The modular building block design allows the formation of containers with programmable sizes, achieving diameters ranging from 100 nm to over 1 µm. The largest containers are large enough to accommodate an entire bacterium. These structures could open up new possibilities for engineering biomolecular robotic systems.
Containers that combine the best of both worlds
Many types of microscopic containers are made from lipids. Although these are versatile and can form nanometer- to micrometer-sized structures, it is challenging to incorporate all the functional modules needed for a biomolecular robot. Other types of biological and synthetic containers are made from proteins or protein-inspired DNA building blocks. Although these can be easily functionalized, their strict self-assembly rules constrain experimentally achievable container sizes and shapes, limiting their usefulness. Christoph Karfusehr, a Max Planck School Matter to Life PhD student working in Prof. Simmel’s lab at TUM, has now developed DNA origami building blocks inspired by lipids, called “Dipids”, which combine the permissive assembly principles of lipids with the programmability and straightforward functionalization of DNA structures.
Mirroring the lipid-lipid interaction profile, Dipids are designed as radially symmetric DNA barrels with a diameter of 30 nm. By tuning the length and sequence of 30 single-stranded DNA strands on the Dipid surface, they can be programmed to self-assemble into planar membranes, hollow tubes, and closed containers. The conserved inner structure enables easy and cost-effective conversion between all Dipid designs.
Mixing in functional modules
Simmel’s team generated 74 container-forming Dipid designs, which were predicted to form structures of varying curvature. The six experimentally validated designs self-assembled into containers ranging in size from HIV capsids to bacteria. The largest of these containers have an average diameter of 1.2 µm and exceed the largest size reported to date for a DNA origami container ( ~300 nm).
Simmel’s team further demonstrated that Dipids can function as a versatile membrane patchy-particle system, capable of reconstituting lipid-membrane organization principles in binary mixtures and as a structural framework supporting integration of diverse functional modules. For example, incorporating functionalized Dipids enabled them to transcribe sequences within containers and to trap the RNA products inside. The porous Dipid membrane allows enzymes such as RNA polymerases to pass through container walls, enabling continuous and post-assembly delivery of molecules below the porosity threshold, which is difficult to achieve with lipid vesicles. Using membrane localization modules, Simmel’s team further demonstrated that they could incorporate non-Dipid DNA origami subcompartments into larger Dipid containers analogous to organelles within cells.
Seeing is believing
To reveal the native 3D organization of Dipid structures, Christoph Karfusehr and Prof. Friedrich Simmel collaborated with Dr Marion Jasnin, head of the Cryoskeleton Lab at the Helmholtz Pioneer Campus and of cryo-electron tomography operations at the Cryo-Electron Microscopy Platform at Helmholtz Munich. Dr Brice Beinsteiner, a postdoctoral researcher in Dr Jasnin’s lab, discovered that Dipids could form intricately folded clusters of stacked 2D membranes. He also demonstrated that containers can be fully closed monolayer structures. Furthermore, he found that, in the smallest containers, Dipids form amorphous membranes with non-trivial monomer arrangements. Dr Brice Beinsteiner also computed the size distribution of the naturally occurring pores in Dipid membranes, determining the membrane porosity thresholds.
Combining the structural programmability of Dipids with the ease of design and the effortless integration of diverse functional modules, the Dipid framework offers a plug-and-play approach for the community-driven development of biomolecular robots.