZ/ALuc26], [6-pi-OH-CTZ/RLuc8.6], and [6-N₃-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.