Figure 5. The electrostatic surface potential of two consecutive duplexes of the [GC(8-BrG)GCGGCGGC]2 structure. Red is negative, blue is positive. Sulphate anions (sticks) are shown interacting in the major groove. CONCLUSIONS This is a third in a series of studies aimed at proﬁling CNG structures at crystallographic resolution to facilitate drug design and provide 3D templates for rationalizing bio- chemical and cytological observations. The need for detailed RNA structures is clearly signalled in the litera- ture on rational design of CNG-binding ligands (55–57). The crystal structures presented here of unmodiﬁed and modiﬁed CGG repeats are consistent and allow a general description of double-stranded CGG tracts and a com- parison with CUG and CAG structures reported previous- ly (29,31). The foremost common feature is that all the known CNG structures form A-helices stabilized by C–G and G–C pairs acting as sturdy struts. The variety is provided in between by the non-canonical pairs. Nucleic Acids Research, 2011, Vol. 39, No. 16 7313 The ‘accommodation problem’ is solved differently for each N–N pair, but in every case the disruption of the helix is surprisingly small. The bulky guanines ﬁt within the helical constraints by a 180 ﬂip about the glycosidic bond of one of the bases. The equally bulky adenines retain the anti-conformation, but are shifted out of the helical axis, towards the major groove. In the one known example of paired pyrimidines, the relatively small uracil rings remain some distance apart, making only one direct hydrogen bond instead of two bonds which they can make in different environments. Despite the general resilience of the A-form, character- istic differences can be observed between the CNG duplex structures. The parameters that seem to be specially sen- sitive are helical twist and major groove width. In CGG helices, we observe local unwinding of the helix around the G–G pairs, as opposed to the more general unwinding in the case of CAG structures. The major groove width may depend on the nature of the ligand which occupies it, bound to the G–G pair. This work provides examples of bound calcium and sulphate ions; another example of sulphate interacting with G–G pairs and possible affect- ing the groove width is provided by Adamiak and col- leagues (58). Thermodynamic stability is an important property that is difﬁcult to investigate in crystallography, but some measure is provided by the nature and the count of hydrogen bonds and the extent of stacking interactions. In this respect, CGG helical structures appear relatively stable compared with CAG and CUG tracts—in agree- ment with calorimetric studies (19,21). This could be an important factor in the RNA’s ligand binding afﬁnity and speciﬁcity. So far, the three known CNG repeats have been observed to bind small ligands in the major groove. Glycerol, sulphate anion and Ca2+ cations were found to be associated with N–N pairs of the CNG duplexes. The interactions depend on hydrogen bonds formed with the functional groups exposed in the major groove, character- istic of each N–N pair and to some extent on the electro- static charge distribution. These can be taken as the main indicators in designing speciﬁc ligands. A detailed comparison of CGG, CAG and CUG duplex structures is provided in Supplementary Table 6. ACCESSION NUMBERS 3R1C, 3R1D, 3R1E. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. FUNDING Ministry of Science and Higher Education (Poland, N-N301-0171634); the EU structural funds (POIG.01.03.01-30-098/08); the European Community – Research Infrastructure Action under the FP6 ‘Structuring the European Research Area’ Programme