A Molecular Beacon Strategy for Real-Time Monitoring of Triplex DNA Formation Kinetics

Abstract

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 TC1 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 (k_ψ) 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 × 10^-3 s^-1 and 6.4 ± 1.8 to 30 ± 5.6 × 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 ΔG 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 ΔH and ΔS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs in 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.