Alchemical Free-Energy Calculations of Watson–Crick and Hoogsteen Base Pairing Interconversion in DNA

[Image: see text] Hoogsteen (HG) base pairs have a transient nature and can be structurally similar to Watson–Crick (WC) base pairs, making their occurrence and thermodynamic stability difficult to determine experimentally. Herein, we employed the restrain–free-energy perturbation–release (R-FEP-R) method to calculate the relative free energy of the WC and HG base pairing modes in isolated and bound DNA systems and predict the glycosyl torsion conformational preference of purine bases. Notably, this method does not require prior knowledge of the transition pathway between the two end states. Remarkably, relatively fast convergence was reached, with results in excellent agreement with experimental data for all the examined DNA systems. The R-REP-R method successfully determined the stability of HG base pairing and more generally, the conformational preference of purine bases, in these systems. Therefore, this computational approach can help to understand the dynamic equilibrium between the WC and HG base pairing modes in DNA.