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Designer nanoscale DNA assemblies programmed from the top down

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Science  24 Jun 2016:
Vol. 352, Issue 6293, pp. 1534
DOI: 10.1126/science.aaf4388
  • 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) aPCR is used to synthesize object-specific ssDNA scaffold for folding. Nanoparticle stability is characterized in cellular media with serum and nanoparticle 3D structure is characterized by single-particle cryo-EM.

  • 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 with 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 ssDNA 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.

    (A) Face-shaded 3D representations of geometric models used as input to the algorithm. (B) Three-dimensional 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. Thirty of the 45 structures shown have scaffolds smaller than the 7249-nt M13mp18, whereas 15 have scaffold lengths that exceed it (table S2).

  • Fig. 3 aPCR strategy to synthesize custom ssDNA scaffolds.

    (A) 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× antisense primer concentration relative to the scaffold concentration. (B) Amplified ssDNA products are purified and analyzed by agarose gel electrophoresis.

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

    (A) Characterization of folding for five platonic solids (52-, 63-, and 73-bp edge-length tetrahedra; 52-bp edge-length octahedron; 52-bp edge-length icosahedron) by agarose gel electrophoresis, 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 agarose gel electrophoresis, AFM, and cryo-EM. M: DNA marker; sc: custom ssDNA scaffold. Scale bars: 20 nm for AFM and cryo-EM and 10 nm for atomic models.

  • Fig. 5 Three-dimensional 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 the model is 0.85 (55). (B) Edges of the 63-bp edge-length tetrahedron reveal outward bowing (arrow) attributable to its acute interior angles that might result in steric hindrance. Cryo-EM resolution is 1.8 to 2.2 nm and correlation with the model is 0.72. (C) A 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 the model is 0.89. (D) A 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 (32). Cryo-EM resolution is 2.9 nm and correlation with the 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) Three-dimensional 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 to 4.5 nm and correlation with the model is 0.74. Scale bars: 5 nm.

  • Fig. 6 Characterization of scaffolded DNA origami nanoparticle folding in variable concentrations of 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% agarose gel electrophoresis 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: 30 nm.

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

    Agarose gel electrophoresis 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 owing 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 observed in agarose gel electrophoresis. Scale bars: 30 nm.

Supplementary Materials

  • Designer nanoscale DNA assemblies programmed from the top down

    Rémi Veneziano, Sakul Ratanalert, Kaiming Zhang, Fei Zhang, Hao Yan, Wah Chiu, Mark Bathe

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Supplementary Text S1 to S7
    • Figs. S1 to S59
    • Tables S1 to S5
    • Captions for tables S6 to S27
    • Captions for movies S1 to S7
    • References (5764)
    Tables S6 to S27
    Table S6. aPCR amplified sequences.
    Table S7. Digested sequences.
    Tables S8 to S27. Staple lists for the scaffolded DNA origami objects synthesized in this paper.

    Images, Video, and Other Media

    Movie S1
    3D rotation of icosahedron cryo-EM map with atomic model superimposed.
    Movie S2
    3D rotation of tetrahedron cryo-EM map with atomic model superimposed.
    Movie S3
    3D rotation of cuboctahedron cryo-EM map with atomic model superimposed.
    Movie S4
    3D rotation of octahedron cryo-EM map with atomic model superimposed.
    Movie S5
    3D rotation of reinforced cube cryo-EM map with atomic model superimposed.
    Movie S6
    3D rotation of nested cube cryo-EM map with atomic model superimposed.
    Movie S7
    3D rotation of octahedron cryo-EM map with alternate atomic model superimposed.

    Additional Data

    DAEDALUS Software Package

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