biostudies-literatureUnknown6(1)Noori ZExpanding 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 <i>Escherichia coli</i> 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 strenACS physical chemistry Au153-162https://www.ebi.ac.uk/biostudies/studies/S-EPMC12856653biostudies-literatureπ-π Stacking Determines the Selectivity of Unnatural DNA Base Pairs Even without Polymerase.PMC12856653Bofill JMPoater JNoori ZBermejo Afalseπ-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 <i>Escherichia coli</i> 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 stren2026-01-01T00:00:00Z2026 Jan2026-06-18T05:49:12.14Z2026-06-18T03:07:36.353ZS-EPMC128566534162470810.1021/acsphyschemau.5c00100