Hybridization of DNA strands and motions of the DNA duplex play fundamental roles in biology and the design of nanotechnology and biotechnology applications, and our understanding in these fields is rooted in the thermodynamic and kinetic models of hybridization developed throughout the twentieth century. Many of these technologies and functions of DNA act in concert with chemical modifications to nucleobases or the backbone that alter the physical and biochemical properties of DNA. The mechanisms by which modifications alter hybridization and interactions within the duplex remain poorly understood and are often not captured by the established thermodynamic and kinetic models of hybridization. Interpreting the effects of modifications instead requires a predictive understanding of the series of molecular events during hybridization and structural changes within the duplex state, for which there is little direct insight even for unmodified DNA. This thesis describes efforts to characterize the molecular dynamics of hybridization and the duplex ensemble in DNA oligonucleotides and the corresponding effects from modifications and lesions. Our approach relies on the development and application of time resolved infrared (IR) spectroscopy in combination with molecular dynamics (MD) simulations to directly interrogate and visualize motion along the free-energy landscape of hybridization with previously unachieved molecular detail. Chapters 2 – 5 introduce the benefits and drawbacks of IR spectroscopy and MD simulations for studying these problems. Investigation of oligonucleotides containing an abasic site (AP site) demonstrates how the loss of a single nucleobase disrupts the cooperativity to hybridization and ultimately destabilizes the duplex. Temperature-jump (T-jump) IR experiments reveal that an AP site creates a free energy barrier for forming base pairs on each side of the modified site, effectively breaking the duplex into two segments with free-energy barriers for nucleation even though the average structure of the duplex is nearly unperturbed relative to canonical DNA. Coarse-grained MD simulations reveal that an AP site constrains the trajectories of hybridization events (i.e. transition paths) to follow a mechanism of nucleating and zippering a stretch of base pairs on one side of the AP site before nucleating and zippering the other segment. The added free-energy penalty for nucleating the second segment destabilizes the duplex beyond what is expected from the well established nearest-neighbor thermodynamic models of DNA. Shifting the position of an AP site within an oligonucleotide moves the location of the second nucleation barrier along the free-energy landscape, tuning the destabilization and dynamics of the duplex. T-jump IR experiments reveal that the barrier induces fraying of the short base-pair segment on nanosecond timescales when the AP site is close to the termini. The short segment gains binding stability as the AP site moves away from the termini and eventually overcomes the nucleation barrier at length scales of 2 – 5 base pairs depending on the sequence. The fully encompassed nucleation barrier maximally destabilizes the duplex and creates metastable configurations with one segment dissociated. Nuclear magnetic resonance (NMR) and two-dimensional IR spectroscopy (2D IR) measurements reveal that certain sequences can circumvent the nucleation barrier by base pairing out-of-register.