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.

Visualizing the structure and motion of the long noncoding RNA HOTAIR.

Long noncoding RNA molecules (lncRNAs) are estimated to account for the majority of eukaryotic genomic transcripts, and have been associated with multiple diseases in humans. However, our understanding of their structure-function relationships is scarce, with structural evidence coming mostly from indirect biochemical approaches or computational predictions. Here we describe direct visualization of the lncRNA HOTAIR (HOx Transcript AntIsense RNA) using atomic force microscopy (AFM) in nucleus-like conditions at 37°. Our observations reveal that HOTAIR has a discernible, although flexible, shape. Fast AFM scanning enabled the quantification of the motion of HOTAIR, and provided visual evidence of physical interactions with genomic DNA segments. Our report provides a biologically plausible description of the anatomy and intrinsic properties of HOTAIR, and presents a framework for studying the structural biology of lncRNAs.

Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy.

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling of human NPC revealed with acute precision how most constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains poorly resolved. Here by atomic force microscopy (AFM) coupled to single molecule localization microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical basket shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations highlight how this structure can adapt and let morphologically diverse cargoes shuttle through NPCs.

Hybrid fluorescence-AFM explores articular surface degeneration in early osteoarthritis across length scales.

Atomic force microscopy (AFM) has become a powerful tool for the characterization of materials at the nanoscale. Nevertheless, its application to hierarchical biological tissue like cartilage is still limited. One reason is that such samples are usually millimeters in size, while the AFM delivers much more localized information. Here a combination of AFM and fluorescence microscopy is presented where features on a millimeter sized tissue sample are selected by fluorescence microscopy on the micrometer scale and then mapped down to nanometer precision by AFM under native conditions. This served us to show that local changes in the organization of fluorescent stained cells, a marker for early osteoarthritis, correlate with a significant local reduction of the elastic modulus, local thinning of the collagen fibers, and a roughening of the articular surface. This approach is not only relevant for cartilage, but in general for the characterization of native biological tissue from the macro- to the nanoscale. STATEMENT OF SIGNIFICANCE: Different length scales have to be studied to understand the function and dysfunction of hierarchically organized biomaterials or tissues. Here we combine a highly stable AFM with fluorescence microscopy and precisely motorized movement to correlate micro- and nanoscopic properties of articular cartilage on a millimeter sized sample under native conditions. This is necessary for unraveling the relationship between microscale organization of chondrocytes, micrometer scale changes in articular cartilage properties and nanoscale organization of collagen (including D-banding). We anticipate that such studies pave the way for a guided design of hierarchical biomaterials.

Imaging collagen type I fibrillogenesis with high spatiotemporal resolution.

Fibrillar collagens, such as collagen type I, belong to the most abundant extracellular matrix proteins and they have received much attention over the last five decades due to their large interactome, complex hierarchical structure and high mechanical stability. Nevertheless, the collagen self-assembly process is still incompletely understood. Determining the real-time kinetics of collagen type I formation is therefore pivotal for better understanding of collagen type I structure and function, but visualising the dynamic self-assembly process of collagen I on the molecular scale requires imaging techniques offering high spatiotemporal resolution. Fast and high-speed scanning atomic force microscopes (AFM) provide the means to study such processes on the timescale of seconds under near-physiological conditions. In this study we have applied fast AFM tip scanning to study the assembly kinetics of fibrillar collagen type I nanomatrices with a temporal resolution reaching eight seconds for a frame size of 500 nm. By modifying the buffer composition and pH value, the kinetics of collagen fibrillogenesis can be adjusted for optimal analysis by fast AFM scanning. We furthermore show that amplitude-modulation imaging can be successfully applied to extract additional structural information from collagen samples even at high scan rates. Fast AFM scanning with controlled amplitude modulation therefore provides a versatile platform for studying dynamic collagen self-assembly processes at high resolution.