Nucleic Acids Research, 2000, Vol. 28, No. 8 E34-00
© 2000 Oxford University Press
Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions
Tsukuba Research Laboratory, Hamamatsu Photonics K. K., 5-9-2 Tokodai, Tsukuba 300-2635, Japan, 1Department of Biochemical Engineering and Science, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Japan, 2Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-machi, Koganei 184-8588, Japan and 3Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32mer and its complementary two sequential 16mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of ~100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 Å (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleotides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 Å. Comparison between the fluorescent properties of a pyrene–perylene pair with those of a widely used fluorescein–rhodamine pair showed that the pyrene–perylene FRET is suitable for hybridization assays.
* To whom correspondence should be addressed. Tel: +81 298 47 5161; Fax: +81 298 47 5266; Email: masuko@hpk.trc-net.co.jp Present address: Akira Shimadzu, FV Project, Canon Inc., 5-1 Morinosato-Wakamiya, Atsugi 243-0193, Japan
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