An intramolecular i-motif: the solution structure and base-pair opening kinetics of d(5mCCT3CCT3ACCT3CC).

We present a high-definition structure of d(5mCCT3CCT3ACCT3CC), a DNA sequence which resembles a four-times repeat of the C-rich strand of telomeres and centromeres. The structure is monomeric. The CC stretches form four hemi-protonated C.C base-pairs, belonging to two parallel-stranded duplexes which intercalate head-to-tail into an i-motif core. The four grooves of the core are similar to those observed previously in i-motif tetramers, with P-P distances around 0.9 nm and 1.4 nm for the narrow and wide grooves, respectively. At 0 degrees C, the structure is formed even at pH 7, despite the required protonation of cytidine pairs, suggesting that it may be biologically relevant.The intercalation topology of the i-motif core is read off the pattern of inter-residue cross-peaks along each groove: between H1' protons across the narrow grooves, and between amino and H2' protons across the wide grooves. In the hemi-protonated C.C pairs, the imino proton is shared equally between the two bases, as shown by the equal intensities of the NOESY cross-peaks between the imino proton and the two cis amino protons of the pair. Short inter-sugar distances and the direction of CH1' bonds are consistent with CH1'...O4' hydrogen bonds across the narrow grooves, as suggested by Berger et al. (1996). Proc. Natl. Acad. Sci. USA, 93, 12116-12121. At one extremity of the i-motif core, the T3A linker loops across one of the two wide grooves. It extends the core by stacking of A11, which also forms a strongly propeller-twisted reverse-Hoogsteen pair with T8. At the other extremity, the two T3 linkers loop side by side across the two narrow grooves, extending the core by stacking of a T5.T16 pair which connects the two linkers. In this T.T pair between parallel strands, the hydrogen bonds are from imino proton to O4, and the base-pair lifetime is 6 ms at 0 degrees C. The structures of segments 1 to 7 and 12 to 18, which form the i-motif core and the T3 loops, are related by a 2-fold pseudo-symmetry: the geometries and environment are so similar that the NOESY spectra are barely resolved. These various interactions illustrate how linker sequences may affect the stability, intercalation topology and folding pattern of the intramolecular i-motif.