Research ArticleAPPLIED SCIENCES AND ENGINEERING

Sequence-programmable covalent bonding of designed DNA assemblies

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Science Advances  17 Aug 2018:
Vol. 4, no. 8, eaau1157
DOI: 10.1126/sciadv.aau1157
  • Fig. 1 Proximal thymidines as sites for cross-linking in DNA nanostructures.

    (A) Left: Chemical structures of two proximal thymidines before UV irradiation. Right: Schematic illustration of a six-helix bundle DNA nanostructure featuring single-stranded thymidines at strand termini (1), at half-crossovers (2), at full crossovers (3), and thymidine loops (4) before UV irradiation. (B) As in (A) but after exposure to light with 310-nm wavelength. CPD bonds are indicated as red ellipsoids.

  • Fig. 2 Proof of concept of UV cross-linking with multilayer DNA origami.

    (A) From left to right: Model of the brick-like DNA origami object featuring additional thymidines at all strand termini and at all strand crossover positions; laser-scanned fluorescent images of 2.0% agarose gels stained with ethidium bromide. Irradiated (135 min at 310 nm) and nonirradiated samples were either incubated for 30 min at different temperatures or incubated for 3 hours at room temperature in double-distilled water (ddH2O) containing successively lower concentrations of monovalent sodium chloride, respectively. p, pocket; u, unfolded species; f, folded species; c, cross-linked staple strands; s, uncross-linked staple strands; L, 1kB ladder; NI and I, nonirradiated and irradiated reference samples in folding buffer with 5 mM MgCl2, respectively. The images of the gels were autoleveled, and the highlighted regions were autoleveled twice; average 2D particle micrograph of the irradiated sample in double-distilled water. (B and C) As in (A) but with the brick-like DNA origami object featuring additional thymidines at all strand termini and at all strand crossover positions and with 5-T loops and the pointer object featuring additional thymidines at all strand termini and at all strand crossover positions, respectively. See fig. S9 for globally autoleveled gel images.

  • Fig. 3 Stability under physiological conditions.

    Laser-scanned fluorescent images of 2.0% agarose gels stained with ethidium bromide. Cross-linked samples were irradiated for 135 min at 310 nm. (A) The brick-like DNA origami object featuring additional thymidines at all strand termini and at all strand crossover positions was incubated for different periods of time in PBS solution at 37°C. (B) The brick-like DNA origami object featuring additional thymidines at all strand termini, at all strand crossover positions, and 5-T loops was incubated for different periods of time in 10% FBS at 37°C. (C) The brick-like DNA origami object from (A) was exposed to a set of different nucleases (100 U/ml) for 24 hours at 37°C. Lanes labeled with a “c” indicate controls in which the sample was dissolved in the corresponding buffers in the absence of nuclease. (D) The brick-like DNA origami object from (B) was exposed to DNase I (0.4 U/ml) for different periods of time at 37°C. (B to D) Nonirradiated and irradiated samples were loaded on the gel alternatingly. All images of the gels were globally autoleved.

  • Fig. 4 Cryo-EM structural analysis before and after UV irradiation.

    (A) Cryo-EM density map of the nonirradiated brick-like object with TT motifs 1 to 3 (Electron Microscopy Data Bank identifier EMD-4354). (B and C) Cryo-EM density map of the irradiated (135 min at 310 nm) brick-like object with TT motifs 1 to 3 in buffer containing 5 mM MgCl2 or in PBS buffer, respectively. The electron density thresholds are chosen in such a way that all crossovers in the top layer are visible, as seen in the side view (Electron Microscopy Data Bank identifier EMD-0027 and EMD-0028, respectively). (D) Slices along the z direction obtained from the three density maps shown in (A) to (C) from top to bottom. To determine the twist angle delta Theta, the first and last slices were chosen. (E) Slices showing the three crossover layers in the reconstructions shown in (A) to (C). (F) Comparison of the global dimensions of the uncross-linked variant in 5 mM MgCl2 buffer and the cross-linked variant in PBS buffer. Color code as shown in (A) to (C).

