Abstract: Throughout their lifetime, biomolecules are subject to a variety of mechanical forces. These forces occur through specific interactions, such as binding and catalysis, and nonspecific ones via the crowded cellular environment. How do biomolecules respond to and generate mechanical forces? Despite the importance of this question, answers have only recently become accessible through the development of single molecule manipulation techniques. This dissertation addresses how RNA molecules unfold via mechanical force, and how a helicase accomplishes the same mechanical task. Using optical tweezers, we have manipulated single Tetrahymena thermophila ribozyme molecules and determined the strength and location of kinetic barriers opposing their local mechanical unfolding. We observed eight unfolding intermediates, corresponding to discrete barriers with lifetimes of seconds and rupture forces of 10-30 piconewtons. Barriers were Mg 2+-dependent, brittle, and corresponded to known intra and inter domain interactions. Although mechanical unfolding of this complex RNA displayed rich kinetic information, it occurred irreversibly and left thermodynamic information inaccessible. We have experimentally tested Jarzynski's equality, relating the irreversible work to the free energy difference. We mechanically stretched a simple RNA structure (P5abc) both reversibly and irreversibly, and application of the equality recovered the free energy landscape to within ∼ k_BT/2. The test and application of Jarzynski's equality has implications extending to a broad range of scientific areas. Lastly, how does a helicase motor utilize fuel to generate mechanical forces to unfold RNA, and how does it respond to local RNA mechanical barriers? We have followed in real-time, with 2-basepair and 20-millisecond resolution, individual RNA translocation and unwinding cycles of the Hepatitis C virus helicase (NS3). We found the cyclic movement of an NS3 monomer to be coordinated by ATP in discrete steps of 11±3 bp, and actual unwinding to occur in rapid smaller substeps of 3.6±1.3 bp, also triggered by ATP binding, suggesting that NS3 moves like an inchworm. When encountering strong mechanical barriers, NS3 fell off more frequently and slowed down. Coupled with tools to mechanically probe substrate biomolecules, the assay developed here should be useful to investigate the mechanical action of a broad range of nucleic acid translocation motors and binding proteins.