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. 2009 May 21;459(7245):414-8.
doi: 10.1038/nature08016.

Self-assembly of DNA Into Nanoscale Three-Dimensional Shapes

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Free PMC article

Self-assembly of DNA Into Nanoscale Three-Dimensional Shapes

Shawn M Douglas et al. Nature. .
Free PMC article

Abstract

Molecular self-assembly offers a 'bottom-up' route to fabrication with subnanometre precision of complex structures from simple components. DNA has proved to be a versatile building block for programmable construction of such objects, including two-dimensional crystals, nanotubes, and three-dimensional wireframe nanopolyhedra. Templated self-assembly of DNA into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase 'scaffold strand' that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide 'staple strands'. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes-monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross-with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometre scale.

Figures

Figure 1
Figure 1. Design of three-dimensional DNA origami
a, Double helices comprised of scaffold (grey) and staple strands (orange, white, blue), run parallel to the z-axis to form an unrolled two-dimensional schematic of the target shape. Phosphate linkages form crossovers between adjacent helices, with staple crossovers bridging different layers shown as semi-circular arcs. b, Cylinder model of a half-rolled conceptual intermediate. Cylinders represent double helices, with loops of unpaired scaffold strand linking the ends of adjacent helices. c, Three-dimensional cylinder model of folded target shape. Honeycomb arrangement of parallel helices is shown in cross-sectional slices (i–iii) parallel to the x–y plane spaced apart at 7 base-pair intervals repeating every 21 base pairs. All potential staple crossovers are shown for each cross-section. d, Atomistic DNA model of shape from c.
Figure 2
Figure 2. Three-dimensional DNA origami shapes
First and second rows, perspective and projection views of cylinder models, with each cylinder representing a DNA double helix. a, monolith. b, square nut. c, railed bridge. d, slotted cross. e, stacked cross. Rows 3–7, transmission electron microscope (TEM) micrographs of typical particles. For imaging, samples were adsorbed (5 min) onto glow-discharged grids pre-treated with 0.5 M MgCl2, stained with 2% uranyl formate, 25mM NaOH (1 min), and visualized with an FEI Tecnai T12 BioTWIN at 120 kV. f, Field of homogeneous and monodisperse stacked-cross particles. g, Integrated-intensity profile (red) of line orthogonal to the longitudinal axis of typical monolith particle, with expected profile (grey) assuming a simple homogeneous cylinder model. h, Gaussian-fitted average peak positions (circles) in such integrated-line profiles for twenty different monolith particles as a function of peak index. The observed average peak-to-peak distance was 3.65 nm (±0.2 nm s.d., ±0.01 nm s.e.m.). This peak-to-peak distance should correspond to 1.5 times the effective diameter d of individual double helices in the monolith structure, hence d=2.4 nm. Solid line: linear fit with a slope of 3.65 nm from peak to peak, corroborating equidistant arrangement of helices across the entire particle width. Error bars (red) indicate mean width of the peaks. Slightly higher variations in peak width at the edges of the particles are most likely due to frayed edges (cf. particles in a, g). i, Analysis as in h repeated for the square-nut shape. j, Histogram of Gaussian-fitted peak-to-peak distances as found for the square-nut particles, with the mean value at 3.18 nm (±0.2 nm s.d., ±0.01 nm s.e.m.), indicating an effective diameter of 2.1 nm per individual double helix. Scale bars: a–e: 20 nm; f: 1 μm (top), 100 nm (bottom).
Figure 3
Figure 3. Gel and TEM analysis of folding conditions for three-dimensional DNA origami
a, Cylinder models of shapes: monolith, stacked cross, railed bridge, and two versions of genie bottle, with corresponding scaffold sequences. b, Shapes were folded using different thermal-annealing ramps (1.2 h: 95°C to 20°C at 1.6 min/°C; 3 h, 6 h, 12 h, 18 h, 37 h, 74 h, 173 h: 80°C to 60°C at 4 min/°C, followed by 60°C to 24°C at 5, 10, 20, 30, 60, 120, or 280 min/°C, respectively) in 5 mM Tris, 1 mM EDTA, and 16 mM MgCl2 and analyzed by gel electrophoresis (2% agarose, 0.5 × TBE, 11 mM MgCl2). c–e, TEM and gel analysis of influence of MgCl2 concentration on folding quality. c, The fastest-migrating bands in the 4 mM MgCl2 lanes were purified and imaged, revealing gross folding defects. d, Shapes were folded with a 173 h ramp in 5 mM Tris, 1 mM EDTA, and MgCl2 concentrations varying from 0 to 30 mM. e, As in c, leading bands were purified from the 16 mM MgCl2 lanes and found to exhibit higher-quality folding when analyzed by TEM. f, Excess NaCl inhibits proper folding. Shapes were folded with 173 h ramp in 5 mM Tris, 1 mM EDTA, 16 mM MgCl2, and varying NaCl concentrations.
Figure 4
Figure 4. Two-step hierarchical assembly of larger three-dimensional structures and polymers
a, Cylinder model of stacked-cross monomer (Fig. 2e), with dotted line indicating direction of assembly. Typical TEM micrographs showing stacked cross polymers. Purified stacked-cross samples were mixed with a 5-fold molar excess of connector staple strands in the presence of 5 mM Tris, 1 mM EDTA, 16 mM MgCl2 at 30°C for 24 hours. Monomers were folded in separate chambers, purified, and mixed with connector staple strands designed to bridge separate monomers. b, Cylinder model and transmission electron micrograph of a double-triangle shape comprised of 20 six-helix bundle half-struts. c, Heterotrimerization of the icosahedra was done with a 1:1:1 mixture of the three unpurified monomers at 50°C for 24 hours. d, Orthographic projection models and TEM data of four icosahedron particles. Scale bars: 100 nm.

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