title: Co-Immobilization of Proteins and DNA Origami Nanoplates to Produce High-Contrast Biomolecular Nanoarrays.
abstract: The biofunctionalization of nanopatterned surfaces with DNA origami nanostructures is an important topic in nanobiotechnology. An unexplored challenge is, however, to co-immobilize proteins with DNA origami at pre-determined substrate sites in high contrast relative to the nontarget areas. The immobilization should, in addition, preferably be achieved on a transparent substrate to allow ultrasensitive optical detection. If successful, specific co-binding would be a step towards stoichiometrically defined arrays with few to individual protein molecules per site. Here, we successfully immobilize with high specificity positively charged avidin proteins and negatively charged DNA origami nanoplates on 100 nm-wide carbon nanoislands while suppressing undesired adsorption to surrounding nontarget areas. The arrays on glass slides achieve unprecedented selectivity factors of up to 4000 and allow ultrasensitive fluorescence read-out. The co-immobilization onto the nanoislands leads to layered biomolecular architectures, which are functional because bound DNA origami influences the number of capturing sites on the nanopatches for other proteins. The novel hybrid DNA origami-protein nanoarrays allow the fabrication of versatile research platforms for applications in biosensing, biophysics, and cell biology, and, in addition, represent an important step towards single-molecule protein arrays.
authors.affiliation: Center for Advanced Bioanalysis GmbH, 4020, Linz, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Hager
authors.affiliation: Department of Chemistry, Institute of Structural and Molecular Biology, University College London, London, UK.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Burns
authors.affiliation: Institute for Semiconductor and Solid State Physics, Johannes Kepler University, 4040, Linz, Austria.
authors.forename: Martyna J
authors.initials: MJ
authors.lastname: Grydlik
authors.affiliation: Institute for Semiconductor and Solid State Physics, Johannes Kepler University, 4040, Linz, Austria.
authors.forename: Alma
authors.initials: A
authors.lastname: Halilovic
authors.affiliation: Center for Advanced Bioanalysis GmbH, 4020, Linz, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselgrübler
authors.affiliation: Institute for Semiconductor and Solid State Physics, Johannes Kepler University, 4040, Linz, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schäffler
authors.affiliation: Center for Advanced Bioanalysis GmbH, 4020, Linz, Austria.|Department of Chemistry, Institute of Structural and Molecular Biology, University College London, London, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Howorka
mesh_terms: D004247:DNA / Q000737:chemistry*; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D004247:DNA
keywords: DNA origami; biofunctionalization; fluorescence microscopy; nanopatterns; proteins