Designer nanoscale DNA assemblies programmed from the top down

Simplifying DNA origami design Many intricate nanostructures have been made with DNA origami. This process occurs when a long DNA scaffold develops a particular shape after hybridization with short staple strands. Most designs, however, require a difficult iterative procedure of refining the base pairing. Veneziano et al. now report algorithms that automate the design of arbitrary DNA wireframe structures. Synthesizing and structurally characterizing a variety of nanostructures allowed for verification of the algorithms' accuracy. Science, this issue p. 1534 A top-down algorithm can program the design of arbitrary three-dimensional DNA structures. INTRODUCTION Synthetic DNA can be programmed by using canonical Watson-Crick base pairing to form highly structured nanometer-scale assemblies that rival many natural protein and RNA assemblies in structural complexity. The use of a single-stranded scaffold DNA molecule that traverses the entire DNA architecture offers near-quantitative yield of target DNA-based objects, in addition to full control over their asymmetric three-dimensional (3D) structure and site-specific functionalization for applications that include cellular delivery, nanoscale photonic materials, single-molecule imaging, and structured metamaterials, among others. Design of these versatile DNA assemblies is currently limited, however, to experts who are knowledgeable in the sequence design rules needed to fold a target DNA shape. RATIONALE A fully autonomous design procedure that computes the single-stranded DNA (ssDNA) sequences needed to fold an arbitrary 3D shape offers the potential for broadening participation in this powerful molecular design paradigm. Toward this end, we present the algorithmic framework DAEDALUS (http://daedalus-dna-origami.org) that uses a simple surface-based representation of target 3D geometry to automatically generate the ssDNA needed to synthesize the object. Target shapes are rendered with parallel DNA duplexes that are folded to form nanoparticles of high structural fidelity that are also biologically compatible. Our approach realizes the fully automatic, top-down sequence design of nearly arbitrary structured 3D DNA assemblies based only on simple geometric representation, offering nonexperts the ability to design synthetic DNA-based molecular architectures. RESULTS We apply our computational algorithm to design a broad palette of 45 complex nanoparticle geometries including Platonic, Archimedean, Catalan, and Johnson solids, as well as more than 10 arbitrarily shaped solids that include both symmetric and asymmetric shapes. We apply asymmetric polymerase chain reaction (aPCR) to generate custom ssDNA scaffolds that result in high-fidelity folding of DNA nanoparticles of diverse shapes and sizes, which are also verified to fold in low salt concentrations due their open wireframe design. We use single-particle cryo–electron microscopy (cryo-EM) to obtain 3D reconstructions of six target objects that are consistent with 3D atomic model predictions within the nanometer-scale resolution obtained, and demonstrate versatility of our algorithm in realizing both rigid and toroidal-like objects. Nanoparticles are demonstrated to be stable in cellular buffers including Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS). Our aPCR procedure offers facile amplification of target natural or synthetic DNA scaffolds for assays requiring high amounts of material, such as the cryo-EM reconstructions realized here. CONCLUSION Inverse sequence design of structured DNA assemblies with full control over asymmetric 3D structure and sequence properties is a long-standing aim of molecular engineering. We present a general solution to this problem that offers the ability for nonspecialists to design and synthesize nearly arbitrary DNA-based nanoparticles using only a simple surface representation of the target object. We provide open-source software together with a versatile synthesis strategy to self-assemble complex nanoparticles that are stable in diverse buffers, including in physiological conditions, which offers the opportunity for their potential use in numerous in vitro as well as biomedical applications. DNA nanoparticle design, synthesis, and characterization. (Top) Top-down geometric specification of the target geometry is followed by fully automatic sequence design and 3D atomic-level structure prediction. (Bottom) aPCR is used to synthesize object-specific ssDNA scaffold for folding. Nanoparticle stability is characterized in cellular media with serum and nanoparticle 3D structure is characterized by single-particle cryo-EM. Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.