bc8b00188_si_001.pdf (1.7 MB)
Azide- and Dye-Conjugated Coelenterazine Analogues for a Multiplex Molecular Imaging Platform
journal contribution
posted on 2018-05-16, 00:00 authored by Ryo Nishihara, Emi Hoshino, Yoshiki Kakudate, Satoshi Kishigami, Naoko Iwasawa, Shin-ichi Sasaki, Takahiro Nakajima, Moritoshi Sato, Shigeru Nishiyama, Daniel Citterio, Koji Suzuki, Sung Bae KimNative
coelenterazine (nCTZ) is a common substrate to most marine
luciferases and photoproteins. In this study, nine novel dye- and
azide-conjugated CTZ analogues were synthesized by conjugating a series
of fluorescent dyes or an azide group to the C-2 or C-6 position of
the nCTZ backbone to obtain bulkiness-driven substrate specificity
and potential chemiluminescence/bioluminescence resonance energy transfer
(C/BRET). The investigation on the optical properties revealed that
azide-conjugated CTZs emit greatly biased bioluminescence to ALucs
and ca. 130 nm blue-shifted bioluminescence with RLuc8.6 in living
animal cells or lysates. The corresponding kinetic study explains
that azide-conjugated CTZ exerts higher catalytic efficiency than
nCTZ. Nile red-conjugated CTZ completely showed red-shifted CRET spectra
and characteristic BRET spectra with artificial luciferase 16 (ALuc16).
No or less spectral overlap occurs among [Furimazine–NanoLuc],
[6-N3-CTZ–ALuc26], [6-pi-OH-CTZ–RLuc8.6],
and [6-N3-CTZ–RLuc8.6] pairs, because of the substrate-driven
luciferase specificity and color shifts, providing a crosstalk-free
multiplex bioassay platform. The unique bioluminescence system appends
a new toolbox to bioassays and multiplex molecular imaging platforms.
This study is the first example that systematically synthesized fluorescent
dye-conjugated CTZs and applied them for a bioluminescence assay system.
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Keywords
bioluminescence system appendsRLuc 8.6Multiplex Molecular Imaging Platform Native coelenterazineazide-conjugated CTZsbioluminescence assay systemDye-Conjugated Coelenterazine AnaloguesC -2imaging platformsanimal cellssubstrate-driven luciferase specificitybulkiness-driven substrate specificityNile red-conjugated CTZC -6 positionazide-conjugated CTZ analoguesdye-conjugated CTZsluciferase 16marine luciferasesBRET spectraazide groupnovel dyeazide-conjugated CTZcolor shiftscrosstalk-free multiplex bioassay platform130 nm blue-shifted bioluminescencenCTZ backbonered-shifted CRET spectra
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