Single molecule techniques have developed into a powerful tool to study the molecular machines involved in many fundamental biological processes. Techniques such as fluorescence localization, F"rster resonance energy transfer (FRET), and optical tweezers have been instrumental in deciphering the mechanism of molecular motors such as myosin and kinesin, DNA and RNA polymerases, and helicases, to name just a few examples. Recently, the development of ultrahigh-resolution optical tweezers has made possible, for the first time, the direct observation of molecular motion on the scale of 1 basepair of DNA (3.4[unreadable]). Despite such advances, single molecule techniques have had important limitations. Although the conformation changes involved in molecular machines are inherently three-dimensional, such techniques typically project all motion onto a single axis and thus cannot capture the full complexity of molecular motion. Furthermore, these techniques have largely been limited to simple systems involving very few components examined in isolation, whereas, in the cellular context, molecular machines consist of highly coordinated multi-component protein assemblies. To address these limitations, we propose to 1) develop the new generation of single molecule instrumentation combining multi-color fluorescence detection and ultrahigh-resolution optical tweezers. Although instruments merging fluorescence and optical traps have been developed previously, achieving basepair resolution remains a grand challenge that will require a new approach. These capabilities nevertheless will be essential to understand the molecular complexes involved in DNA metabolism-transcription, replication, recombination, and repair-that have great biomedical significance. The hybrid instrument we propose will have the ability to measure multiple observables simultaneously, such as internal protein dynamics by FRET or the assembly kinetics of protein complexes by fluorescence localization, combined with detection of motor displacement at basepair resolution by optical tweezers. As a demonstration of this technique, we will 2) monitor translocation and duplex unwinding by E. coli Rep helicase at basepair resolution, simultaneously with conformational changes by FRET and oligomeric state by fluorescence detection. This proposed work involves the collaboration of experts in ultrahigh-resolution optical tweezers (Y. Chemla, PI) and single-molecule fluorescence (T. Ha, co-PI) at the University of Illinois, Urbana-Champaign. Public Health Relevance Statement: We are proposing to develop an instrument that will combine two powerful cutting-edge technologies: single-molecule fluorescence and ultrahigh-resolution optical trapping. Our goal is to study the dynamics of proteins and protein complexes involved in DNA replication, transcription, recombination, and repair at [unreadable]ngstrom level resolution. This proposed technique has the potential to reveal the detailed mechanism of these molecular machines, a problem of great medical interest as defects in their activity have been implicated in a number of human diseases, specifically cancer.