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

Area of Science:

• Synthetic biology
• Molecular biology
• Computational chemistry

Background:

• Expanding the genetic alphabet necessitates understanding the replication fidelity of synthetic bases alongside natural DNA.
• Unnatural base pairs (UBPs) offer a route to expanding the genetic alphabet, but their faithful replication is a challenge.

Purpose of the Study:

• To mechanistically understand the single-nucleotide incorporation selectivity of Hirao's UBPs by a DNA polymerase.
• To provide a quantitative framework for UBP incorporation selectivity using quantum chemical calculations.

Main Methods:

• Quantum chemical studies were performed on the DsPx UBP and compared with canonical Watson-Crick pairs.
• Analysis focused on computed stacking energies within the DNA helix, without explicit polymerase modeling.
• Molecular orbital and energy-decomposition analyses were used to investigate electrostatic and dispersion interactions.

Main Results:

• Computed stacking energies accurately reproduced the experimentally observed incorporation selectivity of the DsPx UBP.
• Electrostatic and dispersion interactions were found to significantly strengthen the affinity of the DsPx pair within the DNA helix.
• The quantum chemical approach successfully explained the superior performance of DsPx compared to other UBPs.

Conclusions:

• Noncovalent interactions, particularly stacking energies, play a crucial role in the stabilization and selective incorporation of UBPs.
• This work provides a computational framework for designing and optimizing UBPs for synthetic genetic systems.
• The findings advance the understanding of DNA replication fidelity and the molecular basis of synthetic base pairing.