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Nucleic Acids Research, 2000, Vol. 28, No. 24 e107
© 2000 Oxford University Press

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

Yukio Okamura1,2,3, Satoshi Kondo1,2, Ichiro Sase1,4, Takayuki Suga1, Kazuyuki Mise5, Iwao Furusawa5, Shigeki Kawakami3 and Yuichiro Watanabe3,*

1Laboratory of Molecular Biophotonics, 5000 Hirakuchi, Hamakita, Shizuoka 434-8555, Japan, 2Bio-research Laboratory, Toyota Motor Corporation, 1 Toyota-cho, Toyota, Aichi 471-8572, Japan, 3Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan, 4Yanagida Project, Kansai Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka, Iwaoka-cho, Nishi-ku, Kobe, Hyogo 551-2492, Japan and 5Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.

* To whom correspondence should be addressed. Tel: +81 3 5454 6776; Fax: +81 3 5454 6776; Email: solan@bio.c.u-tokyo.ac.jp


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