title: Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions.
abstract: 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 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 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 A (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 nucleo-tides 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 A. 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.
authors.affiliation: Tsukuba Research Laboratory, Hamamatsu Photonics K. K., 5-9-2 Tokodai, Tsukuba 300-2635, Japan, masuko@hpk.trc-net.co.jp
authors.forename: M
authors.initials: M
authors.lastname: Masuko
authors.forename: S
authors.initials: S
authors.lastname: Ohuchi
authors.forename: K
authors.initials: K
authors.lastname: Sode
authors.forename: H
authors.initials: H
authors.lastname: Ohtani
authors.forename: A
authors.initials: A
authors.lastname: Shimadzu
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000032:analysis / Q000737:chemistry; D015345:Oligonucleotide Probes; D010569:Perylene / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011721:Pyrenes; D012235:Rhodamines; D012996:Solutions; D010569:Perylene; C030984:pyrene