DNA is constantly being modified by exogenous and endogenous agents, leading to covalent changes to its structure and, if left unrepaired, to permanent changes in sequence. We use a wide variety of biophysical techniques to examine the effect on DNA structure and dynamics of the common base lesion 8-oxoguanine, the cis-syn thymine dimer lesion, single-base mismatches, and bubbles. The stability of duplex formation in the presence of these disturbances is explored using UV melting thermodynamics, calorimetry, and single-molecule force measurements. Duplex structure is analyzed using small molecule reactive probes, circular dichroism, and NOESY spectroscopy. NMR proton exchange experiments reveal locations of dynamic changes to the DNA duplex, including base pair opening and flexibility. By bringing a diverse set of techniques to bear on each non-canonical structure, we have shown the 8oxoG lesion to be surprisingly stable, while the thymine dimer lesion is significantly more labile. Even Hoogsteen-binding wobble mismatches can effect the kinetics and thermodynamics of duplex formation in surprising ways. These results complement and extend the information obtained by crystallographic methods to include dynamic and thermodynamic factors.