Aim of this project is the improvement of imaging speed and suppression of background in PAINT super-resolution microscopy applied to DNA:RNA:protein targets. We will develop local PAINT (L-PAINT) with amino acids probe design that exhibits two binding sites to the target structure. The stronger binding site upconcentrates the probe at the target structure while the weaker binding site equipped with a fluorescent dye is continuously localised while it samples the binding sites in the local environment. We will study the “phasespace” for optimal L-PAINT probe design using DNA origami model structures with defined patterns of binding sites and a landscape of interactions strengths. What should be the interactions strengths of strong and weak binding site? What is the optimal length of the linker? How does the binding kinetics scale with distance to the strong binding site and how does it depend on the mechanical properties of the linker (single-stranded vs double-stranded or “nunchucks” design with alternating single- and double-stranded regions)? We will then use L-PAINT for cell receptor imaging and for single-molecule imaging of aptamers. Most importantly, a new approach for multiplexed detection of RNA and RNA:DNA nanostructures will be developed in cooperation with the Keyser lab.