DNA plays a central role in biological processes, and its interactions with drugs, proteins, and itself occur within a highly dynamic environment. Experimental studies show that Cryptolepine intercalates into DNA, preferentially binding to cytosine–guanine rich regions, though the detailed mechanism of interaction remains unclear. As Cryptolepine exhibits cytotoxicity against cancer cells, atomistic insights into its binding behaviour are of particular interest. Likewise, Hoogsteen bonding involving 8-oxoguanine, an oxidatively damaged form of guanine and a biomarker of cancer, warrants investigation. Experiments suggest that 8-oxoguanine can mimic Watson–Crick

pairs in the syn conformation when mispaired with adenine, potentially leading to its incorporation into DNA. To better understand the selectivity of Cryptolepine’s binding and how 8-oxoguanine can evade fidelity checks and misincorporate, we employ a multiscale approach to examine both systems. Molecular dynamics (MD) simulations combined with umbrella sampling and free energy perturbation were used to characterise Cryptolepine binding across different nucleobase pairs, with energy decomposition analyses revealing key contributions to stability. For 8-oxoguanine, extensive MD simulations (100 per nucleobase pair) probed its behaviour within DNA and in complex with polymerase. Our results show that polymerase fails to significantly mitigate adenine–8-oxoguanine backbone spacing; with the triphosphate structure showing strong stability compared to the template structure. Both pairs closely mimics adenine–thymine. Quantum mechanical calculations further indicate that the adenine–8-oxoguanine Hoogsteen mispair has the lowest Gibbs free energy, with small conformational barriers. Proton transfer occurs stably only in single steps, with Watson–Crick pairing yielding the most stable product. Nuclear–electronic orbital

density functional theory (NEO-DFT), which explicitly treats select protons quantum mechanically alongside electrons, further confirms Watson–Crick pairing as the only stable product state.