Self-assembled biomolecular nanostructures with single molecule techniques

Abstract

Bottom-up approaches using self-assembled biomolecules for nanostructure building promises advantageous features like biocompatibility, nanometer accuracy. In this study, we used lipid and DNA molecules for building block to make nanostructures and monitored the protein binding and folding process on the nanostructure using single molecule techniques. SNARE proteins, the core machinery of membrane fusion drives membrane fusion once they were incorporated into the vesicle. We used single molecule FRET technique to observe their lipid mixing ability, and protein complex formation dynamics. We observed SNARE dependent lipid mixing and SNARE complex formation to reveal that the SNARE complex formation kinetics is actually cooperative. We also developed content mixing assay using self-quenching dyes to observe calcium dependent content mixing by neuronal SNARE and synaptotagmin I. Next we used magnetic tweezers to reveal the folding pathway and intermediate states of DNA origami process. DNA origami provides a versatile tool to design and build DNA based nanostructures with arbitrary shape and modification sites. We used the selective denaturing capability of magnetic tweezers to separate the binding of staple strands from the folding process and simplified the molecular processes during the nanostructure folding. The folding process was finished within 10 minutes which was 100 times faster than conventional thermal folding method.