The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH

UV absorption and CD spectroscopy, along with polyacrylamide gel electropho resis, were used to study conformational properties of DNA fragments contai ning the trinucleotide repeat (GCC)(n) (n=4, 8 or 16), whose expansion is c orrelated with the fragile X chromosome syndrome. We have found that the co nformational spectrum of the (GCC)n strand is wider than has been shown so far. (GCC)n strands adopt the hairpin described in the literature under a w ide range of salt concentrations, but only at alkaline (>7.5) pH values. Ho wever, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodi merize. Our data suggest that the homodimer is a bimolecular tetraplex form ed by two parallel-oriented hairpins held together by hemi-protonated inter molecular C.C+ pairs. The (GCC)(16) strand forms the same tetraplex intramo lecularly. We further show that below pH 5 (GCC)(n) strands generate interc alated cytosine tetraplexes, whose molecularity depends on DNA strand lengt h. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and mon omolecular with (GCC)(16). i-Tetraplex formation is a complex and slow proc ess. The neutral tetraplex, on the other hand, arises with fast kinetics un der physiological conditions. Thus it is a conformational alternative of th e (GCC)(n) strand duplex with a complementary (GGC)(n) strand.