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Fluorescence Resonance Energy Transfer and Complex Formation Between Thiazole Orange and Various Dye-DNA Conjugates: Implications in Signaling Nucleic Acid Hybridization

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Abstract

Fluorescence resonance energy transfer (FRET) was investigated between the intercalating dye thiazole orange (TO), and the dyes Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black FQ (IabFQ), and Iowa Black RQ (IabRQ), which were covalently immobilized at the end of dsDNA oligonucleotides. In addition to determining that TO was an effective energy donor, FRET efficiency data obtained from fluorescence lifetime measurements indicated that TO intercalated near the middle of the 19mer oligonucleotide sequence that was used in this study. Discrepancies in FRET efficiencies obtained from intensity and lifetime measurements led to the investigation of non-fluorescent complex formation between TAMRA and modified TO. The hydrophobicity of TO was modified by the addition of either an alkyl or polyethylene glycol (PEG) side-chain to study effects of dimer and aggregate formation. It was found that at stoichiometric excesses of modified TO, fluorescence quenching of TAMRA was observed, and that this could be correlated to the hydrophobicity of a TO-chain species. The TAMRA:TO-chain association constant for the TO-alkyl system was 0.043±0.002 M−1, while that obtained for the TO-PEG was 0.037±0.002 M−1. From the perspective of method development for the transduction of hybridization events, we present and evaluate a variety of schemes based on energy transfer between TO and an acceptor dye, and discuss the implications of complex formation in such schemes.

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Acknowledgements

We are grateful to Dr. Paul Piunno, Dr. Sergei Musikhin, and Dr. Arkady Major for their assistance in acquiring fluorescence lifetimes. In addition, we thank NSERC for financial support of this research work, and for provision of a research fellowship to WRA.

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  1. Chemical Sensors Group, Department of Chemical & Physical Sciences, University of Toronto at Mississauga, South Building, Room 2035, 3359 Mississauga Road North, Mississauga, Ontario, L5L 1C6, Canada

    W. Russ Algar, Melissa Massey & Ulrich J. Krull

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  1. W. Russ Algar
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  2. Melissa Massey
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  3. Ulrich J. Krull
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Correspondence to Ulrich J. Krull.

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The authors wish it to be known that they have participated equally in the experiments and preparation of this manuscript and should, in their opinion, be considered joint first authors.

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Algar, W.R., Massey, M. & Krull, U.J. Fluorescence Resonance Energy Transfer and Complex Formation Between Thiazole Orange and Various Dye-DNA Conjugates: Implications in Signaling Nucleic Acid Hybridization. J Fluoresc 16, 555–567 (2006). https://doi.org/10.1007/s10895-006-0091-y

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