Cryo-EM structure of a 3D DNA-origami object

Abstract

A key goal for nanotechnology is to design synthetic objects that may ultimately achieve functionalities known today only from natural macromolecular complexes. Molecular self-assembly with DNA has shown potential for creating user-defined 3D scaffolds, but the level of attainable positional accuracy has been unclear. Here we report the cryo-EM structure and a full pseudoatomic model of a discrete DNA object that is almost twice the size of a prokaryotic ribosome. The structure provides a variety of stable, previously undescribed DNA topologies for future use in nanotechnology and experimental evidence that discrete 3D DNA scaffolds allow the positioning of user-defined structural motifs with an accuracy that is similar to that observed in natural macromolecules. Thereby, our results indicate an attractive route to fabricate nanoscale devices that achieve complex functionalities by DNA-templated design steered by structural feedback.

Fig. 1.

Cryo-EM reconstruction of a designed, densely packed DNA object. (A) Schematic representation of the designed rectangular lattice comprising 82 parallel dsDNA helices (gray circles). (B) Representative part of an electron micrograph. (Scale bar, 50 nm.) (C) Reference-free 2D class averages. (D) Six orthogonal views of the 3D reconstruction, shown as iso-density surface at density level 0.1.

Fig. 2.

Pseudoatomic model. Six orthogonal views of the pseudoatomic model that was fitted into the EM density map.

Fig. 3.

Analysis of internal geometry. (A) Central slice through the object at row 6, showing the cryo-EM density map (transparent gray) and the fitted pseudoatomic model as ribbon/slab model. For part of the model, the scaffold is shown in blue and the staples in red. Vertical arrows indicate a vertical stack of three cross-overs. (B) Close-up of colored area in A, but rotated around the z axis by 180°. White crosses indicate out-of-plane cross-over positions. (C) Side view of a single cross-over. (D) Top view of the same cross-over as in C. (E and F) Schematic representation of the 3D chickenwire-like pattern found in the structure, depicting dsDNA helical stretches in gray and cross-overs in red. The pattern was computed using the coordinates of base pair midpoints in the pseudoatomic model. The midpoints of neighboring dsDNA helices move on average from a minimum distance <d_min> = 18.5 Å at the cross-over to a maximum distance of <d_max> = 36 Å away from each other. Cross-overs marked in blue indicate scaffold-based strand cross-overs. (G) Definition of the angles enclosed by the four helical legs of a cross-over. Vectors are computed using the coordinates of base pair midpoints at the cross-over position and 2 bp away from the cross-over in each leg. The cross-over vector x is computed from the coordinates of the midpoints between the two base pairs in each of the two helices at the cross-over position and is normal to what we call the cross-over plane. The subscript “||” indicates vectorial projections into the cross-over plane. The angle β is also computed as indicated for vectors C and D. (H) Observed distribution of angles between the legs for cross-overs with (light gray) and without (dark gray) nearby nicks for 377 cross-overs in the structure, where each cross-over contributed two γ, α, and β angle values each. (I) Schematic representation of a revised cross-over model for DNA nanostructure design. (J) 3D chickenwire-like pattern formed by multiple instances of the revised cross-over model when rotated and translated according to the square-lattice packing connectivity scheme.

Fig. 4.

Motifs beyond B-form DNA for nanotechnology. (A) A vertical stack of five Holliday junctions. (B) A pseudohelical structure that runs along the direction of the base pairs. (C) A bent helix and distorted cross-over due to an omitted base pair (Lower) and a typical cross-over without omission (Upper). (D) Crevices formed by splayed-out helices due to the absence of stabilizing cross-overs.