π-π Stacking Determines the Selectivity of Unnatural DNA Base Pairs Even without Polymerase

Expanding the genetic alphabet requires a mechanistic understanding of how synthetic bases are faithfully replicated alongside natural DNA. We present a quantum chemical study reproducing the experimentally observed single-nucleotide incorporation selectivity of Hirao's unnatural base pairs (UBPs) by the 3'-5' exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. Our analysis focuses on the highly selective DsPx pair, benchmarking its behavior against canonical Watson-Crick pairs and other UBPs. Strikingly, the observed selectivity emerges without explicitly modeling the polymerase, relying solely on computed stacking energies within the DNA helix. Molecular orbital and energy-decomposition analyses show that both electrostatic and dispersion interactions strengthen DsPx's affinity more, capturing experimental fidelity trends and explaining its superior performance relative to related systems. We further evaluate other selective UBPs, including QPa, DsPa, and DsPn. Together, these results provide a quantitative framework for UBP incorporation selectivity and highlight the crucial role of noncovalent interactions in stabilizing synthetic bases within DNA. By bridging computation and experiment, this work advances design principles for synthetic genetic systems and contributes to unraveling the molecular origins of DNA replication fidelity.