Automated Sequence Design of 3D Polyhedral Wireframe DNA Origami with Honeycomb Edges

Abstract

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.

Keywords: 3D cryo-EM reconstruction; DNA nanotechnology; molecular dynamics; scaffolded DNA origami; six-helix bundle; wireframe origami.

Figure 1.

Overview of the top-down sequence design procedure TALOS for scaffolded 6HB DNA-NPs. The arbitrary target geometry is based on a polyhedral mesh, with discretized line segments (step (i)) to represent six DNA duplexes per wireframe edge with the endpoints joined (step (ii)) to form closed loops with geometrically allowable scaffold double crossovers between them. The dual graph of the loop–crossover structure is obtained (step (iii)) by converting each closed scaffold loop to a node and connecting each possible scaffold double crossover to an edge. The minimum spanning tree of the dual graph was then determined and inverted (step (iv)), defining the DNA scaffold routing. On the basis of the user-defined staple length (for this example, 20 to 60 nt (a) and 15 to 49 nt (b)), staple sequences generated (step (v)) by the algorithm were used with the input scaffold to synthesize (step (vi)) the 3D DNA-NP in one-pot thermal annealing.

Figure 2.

Validation of 6HB DNA-NP origami objects synthesized using TALOS sequence designs. (a–c) In the FV case, the routed structure is generated such that each wireframe edge is connected covalently to its neighboring edges by one scaffold and staple crossing. A circular map is rendered, in which the outer circle representing the scaffold has points assigned in the center of each double-stranded DNA domain with staple connections between regions rendered as lines traversing the circle (see also Figures S14–S18). Characterization of folding for FV tetrahedra of 63-bp and 84-bp edge lengths and an FV octahedron of 84-bp edge length with agarose gel mobility shift assays ((b); uncropped gel images in Figure S48) and cryo-EM (c). (d–f) In the MV case, the routed structure is generated such that each wireframe edge is connected covalently to its neighboring edges by three scaffold and staple crossings. Characterization of folding for MV tetrahedra of 63- and 84-bp edge lengths and an MV octahedron of 84-bp edge length with agarose gel mobility shift assays ((e); uncropped gel images in Figure S48) and cryo-EM (f). (g) Wide-field TEM micrograph shows monodisperse MV octahedra of 84-bp edge length (see also Figures S51–S64). Scale bars are 5 nm in atomic models and 20 nm in cryo-EM and 200 nm in the wide-field TEM.

Figure 3.

3D characterization of 6HB DNA-NPs using cryo-EM reconstructions compared with predicted atomic models. (a) The FV tetrahedron of 84-bp edge length shows straight edges with a distinctive FV type and a 3° left-handed twist visible at each vertex, with a clear signature of a 6HB along the edge (arrow). (b) The FV octahedron of 84-bp edge length has straight edges and regular programmed vertices with characteristic open vertices and no detectable deviation along the edge compared to the atomic model, with a clear signature of a 6HB along the edge (arrow). (c) The MV tetrahedron of 84-bp edge length shows the characteristic programmed electron-dense vertex (arrow). (d) The MV octahedron of 84-bp edge length has straight edges and electron-dense vertices with approximately a 1 nm deviation along the edge from the predicted atomic model (arrow). Scale bars are 5 nm.

Figure 4.

Molecular dynamics simulations of tetrahedra. (a,b) Superposition of molecular dynamics snapshots of FV (a) and MV (b) tetrahedra of 42-bp edge length at 0, 100, and 200 ns. Initial atomic models were generated by TALOS. (c) Total RMSD of all nucleic acid atoms based on the ground-state atomic model generated by TALOS. The FV and DX tetrahedra of 42-bp edge lengths show additional dynamical motion due to vertex fluctuations compared with the MV design. (d) All-atom RMSFs were calculated for the FV, MV, and DX tetrahedra over the 200 ns simulation and mapped as a white-to-red color gradient on each of the structures.

Figure 5.

Fully automatic sequence design of diverse scaffolded 6HB DNA-NPs. (a) 3D representations of geometric models as input to the algorithm with associated 3D atomic models and circular map of the DNA-NPs for a tetrahedron, cube, octahedron, and pentagonal bipyramid using the automatic scaffold routing and sequence design procedure for the FV and MV cases. (b) 3D representations of geometric and atomic models for two Platonic-, Archimedean-, Johnson-, and Catalan-type geometries, with additional DNA-NPs shown in Figures S19–S31. All DNA-NPs in panels (a) and (b) have a minimum 42-bp edge length, and the required scaffold lengths are shown in Table S5. (c) The generality of the MV design for arbitrary edge length and arbitrary vertex angle with a fully asymmetric tetrahedron (left) and a twisted triangular prism (right). For both objects, the inner, middle, and outer layers of helices are shown with helical extensions shown in gray and vertices zoomed-in to show the 6HB extensions (gray) to achieve the modeled angles. Individual particles are not shown to scale.