Netropsin Binding as a Thermodynamic Probe of the Grooves of Parallel DNA

We use the minor groove ligand netropsin as a thermodynamic probe of the grooves of parallel DNA. A combination of circular dichroism spectroscopy, temperature-dependent UV spectroscopy, and isothermal titration calorimetry has been employed to characterize the association of netropsin with two sets of DNA 25-mer duplexes: one set contains duplexes with exclusively dA·dT base pairs; the other, duplexes with four dG·dC base pairs in parallel (ps-Dl·D2 and ps-D5·D6) and antiparallel (aps-Dl·D3 and aps-D5·D7) orientation. Circular dichroism and calorimetrie titration curves show overall stoichiometries of 4:1 ([netropsin]:[duplex]) for the netropsin complexes with the antiparallel duplexes and with ps-Dl·D2, and 3:1 with ps-D5·D6. Fully saturated netropsin-DNA complexes melt with transition temperatures 16–28 °C higher than those of the respective free duplexes. These ligand-induced thermal stabilizations correspond to binding affinities, K_b, of ∼5 × 10⁷ M⁻¹ for the parallel duplexes and ∼1 × 10⁸ M⁻¹ for the antiparallel duplexes. Both values correspond to highly specific netropsin binding sites, presumably Α·Τ base pairs. The similarity in values suggests that netropsin recognizes one of the grooves of parallel DNA in a similar manner to the minor groove of antiparallel B-DNA. However, for the set of duplexes containing all Α·Τ base pairs, we obtained binding enthalpies, ΔH°_b, of−6.6 (ps-Dl·D2) and −2.2 kcal mol⁻¹ (aps-Dl·D3), while ΔH°_b values of−8.9 (ps-D5·D6) and −7.9 kcal mol⁻¹ (aps-D5·D7) were obtained for duplexes containing dG·dC base pairs. Binding of netropsin to the first set of duplexes was accompanied by similar release of counterions equal to 1.6 mol of Na⁺/mol of ligand. Therefore, the ΔΔH°_b of 4.4 kcal in this set can be interpreted as a net hydration difference of three water molecules per base pair, with the parallel duplex being less hydrated.