Figure 2. The G(syn) residue in a helical context is nearly co-planar with the adjacent cytosine (pink). The torsion angles a, g (cyan) are ﬂipped with respect to typical values for A-form and the helix is locally unwound. residues, ranging 152 to 152 . This is 120 from the usual +sc. This amounts to ﬂipping of the O50-C50 bond and a local ‘straightening’ of the sugar-phosphate backbone (Figure 2). The unusual torsion of the backbone is similar to the ‘extended’ conformation observed by Haran et al. (49) in a CG step of a DNA helix. In the present study this conformation seems to be necessary for the simultaneous (i) Hoogsteen–Watson– Crick pairing of the guanosines; (ii) the H-bond between exo-amino group of G(syn) with the phosphate O; and (iii) stacking against the neighbouring cytosine. These effects explain the accommodation of the G–G pair within the helix. The local straightening of the sugar-phosphate backbone, amounting to a local unwinding of the helix, is compensated elsewhere along the duplex, and the overall statistics do not deviate from typical (Supplementary Tables 1–4). This is different from the case of CAG repeats (31) where a similar inversion of the a and g angles is associated with the overall unwinding of the helix and broadening of the major groove to >20 A˚ (measured as the distance between lines connecting P atoms). For the native CGG-containing duplexes, the average width of the major groove was 17.9 ± 0.9 A˚ , for the longer Br-modiﬁed duplex it was 17.8 ± 2.5 A˚ and for the shorter modiﬁed structure 14.3 A˚ . The values for the minor groove were 15.8 ± 0.5 A˚ , 15.4 ± 0.5 A˚ and 16.1 A˚ , respectively. The values are not out of the ordinary for the A form. Another effect of the G(syn) conformation is that the guanine has no stacking interaction with the downstream G–C pair and it stacks against the preceding pair of residues (Figure 3). The effect of bromination and the distribution of G–G conformers All the 8-Br modiﬁed guanosine residues are in the syn conformation, therefore the pairs are 8-BrG(syn)- G(anti). As observed before, duplexes containing the modiﬁed residues are more stable than the Nucleic Acids Research, 2011, Vol. 39, No. 16 7311 corresponding native duplexes. The melting temperature of [GC(8-BrG)GCGGC]2 is 13 C higher than for (GCGG CGGC)2 (21). In structural terms this is probably due to a restricting effect of Br on the conformational freedom and excluding the unfavourable G(anti)-G(anti) interactions. The crystallographic structures bear this out in the sense that the modiﬁed duplexes contain only well ordered 8-BrG(syn)-G(anti) pairs, while in the native duplexes each G–G pair is observed in one of three possible ar- rangements: G(syn)-G(anti), G(anti)-G(syn) or a statically disordered mixture of the two (in 2 out of 36 pairs). The three base-pairing arrangements occur with different frequencies and in various combinations of pairs along the native duplex. Symmetric arrangements are clearly favoured: anti-syn followed by syn-anti or vice versa (in 14 out of 18 cases). There is a slight preference, which can be fortuitous, for the former arrangement (8 cases as opposed to 6). Of the remaining four duplexes, two are clearly asymmetric and two contains statically disordered G–G pairs (Supplementary Table 5). In the longer modiﬁed duplexes, containing unmodiﬁed G–G pair in the middle, two of the native G–G pairs are disordered, showing both possible conformations; the third is ordered. Apart from restricting the conformational freedom, and thus deﬁning the conformation of the G–G pair, there is little crystallographic evidence that bromination alters the structure compared with the native RNA. The native and brominated duplexes can be superposed with an r.m.s. deviation of 1 A˚. The similarity is also reﬂected in helical parameters. In terms of interactions with the solvent, the Br atom seems to displace a water molecule, which in native G(syn) is located 3.2 A˚ from C8, in the minor groove, but its main effect appears to be steric. In terms of H-bonding capacity, bromination alters the pKa of guanosine from 9.3 to 8.4 (50), but this is likely to be insigniﬁcant. Solvent interactions and hydration The exposed Watson–Crick edges of G(syn) residues interact with sulphate anions. In some instances in the native RNA structure and in the longer modiﬁed duplex, the sulphate appears well ordered, but in most cases its orientation is poorly deﬁned. The anions can be distin- guished from water molecules by the size and shape of the electron density and the interaction distance from the RNA (Figure 4b). In the high resolution structure, where there was no sulphate in the crystallization medium, an inner complex is observed involving the carbonyl oxygen atom with a hydrated calcium cation (Figure 4a). The conditions in the crystallization medium are far from physiological, nevertheless the observed complexes indicate a potential of the solvent-exposed Hoogsteen–Watson–Crick edge for at- tracting charged species, especially as no interactions with ions are observed elsewhere in the structures. In the absence of sulphate, the G(syn) Watson–Crick edge is hydrated by three water molecules that form a crest co-planar with the guanine. In the absence of Ca2+, the G(anti) H-edge is hydrated by two- or three-ordered water molecules.