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Abstract

The work presented in this thesis utilized experimental and computational methods to investigate the association and dissociation of small DNA oligonucleotides. Fourier transform infrared spectroscopy (FTIR) and temperature-jump (T-jump) infrared (IR) spectroscopy were used to investigate the thermodynamics, mechanism, dynamics, and kinetics of DNA oligos with the sequence 5'-C(AT)nG-3' where n = 2-6. To compliment the experiments a Markov state Monte Carlo kinetic model, intended to be accessible to experimentally focused researchers with regards to the model's complexity and computational expense, was built to simulate association and dissociation trajectories of these sequences plus 5'-ATATGCATAT-3' (GC-core) and 5'-ATATATATAT-3'. These sequences were selected to make a first attempt at separating the different factors that impact DNA dynamics and kinetics focusing initially on sequence length and composition. IR spectroscopy is ideal for studying DNA due to its ability to resolve adenine-thymine (A:T) and guanine-cytosine (G:C) base pairs. Additionally, the kinetics of the sequences studied here fall within the nanosecond to millisecond time window the T-jump instrument can resolve. The Markov state Monte Carlo model provides improved base pair resolution by independently tracking each base pair providing new insights into the mechanism and dynamics of association and dissociation. The experimental results of the 5'-C(AT)nG-3' series were analyzed using an Eyring analysis of a two-state model providing a clearer interpretation of the reaction energetics by extracting the activation entropy, activation enthalpy, and activation free energy. Global analysis links the thermodynamic and kinetic parameters utilizing a linear dependence on oligo length of the entropic and enthalpic activation barriers. Analysis incorporating the thermodynamic nearest neighbor parameters and the experimentally determined activation enthalpy found that the critical nucleus, the minimum number of base pairs such that the partially formed duplex is stable and will proceed downhill to the fully formed dimer, increases in size with increasing temperature and sequence length. Association and dissociation trajectories from the kinetic model were analyzed directly and utilizing transition pathway theory (TPT). The dominant association pathways, isolated by TPT, showed two primary motifs: initiating at or next to a G:C base pair, which is enthalpically driven, and initiating in the center of the sequence, which is entropically driven. For GC-core these motifs overlap resulting in a strong preference for initiating association at the central G:C base pairs. For 5'-C(AT)nG-3' sequences the paths compete resulting in a preference for initiating association events either at or next to a terminal G:C base pair or in the center. Configurations in the transition state ensemble were found to increase in size with increasing sequence length and temperature, in good agreement with the literature and the experimentally determined critical nucleus size. Finally, terminal end fraying experimentally observed in GC-core was replicated by the model and shown to be driven by increased thermodynamic accessibility of the frayed states after the T-jump. This was compared to fast dynamics observed for longer 5'-C(AT)nG-3' sequences, the physical origins of which were not previously clear, and suggests that this fast response is also due to thermodynamically driven end fraying.

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