Effect of sodium ion on the high-resolution melting of lambda DNA.

A series of high-resolution melting curves were obtained by the continuous direct-derivative method [Blake, R. D. & Lefoley, S. G. (1978) Biochim. Biophys. Acta 518 , 233–246] on lambda DNA (cI₈₅₇S₇ strain) under varying conditions of [Na⁺]. Examination of the denaturation patterns at close intervals of [Na⁺] indicates that frequent changes in mechanism occur below 0.04M Na⁺, while almost none occurs above 0.1M Na⁺. Changes at low [Na⁺] generally occur in an abrupt fashion, in most cases within a 3 mM change in [Na⁺], and in at least one case within 0.6 mM, indicating the balance between alternative mechanisms is frequently quite delicate. These changes involve segments of between 900 and 1500 or more base pairs in length and are therefore not insignificant. Changes at low [Na⁺] reflect a perturbation of the energetic balance between competing mechanisms by weakly screened long-range electrostatic forces. Some perturbation probably also arises from variations in the linear charge density of the double helix induced by the proximity of premelted loop segments; however, this contribution cannot be evaluated without a detailed denaturation map. At high [Na⁺] the mechanism of melting is more conserved, permitting the dependence of subtrasitional melting temperature t_m⁽ⁱ⁾ on [Na⁺] to be examined for almost all 34 ± 2 subtransitions. The G + C composition of segments responsible for each subtransition was determined by a quantitative spectral method. Analysis according to the Manning-Record expression [Manning, G. (1972) Biopolymers 11 , 937–949; Record, M. T., Jr., Anderson, C. F. & Lohman, T. M. (1978) Q. Rev. Biophysics 11 , 103–178] relating ΔH_m and dt_m⁽ⁱ⁾/d log[Na⁺] to the fraction of Na⁺ released during melting, appears to indicate almost 40% more Na⁺ is bound to the single-stranded G and/or C residues than to A and T residues. This is consistent with a much shorter mean axial spacing and higher charge density in the former, particularly single-stranded G residues, which have an extraordinary tendency to stack.