Decoding Stability and Selectivity in Unnatural DNA Base Pairs with Spatially Separated Recognition Interfaces

Abstract Unnatural base pairs (UBPs) with spatially separated recognition interfaces offer a promising route to expand the genetic alphabet, yet the origin of their enhanced stability remains unclear. Herein, we present a quantum chemical investigation of alkynylated purine–pyridazine UBPs, combining Kohn–Sham molecular orbital and energy decomposition analyses to characterize both base-pairing and π–π stacking interactions. The UBPs display slightly stronger base-pair interaction energies than the canonical G–C pair, arising from a favorable balance of electrostatic and dispersion interactions that compensate for the increased Pauli repulsion. Importantly, modeling of experimentally relevant DNA trimers reveals a quite good correlation between increasing UBP content, enhanced stacking interaction energies, and experimentally observed increases in melting temperature. This agreement highlights the central role of dispersion-driven stabilization and supports the reliability of the computational approach in reproducing the observed trends. These results provide key insights into the factors governing UBP stability and establish a foundation for the rational design of expanded genetic systems.