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

## TL;DR

This study explains how synthetic DNA base pairs can be selectively incorporated into DNA using quantum chemistry, without needing a polymerase enzyme.

## Contribution

The study shows that π–π stacking interactions alone determine the selectivity of unnatural DNA base pairs.

## Key findings

- Stacking energies within DNA helix explain the selectivity of DsPx base pairs.
- Electrostatic and dispersion interactions enhance DsPx's affinity compared to other base pairs.
- The framework helps design synthetic genetic systems with improved replication fidelity.

## Abstract

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.