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(CCCTA₂CCCTA₂CCCTA₂CCCT), 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 TA₂ 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 TA₂ 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(CCCTA₂5mCCCTA₂CCCUA₂CCCT). 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.