Two-Photon Excited Fluorescence Energy Transfer: A Study Based on Oligonucleotide Rulers |
| |
Authors: | Rina Wahlroos Juha Toivonen Marko Tirri Pekka Hänninen |
| |
Affiliation: | (1) Laboratory of Biophysics, Institute of Biomedicine, University of Turku, P.O. Box 123, 20521 Turku, Finland |
| |
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. |
| |
Keywords: | Two-photon excitation FRET Spectroscopy Fluorescent labels DNA Oligonucleotide Hybridization |
本文献已被 PubMed SpringerLink 等数据库收录! |