A New Fluorescence Resonance Energy Transfer Pair and Its Application to Oligonucleotide Labeling and Fluorescence Resonance Energy Transfer Hybridization Studies
Tóm tắt
We describe two new fluorescence resonance energy transfer (FRET) compatible labels, their covalent linkage to oligonucleotides, and their use as donor and acceptor, respectively, in FRET hybridization studies. The dyes belong to the cyanine dyes, and water solubility is imparted by a phosphonate which represents a new solubilizing group in DNA labels. They were linked to amino-modified synthetic oligonucleotides via oxysuccinimide (OSI) esters. The studies performed include binding assays, determinations of molecular distances, homogeneous competitive assays, and limits of detection, which are in the order of 5 pmol/L for a 15-mer.
Tài liệu tham khảo
B. Oswald, M. Gruber, M. B¨hmer, F. Lehmann, M. Probst, and O. S. Wolfbeis (2001). Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy immunoassay. Photochem. Photobiol. 74, 237–245.
O. S. Wolfbeis, M. B¨hmer, A. D¨rkop, J. Enderlein, M. Gruber, I. Klimant, C. Krause, J. K¨rner, G. Liebsch, Z. Lin, B. Oswald, and M. Wu (2002). In R. Kraayenhof (Ed.), Advanced Luminescent Labels, Probes, and Beads, and Their Application to Luminescence Bioassay, and Imaging in Springer Series in Fluorescence, vol. 2. Springer Verlag, Berlin-Heidelberg.
J. R. Lakowicz (1999). Principles of Fluorescence Spectroscopy, 2nd ed. Plenum, New York.
R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis, and A. S. Waggoner (1993). Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjugate Chem. 4, 105–111.
F. M. Hamer (1949). Some unsymmetrical pentamethinecyanine dyes and their tetramethine intermediates. J. Chem. Soc. 32–37.
G. T. Hermanson (1996). Bioconjugate Techniques. Academic Press, San Diego, p. 13.
Oligonucleotide Phosphate Labeling Protocol, Molecular Probes (Eugene, Oregon), http://www.probes.com/handbook/sections/0802.html.
Fluoro Link Cy5, Product Description, Cat. No. PA25001. Amersham Life Science, Inc., IL.
J. N. Demas and G. A. Crosby (1971). The measurement of photoluminescence quantum yields. A review. J. Phys. Chem. 75, 991– 1024.
S. Tyagi and F. R. Kramer (1996). Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303– 308.
S. A. E. Marras, F. R. Kramer, and S. Tyagi (2002). Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, e122-2 (electronic paper).
P. J. O’Sullivan, M. Burke, A. E. Soini, and D. B. Papkovsky (2002). Synthesis and evaluation of phosphorescent oligonucleotide probes for hybridisation assays. Nucleic Acids Res. 30, e114 (electronic paper).
S. Tyagi (2000). Wavelength-shifting molecular beacons. Nature Biotechnol. 18, 1191–1196.
M. Masuko, S. Ohuchi, K. Sode, H. Ohtani, and A. Shimadzu (2000). Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions. Nucleic Acids Res. 28, e34, 2–8.
R. Narayanaswamy and O. S. Wolfbeis (2003). Optical Sensors: Industrial, Environmental and Diagnostic Applications. Springer, Berlin, Heidelberg, chapters 4–6.