The High-Resolution Crystal Structure of a Parallel-Stranded Guanine Tetraplex

DNA and RNA containing runs of consecutive guanine bases may adopt four- stranded conformations based on the hydrogen-bonded guanine tetrad, or G quartet. The character of the unusual conformations adopted by polymers containing guanosine as well as guanosine mononucleotides were first investigated in the 1960s. In more recent years interest has been rekindled by the discovery that telomeric sequences in Oxytricha and Tetrahymena, which typically contain repeat tracts of guanine bases, adopt higher order structures in vivo and unusual conformations in vitro. Nucleic acid sequences containing guanine repeats have been located in a number of biological areas including chromosomal telomeres, immunoglobulin switch regions and the human immunodeficiency virus (HIV) genome, as well as in a variety of recombination sites. The oligonucleotide d(T2G4T2) has recently been demonstrated to be a potent inhibitor of HIV viral-mediated cell fusion. Probing of the structures adopted by these sequences in vitro has revealed a capacity for several non-canonical structures, all of which involve the formation of four-stranded DNA based upon a guanine tetrad with a highly distinctive hydrogen-bonding arrangement. Structural investigations have indicated at least two major classes of non-canonical conformation assumed by guanine-rich DNA deploying the guanine tetrad. The first class involves the folding back of a tandem sequence, or the adoption of a hairpin dimer. This results in the anti-parallel alignment of strands and the consequent formation of both syn and anti glycosyl conformations for the guanine residues within a strand. The second class are defined by the association of four independent strands in a parallel formation, and in this case it is anticipated, on the basis of results from fibre diffraction studies, that the glycosyl linkages of the guanines all adopt an anti conformation. Both structural classes require the presence of group I metal ions, in particular sodium and potassium, and a number of hypotheses have been promoted to account for the role of these ions in stabilising the structures adopted. Recent high-resolution NMR and X-ray crystallographic studies have succeeded in determining the key structural features of the structures of the foldback form. In the last couple of years the isolation of proteins that bind to and promote the formation of both structural classes has lent weight to their functional importance in living systems. This thesis describes my work in determining the three dimensional crystal structure of the parallel-stranded tetraplex formed by the oligonucleotide d(TG4T) in the presence of sodium cations. Samples of the oligonucleotide were synthesised by solid- state methods and the integrity of the final products checked by MALDI-TOF mass spectrometry. Single crystals suitable for X-ray diffraction studies were obtained using the hanging drop vapour diffusion method. Characterisation of the crystals revealed the space group to be PI with unit cell dimensions a=28.76A, b=35.47A, c=56.77A, alpha=74.3