It was first suggested 1 more than 30 years ago that Watson-Crick base pairing might be used to rationally design nanoscale structures from nucleic acids. Since then, and especially since introduction of the origami technique 2 , DNA nanotechnology has seen astonishing developments and increasingly more complex structures are being produced [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] . But even though general approaches for creating DNA origami polygonal meshes and design software are available 14,16,17,[19][20][21] , constraints arising from DNA geometry and sense/antisense pairing still impose important restrictions and necessitate a fair amount of manual adjustment during the design process. Here we present a general method for folding arbitrary polygonal digital meshes in DNA that readily produces structures that would have been very difficult to realize with previous approaches. This is achieved with a high level of automation of the design process, which uses a routing algorithm based on graph theory and a relaxation simulation to trace scaffold strands through the target structures. Moreover, unlike conventional origami designs built from closed-packed helices, our structures have a more open conformation with one helix per edge and are thus stable in salt conditions commonly used in biological assays.The starting point of the present method is a 3D mesh representing the geometry one wishes to realize at the nanoscale. Focusing only on polyhedral meshes, i.e. meshes which enclose a volume inflatable to a ball, and in contrast to several previous approaches 14,17,19 (see Extended Data Fig. 1) we aim to replace the edges of the mesh by single DNA double helices such that the scaffold strand traverses each of these edges once. This problem is closely related to the Chinese Postman Tour problem 22 in graph theory, which we use to find solutions as doing so by hand would be practically impossible for most meshes. The main principles underpinning our design paradigm are that the technique should allow meshes to be triangulated to optimize structural rigidity; that each edge should be represented by one double helix to enable construction of large structures using as little DNA as possible (though some meshes require two helices to render certain edges as discussed below); and that vertices should be nonBenson et. al, -DNA Rendering of Polyhedral Meshes at the Nanoscale Confidential 2 crossing (i.e. the scaffold should not cross itself in the vertices to ensure non-knotted paths with fewer topological-and kinetic traps during folding, and planar vertex junctions that avoid mesh protrusions due to stacking of crossing helices at each vertex).The overall design scheme is split into four discrete steps: i) Drawing of a 3D polygon mesh in a 3D software, Fig. 1a. ii) Generating an appropriate routing of the long scaffold strand through all the edges of the mesh, Fig. 1b-e. iii) Determining the least strained DNA helix arrangement realizing the 3D mesh, Fig. 1f-i. And iv), Optional fine tuning of the des...

In the assembly of DNA nanostructures, the specificity of Watson-Crick base pairing is used to control matter at the nanoscale. Using this technology for drug delivery is a promising route toward the magic bullet concept, as it would allow the realization of complex assemblies that co-localize drugs, targeting ligands and other functionalities in one nanostructure. Anthracyclines' mechanism of action in cancer therapy is to intercalate DNA, and since DNA nanotechnology allows for such a high degree of customization, we hypothesized that this would allow us to tune the DNA nanostructures for optimal delivery of the anthracycline doxorubicin (Dox) to human breast cancer cells. We have tested two DNA origami nanostructures on three different breast cancer cell lines (MDA-MB-231, MDA-MB-468, and MCF-7). The different nanostructures were designed to exhibit varying degrees of global twist, leading to different amounts of relaxation in the DNA double-helix structure. By tuning the nanostructure design we are able to (i) tune the encapsulation efficiency and the release rate of the drug and (ii) increase the cytotoxicity and lower the intracellular elimination rate when compared to free Dox. Enhanced apoptosis induced by the delivery system in breast cancer cells was investigated using flow cytometry. The findings indicate that DNA origami nanostructures represent an efficient delivery system for Dox, resulting in high degrees of internalization and increased induction of programmed cell death in breast cancer cells. In addition, by designing the structures to exhibit different degrees of twist, we are able to rationally control and tailor the drug release kinetics.

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor-mediated signaling.

