Tautomeric Equilibria of Nucleobases in the Hachimoji Expanded Genetic Alphabet

Evolution
has yielded biopolymers that are constructed from exactly
four building blocks and are able to support Darwinian evolution.
Synthetic biology aims to extend this alphabet, and we recently showed
that 8-letter (hachimoji) DNA can support rule-based information encoding.
One source of replicative error in non-natural DNA-like systems, however,
is the occurrence of alternative tautomeric forms, which pair differently.
Unfortunately, little is known about how structural modifications
impact free-energy differences between tautomers of the non-natural
nucleobases used in the hachimoji expanded genetic alphabet. Determining
experimental tautomer ratios is technically difficult, and so, strategies
for improving hachimoji DNA replication efficiency will benefit from
accurate computational predictions of equilibrium tautomeric ratios.
We now report that high-level quantum-chemical calculations in aqueous
solution by the embedded cluster reference interaction site model,
benchmarked against free-energy molecular simulations for solvation
thermodynamics, provide useful quantitative information on the tautomer
ratios of both Watson–Crick and hachimoji nucleobases. In agreement
with previous computational studies, all four Watson–Crick
nucleobases adopt essentially only one tautomer in water. This is
not the case, however, for non-natural nucleobases and their analogues.
For example, although the enols of isoguanine and a series of related
purines are not populated in water, these heterocycles possess N1–H and N3–H keto tautomers that are
similar in energy, thereby adversely impacting accurate nucleobase
pairing. These robust computational strategies offer a firm basis
for improving experimental measurements of tautomeric ratios, which
are currently limited to studying molecules that exist only as two
tautomers in solution.