The main feature of the i-motif structure are cytosine base pairs, each of them made of a protonated and an unprotonated cytosine connected with three hydrogen bonds. I-motifs can be formed from nucleotide sequences comprised of four stretches of cytosine-rich sequences interspersed with loop nucleotides. As the protonation of cytosines is crucial for formation of cytosine base pairs and it occurs at pH ~ 5,0, which is far below the physiological pH, it had long been speculated whether these structures do exist in vivo. Nevertheless, further studies confirmed their presence in living cells and brought up speculations about their role in processes such as DNA replication, gene expression and telomere lengthening. The aim of this research is to determine mechanism and driving forces of i-motif folding for various nucleotide sequences. Changes in conformation of the oligonucleotides depending on pH were determined using CD spectroscopy. As expected, i-motifs were mainly formed at pH ~ 5,0 except for the sequence 8bv6 which already at the physiological pH adopted one of its two possible i-motif conformations. Experiments were conducted in the pH range between 7,0 and 4,5. Reversibility of the process was confirmed by changing pH in the opposite direction. Furthermore, thermal denaturation was assessed with UV absorption spectroscopy. We concluded that the process is irreversible for all i-motifs. Isothermal titration calorimetry was used to measure heat effects accompanying proton binding and consequent folding of the nucleotides into i-motifs in the solution whose pH we were gradually lowering. The results of CD spectroscopy and isothermal titration calorimetry were used to determine thermodynamic parameters in a model-independent manner. Additionally, obtained pH and ITC titration curves were used for model analysis that provided insight into the folding mechanism. We assumed that folding is gradual, with protons binding singly. Based on the model we also determined driving forces of the i-motif formation and distribution of populations of partly folded i-motif species depending on the pH of the solution.
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