SpringerProtocols: Abstract: 4. Detecting RNA/DNA Hybridization Using Double-Labeled Donor Probes With Enhanced Fluorescence Resonance Energy Transfer Signals

4. Detecting RNA/DNA Hybridization Using Double-Labeled Donor Probes With Enhanced Fluorescence Resonance Energy Transfer Signals

By: Yukio Okamura, Yuichiro Watanabe

Abstract

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Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity, and the emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore. Using such fluorescently labeled oligonucleotides as FRET probes, makes possible specific detection of RNA molecules even if similar sequences are present in the environment. A higher ratio of signal to background fluorescence is required for more sensitive probe detection. We found that double-labeled donor probes labeled with BODIPY dye resulted in a remarkable increase in fluorescence intensity compared to single-labeled donor probes used in conventional FRET. Application of this double-labeled donor system can improve a variety of FRET techniques.

Book Title: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols

Series: Methods in Molecular Biology | Volume: 335 | Pub. Date: Apr-01-2006 | Page Range: 43-56 | DOI: 10.1385/1-59745-069-3:43

Subject: Biochemistry

Key Words: FRET - nucleic acid - acceptor probe - donor probe - BODIPY 493/503 - Cy5 - target RNA - plant virus - viral RNA

References

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1.	Clegg, R. M. (1996) Fluorescence resonance energy transfer, in Fluorescence Imaging Spectroscopy and Microscopy, (Wang X. F. and Herman B. eds.), Wiley-Interscience Publication, New York, NY, pp. 179–252.

2.	Herman, B. (1989) Resonance engergy transfer microscopy, in Fluorescence Microscopy of Living Cells in Culture (Taylor D. L. and Wang Y.-L. eds.), Academic Press, San Diego, CA, pp. 219–243.

3.	Cardullo, R. A., Agrawal, S., Flores, C., Zamecnik, P. C., and Wolf, D. E. (1988) Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85, 8790–8794.

4.	Morrison, L. E., Halder, T. C., and Stols, L. M. (1989) Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal. Biochem. 183, 231–244.

5.	Mergny, J. L., Boutorine, A. S., Garestier, T., et al. (1994) Fluorescence energy transfer as a probe for nucleic acid structures and sequences. Nucleic Acids Res. 22, 920–928.

6.	Sokol, D. L., Zhang, X., Lu, P., and Gewirtz, A. M. (1998) Real time detection of DNA.RNA hybridization in living cells. Proc. Natl. Acad. Sci. USA 95, 11,538–11,543.

7.	Matsuo, T. (1998) In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim. Biophys. Acta 1379, 178–184.

8.	Sixou, S., SzokaJr, F. C., Green, G. A., Giusti, B., Zon, G., and Chin, D. J. (1994) Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET). Nucleic Acids Res. 22, 662–668.

9.	Okamura, Y., Kondo, S., Sase, I., et al. (2000) Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization. Nucleic Acids Res. 28, e107.

10.	Okamura, Y., Kondo, S., Suga, T., et al. (2003) Visualization of viral RNA localization in a living tobacco protoplast by in vivo hybridization. Submitted.

11.	Lawson, T. G., Ray, B. K., Dodds, J. T., et al. (1986) Influence of 5′ proximal secondary structure on the translational efficiency of eukaryotic mRNAs and on their interaction with initiation factors. J. Biol. Chem. 261, 13,979–13,989.

12.	Ota, N., Hirano, K., Warashina, M., et al. (1998) Determination of interactions between structured nucleic acids by fluorescence resonance energy transfer (FRET): selection of target sites for functional nucleic acids. Nucleic Acids Res. 26, 735–743.

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