The goal of this Phase I SBIR project is to develop a set of high resolution antibody mimetics (nanoCLAMPs) optimized for super-resolution imaging of a broad range of subcellular structures. Advances in super-resolution microscopy have enabled the achievement of resolutions well below the diffraction limit with current techniques now approaching 20 nm. For immunostaining, the 25 nm size of antibody complexes now limits the achievable resolution. With a much smaller size of 3 nm, alternatives to antibodies such as aptamers and nanobodies have been shown to improve resolution significantly with STORM and STED techniques. Unfortunately, only a limited number of targets have validated, high resolution affinity reagents available. nanoCLAMPs are a newly described class of single domain affinity reagents with high affinity, high specificity and the rare property of polyol-responsiveness. nanoCLAMPs have the ability to release functionally and structurally intact targets upon exposure to buffer containing polyol and salt. Panels of nanoCLAMPs to new protein targets are easily generated from our validated synthetic phage display library in a few weeks, a process that eliminates the need for animal immunization and provides multiple binders per target. nanoCLAMPs have the potential to improve the performance of super-resolution imaging because 1) their small size and lack of endogenous cysteines allows fluorescent labels to be positioned within nanometers of the target structure and 2) their polyol-responsiveness enables the development of gentle stripping and restaining methods. Successful completion of this project will generate commercially available, open-source research tools that will improve the achievable resolution of super-resolution imaging with reagents marking a broad range of subcellular structures.
Project Terms: Achievement; Affinity; Animals; Antibodies; Antibody Formation; aptamer; base; Behavior; Benchmarking; Binding; Biophysical Process; Buffers; Carbohydrates; Cellular Structures; Complex; Cysteine; density; Development; Direct immunofluorescence; Disease; effective therapy; Engineering; Exposure to; fluorophore; Generations; Goals; Hyaluronidase; Image; Imagery; Immunization; Immunofluorescence Microscopy; Immunoglobulins; improved; Investigation; Label; Length; Libraries; Methods; Microscopy; mimetics; nanobodies; nanometer; Nature; novel; open source; Performance; Phage Display; Phase; polyol; Positioning Attribute; Process; Property; Proteins; Protocols documentation; Reagent; Research; Resolution; scaffold; Small Business Innovation Research Grant; Sodium Chloride; Specificity; Stains; Structure; Subcellular structure; Surface; Techniques; Technology; Testing; tool; Variant;
