Nanoscale manipulation of individual biomolecules, using such techniques as the atomic force microscope (AFM) and laser optical tweezers (LOT), has increased the scope and detail with which important biological interactions, such as protein folding, receptor-ligand binding, and double-stranded DNA melting, can be studied. In recent years, single molecule manipulation via AFM has been used to characterize the mechanical properties of various proteins. However, since single molecule manipulation experiments are typically performed under nonequilibrium conditions, extracting thermodynamic properties from these measurements has proven difficult. The derivation of Jarzynski's equality, which relates nonequilibrium work fluctuations to equilibrium free energy differences, provides the possibility for extracting equilibrium information from single molecule manipulation data. Here, single molecule force measurements of the stretching and unfolding of the titin I27 protein are analyzed using Jarzynski's equality to reconstruct the underlying free energy landscape associated with this process. We describe the procedures for the automated selection, alignment, and Jarzynski analysis of single molecule data. We use the recovered equilibrium free energy landscape to estimate thermodynamic properties such as the unfolding free energy barrier, which is in good agreement with estimates from bulk kinetics studies and various simulations. Also, the convergence behavior of Jarzynski's equality with respect to pulling velocity is studied experimentally. We demonstrate that with enough pulling trajectories, Jarzynski's equality will indeed recover the equilibrium free energy landscape for a given process. The number of trajectories required to recover the equilibrium free energy for a given pulling velocity is used to quantify this convergence behavior and identify a range of pulling velocities that is most efficient for thermodynamic analysis of single molecule manipulation data. Single molecule manipulation is also used to characterize DNA melting transitions by repeatedly stretching and relaxing an individual λ-DNA molecule. Here, a force induced transition between B form DNA ( B -DNA) and S form DNA ( S -DNA), prior to dsDNA melting, is observed. The mechanical properties of the various conformations, B -DNA, S -DNA, and single-stranded DNA, are quantified using polymer elasticity models, and are shown to agree well with expectations from previous experiments and theory. Fabrication of DNA-based nanostructures, particularly DNA-gold nanoparticle assemblies, has generated significant interest due to their interesting optical and phase transition properties. Here, the effects of various types of disorder within the DNA-gold nanoparticle system are studied and quantified. We show that the stability of these complex fluids can not be quantified using expectations from free DNA hybridization. For example, when linking pairs of gold nanoparticle probes using a DNA linker sequence, a lack of base-pairing symmetry between the probes creates a disorder that decreases the overall stability of the system. This occurs despite the energy contribution that is gained by adding a single base pair to one probe, creating the lack of symmetry. The assumption that base-pairing defects will lower the stability of the nanoparticle aggregates due a loss of hybridization energy is also found to be violated with surprising frequency. In some cases, nonspecific binding between the gold particle surface and a free DNA base can result in a system with increased stability, despite the loss in energy resulting from a mismatched or deleted base. These observations demonstrate that the DNA interactions within these nanostructures are highly complex and that the system stability is not always governed by free DNA hybridization.