pH-induced intramolecular folding dynamics of i-motif DNA

Using the combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) technique, we investigate the mechanism and dynamics of the pH-induced conformational change of i-motif DNA in the bulk phases and at the single-molecule level. Despite numerous studies on i-motif that is formed from cytosine (C)-rich strand at slightly acidic pH, its detailed conformational dynamics have been rarely reported. Using the FRET technique to provide valuable information on the structure of biomolecules such as a protein and DNA, we clearly show that the partially folded species as well as the single-stranded structure coexist at neutral pH, supporting that the partially folded species may exist substantially in vivo and play an important role in a process of gene expression. By measuring the FCS curves of i-motif, we observed the gradual decrease of the diffusion coefficient of i-motif with increasing pH. The quantitative analysis of FCS curves supports that the gradual decrease of diffusion coefficient (D) associated with the conformational change of i-motif is not only due to the change in the intermolecular interaction between i-motif and solvent accompanied by the increase of pH but also due to the change of the shape of DNA. Furthermore, FCS analysis showed that the intrachain contact formation and dissociation for i-motif are 5-10 times faster than that for the open form. The fast dynamics of i-motif with a compact tetraplex is due to the intrinsic conformational changes at the fluorescent site including the motion of alkyl chain connecting the dye to DNA, whereas the slow intrachain contact formation observed from the open form is due to the DNA motion corresponding to an early stage interaction in the folding process of the unstructured open form.