Skip to main content
SpringerLink
Log in

Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences†

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

We report the design, synthesis, and characterization of a molecular beacon (MB) consisting of two fluorescent dyes (Alexa 488 and RedX) for DNA and RNA analysis. In the absence of the target DNA or RNA the MB is in its stem-closed form and shows efficient energy transfer from the donor (Alexa) to the acceptor (RedX), generating mostly fluorescence from RedX. In the presence of the complementary target DNA the MB opened efficiently, hybridizes with the target DNA, and energy transfer is blocked in the stem-open form. This attachment to the target generates a fluorescence signature, which is clearly distinguishable from the fluorescence signature of the stem-closed form, allowing for ratiometric analysis of the fluorescence signal. In addition to steady-state fluorescence analysis, time resolved fluorescence (ps time range) and fluorescence depolarization studies were performed. We show that fluorescence lifetime and fluorescence depolarization measurements are useful analytical tools to optimize the MB design.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. W. Tan, X. Fang, J. Li and X. Liu, Molecular beacons: a novel DNA probe for nucleic acid and protein studies, Chem.–Eur. J., 2000, 6, 1107–1111.

    Article  CAS  Google Scholar 

  2. T. Antony and V. Subramaniam, Molecular Beacons: Nucleic Acid Hybridization and Emerging Applications, J. Biomol. Struct. Dyn., 2001, 19, 4997–5004.

    Article  Google Scholar 

  3. G. Bonnet, S. Tyagi, A. Libchaber and F. R. Kramer, Thermodynamic basis of the enhanced specificity of structured DNA probes, Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 6171–6176.

    Article  CAS  Google Scholar 

  4. S. Tyagi and F. R. Kramer, Molecular Beacons: Probes that Fluoresce upon Hybridization, Nat. Biotechnol., 1996, 14, 303–308.

    Article  CAS  Google Scholar 

  5. S. Tyagi, D. P. Bratu and F. R. Kramer, Multicolor molecular beacons for allele discrimination, Nat. Biotechnol., 1998, 16, 49–58.

    Article  CAS  Google Scholar 

  6. K. Faulds, L. Fruk, D. C. Robson, D. G. Thompson, A. Enright, W. E. Smith and D. Graham, A new approach for DNA detection by SERRS, Faraday Discuss., 2006, DO: 10.1039/b506219e.

    Google Scholar 

  7. G. Bonnet, O. Krichevsky and A. Libchaber, Kinetics of confor-mational fluctuations in DNA hairpin-loops, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 8602–8606.

    Article  CAS  Google Scholar 

  8. S. A. E. Marras, F. R. Kramer and S. Tyagi, Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes, Nucleic Acids Res., 2002, 30, e122.

    Article  Google Scholar 

  9. P. Zhang, T. Beck and W. Tan, Design of a Molecular Beacon DNA Probe with Two Fluorophores, Angew. Chem., Int. Ed., 2001, 40, 402–405.

    Article  CAS  Google Scholar 

  10. A. A. Martí, S. Jockusch, Z. Li, J. Ju and N. J. Turro, Molecular Beacons with intrinsically fluorescent nucleotides, Nucleic Acids Res., 2006, accepted, DOI: 10.1093/nar/gkl134.

    Google Scholar 

  11. K. Fujimoto, H. Shimizu and M. Inouye, Unambiguous Detection of Target DNAs by Excimer-Monomer Switching Molecular Beacons, J. Org. Chem., 2004, 69, 3271–3275.

    Article  CAS  Google Scholar 

  12. C. J. Yang, S. Jockusch, M. Vincens, N. J. Turro and W. Tan, Light-switching excimer probes for rapid protein monitoring in complex biological fluids, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 17278–17283.

    Article  CAS  Google Scholar 

  13. S. A. E. Marras, F. R. Kramer and S. Tyagi, Genotyping single nucleotide polymorphisms with molecular beacons, The Humana Press Inc., Towada, NJ, 2003, vol. 212, pp. 111–128.

    Google Scholar 

  14. J.-F. Brunet, E. Shapiro, S. A. Foster, E. R. Kandel and Y. Iino, Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening, Science, 1991, 252, 856–859.

