Protein-Assisted Large-Scale Assembly and Differential Patterning of DNA Origami Lattices.

Nanofabrication has experienced a big boost with the invention of DNA origami, enabling the production and assembly of complex nanoscale structures that may be able to unlock fully new functionalities in biology and beyond. The remarkable precision with which these structures can be designed and produced is, however, not yet matched by their assembly dynamics, which can be extremely slow, particularly when attached to biological templates, such as membranes. Here, the rapid and controlled formation of DNA origami lattices on the scale of hundreds of micrometers in as little as 30 minutes is demonstrated, utilizing active patterning by the E.coli Min protein system, thereby yielding a remarkable improvement over conventional passive diffusion-based assembly methods. Various patterns, including spots, inverse spots, mazes, and meshes can be produced at different scales, tailored through the shape and density of the assembled structures. The differential positioning accomplished by Min-induced diffusiophoresis even allows the introduction of "pseudo-colors", i.e., complex core-shell patterns, by simultaneously patterning different DNA origami species. Beyond the targeted functionalization of biological surfaces, this approach may also be promising for applications in plasmonics, catalysis, and molecular sensing.

Light-Activated Synthetic Rotary Motors in Lipid Membranes Induce Shape Changes Through Membrane Expansion.

Membranes are the key structures to separate and spatially organize cellular systems. Their rich dynamics and transformations during the cell cycle are orchestrated by specific membrane-targeted molecular machineries, many of which operate through energy dissipation. Likewise, man-made light-activated molecular rotary motors have previously shown drastic effects on cellular systems, but their physical roles on and within lipid membranes remain largely unexplored. Here, the impact of rotary motors on well-defined biological membranes is systematically investigated. Notably, dramatic mechanical transformations are observed in these systems upon motor irradiation, indicative of motor-induced membrane expansion. The influence of several factors on this phenomenon is systematically explored, such as motor concentration and membrane composition., Membrane fluidity is found to play a crucial role in motor-induced deformations, while only minor contributions from local heating and singlet oxygen generation are observed. Most remarkably, the membrane area expansion under the influence of the motors continues as long as irradiation is maintained, and the system stays out-of-equilibrium. Overall, this research contributes to a comprehensive understanding of molecular motors interacting with biological membranes, elucidating the multifaceted factors that govern membrane responses and shape transitions in the presence of these remarkable molecular machines, thereby supporting their future applications in chemical biology.

Protein-Based Patterning to Spatially Functionalize Biomimetic Membranes.

The bottom-up reconstitution of proteins for their modular engineering into synthetic cellular systems can reveal hidden protein functions in vitro. This is particularly evident for the bacterial Min proteins, a paradigm for self-organizing reaction-diffusion systems that displays an unexpected functionality of potential interest for bioengineering: the directional active transport of any diffusible cargo molecule on membranes. Here, the MinDE protein system is reported as a versatile surface patterning tool for the rational design of synthetically assembled 3D systems. Employing two-photon lithography, microswimmer-like structures coated with tailored lipid bilayers are fabricated and demonstrate that Min proteins can uniformly pattern bioactive molecules on their surface. Moreover, it is shown that the MinDE system can form stationary patterns inside lipid vesicles, which allow the targeting and distinctive clustering of higher-order protein structures on their inner leaflet. Given their facile use and robust function, Min proteins thus constitute a valuable molecular toolkit for spatially patterned functionalization of artificial biosystems like cell mimics and microcarriers.

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes.

Nanotechnology often exploits DNA origami nanostructures assembled into even larger superstructures up to micrometer sizes with nanometer shape precision. However, large-scale assembly of such structures is very time-consuming. Here, we investigated the efficiency of superstructure assembly on surfaces using indirect cross-linking through low-complexity connector strands binding staple strand extensions, instead of connector strands binding to scaffold loops. Using single-molecule imaging techniques, including fluorescence microscopy and atomic force microscopy, we show that low sequence complexity connector strands allow formation of DNA origami superstructures on lipid membranes, with an order-of-magnitude enhancement in the assembly speed of superstructures. A number of effects, including suppression of DNA hairpin formation, high local effective binding site concentration, and multivalency are proposed to contribute to the acceleration. Thus, the use of low-complexity sequences for DNA origami higher-order assembly offers a very simple but efficient way of improving throughput in DNA origami design.