Several studies by X-ray crystallography, NMR and molecular simulations have suggested that monovalent counterions can penetrate deeply into the minor groove of B-DNA AT-tracts. However, the extent, and in some cases even the existence, of such binding remains controversial. We have recently addressed this issue by ²³Na magnetic relaxation dispersion (MRD) (1). That study unambiguously identified Na⁺ ions in long-lived (>> 1 ns) association with several B-DNA dodecamers. Since counterions at the DNA surface are expected to be highly mobile, groove binding was implicated. This was confirmed more directly by demonstrating that the extent of long-lived Na⁺ binding correlates with the length of the AT-tract and that it is virtually abolished on addition of the AT-tract specific minor-groove binding drug netropsin (1). Competitive ion binding was also studied, albeit at a single frequency, and indicated that K⁺, Rb⁺, Cs⁺ bind with similar or slightly higher affinity than Na⁺ (1).

Here, we report the result of a second MRD study of ion binding to the minor groove of the [d(CGCGAATTCGCG)]₂ duplex, extending the previous MRD study in important ways. First, DNA of very high purity (97% by HPLC) was used at a fivefold lower concentration (1.5 mM duplex). Second, the longitudinal and transverse relaxation dispersion profiles were recorded for both ²³Na⁺ and ⁸⁷Rb⁺ in the same mixed-salt (0.2 M NaCl, 0.5 M RbCl) sample before and after addition of netropsin. The new results clearly confirm low-affinity binding of Na⁺ to the minor groove AT-tract. Moreover, they show directly that Rb⁺ and Na⁺ ions compete for the same binding sites in the minor groove AT-tract.