Fluorescence Imaging Study of Film Coating Structure and Composition Effects on DNA Hybridization

Hybridization of surface‐bound DNA with complementary strands is the basis of many biotechnological applications. Herein, the structure of interfacial coatings between substrate and bound DNA is a crucial element for hybridization behavior. Herein, three reactive surfaces for constructing DNA‐sensing platforms, namely, plain gold films on silicon, poly(bisphenolA‐co‐epichlorohydrin) (PBAG) surfaces with a brush‐like bilayer structure, and dibenzocyclooctyne monolayers (both on glass), are compared. Fluorescence imaging is employed to survey the effect of coating structure and conformation on hybridization performance. To better understand the interfacial structural properties and chemistry of the coated films, atomic force microscopy, water contact angle measurements, and X‐ray photoelectron spectroscopy are employed to characterize the surface morphology. DNA probe microarrays are created on the different platforms via microchannel cantilever spotting, and their performance for hybridizing with the DNA counterparts is assessed. While all three platforms work reliable for DNA detection, a protein‐binding assay reveals that PBAG surfaces offer the highest hybridization efficiency among these approaches. The results of the present work have significant implications for comprehension of the interactions between the DNA hybridization efficiency and the physico‐chemical properties of surface coatings and can inform the fabrication of DNA sensors

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

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

Fluorescence Imaging Study of Film Coating Structure and Composition Effects on DNA Hybridization

Hybridization of surface‐bound DNA with complementary strands is the basis of many biotechnological applications. Herein, the structure of interfacial coatings between substrate and bound DNA is a crucial element for hybridization behavior. Herein, three reactive surfaces for constructing DNA‐sensing platforms, namely, plain gold films on silicon, poly(bisphenolA‐co‐epichlorohydrin) (PBAG) surfaces with a brush‐like bilayer structure, and dibenzocyclooctyne monolayers (both on glass), are compared. Fluorescence imaging is employed to survey the effect of coating structure and conformation on hybridization performance. To better understand the interfacial structural properties and chemistry of the coated films, atomic force microscopy, water contact angle measurements, and X‐ray photoelectron spectroscopy are employed to characterize the surface morphology. DNA probe microarrays are created on the different platforms via microchannel cantilever spotting, and their performance for hybridizing with the DNA counterparts is assessed. While all three platforms work reliable for DNA detection, a protein‐binding assay reveals that PBAG surfaces offer the highest hybridization efficiency among these approaches. The results of the present work have significant implications for comprehension of the interactions between the DNA hybridization efficiency and the physico‐chemical properties of surface coatings and can inform the fabrication of DNA sensors

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

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate “Multiscale Origami Structures As Interface for Cells” (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30–40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades