Two-photon excited fluorescence energy transfer: a study based on oligonucleotide rulers.
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. 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.
Wahlroos
Rina
R
Laboratory of Biophysics, Institute of Biomedicine, University of Turku, P.O. Box 123, 20521 Turku, Finland. rina.wahlroos@utu.fi
Toivonen
Juha
J
Tirri
Marko
M
Hänninen
Pekka
P
D004277:DNA, Single-Stranded / Q000737:chemistry; D004735:Energy Transfer; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D017785:Photons; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D009841:Oligonucleotides