Linker Engineering of Ligand‐Decorated DNA Origami Nanostructures Affects Biological Activity

Abstract

News from an old acquaintance: The streptavidin (STV)‐biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono‐ or bi‐dentate linker architectures on DON with a novel high‐speed atomic force microscope (HS‐AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub‐100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker‐specific STV/DON interactions observed by HS‐AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes.

Hapten-Decorated DNA Nanostructures Decipher the Antigen-Mediated Spatial Organization of Antibodies Involved in Mast Cell Activation

“…Rectangular DNA origami nanostructures (DON) were designed using the single-stranded scaffold p7560 (Tilibit nanosystems) and 240 staple-strand oligonucleotides (Sigma-Aldrich) as described previously. 33 Several staple strand oligonucleotides were modified at the 5′ end to incorporate Cy5 fluorophores and 2,4-dinitrophenol (DNP) ligands on the upper side of the DON and protruding single-stranded oligonucleotides 24 bases long on the lower side of the DNA origami for immobilization of the DON via hybridization. Lower and upper side refer to the position on the DON plane after binding on a surface.…”

“…In recent years, Rothemund’s scaffolded origami technique has emerged as a promising tool for the study of biological processes, such as cell adhesion and activation. ,− DNA origami nanostructures (DON) can be used as molecular pegboards for presenting immobilized bioactive ligands to cells and tissues, thus enabling complete control over the number and spatial arrangement of ligands at the lower nanoscale. Here, we report on the use of DNP-decorated DON in order to investigate the spatial nanoscale tolerance for bivalent binding of anti-DNP IgE and anti-DNP IgG, as well as cell membrane-associated IgE antibodies.…”

Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation

“…Characterization of these rulers by AFM revealed surface coverages of about 70% (Figures 2b and S5), as is typically observed for this type of protein-decorated DON constructs with standard biotinylated linkers. 24,39 MOSAIC surfaces were prepared from these constructs as described above and used to stimulate EGFR activation in MCF7 cells (Figure S6 for representative fluorescence micrographs). Data analysis was performed using the software tool described above.…”

“…Because DNA origami nanostructures (DON) can be easily and efficiently modified with proteins and other components at near molecular resolution, they are increasingly proving to be a powerful tool for studying biological processes such as cell adhesion and activation. − Our group has developed the so-called “Multiscale Origami Structures As Interface for Cells” (MOSAIC) technique to overcome the problems of alternative techniques described above. MOSAIC takes advantage of top-down printed DNA patterns of ∼5 μm spots, i.e., with subcellular dimensions, which are used for the DNA-directed immobilization (DDI) of double-sided functionalized DONs carrying anchor strands and ligand patterns on the bottom and top side, respectively, of a quasi-two-dimensional rectangular scaffold plate (Figure a,b).…”

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher–SpyTag interaction

“…6 Both the SNAP- 7 and Halo-tag 8 have already been refined to the point of achieving a coupling efficiency of ∼80% with DNA origami nanostructures (DON), which is quantitatively comparable to the frequently used streptavidin–biotin binding system. 9 To expand the toolbox of genetically encodable connectors for DNA scaffold modification, the SpyCatcher–SpyTag (SC–ST) bioconjugation system 10 should be particularly suitable due to the rapid and quantitative nature of isopeptide bond formation. This system consists of the 116 amino acid (aa) SpyCatcher (SC) protein, which spontaneously forms a covalent amide bond between one of its lysine residues and an aspartic acid residue of the short SpyTag (ST, 13 aa) peptide under physiological conditions (Fig.…”