Quadruplex-forming properties of FRAXA (CGG) repeats interrupted by (AGG) triplets.

The (CGG) repeats associated with X-chromosome fragility are generally believed to form quadruplexes. This notion has persisted although it had been shown that only very short (CGG)(n) sequences form quadruplexes and that this quadruplex formation occurs in conditions far from physiological. We have now studied, using CD and absorption spectroscopies, quadruplex formation of (CGG)(n) (n=4, 7, 8, or 16) and their analogs interrupted by (AGG) triplets under various solvent conditions. In healthy individuals, (AGG) triplets are interspersed throughout the (CGG) repeat regions and appear to hinder (CGG)(n) motif expansion. Here we show that (CGG) repeats do not form quadruplexes under physiological conditions in aqueous solution but, interestingly, quadruplexes are readily formed in water-ethanol solutions. The presence of (AGG) triplets markedly stabilized quadruplex formation. Quadruplexes may thus hinder rather than support (CGG)(n) motif expansion.

Conformational properties of DNA containing (CCA)n and (TGG)n trinucleotide repeats.

We have used CD spectroscopy, polyacrylamide gel electrophoresis, and UV absorption spectroscopy to study conformational properties of DNA fragments containing (CCA)n and (TGG)n repeats, which are the most length-polymorphic microsatellite sequences of the human genome. The (CCA)n fragments are random single strands at neutral and alkaline pH but they fold into intramolecular intercalated cytosine tetraplexes at mildly acid pH values. More acid values stabilize intermolecular tetraplex formation. The behavior of (TGG)n repeats is more complex. They form hairpins or antiparallel homoduplexes in low salt solutions which, however, are transformed into parallel-stranded guanine tetraplexes at physiological KCl concentrations. Their molecularity depends on the repeat number: (TGG)4 associates into an octameric complex, (TGG)8 forms tetramolecular complexes. (TGG)n with odd repeat numbers (5, 7, and 9) generate bimolecular and tetramolecular tetraplexes. The only (TGG)7 folds into an intramolecular tetraplex at low KCl concentrations, which is antiparallel-stranded. Moreover, the (TGG)(n) fragments provide various mutually slipped conformers whose population increases with salt concentration and with the increasing repeat number. However, the self-structures of both strands disappear in the presence of the complementary strand because both (TGG)n and (CCA)n prefer to associate into the classical heteroduplex. We suppose that the extreme conformational variability of the DNA strands stands behind the length polymorphism which the (CCA)n/(TGG)n repeats exhibit in the human genome.