NMR study has shown that DNA oligonucleotide d(G(3)T(4)G(4)) adopts an asymmetric bimolecular G-quadruplex structure in solution. The structure of d(G(3)T(4)G(4))(2) is composed of three G-quartets, overhanging G11 residue and G3, which is part of the loop. Unique structural feature of d(G(3)T(4)G(4))(2) fold is the orientation of the two loops. Thymidine residues T4-T7 form a diagonal loop, whereas T15-T18 form an edge type loop. The G-quadruplex core of d(G(3)T(4)G(4))(2) consists of two stacked G-quartets with syn-anti-anti-anti alternation of dG residues and one G-quartet with syn-syn-anti-anti alternation. Another unusual structural feature of d(G(3)T(4)G(4))(2) is a leap between G19 and G20 over the middle G-quartet and chain reversal between G19 and G20 residues. The presence of one antiparallel and three parallel strands reveals the hitherto unknown G-quadruplex folding motif consisting of antiparallel/parallel strands and diagonal as well as edge type loops. Further examination of the influence of different monovalent cations on the folding of d(G(3)T(4)G(4)) showed that it forms a bimolecular G-quadruplex in the presence of K+, Na+, and NH4+ ions with the same general fold.

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2D NMR studies demonstrate that the residence lifetime of 15NH4+ ions within the bimolecular G-quadruplex adopted by d(G4T4G4) is reduced from 270 ms in the presence of ammonium ions alone to 36 ms in the presence of Na+ ions.

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Variable temperature-and pH-dependent 1 H NMR conformational analyses of 3 J HH coupling constants and NOE enhancements in the 4Ј-thionucleosides 1-10 in D 2 O, complemented by ab initio calculations, have given insight into the interplay of anomeric and other stereoelectronic effects that are modulated by the substitution of ring oxygen by sulfur in natural nucleosides. The N S pseudorotational equilibrium of the 2Ј-deoxy-4Ј-thionucleosides 1-4 is slightly shifted towards S-type conformers, while their ribo analogues 5-8 exhibit ca. 50 : 50 ratio at 278 K and neutral pH. α-4Ј-Thionucleosides 9 and 10 display a strong preference for N-type conformers. The S-C-N anomeric effect in 1-4 is stronger in purine than in pyrimidine 4Ј-thionucleosides, which is opposite to natural 4Ј-oxonucleosides, and increases in the following order: thymine < cytosine < guanine < adenine. The S-C-N anomeric effect in 1-4 is weaker than the O-C-N anomeric effect in their 4Ј-oxo counterparts. We have observed considerable population of up to 40% of γ Ϫ rotamers across the C4Ј-C5Ј bond, which has been attributed to the preference of the [S4Ј-C4Ј-C5Ј-O5Ј] fragments in 1-10 for trans over gauche conformation. Similarly, the 3Ј-OH group drives the N S equilibrium in 1-10 towards N where the [S4Ј-C4Ј-C3Ј-O3Ј] fragment adopts trans conformation. The 2Ј-OH group has been found to preferentially stabilise N-type sugar conformation in the 4Ј-thioribonucleosides 5-8 where it occupies a pseudoaxial orientation. The pK a values in 1-10 are almost identical to the pK a values of their natural counterparts, which shows that the acid-base character of the constituent heterocyclic moieties does not change upon substitution of oxygen with sulfur atom. The shift of the N S pseudorotational equilibrium in 1-8 towards N upon protonation and towards S upon deprotonation of the nucleobase is smaller by up to 10 percentage points in comparison to their 4Ј-oxo counterparts. This can be attributed to less efficient tuning of the S-C-N anomeric effect in 1-8 by protonation and deprotonation of the nucleobase. 1D difference NOE experiments indicated predominant anti orientation of the nucleobase in 2-10. Ab initio calculations at up to MP2/6-31G**//6-31G** level have shown two energy minima in the North and South regions of conformational space with the energy barriers between 17.5 and 28.5 kJ mol Ϫ1 in the East region. Interestingly, the energy barrier in the West region is comparable or even lower than the barrier in the East region of conformational space in 1-10, which is in contrast to natural nucleosides.