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

The use of DNAa sananoscale construction material has been ar apidly developing field since the 1980s, in particular since the introduction of scaffolded DNAorigami in 2006. Although software is available for DNAo rigami design, the user is generally limited to architectures where finding the scaffold path through the object is trivial. Herein, we demonstrate the automated conversion of arbitrary twodimensional sheets in the form of digital meshes into scaffolded DNAn anostructures.W ei nvestigate the properties of DNA meshes based on three different internal frameworks in standardf olding buffer and physiological salt buffers.W e then employ the triangulated internal framework and produce four 2D structures with complex outlines and internal features. We demonstrate that this highly automated technique is capable of producing complex DNAn anostructures that fold with high yield to their programmed configurations,c overing around 70 %more surface area than classic origami flat sheets.Since its introduction in the 1980s, [1] DNAn anotechnology has been arapidly growing and diversifying field. This growth has accelerated since the introduction of scaffolded DNA origami in 2006.[2] In aDNA origami structure,along strand, called the scaffold, traverses the entire structure pairing with hundreds of oligonucleotides,c alled staple strands,t hat hold the structure together.The structures are often based around as quare or honeycomb lattice [3] where finding the scaffold path and designing staples is relatively easy,e specially when using software like caDNAno.[4] DNAn anostructures based on small polyhedra have been demonstrated with both scaffolded [5] and non-scaffolded [6] designs.S caffolded DNA nanostructures based on meshwork designs have also been demonstrated with crossing four-arm junctions, [7] with others containing meshes with two DNAd ouble helices per edge. [8] However,n og eneral strategy for producing arbitrary wireframe 2D structures has been demonstrated.Am ajor branch of research has been the addition of functional elements to DNAn anostructures to give them novel properties.C arbon nanotubes and metal nanoparticles have been added for electronic [9] and plasmonic [10] applications.P roteins have been added for templating enzymatic reactions [11] or cell signaling studies. [12] Fluorophores have been added to study energy transfer [13] and to create nanoscale barcodes.[14] DNAo rigami structures have also been used to control the shape of metal particles [15] and graphene sheets.[16] Demonstrations of drug loading [17] and lipid encapsulation [18] indicate that DNAn anostructures could serve as drug delivery tools.M any applications rely on single layer DNAo bjects as they offer the largest 2D canvas for functionalization and are rigid when immobilized on surfaces.Building on Rothemunds method [2] fors caffolded DNA nanostructures,w er ecently developed am ethod for automatically generating wireframe structures from polyhedral meshes. [19] This method relies on an algorithm for finding an E...

DNA origami is a powerful method for the creation of 3D nanoscale objects, and in the past few years, interest in wireframe origami designs has increased due to their potential for biomedical applications. In DNA wireframe designs, the construction material is double-stranded DNA, which has a persistence length of around 50 nm. In this work, we study the effect of various design choices on the stiffness versus final size of nanoscale wireframe rods, given the constraints on origami designs set by the DNA origami scaffold size. An initial theoretical analysis predicts two competing mechanisms limiting rod stiffness, whose balancing results in an optimal edge length. For small edge lengths, the bending of the rod's overall frame geometry is the dominant factor, while the flexibility of individual DNA edges has a greater contribution at larger edge lengths. We evaluate our design choices through simulations and experiments and find that the stiffness of the structures increases with the number of sides in the cross-section polygon and that there are indications of an optimal member edge length. We also ascertain the effect of nicked DNA edges on the stiffness of the wireframe rods and demonstrate that ligation of the staple breakpoint nicks reduces the observed flexibility. Our simulations also indicate that the persistence length of wireframe DNA structures significantly decreases with increasing monovalent salt concentration.

As DNA origami applications in biomedicine are expanding, more knowledge is needed to assess these structures’ interaction with biological systems. Here, uptake and penetration in cell and cell spheroid tissue models (CSTMs) are studied to elucidate whether differences in internal structure can be a factor in the efficacy of DNA‐origami‐based delivery. Two structures bearing largely similar features in terms of both geometry and molecular weight, but with different internal designs—being either compact, lattice‐based origami or following an open, wireframe design—are designed. In CSTMs, wireframe rods are able to penetrate deeper than close‐packed rods. Moreover, doxorubicin‐loaded wireframe rods show a higher cytotoxicity in CSTMs. These results can be explained by differences in structural mechanics, local deformability, local material density, and accessibility to cell receptors between these two DNA origami design paradigms. In particular, it is suggested that the main reason for the difference in penetration dynamic arises from differences in interaction with scavenger receptors where lattice‐based structures appear to be internalized to a higher degree than polygonal structures of the same size and shape. It is thus argued that the choice of structural design method constitutes a crucial parameter for the application of DNA origami in drug delivery.

In the past decade, DNA nanostructures have made the leap from small assemblies of a handful of oligonucleotides to megadalton objects assembled from hundreds or thousands of component DNA strands. Most DNA designs today are either lattice based with simple and reliable design tools or lattice free with a larger shape space but more challenging design and lower rigidity. In parallel with the development of DNA nanostructures, software packages for the simulation of nucleic acids have seen rapid development allowing for the simulation of the dynamics of full DNA nanostructure assemblies. Here, we implement an unsupervised software based on the coarse-grained molecular dynamics package oxDNA to simulate DNA origami structures and evaluate their rigidity. From this, the software autonomously produces mutant structures by adding or removing base pairs or modifying the positions of internal supports. These mutant structures are iteratively generated and evaluated by simulation to create an in silico evolution toward more rigid DNA nanostructures.