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Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers

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Abstract

The use of two-photon excitation of fluorescence for detection of fluorescence resonance energy transfer (FRET) was studied for a selected fluorescent donor–acceptor pair. A method based on labeled DNA was developed for controlling the distance between the donor and the acceptor molecules. The method consists of hybridization of fluorescent oligonucleotides to a complementary single-stranded target DNA. As the efficiency of FRET is strongly distance dependent, energy transfer does not occur unless the fluorescent oligonucleotides and the target DNA are hybridized. A high degree of DNA hybridization and an excellent FRET efficiency were verified with one-photon excited fluorescence studies. Excitation spectra of fluorophores are usually wider in case of two-photon excitation than in the case of one-photon excitation [1]. This makes the selective excitation of donor difficult and might cause errors in detection of FRET with two-photon excited fluorescence. Different techniques to analyze the FRET efficiency from two-photon excited fluorescence data are discussed. The quenching of the donor fluorescence intensity turned to be the most consistent way to detect the FRET efficiency. The two-photon excited FRET is shown to give a good response to the distance between the donor and the acceptor molecules.

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Acknowledgments

The authors thank M.Sc. Jonne Vaarno, University of Turku for his technical assistance and discussions concerning DNA. This work was supported by Academy of Finland, and a European Comission FP6 program.

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Authors and Affiliations

  1. Laboratory of Biophysics, Institute of Biomedicine, University of Turku, P.O. Box 123, 20521, Turku, Finland

    Rina Wahlroos, Juha Toivonen, Marko Tirri & Pekka Hänninen

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  1. Rina Wahlroos
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  2. Juha Toivonen
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Correspondence to Rina Wahlroos.

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Wahlroos, R., Toivonen, J., Tirri, M. et al. Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers. J Fluoresc 16, 379–386 (2006). https://doi.org/10.1007/s10895-006-0084-x

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