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 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

Rapid genotyping using pyrene−perylene locked nucleic acid complexes

“…3). 28,44,45 The calculations were performed assuming dipole-dipole orientation factor χ 2 = 2/3 due to emission dipole of the donor and the absorption dipole of the acceptor randomized by segmental motions of the nucleic acid components, while the average dipole orientations were favorable for FRET within the complementary complexes. 28 45 Second, dependence of the FRET efficiency values E on the distance between the donor and acceptor FRET was studied using a series of DNA/RNA targets complementary to ON1-ON4 and containing increasing number of separating nucleotides dT n (rU n ) between the modified monomers (n = 0-9; Fig.…”

“…3A). 44 Taking length of one nucleotide unit 3.5 Å, 46 the single-stranded gap model allowed measurements of FRET efficiency when the distance between the modified monomers upon hybridization of ON1-ON4 to the targets varied from zero to 31.5 Å (Fig. 3B).…”

“…3B). This assay format was applied because of small Förster distance of the pyrene-perylene FRET pair, 44 although the single-stranded gap model could not disclose orientation dependence of FRET pairs M 2 /M 3 -M 4 within long double-stranded complexes. 45 Having annealed the FRET probes to the series of DNA/RNA targets and having measured the FRET efficiency values E of the resulting complexes as described above, the resulting E values were plotted against the distance between the monomers M 1 -M 2 and M 3 -M 4 ( Fig.…”

“…The resulting samples were annealed at 85°C for 5 min followed by cooling to rt over 1 h. Fluorescence measurements were performed using excitation wavelength of 340 nm, and monitoring emission at 455 nm and 373 nm. Fluorescence response was analyzed using the following ratiometric equations: 44 ). Molecular modeling.…”

Retraction: Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single‐Silver‐Nanoparticle Counting

“…The homogeneous hybridization format has long attracted great attention because of its fast hybridization rate and simple operation without washing steps. So far, certain methods have been used in homogeneous nucleic acid hybridization assays, including fluorescence resonance energy transfer, [7][8][9][10][11] molecular-beacon techniques, [12] and fluorescence correlation spectroscopy (FCS), [13][14][15] but the sensitivity of these homoge-A C H T U N G T R E N N U N G neous methods is still unsatisfactory.…”