title: Assembly of multienzyme complexes on DNA nanostructures.
abstract: In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths. The protocol describes the preparation and purification of DNA-conjugated enzymes and cofactors, along with the assembly of these prepared complexes on DNA nanostructures. The architecture of assembled enzyme complexes is then readily characterized using a broad selection of techniques from routine gel electrophoresis to advanced single-molecule imaging. We also describe methods of purifying these nano-assemblies and testing them with functional assays based on either bulk or single-molecule fluorescence measurements. The entire assembly and characterization of a multienzyme complex can be completed within 1-2 weeks.
authors.affiliation: Department of Chemistry, Rutgers University-Camden, Camden, New Jersey, USA.|Center for Computational and Integrative Biology, Rutgers University-Camden, Camden, New Jersey, USA.
authors.forename: Jinglin
authors.initials: J
authors.lastname: Fu
authors.affiliation: Center for Molecular Design and Biomimetics, Arizona State University, Tempe, Arizona, USA.|School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
authors.forename: Yuhe Renee
authors.initials: YR
authors.lastname: Yang
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Soma
authors.initials: S
authors.lastname: Dhakal
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Center for Molecular Design and Biomimetics, Arizona State University, Tempe, Arizona, USA.|School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
authors.forename: Minghui
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Rutgers University-Camden, Camden, New Jersey, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
authors.affiliation: Center for Molecular Design and Biomimetics, Arizona State University, Tempe, Arizona, USA.|School of Molecular Sciences, Arizona State University, Tempe, Arizona, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Yan
mesh_terms: D003067:Coenzymes / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D009690:Nucleic Acid Conformation; D011487:Protein Conformation
chemical_list: D003067:Coenzymes; D009097:Multienzyme Complexes; D004247:DNA