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.

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Self‐assembly of biological molecules and structures is a fundamental property of life. Whereas most biological functions are based on self‐assembled proteins and protein complexes, the self‐assembly of lipids is important for the spatial organization of heterogeneous cellular reaction environments and to catalyze cooperative interactions on/with membranes. Lipid domains or “rafts”, which are known to selectively recruit proteins, play an important functional role in sorting and trafficking of membrane components between subcellular organelles. However, how the recruitment and interactions of proteins in turn contributes to the formation and turnover of these structures has not been systematically addressed, due to the large variety in membrane–protein features and their spatiotemporal dynamics. The small size and transient nature of lipid domains adds to the complexity in visualizing them in living cells. Here, DNA origami is presented as a programmable tool to mimic protein clustering and assembly on membranes and illustrate how nanometer sized lipid domains coalesce into visible domains upon origami self‐assembly in defined patterns. Hence, the local membrane composition can be efficiently regulated by the self‐assembly of peripheral membrane binders. This reinforces the hypothesis that lipid rafts in cells occur as a result of membrane–protein interactions and, in particular, protein self‐assembly.