Reviewing the various methods and effectivity to stabilize DNA origami in biological environments.

Understanding hierarchical self‐assembly of biological structures requires real‐time measurement of the self‐assembly process over a broad range of length‐ and timescales. The success of high‐speed atomic force microscopy (HS‐AFM) in imaging small‐scale molecular interactions has fueled attempts to introduce this method as a routine technique for studying biological and artificial self‐assembly processes. Current state‐of‐the‐art HS‐AFM scanners achieve their high imaging speed by trading achievable field of view for bandwidth. This limits their suitability when studying larger biological structures. In ambient conditions, large‐range scanners with lower resonance frequencies offer a solution when combined with first principle model–based schemes. For imaging molecular self‐assembly processes in fluid, however, such traditional control techniques are less suited. In liquid, the time‐varying changes in the behavior of the complex system necessitate frequent update of the compensating controller. Recent developments in data‐driven control theory offer a model‐free, automatable approach to compensate the complex system behavior and its changes. Here, a data‐driven control design method is presented to extend the imaging speed of a conventional AFM tube scanner by one order of magnitude. This enables the recording of the self‐assembly process of DNA tripods into a hexagonal lattice at multiple length scales.

The most basic function of synthetic microenvironments for tissue engineering is to act as a physical substrate for cell attachment, migration, and proliferation, similar to the natural cell environment. Functionalization of supramolecular materials with guest compounds that display the same recognition moieties is a common strategy to introduce biofunctionality. However, besides a robust interaction with the material, a certain level of dynamics needs to be conserved for an adaptive interface toward the living environment. A balance between robust material functionalization and dynamic cell interaction needs to be met. The detailed analysis hereof using a ureido‐pyrimidinone (UPy) poly(ethylene glycol) system in dilute and transient network regime is demonstrated. Monovalent and bivalent UPy‐functionalized guest molecules are designed and their interaction with UPy‐host fibers is evaluated. Analysis of guest interaction in the dilute state by microfluidics, and in the gel state, by fluorescence recovery after photobleaching and fluorescence resonance energy transfer is proven to be suitable to quantify the local and ensemble guest mobility. The results demonstrate that the interaction of bioactive moieties through supramolecular host–guest chemistry yields a dynamic system, which is stronger for divalent guests but risks unintended leakage in the case of functional monomeric units.

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5β1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.