(360z) Understanding DNA Hybridization through Thermodynamics and Kinetics of Abasic Oligomers

The genetic and catalytic responsibilities of nucleic acids, as well as their more recent nanotechnology applications, rely on cooperative transitions of (un)folding and strand (de)hybridization. Nucleobase sequence plays a key role in all of these processes as it tunes the thermodynamic and time-dependent properties of nucleic acids, and a primary goal has been to develop a predictive understanding of sequence-dependent (de)hybridization. To interrogate this question, we performed computational and experimental analysis on a set of four DNA oligomers with a nucleobase removed from one of three sites along the duplex. This approach, analogous to residue mutations of proteins, allowed us to probe the role of individual nucleobases in the hybridization process as a function of sequence and relative location in the duplex. Using metadynamics and coarse-grained molecular dynamics, we converged a temperature-dependent free energy surface for each system, determined the relationship between the energy landscape and sequence and abasic location, and validated our finding using temperature-dependent IR spectroscopy and isothermal titration calorimetry. Furthermore, we constructed deep-learning optimized Markov state models and translated the collective system dynamics into interpretable mechanistic features with a distinct hierarchy of timescales. These features and timescales were compared against temperature-jump IR relaxation profiles and used to determine each oligomer’s kinetic landscape and metastable state populations during (de)hybridization. Our combined experimental and simulation approach furnishes a detailed characterization of how an abasic site alters base pairing globally across small DNA oligonucleotide systems and provides insight into the contribution of individual nucleobases to DNA duplex hybridization. Furthermore, our findings can inform DNA nanotechnology design and provide insight into potential engineering applications for abasic DNA.