Kinetics and thermodynamics of i-DNA formation: phosphodiester versus modified oligodeoxynucleotides

At slightly acidic or even neutral pH, oligodeoxynucleotides that include a stretch of cytidines have been shown to form a tetrameric structure in which two parallel-stranded duplexes have their hemiprotonated C.C+base pairs face to face and fully intercalated, in a so-called i-motif. Cytosine-rich pyrimidine oligodeoxynucleotides can form an intramolecular i-motif. We have studied the ability of several DNA analogs to fold into this structure. Evidence for folding was provided by thermal denaturation. We have shown that phosphorothioate and phosphodiester oligodeoxynucleotides, but not methylphosphonate or PNA oligomers, may form the i-motif. Four different PS oligodeoxynucleotides were compared with their PO counterparts. In all cases, the melting temperature (Tm) of the phosphorothioate oligomer was equal or slightly inferior (by 2-3 degreesC) to the Tmof the natural oligodeoxynucleotide. For long oligodeoxynucleotides, a small change of pH leads to a completely different melting profile: the curves are reversible at pH 6.4 or lower, and a hysteresis is obtained at pH 6.8 or higher; cooling and heating curves were not superimposed, allowing us to determine the rate constants of association (kon) and dissociation (koff) as a function of the temperature: these rate constants give linear Arrhenius plots, in agreement with the prediction of the two-state model of association-dissociation. The activation energy Eonis strongly negative and, at neutral pH, the phosphorothioate associates and dissociates nine times faster than the phosphodiester oligodeoxynucleotide of identical sequence.