The oncogenic human immunodeficiency virus (HIV) currently infects an estimated 33 million individuals and has claimed more than 25 million lives. The primary treatment of HIV consists of a cocktail of several classes of antiretroviral drugs targeting various components of the HIV virus. The most utilized target of antiretroviral drugs is reverse transcriptase (RT), an enzyme that transforms the viral genome from ssRNA to dsDNA. Unfortunately, significant gaps remain in our understanding of how RT functions. Using single molecule techniques, I have previously shown that the orientation of RT while associated with nucleic acids is highly dynamic and characterized by both flipping and sliding transitions. These transitions are associated with processing of the poly-purine tract (PPT) sequence, the rapid targeting of RT to the ends of long regions of double stranded nucleic acids, and strand displacement synthesis. In this grant, I propose to develop additional single molecule assays to further study the dynamic interactions of RT with physiologically relevant nucleic acid substrates. These new assays will allow my lab to address specific questions relevant to the understanding of this key enzyme. First, we will determine the mechanisms of the flipping and sliding transitions and test whether these transitions are required for the successful completion of key steps in the reverse transcription reaction. Second, we will probe the detailed mechanism of strand displacement synthesis and determine the energetic contacts RT uses to stabilize the annealed primer strand. Finally, we will also measure the effects of antiretroviral drugs in preventing the completion of individual steps of the reverse transcription process to identify potentially novel mechanisms of inhibition. Taken together, this research offer fundamental insights into protein and nucleic acid interactions and help to define the next generation of anti-HIV drugs.