Effect of Interior Loop Length on the Thermal Stability and pK_a of i‑Motif DNA

The four-stranded i-motif (iM) conformation of cytosine-rich DNA is important in a wide variety of biochemical systems ranging from its use in nanomaterials to a potential role in oncogene regulation. An iM is stabilized by acidic pH that allows hemiprotonated cytidines to form a C·C⁺ base pair. Fundamental studies that aim to understand how the lengths of loops connecting the protonated C·C⁺ pairs affect intramolecular iM physical properties are described here. We characterized both the thermal stability and the pK_a of intramolecular iMs with differing loop lengths, in both dilute solutions and solutions containing molecular crowding agents. Our results showed that intramolecular iMs with longer central loops form at pHs and temperatures higher than those of iMs with longer outer loops. Our studies also showed that increases in thermal stability of iMs when molecular crowding agents are present are dependent on the loop that is lengthened. However, the increase in pK_a for iMs when molecular crowding agents are present is insensitive to loop length. Importantly, we also determined the proton activity of solutions containing high concentrations of molecular crowding agents to ascertain whether the increase in pK_a of an iM is caused by alteration of this activity in buffered solutions. We determined that crowding agents alone increase the apparent pK_a of a number of small molecules as well as iMs but that increases to iM pK_a were greater than that expected from a shift in proton activity.