  • Fig. 5 Covalent bonding of conformational states and higher-order assemblies.

    (A) Schematics of the two-state switch that consists of two rigid beams flexibly connected in the middle by an immobile Holliday junction. Cylinders in the models represent double-helical DNA domains, and shape-complementary surface features are highlighted in red and blue. Insets show blow-ups of the blunt-ended interfaces of protruding (red) and recessive (blue) surface features. Thymidines directly located at the blunt-end site can be cross-linked upon UV light irradiation. The resulting CPD bond is indicated as a red ellipsoid. (B) Laser-scanned fluorescent image of 2.0% agarose gel stained with ethidium bromide. Switch samples were irradiated at 310 nm for different periods of time and loaded on the gel. o and c, species of particles populating open and closed state, respectively. (C) Plot of the fraction of cross-linked switch particles as a function of time obtained from the gel in (B). The experiment was performed in triplicate; data points represent the mean, and error bars represent the SD. (D) Exemplary TEM micrographs. Top: Nonirradiated sample with particles populating the open state. Bottom: Irradiated (20 min at 310 nm) sample with particles locked in the closed conformational state. Scale bars, 100 nm. Inset: Average 2D particle micrograph of cross-linked particles. Scale bar, 20 nm. (E) Top left: Model of the multilayer DNA origami brick that polymerizes into linear filaments. Fields of view of TEM micrographs recorded at the indicated conditions. Scale bars, 100 nm.

  • Table 1 One-pot self-assembly of DNA origami objects.
    Object nameDenaturation temperature for 15 min (°C)Folding rampStorage temperature (°C)Scaffold
    Brick-like, TT motifs 1–365[60–20°C]; 60 min/1°C20p7560
    Brick-like, TT motifs 1–465[60–20°C]; 60 min/1°C20p7560
    Pointer65[60–20°C]; 60 min/1°C20p7249
    Switch65[58–55°C]; 90 min/1°C25p8064
    Polymerization brick65[60–44°C]; 60 min/1°C25p8064
  • Table 2 Negative-stain TEM settings.
    MicroscopeOperating voltage (kV)CameraObjects
    Philips CM 100100AMT 4-megapixel charge-coupled device cameraSwitch; polymerization brick
    FEI Tecnai 120120Tietz TemCam-F416 (4k × 4k)Brick-like, TT motifs 1–3
    Brick-like, TT motifs 1–4
    Pointer

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaau1157/DC1

    Fig. S1. Design diagram of the brick-like (TT motifs 1 to 3) object prepared using caDNAno.

    Fig. S2. Exemplary negative-stained TEM micrographs of the brick-like object (TT motifs 1 to 4) in different buffers/solvents.

    Fig. S3. Laser-scanned fluorescent image of a 2.0% agarose gel that was run in an ice-cooled water bath.

    Fig. S4. Laser-scanned fluorescent image of a 2.0% agarose gel that was run in an ambient temperature water bath.

    Fig. S5. Design diagram of the brick-like (TT motifs 1 to 4) object prepared using caDNAno.

    Fig. S6. Laser-scanned fluorescent image of a 2.0% agarose gel placed in an ice-cooled water bath.

    Fig. S7. Design diagram of the pointer object.

    Fig. S8. Exemplary negative-stained TEM micrographs of the pointer object in different buffers/solvents.

    Fig. S9. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S10. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S11. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S12. Laser-scanned fluorescent image of a 2.0% agarose gel placed in an ice-cooled water bath.

    Fig. S13. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S14. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S15. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S16. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.

    Fig. S17. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) before crosslinking in folding buffer.

    Fig. S18. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) after crosslinking in folding buffer.

    Fig. S19. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) after crosslinking in phosphate-buffered saline (PBS).

    Fig. S20. Slice-by-slice visualization of cryo-EM maps determined from brick samples.

    Fig. S21. Cryo-EM data of the brick-like object with TT-motifs (1) – (4) before crosslinking in folding buffer.

    Fig. S22. Cryo-EM data of the brick-like object with TT-motifs (1) – (4) after crosslinking in folding buffer.

    Fig. S23. Design diagram of the switch object.

