Designer nanoscale DNA assemblies programmed from the top down

Abstract

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.

Fig. 0. 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) The asymmetric PCR is used to synthesize object-specific single-stranded DNA scaffold for folding. Nanoparticle stability is characterized in cellular media with serum and nanoparticle 3D structure is characterized using single-particle cryo-electron microscopy.

Fig. 1. Top-down sequence design procedure for scaffolded DNA origami nanoparticles of arbitrary shape

Specification of the arbitrary target geometry is based on a continuous, closed surface that is discretized using polyhedra. This discrete representation is used (step i) to compute the corresponding 3D graph and (step ii) spanning tree. The spanning tree is used (step iii) to route the single-stranded DNA scaffold throughout the entire origami object automatically, which then enables (step iv) the assignment of complementary staple strands. Finally, (step v) a 3D atomic-level structural model is generated assuming canonical B-form DNA geometry, which is validated using 3D cryo-EM reconstruction.

Fig. 2. Fully automatic sequence design of 45 diverse scaffolded DNA origami nanoparticles

(Top) Face-shaded 3D representations of geometric models used as input to the algorithm. (Bottom) 3D atomic models of DNA-rendered nanoparticles for (blue) Platonic, (red) Archimedean, (green) Johnson, (orange) Catalan, and (violet) miscellaneous polyhedra generated using the automatic scaffold routing and sequence design procedure (particles are not shown to scale). Miscellaneous polyhedra include (first column) heptagonal bipyramid; enneagonal trapezohedron; small stellated dodecahedron, a type of Kepler-Poinsot solid; rhombic hexecontahedron, a type of zonohedron; Goldberg polyhedron G(2,1) with symmetry of Papillomaviridae; (second column) double helix; nested cube; nested octahedron; torus; and double torus. Platonic, Archimedean, and Johnson solids each have 52-bp edge length, Catalan solids and the first column of miscellaneous polyhedra have minimum 42-bp edge length, and the second column of miscellaneous polyhedra have minimum 31-bp edge length. 30 of the 45 structures shown have scaffolds smaller than the 7,249-nt M13mp18 whereas 15 have scaffold lengths that exceed it (Table S2).

Fig. 3. aPCR strategy to synthesize custom single-stranded DNA scaffolds

(A) Single-stranded DNA (ssDNA) scaffolds of custom length and sequence for each target structure are amplified using either a single- or double-stranded DNA template mixed with appropriate primer pairs consisting of 50× sense primer and 1× anti-sense primer concentration relative to the scaffold concentration. (B) Amplified ssDNA products are purified and analyzed using agarose gel electrophoresis.

Fig. 4. Folding and 2D structural characterization of scaffolded DNA origami nanoparticles

(A) Characterization of folding for five platonic solids (52-bp, 63-bp, and 73-bp edge-length tetrahedra; 52-bp edge-length octahedron; 52-bp edge-length icosahedron) using AGE, AFM and cryo-EM. (B) Characterization of folding for one Archimedean solid (52-bp edge-length cuboctahedron), one miscellaneous solid (reinforced cube with 52- and 73-bp edge lengths), and one Johnson solid (42- and 52-bp edge-length pentagonal bipyramid); using AGE, AFM and cryo-EM. M: DNA marker; sc: custom ssDNA scaffold. Scale bars are 20 nm for AFM and cryo-EM and 10 nm for atomic models.

Fig. 5. 3D structural characterization of scaffolded DNA origami nanoparticles using cryo-EM reconstruction and comparison with model predictions

(A) Programmed edges of the 52-bp edge-length icosahedron are straight and vertices are rotationally symmetric, as designed. Cryo-EM resolution is 2.0 nm and correlation with model is 0.85 (54). (B) Edges of the 63-bp edge-length tetrahedron reveal significant outward bowing (arrow) that is attributable to its acute interior angles that might result in steric hindrance. Cryo-EM resolution is 1.8–2.2 nm and correlation with model is 0.72. (C) 15° right-handed twist is visible at each vertex (arrow) of the 52-bp edge-length octahedron, which suggests that the structure folds as prescribed rather than “inside-out.” Cryo-EM resolution is 2.5 nm and correlation with model is 0.89. (D) 52-bp edge-length cuboctahedron has unequal angles between edges that meet at vertices (arrows), which supports a rigid-duplex model in which phosphate backbone stretch is minimized (31). Cryo-EM resolution is 2.9 nm and correlation with model is 0.92. (E) The addition of 73-bp reinforcing struts to a simple cube of 52-bp edge-length increases its structural homogeneity to produce a 3D reconstruction with 915 particles. With the reinforcement, the particles maintain right-angled vertices (upper arrow). The diagonal edges form a tetrahedral symmetry that exhibits outward bowing (lower arrow). Cryo-EM resolution is 2.7 nm and correlation with model is 0.72. (F) 3D reconstruction of a nested cube within a cube that has nonspherical topology. The 73-bp edge-length outer cube is connected to a 32-bp edge-length inner cube by eight 31-bp edge-length diagonals. Cryo-EM resolution is 4.0–4.5 nm and correlation with model is 0.74. Scale bars are 5 nm.

Fig. 6. Characterization of scaffolded DNA origami nanoparticle folding in variable added salt

Characterization of folding of the 52-bp edge-length pentagonal bipyramid in increasing magnesium chloride (MgCl₂) and sodium chloride (NaCl) concentration using 2% AGE and AFM imaging. Critical concentrations for folding are 4 mM MgCl₂ and 500 mM NaCl in TRIS-acetate pH 8.0. M: DNA marker; sc: custom ssDNA scaffold. Scale bars are 30 nm.

Fig. 7. Characterization of scaffolded DNA origami nanoparticle stability in physiological buffer and serum

AGE and AFM structural characterization of the 52-bp edge-length pentagonal bipyramid after 6 hours in PBS, TAE (without added NaCl or MgCl₂), and DMEM buffer with increasing concentration of FBS (0, 2, and 10%) after folding in TAE-Mg²⁺ buffer (12 mM MgCl₂) followed by buffer exchange. Stability is observed for structures in PBS buffer but not in TAE due to the absence of salt, which demonstrates the importance of a minimal salt concentration for stability. AFM imaging reveals the presence of intact objects after 6 hours in DMEM media in the presence of 2 to 10% FBS despite partial degradation is observed in AGE. Scale bars are 30 nm.