7312 Nucleic Acids Research, 2011, Vol. 39, No. 16 Figure 3. Stacking interactions in the CGG duplex structure. (A and B)The non-canonical G–G pairs (aquamarine); (C) the canonical CG/GC step. Figure 4. The hydration of G(syn)–G(anti) pairs in the GC(8-BrG)GCGGC (A) and in the GC(8-BrG)GCGGCGGC (B) structures. Ca2+ (green) is bound directly to the carbonyl oxygen atom of G(anti); a sulphate anion interacts with the WC edge of G(syn). The 2Fo-Fc electron density map is contoured at 1s level. Interestingly, in all the examined instances of the struc- ture which have sulphate anions in the major groove, the width of the grooves are similar and close to 18 A˚ (see above), as opposed to the Ca2+-containing structure, in which the groove is narrower by nearly 4 A˚ . It is possible that the presence of the anions stabilizes the width of the major groove. Electrostatic surface potential The electrostatic surface potential shows the already familiar pattern of alternating stripes of positive and negative potential in the minor groove, similar to the pre- viously observed distribution in CUG and CAG repeats (Figure 5). The pattern is due primarily to the C–G and G–C pairs rather than to the interposed N–N pairs. The major groove in the CGG structures is mostly electronega- tive with positive areas generated each by the Watson– Crick edge of G(syn) and the exo-amino group of the preceding cytidine residue. The binding of the sulphate ions corresponds very closely with the electropositive features associated with G(syn) residues and their calculated surface potential indeed appears higher than for the adjacent cytosines, which have not attracted any ions. The difference in binding potential for ligands can be explained by the exposed Watson–Crick edge of the guanine in this position. In addition, the guanines have stacking interactions only on one side, while the cytosines are engaged on both their surfaces. It is harder to explain, just by examining the electrostat- ic potential, what distinguishes CGG from CUG or CAG, and why CGG is not recognized by MBNL1 protein (51–53). An answer could lie in the stability of the CGG duplex. The non-canonical G–G pair contains two H-bonds, as opposed to a single bond in U–U and a weak C–H···N bond in A–A. Therefore the CGG tracts could be more stable in the duplex form and less accessible to the protein which appears to bind single-stranded RNA (54). This is consistent with thermo- dynamic parameters: G of duplexes containing CGG is markedly higher than for the other three repeats, which are similar (18,19,21).