Summary The structure of important biomolecules is intrinsically dynamic, and there is an important need for optical tools that can probe the structural dynamics of biopolymers at the single molecule level with high temporal resolution and without limitation in maximum observation time. Dynamic molecular rulers, such as Fluorescence Resonance Energy Transfer (FRET) dye pairs or plasmon rulers (PRs), as well as tethered particle assays are currently available optical methods to probe the structural dynamics of individual molecules. FRET is, however, plagued by the limited photophysical stability of conventional organic dyes that serve as energy donor and acceptor. Photobleaching limits the maximum number of photoexcitation and emission cycles and, thus, defines fundamental limitations for a continuous monitoring of molecular structure. Conventional PRs and tethered particle assays can provide high signal intensities without blinking or limitation in observation time. The caveat of these approaches is, however, the large size of the particles with typical dimensions on the order of tens of nanometers or larger. This proposal develops a new class of PRs that is based on the phenomenon that the distance-dependent coupling of a gold nanoparticle (NP) tethered to a gold film through a biopolymer modulates the interferometric scattering signal of the NP. This new PR is based on an interferometric detection of plasmon coupling and allows the use of NPs with dimensions as small as 5 nm as probes. The interferometric PRs will make it possible to monitor the structural dynamics of individual biopolymers with high temporal resolution and with no need to compromise between temporal resolution and the duration of the observation. The work described in this proposal will implement interferometric PRs using DNA as biopolymer and characterize their performance. After validating the interferometric PR concept with DNA, the interferometric PR platform will be expanded to allow the characterization of the structural dynamics of the intrinsically disordered tau protein in the presence of a lipid membrane of defined composition. The ability of the interferometric PR to monitor the structural dynamics of a single tau molecule and to detect membrane-induced changes in the structure and dynamics of the biopolymer will be tested. The research described in this proposal will result in a new dynamic molecular ruler technology that overcomes longstanding limitations of conventional optical molecular rulers in terms of the size and photophysical stability of the probes. The specific Aims of this proposal are to: Aim1: Implement the Interferometric PR and Test Its Applicability to Characterize Structural Fluctuations in Single DNA Molecules Aim2: Implement and Validate an Interferometric PR for Probing the Structural Dynamics of a Single Tau Protein in the Vicinity of a Membrane