Free Energy Landscape between Watson–Crick to Hoogsteen Base Pairing Transitions

The ability of individual Watson-Crick (WC) base pair (bp) in duplex DNA to transition to alternative pairing modes, such as Hoogsteen (HG) bp, on biologically relevant time scales has significantly influenced DNA research for many years. Such bp transitions in the presence of proteins are not well documented in the literature. Here, we employ the well-tempered metadynamics (WT-MetaD) method to determine the lowest free energy path for the transition between WC and HG bp in naked DNA to standardize our protocol of using the glycosidic angle (χ) as collective variable (CV) and reproduce previously reported experimental and simulation data. We use these protocols for the bp transition in the presence of proteins. For the transition between WC → HG bp, the energy barrier decreases to approximately 8 kcal/mol in the presence of proteins compared to naked DNA, and the HG bp becomes ∼4.0 kcal/mol more stable than the WC bp. In contrast, for the reverse HG → WC transition, the energy barrier increases to nearly 12 kcal/mol, with the HG bp remaining ∼3 kcal/mol more stable than its WC counterpart.