title: A Rationally Designed Connector for Assembly of Protein-Functionalized DNA Nanostructures.
abstract: We report on the rational engineering of the binding interface of the self-ligating HaloTag protein to generate an optimized linker for DNA nanostructures. Five amino acids positioned around the active-site entry channel for the chlorohexyl ligand (CH) of the HaloTag protein were exchanged for positively charged lysine amino acids to produce the HOB (halo-based oligonucleotide binder) protein. HOB was genetically fused with the enzyme cytochrome P450 BM3, as well as with BMR, the separated reductase domain of BM3. The resulting HOB-fusion proteins revealed significantly improved rates in ligation with CH-modified oligonucleotides and DNA origami nanostructures. These results suggest that the efficient self-assembly of protein-decorated DNA structures can be greatly improved by fine-tuning of the electrostatic interactions between proteins and the negatively charged nucleic acid nanostructures.
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Katja J
authors.initials: KJ
authors.lastname: Koßmann
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Ziegler
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Angelin
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Meyer
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Skoupi
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany.
authors.forename: Kersten S
authors.initials: KS
authors.lastname: Rabe
authors.affiliation: Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz 1, 76344, Eggenstein-Leopoldshafen, Germany. niemeyer@kit.edu.
authors.forename: Christof M
authors.initials: CM
authors.lastname: Niemeyer
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D049329:Nanostructures / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
chemical_list: D009841:Oligonucleotides; D011994:Recombinant Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System
keywords: DNA nanostructures; bioconjugates; enzymes; self-assembly