Generation of High Resolution Antibody Mimetics to Improve the Visualization of Subcellular Structures by Super Resolution Microscopy

The goal of this Phase I SBIR project is to develop a set of high resolution antibody mimeticsnanoCLAMPsoptimized for super resolution imaging of a broad range of subcellular structuresAdvances in super resolution microscopy have enabled the achievement of resolutions well below the diffraction limit with current techniques now approachingnmFor immunostainingthenm size of antibody complexes now limits the achievable resolutionWith a much smaller size ofnmalternatives to antibodies such as aptamers and nanobodies have been shown to improve resolution significantly with STORM and STED techniquesUnfortunatelyonly a limited number of targets have validatedhigh resolution affinity reagents availablenanoCLAMPs are a newly described class of single domain affinity reagents with high affinityhigh specificity and the rare property of polyol responsivenessnanoCLAMPs have the ability to release functionally and structurally intact targets upon exposure to buffer containing polyol and saltPanels of nanoCLAMPs to new protein targets are easily generated from our validated synthetic phage display library in a few weeksa process that eliminates the need for animal immunization and provides multiple binders per targetnanoCLAMPs have the potential to improve the performance of super resolution imaging becausetheir small size and lack of endogenous cysteines allows fluorescent labels to be positioned within nanometers of the target structure andtheir polyol responsiveness enables the development of gentle stripping and restaining methodsSuccessful completion of this project will generate commercially availableopen source research tools that will improve the achievable resolution of super resolution imaging with reagents marking a broad range of subcellular structures The development of novel tools to improve the resolution of sub cellular structures will enable the investigation of the nature and behavior of those structures at near atomic levelThis will allow more precise understanding and modulation of biophysical processes leading to more effective treatments for disease