Intracellular tags for electron cryotomography: using DNA nanostructures as cryoEM's equivalent of GFP

Through electron cryotomography (cryoET) we can visualise cell environments at molecular resolution, revealing the structures and interactions that underpin cellular function. The full potential of cryoET has not yet been realised, in large part because specific molecules are challenging to identify. There is an unmet need for cryoET tags, analogous to fluorescent proteins in light microscopy. I previously pioneered a tool, SPOTs, to identify proteins in tomograms. SPOTs are DNA nanostructures, easily visualised by cryoET, which bind specific proteins. The existing technique is applicable to the outside of viruses and cells. In this proposal I will design a widely applicable, modular toolkit for labelling inside cells, building substantially on my existing technology through three aims. I will design tags which undergo a conformational change upon binding their target, providing a visual and fluorescent readout of binding in vitro and in vivo. I will optimise nanostructure contrast by cryoET and cell delivery efficiency, producing a generalised toolkit that can be adopted by any structural biology lab to address far-reaching research questions. I will demonstrate this toolkit on two medically important systems: latrophilin signalling which is linked to Attention-Deficit/Hyperactivity Disorder in adults, and polycystin-1 auto-proteolysis which is linked to kidney disease.