title: Electron Microscopic Visualization of Protein Assemblies on Flattened DNA Origami.
abstract: DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ∼2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.
authors.forename: Leena
authors.initials: L
authors.lastname: Mallik
authors.forename: Soma
authors.initials: S
authors.lastname: Dhakal
authors.forename: Joseph
authors.initials: J
authors.lastname: Nichols
authors.forename: Jacob
authors.initials: J
authors.lastname: Mahoney
authors.forename: Anne M
authors.initials: AM
authors.lastname: Dosey
authors.affiliation: ∥The Biodesign Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, United States.
authors.forename: Shuoxing
authors.initials: S
authors.lastname: Jiang
authors.forename: Roger K
authors.initials: RK
authors.lastname: Sunahara
authors.forename: Georgios
authors.initials: G
authors.lastname: Skiniotis
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D008854:Microscopy, Electron / Q000379:methods; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D009690:Nucleic Acid Conformation
chemical_list: D004247:DNA; D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase
keywords: DNA nanotechnology; DNA scaffolding; cocaine esterase; protein−DNA assembly; single-particle electron microscopy