The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C(+) basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C(+) hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemi-protonation of cytosine results in a stable C-Cþ base pair. Fundamental studies to understand iM formation from C-rich strands of DNA are described. We present a systematic characterization of the consequences of epigenetic modifications, molecular crowding, degree of hydration, and DNA sequence on the stabilities of iM-forming sequences. We used a number of biophysical techniques to characterize both the folded iM and the folding kinetics of an iM. We established a mechanism for the folding. We observed that the C-Cþ hydrogen bonding of certain bases initiates the folding of the iM structure. We also observed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding as compared to those bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded form. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC and PdC can be used to monitor individual residues of an iM structure and can be used to determine the pKa of an iM. Our results indicate that 5-hydroxymethylation of cytosine destabilized the iMs against thermal and pH-dependent melting, while 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions, the thermal stability of iMs increased and the pKa was raised to near 7.0. Taken together, our work has laid the foundation for examining folding and structural changes in more complex iMs.