Structure, internal motions and association-dissociation kinetics of the i-motif dimer of d(5mCCTCACTCC)

At slightly acidic pH, the association of two d(5mCCTCACTCC) strands results in the formation of an i-motif dimer. Using NMR methods, we investigated the structure of [d(5mCCTCACTCC)](2), the internal motion of the base pairs stacked in the i-motif core, the dimer formation and dissociation kinetics versus pH. The excellent resolution of the H-1 and P-31 spectra provided the determination of dihedral angles, which together with a large set of distance restraints, improve substantially the definition of the sugar-phosphate backbone by comparison with previous NMR studies of i-motif structures. [d(5mCCTCACTCC)](2) is built by intercalation of two symmetrical hairpins held together by six symmetrical C center dot C+ pairs and by pair T7 center dot T7. The hairpin loops that are formed by a single residue, A5, cross the narrow grooves on the same side of the i-motif core. The base pair intercalation order is C9 center dot C9(+)/5mC1 center dot 5mC1(+)/C8 center dot C8(+)/C2 center dot C2(+)/T7.T7/C6 center dot C6(+)/C4 center dot C4(+). The T3 bases are flipped out in the wide grooves. The core of the structure includes four long-lived pairs whose lifetimes at 15 degrees C range from 100s (C8 center dot C8(+)) to 0.18s (T7 center dot T7). The formation rate and the lifetime of [d(5mCCTCACTCC)](2) were measured between pH 6.8 and 4.8. The dimer formation rate is three to four magnitude orders slower than that of a B-DNA duplex. It depends on pH, as it must occur for a bimolecular process involving non cooperative association of neutral and protonated residues. In the range of pH investigated, the dimer lifetime, 500 s at 0 degrees C, pH 6.8, varies approximately as 10(-pH).