Determination of Ion Atmosphere Effects on the Nucleic Acid Electrostatic Potential and Ligand Association Using AH(+)·C Wobble Formation in Double-Stranded DNA

[Image: see text] The high charge density of nucleic acids and resulting ion atmosphere profoundly influence the conformational landscape of RNA and DNA and their association with small molecules and proteins. Electrostatic theories have been applied to quantitatively model the electrostatic potential surrounding nucleic acids and the effects of the surrounding ion atmosphere, but experimental measures of the potential and tests of these models have often been complicated by conformational changes and multisite binding equilibria, among other factors. We sought a simple system to further test the basic predictions from electrostatics theory and to measure the energetic consequences of the nucleic acid electrostatic field. We turned to a DNA system developed by Bevilacqua and co-workers that involves a proton as a ligand whose binding is accompanied by formation of an internal AH(+)·C wobble pair [Siegfried, N. A., et al. Biochemistry, 2010, 49, 3225]. Consistent with predictions from polyelectrolyte models, we observed logarithmic dependences of proton affinity versus salt concentration of −0.96 ± 0.03 and −0.52 ± 0.01 with monovalent and divalent cations, respectively, and these results help clarify prior results that appeared to conflict with these fundamental models. Strikingly, quantitation of the ion atmosphere content indicates that divalent cations are preferentially lost over monovalent cations upon A·C protonation, providing experimental indication of the preferential localization of more highly charged cations to the inner shell of the ion atmosphere. The internal AH(+)·C wobble system further allowed us to parse energetic contributions and extract estimates for the electrostatic potential at the position of protonation. The results give a potential near the DNA surface at 20 mM Mg(2+) that is much less substantial than at 20 mM K(+) (−120 mV vs −210 mV). These values and difference are similar to predictions from theory, and the potential is substantially reduced at higher salt, also as predicted; however, even at 1 M K(+) the potential remains substantial, counter to common assumptions. The A·C protonation module allows extraction of new properties of the ion atmosphere and provides an electrostatic meter that will allow local electrostatic potential and energetics to be measured within nucleic acids and their complexes with proteins.