    Fig. S24. Design diagram of the polymerization brick object.

    Table S1. Sequences of staple strands for all DNA objects used in this work.

    Movie S1. Tomogram obtained from cryo-EM of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites.

    Movie S2. Corresponding movie to fig. S20 for the nonirradiated (uncross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in folding buffer and in the presence of 5 mM MgCl2.

    Movie S3. Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in folding buffer and in the presence of 5 mM MgCl2.

    Movie S4. Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in PBS buffer and in the absence of MgCl2.

    Movie S5. Corresponding movie to fig. S20 for the nonirradiated (uncross-linked) sample of the brick-like variant with thymines at all staple termini, with TT motifs at all crossover sites and additional 5-T loops in folding buffer and in the presence of 5 mM MgCl2.

    Movie S6. Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini, with TT motifs at all crossover sites and additional 5-T loops in folding buffer and in the presence of 5 mM MgCl2.

    Reference (70)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Design diagram of the brick-like (TT motifs 1 to 3) object prepared using caDNAno.
    • Fig. S2. Exemplary negative-stained TEM micrographs of the brick-like object (TT motifs 1 to 4) in different buffers/solvents.
    • Fig. S3. Laser-scanned fluorescent image of a 2.0% agarose gel that was run in an ice-cooled water bath.
    • Fig. S4. Laser-scanned fluorescent image of a 2.0% agarose gel that was run in an ambient temperature water bath.
    • Fig. S5. Design diagram of the brick-like (TT motifs 1 to 4) object prepared using caDNAno.
    • Fig. S6. Laser-scanned fluorescent image of a 2.0% agarose gel placed in an ice-cooled water bath.
    • Fig. S7. Design diagram of the pointer object.
    • Fig. S8. Exemplary negative-stained TEM micrographs of the pointer object in different buffers/solvents.
    • Fig. S9. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S10. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S11. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S12. Laser-scanned fluorescent image of a 2.0% agarose gel placed in an ice-cooled water bath.
    • Fig. S13. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S14. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S15. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S16. Laser-scanned fluorescent image of a 2.0% agarose gel placed in a water bath.
    • Fig. S17. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) before crosslinking in folding buffer.
    • Fig. S18. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) after crosslinking in folding buffer.
    • Fig. S19. Cryo-EM data of the brick-like object with TT-motifs (1) – (3) after crosslinking in phosphate-buffered saline (PBS).
    • Fig. S20. Slice-by-slice visualization of cryo-EM maps determined from brick samples.
    • Fig. S21. Cryo-EM data of the brick-like object with TT-motifs (1) – (4) before crosslinking in folding buffer.
    • Fig. S22. Cryo-EM data of the brick-like object with TT-motifs (1) – (4) after crosslinking in folding buffer.
    • Fig. S23. Design diagram of the switch object.
    • Fig. S24. Design diagram of the polymerization brick object.
    • Legend for table S1
    • Legends for movies S1 to S6
    • Reference (70)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Sequences of staple strands for all DNA objects used in this work.
    • Movie S1 (.mov format). Tomogram obtained from cryo-EM of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites.
    • Movie S2 (.mov format). Corresponding movie to fig. S20 for the nonirradiated (uncross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in folding buffer and in the presence of 5 mM MgCl2.
    • Movie S3 (.mov format). Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in folding buffer and in the presence of 5 mM MgCl2.
    • Movie S4 (.mov format). Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini and with TT motifs at all crossover sites in PBS buffer and in the absence of MgCl2.
    • Movie S5 (.mov format). Corresponding movie to fig. S20 for the nonirradiated (uncross-linked) sample of the brick-like variant with thymines at all staple termini, with TT motifs at all crossover sites and additional 5-T loops in folding buffer and in the presence of 5 mM MgCl2.
    • Movie S6 (.mov format). Corresponding movie to fig. S20 for the irradiated (cross-linked) sample of the brick-like variant with thymines at all staple termini, with TT motifs at all crossover sites and additional 5-T loops in folding buffer and in the presence of 5 mM MgCl2.

    Files in this Data Supplement:

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