Analytical Sciences

Fluorescence-Quenching Phenomenon by Photoinduced Electron Transfer between a Fluorescent Dye and a Nucleotide Base

Masaki TORIMURA,*  Shinya KURATA,**^,*** Kazutaka YAMADA,** Toyokazu YOKOMAKU,** Yoichi KAMAGATA,* Takahiro KANAGAWA,*^,*** and Ryuichiro KURANE*

*National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
**Kankyo Engineering Corporation, 1 Kimitsu, Kimitsu, Chiba 299-1141, Japan
***Department of Molecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori, Yokohama 226-8501, Japan

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C₁₀T₆ were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A₆G₁₀. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.

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