DNA can be used to fabricate any free-form 2D drawing at the nanometer scale.

3D polyhedral wireframe DNA nanoparticles (DNA-NPs) fabricated using scaffolded DNA origami offer complete and independent control over NP size, structure, and asymmetric functionalization on the 10–100 nm scale. However, the complex DNA sequence design needed for the synthesis of these versatile DNA-NPs has limited their widespread use to date. While the automated sequence design algorithms DAEDALUS and vHelix-BSCOR apply to DNA-NPs synthesized using either uniformly dual or hybrid single-dual duplex edges, respectively, these DNA-NPs are relatively compliant mechanically and are therefore of limited utility for some applications. Further, these algorithms are incapable of handling DNA-NP edge designs composed of more than two duplexes, which are needed to enhance DNA-NP mechanical stiffness. As an alternative, here we introduce the scaffolded DNA origami sequence design algorithm TALOS, which is a generalized procedure for the fully automated design of wireframe 3D polyhedra composed of edges of any cross section with an even number of duplexes, and apply it to DNA-NPs composed uniformly of single honeycomb edges. We also introduce a multiway vertex design that enables the fabrication of DNA-NPs with arbitrary edge lengths and vertex angles and apply it to synthesize a highly asymmetric origami object. Sequence designs are demonstrated to fold robustly into target DNA-NP shapes with high folding efficiency and structural fidelity that is verified using single particle cryo-electron microscopy and 3D reconstruction. In order to test its generality, we apply TALOS to design an in silico library of over 200 DNA-NPs of distinct symmetries and sizes, and for broad impact, we also provide the software as open source for the generation of custom NP designs.

Wireframe DNA origami has emerged as a powerful approach to fabricating nearly arbitrary 2D and 3D geometries at the nanometer-scale. Complex scaffold and staple routing needed to design wireframe DNA origami objects, however, render fully automated, geometry-based sequence design approaches essential for their synthesis. And wireframe DNA origami structural fidelity can be limited by wireframe edges that are composed only of one or two duplexes. Here we introduce a fully automated computational approach that programs 2D wireframe origami assemblies using honeycomb edges composed of six parallel duplexes. These wireframe assemblies show enhanced structural fidelity from electron microscopybased measurement of programmed angles compared with identical geometries programmed using dual-duplex edges. Molecular dynamics provides additional theoretical support for the enhanced structural fidelity observed. Application of our top-down sequence design procedure to a variety of complex objects demonstrates its broad utility for programmable 2D nanoscale materials.

Structural DNA nanotechnology is beginning to emerge as a widely accessible research tool to mechanistically study diverse biophysical processes. Enabled by scaffolded DNA origami in which a long single strand of DNA is weaved throughout an entire target nucleic acid assembly to ensure its proper folding, assemblies of nearly any geometric shape can now be programmed in a fully automatic manner to interface with biology on the 1–100-nm scale. Here, we review the major design and synthesis principles that have enabled the fabrication of a specific subclass of scaffolded DNA origami objects called wireframe assemblies. These objects offer unprecedented control over the nanoscale organization of biomolecules, including biomolecular copy numbers, presentation on convex or concave geometries, and internal versus external functionalization, in addition to stability in physiological buffer. To highlight the power and versatility of this synthetic structural biology approach to probing molecular and cellular biophysics, we feature its application to three leading areas of investigation: light harvesting and nanoscale energy transport, RNA structural biology, and immune receptor signaling, with an outlook toward unique mechanistic insight that may be gained in these areas in the coming decade.

Wireframe DNA origami assemblies can now be programmed automatically from the top-down using simple wireframe target geometries, or meshes, in 2D and 3D, using either rigid, six-helix bundle (6HB) or more compliant, two-helix bundle (DX) edges. While these assemblies have numerous applications in nanoscale materials fabrication due to their nanoscale spatial addressability and high degree of customization, no easy-to-use graphical user interface software yet exists to deploy these algorithmic approaches within a single, standalone interface. Further, top-down sequence design of 3D DX-based objects previously enabled by DAEDALUS was limited to discrete edge lengths and uniform vertex angles, limiting the scope of objects that can be designed. Here, we introduce the open-source software package ATHENA with a graphical user interface that automatically renders single-stranded DNA scaffold routing and staple strand sequences for any target wireframe DNA origami using DX or 6HB edges, including irregular, asymmetric DX-based polyhedra with variable edge lengths and vertices demonstrated experimentally, which significantly expands the set of possible 3D DNA-based assemblies that can be designed. ATHENA also enables external editing of sequences using caDNAno, demonstrated using asymmetric nanoscale positioning of gold nanoparticles, as well as providing atomic-level models for molecular dynamics, coarse-grained dynamics with oxDNA, and other computational chemistry simulation approaches.