A molecular beacon strategy for real-time monitoring of triplex DNA formation kinetics.
We used a molecular beacon (MB) containing a 15-mer triplex-forming oligonucleotide (TFO) to probe in real-time the kinetics of triplex DNA formation in the left side of the TCl tract (502-516) of the c-src proto-oncogene in vitro. The metal ions Na+, K+, and Mg2+ stabilized triplex DNA at this site. The pseudo-first-order rate constant (kpsi) and the second-order association rate constant (k1) for the binding of the MB to the target duplex in 10 mM sodium phosphate buffer, pH 7.3, increased from 3.2 +/- 0.9 to 15 +/- 2.8 x 10(-3) s(-1) and 6.4 +/- 1.8 to 30 +/- 5.6 x 102 M(-1) s(-1), respectively, on increasing the MgCl2 concentration from 1 to 2.5 mM. Similar values were obtained for the triplex DNA stabilized by NaCl (100-250 mM). Surprisingly, the values were around 2 times higher in the presence of KCl. The AG of triplex formation in the presence of 1 mM MgCl2, 150 mM NaCl, and 150 mM KCl were -7.8 +/- 0.3, -8.2 +/- 0.3 and -8.7 +/- 0.7 kcal/mol respectively, despite significant differences in the values of deltaH and deltaS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs ih probing triplex DNA formation and in evaluating kinetic and thermodynamic parameters important for the design and development of TFOs as triplex DNA-based therapeutic agents.
Antony
Thomas
T
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Subramaniam
Vinod
V
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
C051802:triplex DNA; D015636:Magnesium Chloride; D012965:Sodium Chloride; D011189:Potassium Chloride; D004247:DNA