Aminoluciferins as functional bioluminogenic substrates of firefly luciferase

Hideo Takakura, Ryosuke Kojima, Yasuteru Urano, Takuya Terai, Kenjiro Hanaoka, Tetsuo Nagano

Research output: Contribution to journalArticlepeer-review

45 Citations (Scopus)

Abstract

Firefly luciferase is widely used as a reporter gene in assays to study gene expression, gene delivery, and so on because of its extremely high signal-to-noise ratio. The availability of a range of bioluminogenic substrates would greatly extend the applicability of the luciferin-luciferase system. Herein, we describe a design concept for functional bioluminogenic substrates based on the aminoluciferin (AL) scaffold, together with a convenient, high-yield method for synthesizing N-alkylated ALs. We confirmed the usefulness of ALs as bioluminogenic substrates by synthesizing three probes. The first was a conjugate of AL with glutamate, Glu-AL. When Glu-AL, the first membrane-impermeable bioluminogenic substrate of luciferases, was applied to cells transfected with luciferase, luminescence was not observed; that is, by using Glu-AL, we can distinguish between intracellular and extracellular events. The second was Cy5-AL, which consisted of Cy5, a near-infrared (NIR) cyanine fluorescent dye, and AL, and emitted NIR light. When Cy5-AL reacted with luciferase, luminescence derived from Cy5 was observed as a result of bioluminescence resonance energy transfer (BRET) from AL to Cy5. The NIR emission wavelength would allow a signal to be observed from deeper tissues in bioluminescence in vivo imaging. The third was biotin-DEVD-AL (DEVD=the amino acid sequence Asp-Glu-Val-Asp), which employed a caspase-3 substrate peptide as a switch to control the accessibility of the substrate to luciferase, and could detect the activity of caspase-3 in a time-dependent manner. This generalized design strategy should be applicable to other proteases. Our results indicate that the AL scaffold is appropriate for a range of functional luminophores and represents a useful alternative substrate to luciferin.

Original languageEnglish
Pages (from-to)1800-1810
Number of pages11
JournalChemistry - An Asian Journal
Volume6
Issue number7
DOIs
Publication statusPublished - 2011 Jul 4
Externally publishedYes

Keywords

  • aminoluciferin
  • bioluminescent probes
  • biosensors
  • firefly luciferase
  • luminescence

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry

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