Abstract
Applications associated with nucleobase protonation events are grounded in their fundamental impact on DNA thermodynamics, structure, and hybridization dynamics. Of the canonical nucleobases, N3 protonation of cytosine (C) is the most widely utilized in both biology and nanotechnology. Naturally occurring C derivatives that shift the N3 pK(a) introduce an additional level of tunability. The epigenetic nucleobase 5-carboxylcytosine (caC) presents a particularly interesting example since this derivative forms Watson-Crick base pairs of similar stability and displays pH-dependent behavior over the same range as the canonical nucleobase. However, the titratable group in caC corresponds to the exocyclic carboxyl group rather than N3, and the implications of these divergent protonation events toward DNA hybridization thermodynamics, kinetics, and base pairing dynamics remain poorly understood. Here, we study the pH dependence of these physical properties using model oligonucleotides containing C and caC with FTIR and temperature-jump IR spectroscopy. We demonstrate that N3 protonation of C completely disrupts duplex stability, leading to large shifts in the duplex/single-strand equilibrium, a reduction in the cooperativity of melting, and an acceleration in the rate of duplex dissociation. In contrast, while increasing 5-carboxyl protonation in caC-containing duplexes induces an increase in base pair fluctuations, the DNA duplex can tolerate substantial protonation without significant perturbation to the duplex/single-strand equilibrium. However, 5-carboxyl protonation has a large impact on hybridization kinetics by reducing the transition state free energy. Our thermodynamic and kinetic analysis provides new insight on the impact of two divergent protonation mechanisms in naturally occurring nucleobases on the biophysical properties of DNA.