    Article  CAS  Google Scholar 

  15. N. Panchuk-Voloshina, R. P. Haugland, J. Bishop-Stewart, M. K. Bhalgat, P. J. Millard,.F Mao, W.-Y. Leung and R. P. Haugland, Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates, J. Histochem. Cytochem., 1999, 47, 1179–1188.

    Article  CAS  Google Scholar 

  16. A. M. Iribarren, B. S. Sproat, P. Neuner, I. Sulston, U. Ryder and A. I. Lamond, 2’-O-alkyl oligoribonucleotides as antisense probes, Proc. Natl. Acad. Sci. U. S. A., 1990, 87, 7747–7751.

    Article  CAS  Google Scholar 

  17. P. Bratu Diana, B.-J. Cha, M. Mhlanga Musa, R. Kramer Fred and S. Tyagi, Visualizing the distribution and transport of mRNAs in living cells, Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 13308–13313.

    Article  CAS  Google Scholar 

  18. A. I. Lamond and B. S. Sproat, Antisense oligonucleotide made of 2’-O-AlkylRNA: their properties and applications in RNA biochemistry, FEBS Lett., 1993, 1-2, 123–127.

    Article  Google Scholar 

  19. T. Heinlein, J.-P. Knemeyer, O. Piestert and M. Sauer, Photoinduced Electron Transfer between Fluorescent Dyes and Guanosine Residues in DNA-Hairpins, J. Phys. Chem. B, 2003, 107, 7957–7964.

    Article  CAS  Google Scholar 

  20. K. Stoer, B. Haefner, O. Nolte, J. Wolfrum, M. Sauer and D.-P. Herten, Species-Specific Identification of Mycobacterial 16S rRNAS PCR Amplificons Using Smart Probes, Anal. Chem., 2005, 77, 7195–7203.

    Article  Google Scholar 

  21. Nazarenko, R. Pires, B. Lowe, M. Obaidy and A. Rashtchian, Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes, Nucleic Acids Res., 2002, 30, 2089–2195.

    Article  CAS  Google Scholar 

  22. E. Rusinova, V. Tretyachenko-Ladokhina, O. E. Vele, D. F. Senear and J. B. A. Ross, Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein-protein and protein-nucleic acid interactions, Anal. Biochem., 2002, 308, 18–25.

    Article  CAS  Google Scholar 

  23. R. A. Hochstrasser, S.-M. Chen and D. P. Millar, Distance distribution in a dye-linked oligonucleotide determined by time-resolved fluorescence energy transfer, Biophys. Chem., 1992, 45, 133–141.

    Article  CAS  Google Scholar 

  24. J. R. Lakowicz, Topics in Fluorescence Spectroscopy Volume 5: Nonlinear and Two-Photon-Induced Fluorescence, Plenum Press, New York, 1997, vol. 5, 544 pp.

    Google Scholar 

  25. A. Tsourkas, M. A. Behlke, Y. Xu and G. Bao, Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons, Anal. Chem., 2003, 75, 3697–3703.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Chemistry, Columbia University, New York, NY, 10027, USA

    Steffen Jockusch, Angel A. Martí & Nicholas J. Turro

  2. Departments of Chemical Engineering, Columbia University, New York, NY, 10027, USA

    Nicholas J. Turro, Zengmin Li & Jingyue Ju

  3. Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY, 10032, USA

    Zengmin Li, Xiaoxu Li & Jingyue Ju

  4. Department of Chemistry, City College, City University of New York, NY, 10031, USA

    Nathan Stevens & Daniel L. Akins

Authors
  1. Steffen Jockusch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angel A. Martí
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nicholas J. Turro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zengmin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaoxu Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jingyue Ju
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Nathan Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Daniel L. Akins
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Electronic supplementary information (ESI) available: Details of the synthesis of the MB, UV-Vis absorption spectra of the MBs (Fig. S1), fluorescence spectra of Alexa–MB and MB–RedX (Fig. S2), target DNA concentration dependence (Fig. S3), and no interaction of the MB with non-complementary target DNA (Fig. S4). See do]10.1039/b600213g

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jockusch, S., Martí, A.A., Turro, N.J. et al. Spectroscopic investigation of a FRET molecular beacon containing two fluorophores for probing DNA/RNA sequences†. Photochem Photobiol Sci 5, 493–498 (2006). https://doi.org/10.1039/b600213g

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/b600213g

Search

Navigation