Intramolecular i-Motif Structures of Telomeric DNA

Abstract The i-motif is an intercalated structure formed by association in a head to tail orientation of two parallel duplexes whose strands are held together by hemiprotonated C·C(+) pairs. The i-motif may be formed by a single strand containing four cytidine repeats, by association of two strands containing two cytidine repeats or by four strands containing a single cytidine stretch. The repeated C-rich sequences of centromeric and telomeric regions can potentially fold into an intramolecular i-motif. We have investigated by NMR spectroscopy the structure of d(CCCTA(2)CCCTA(2)CCCTA(2)CCCT), a fragment of the vertebrate telomere. It includes an i-motif core of six intercalated C·C(+) pairs. At one end (the "top"), the central TA(2) linker loops across one of the narrow grooves, and the core is extended by base stacking in the loop. At the bottom, where the two other TA(2) linkers loop across the wide grooves, the NMR spectra reveal motions in the microsecond to millisecond scale. The pseudo-symmetry of the structure, which results in degenerate spectra and poor resolution, was broken by appropriate substitution of T by U and of C by 5-methylcytidine (5mC). This allowed us to solve the structure of d(CCCTA(2)5mCCCTA(2)CCCUA(2)CCCT). The motion is restricted to a flip of A18 around the glycosidic bond. Returning to the pseudo-symmetrical sequence, we find that each of the bottom loops switches between the structures of the first and third loops of the non-symmetrical sequence. We also analyzed the effects of the loop sequence and of the length of the C-stretches on the topology and stability of the intramolecular i-motif structure.