Incorporation of 7вЂ‰Г—вЂ‰Atto488-modified staples on positions T0-6 enabled fluorescence detection of the origami structures (Fig. We assessed the interaction of curved DNA nanostructures with lipid membranes, mainly giant unilamellar vesicles (GUVs) composed of 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), via fluorescence confocal microscopy (Fig. As we explore reconstitution assays with model membranes similar to the ones employed for studying scaffolding proteins in vitro 8, 9, 10, direct comparison with the mechanism of action of BAR domain proteins can be drawn.Д®fficient binding of curved DNA origami to membranes We determined quantitatively the requirements in terms of shape, membrane-attachment and oligomerization needed for a synthetic scaffold to induce specific membrane curvature. Thereupon, we studied the interaction of the DNA origami-based scaffolds with lipid model systems and demonstrate the ability for membrane bending in vitro. Each design further includes positions at the different curved facets for attaching fluorophores, membrane-anchoring moieties or for oligomerizing the objects laterally (Fig.

в€ј 20вЂ‰nm, respectively), these origami structures (H, Q and L) mimic the typical shapes of highly-curved BAR/N-BAR dimers, moderately curved F-BAR dimers and flat PinkBAR/I-BAR dimers, respectively (Fig. Despite their fivefold increased length when compared to BAR proteins ( в€ј110вЂ‰nm vs. 1): (i) a вЂsemi-circleвЂ™ named HALF (origami H) with curvature ( C)вЂ‰в‰€вЂ‰21.7вЂ‰Ојm в€’1 (ii) a вЂquarter-circleвЂ™ named QUARTER (origami Q) with CвЂ‰в‰€вЂ‰11.6вЂ‰Ојm в€’1 and (iii) a вЂstickвЂ™ named LINEAR (origami L) with CвЂ‰в‰€вЂ‰0 (Fig. 2вЂ“ 4 and Supplementary Tables 1вЂ“ 3) were here developed (Fig. Taking inspiration from the different degrees of curvature covered by BAR domain proteins, three DNA origami designs (20-helix bundles with hexagonal lattice Supplementary Figs. Subsequently, in this work, we achieve the transformation of membrane shape on much larger scales, reminiscent of deformations observed in cells 2, 3. In contrast to DNA origami nanocages 24, 25 that template small liposomes via detergent removal, our designed origami structures act on preexisting cell-sized vesicles, imitating the mechanism of action of protein coats. DNA origami has been previously employed to create nanoscale channels in lipid membranes 20, 21 and to guide the assembly of nanoscale lipid compartments 22, 23, 24, 25. This molecular toolkit now serves as the starting point for our goal of constructing membrane-sculpting machinery from the bottom-up. Programmable self-assembly with DNA origami may be employed to produce a variety of two-dimensional and three-dimensional structures on the nanometer-scale, including objects with custom curvature 15, 16, 17, 18, 19. 1), in order to decipher the essential properties of artificial scaffolds for curving lipid membranes. The goal of this work is to mimic structural and functional features of BAR domain proteins by rationally designed DNA origami objects (Supplementary Fig. From the minimalistic perspective of bottom-up synthetic biology 13, 14, it is tempting to speculate about the simplest way to induce specific membrane curvatures, and thus engineer a minimal membrane sculpting machinery de novo. By using BAR domains as model proteins 11, 12, recent studies emphasized the relevance of physical-chemical foundations for membrane bending. BAR proteins presumably rely on their curved shape for their activity: different types of BAR modules adopt folds with different degrees of curvature 4, 5. Several BAR proteins were shown to tubulate membranes in vitro 7, 8, 9, 10. When dimerized, BAR proteins form characteristic banana-shaped scaffolds that induce and stabilize membrane curvature through electrostatic and hydrophobic interactions 4, 5, 6.

An important class of scaffolding proteins which presumably imprint their shape on lipid membranes is the BAR (Bin/Amphiphysin/Rvs) domain superfamily 4, 5. Numerous factors that regulate membrane curvature have been identified, with scaffolding proteins being the most obvious ones 1, 2, 3. The transformation of membranes from one shape to another, for example during cell division, belongs to the most fundamental processes in living cells. The curvatures of biological membranes vary strongly, from predominantly flat in the plasma membrane to highly curved in the endoplasmatic reticulum or in the Golgi apparatus.

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