Albany 2001

category image Biomolecular
SUNY at Albany
June 19-23, 2001

Analysis of Triplex DNA Formation in the Promoter Region of Human c-src Proto-Oncogene - A Molecular Beacon Approach

Molecular beacons are an emerging class of nucleic acid probes that make use of sequence specific interaction of nucleic acid bases and the phenomenon of fluorescence quenching due to energy transfer [1]. They are designed to have a stem and loop structure, in which the donor and acceptor moieties are kept in close proximity by the stem, yielding a non-fluorescent or weakly fluorescent molecule. However, when the donor and acceptor are far apart, (for example, upon binding to a target sequence), it becomes fluorescent [2]. We used this property of the beacon to study triplex DNA formation in the left side of the TC1 tract in the promoter region of human c-src proto-oncogene [3]. The beacon has a donor fluorescein moiety at the 5? end, attached to the triplex forming oligonucleotide (TFO) by a CGCTC stretch and a quencher Dabcyl at the 3?-end by the complimentary GAGCG sequence. The base sequence of the beacon and the single strands of the target duplex were:

Triplex DNA formation was studied by monitoring the increase in fluorescence emission intensity of the beacon at 515 nm (lex=485nm) upon titration with the target duplex. The metal ions Mg2+ and Na+ promoted triplex DNA formation and resulted in ~15 fold increase in emission intensity in the presence of 2 mM MgCl2/100 mM NaCl. The pseudo-first order rate constant and the second?order association rate constant for the binding of beacon to the target duplex in the presence of 2 mM MgCl2 were 4.8 x10-3 S-1 and 9.6 x 102 M-1 S-1 respectively. Similar values were obtained for the triplex DNA stabilized by 100 mM NaCl. These studies show that the TFO used in this study forms a stable triplex with the targeted sequence in the promoter site of c-src proto-oncogene and hence would be useful for developing triplex DNA based anti-gene strategies for controlling the expression of this gene. It also shows the suitability of the molecular beacon approach for the analysis of triplex DNA.

References and Footnotes

  1. Tyagi, S. and Kramer, F.R., Nature Biotechnology 14, 303-308 (1996).
  2. Antony, T., Thomas, T., Sigal, L.H., Shirahata, A. and Thomas, T.J (Communicated).
  3. Aich, P., Ritchie, S., Bonham, K and Lee, J.S., Nucleic Acids Res. 26, 4173-4177 (1998).

Thomas Antony and Vinod Subramaniam

Dep./Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Ph: +49-551-201-1383, F: +49-551-201-1467 email: vsubram@gwdg.de