Research ArticleAPPLIED SCIENCES AND ENGINEERING

The sequence of events during folding of a DNA origami

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Science Advances  03 May 2019:
Vol. 5, no. 5, eaaw1412
DOI: 10.1126/sciadv.aaw1412
  • Fig. 1 Illustration of the DNA origami test object and the fluorometric folding assays with sample data.

    (A) Left: CanDo model (30, 31) of the investigated DNA origami test object, a 42-helix bundle, designed in honeycomb lattice consisting of 140 staple strands. Middle: Image of an agarose gel lane on which folding products of the 42-helix bundle were electrophoresed. F, folded species; P, gel pocket. Right: Class averages of negative-staining TEM images. dsDNA, double-stranded DNA. (B) Illustration of the intrastrand terminal proximity folding assay. Segmented arrows indicate individual staple strands; segments illustrate sequence stretches that will form continuous double-helical domains with the scaffold. Cy3, cyanine 3; BHQ2, Black Hole Quencher 2. (C) Raw fluorescent data of sample folding reactions of the intrastrand assay. (D) Illustration of the interstrand terminal proximity folding assay. Cy5, cyanine 5. (E) Raw fluorescent data of sample folding reactions of the interstrand assay. FRET, fluorescence resonance energy transfer. (C and E) Orange circles, complete folding reaction mixtures; gray symbols, folding reaction mixtures missing essential components (empty circles, scaffold missing; empty diamonds, full set of staple strands missing; gray diamonds, full set of staple strands and scaffold missing). The fluorescently labeled staple strands are always included. cts, counts.

  • Fig. 2 Time-dependent reaction progress and fit parameters.

    (A) Measured reaction progress data obtained with the intrastrand folding assay for the 42-helix bundle test objects, sequence permutation-0. Each column is the average of three replicates of fluorescence time traces obtained for a particularly labeled folding reaction mixture. Traces are sorted according to the mean folding time. a.u., arbitrary units. (B) Sample curves obtained from the intrastrand assay to illustrate the variety of incorporation behavior. Shaded areas indicate the SD of the averaged data. (C) Measured reaction progress data obtained with the interstrand folding assay. Data are sorted according to the time needed to reach 80% of the maximum intensity. (D) Sample traces obtained with the interstrand assay to illustrate the variety of incorporation behavior. Gray circles, a single trace to illustrate noise level. (E) A set of sample pair probe measurements with the same staple strand labeled with a Cy3 and varying Cy5-labeled staple strands in which the donor-carrying strand limits the kinetics of signal evolution. (F) A set of sample pair probe measurements in which the kinetics of the acceptor-carrying strand can be discriminated and sorted (relative to the donor carrier). (G) Time constants Tau1 (blue) and Tau2 (red) obtained by fitting double exponentials to the intrastrand assay data. (H) Time constants Tau1 (blue) and Tau2 (red) values obtained by fitting double-exponential fits to the interstrand assay data.

  • Fig. 3 Single segment binding probabilities.

    (A) Sample time traces illustrating the inference of single-strand terminal binding probabilities (right) from the pair probe interstrand assay reaction progress data (left). (B and C) Inferred single-strand 5′ and 3′ terminal binding probabilities, respectively. Data are sorted according to the mean folding time of the 5′ termini. Arrows indicate the 10 (20) fastest (slowest) staple strand termini. (D) The mean folding time of the 3′ terminal staple strand segments versus 5′ termini mean folding time. (E) Histogram of the distribution of mean folding time for the 5′ termini (black) and the 3′ termini (red). (F) Time-resolved gel electrophoretic folding analysis of the default folding reaction (top), with the 20 fastest (middle) and 20 slowest (bottom) 5′ staple strand termini truncated. Three-dimensional (3D) representation was chosen to increase visibility of weak bands. Arrows indicate the occurrence of a compacted intermediate (1) and folded species (2).

  • Fig. 4 Analysis of the mean folding times.

    (A) The intrastrand assay mean folding time versus the mean folding time resolved from the interstrand assay including 5′ and 3′ termini incorporation. (B) The intrastrand assay mean folding time versus the total binding energy of the staple strands. (C) The mean folding time of 5′ and 3′ terminal staple strands resolved from the interstrand assay versus the binding energy of the terminal staple strand segments. (D) The mean folding time of the intrastrand assay mean folding times obtained for circular sequence permutation-1 versus permutation-0 objects. The solid line is merely a guide to the eye. (E) Time-resolved gel electrophoresis of folding reactions where the 20 5′ terminal staple strand segments with the highest (top) and lowest (middle) binding energy have been truncated. Lower gel shows a folding reaction missing the 11 longest (21 bases) staple strand segments. (F) Gel electrophoresis of permutation-0 (top) and permutation-1 (bottom) after 6 hours of incubation at 50°C. Folding reactions have been prepared without the 10 (20) fastest (F1 and F2) and slowest (S1 and S2) staple strands, respectively, and with 10 randomly omitted staple strands.

  • Fig. 5 DNA origami strand routing and folding pathway.

    (A) Layer-by-layer 3D models of the 42-helix bundle, colored according to the experimentally measured mean folding times of the staple strand segments. (B) Arcs denote staple strand crossover bridging the terminal staple strand segments to the next-to-terminal segment. Discs indicate the terminal position. Numbers give scaffold strand base indices. Color code indicates the mean folding time of the terminal staple strand segment resolved from the interstrand assay. (C) Estimation of the evolution of the average interbase distance on the scaffold over time. Each circle indicates a new compaction event produced by binding of a new staple segment at the mean folding time obtained from the interstrand assay.

  • Table 1 Temperature settings for the folding reactions.

    Each folding reaction consisted of three temperature plateaus. The first step serves instrument control; the second step serves strand annealing, while the folding takes place during the 50°C incubation.

    Temperature (°C)Time (min)
    252.25
    65120
    50840

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaaw1412/DC1

    Fig. S1. Design diagram of the brick-like test object variant 0, prepared using cadnano.

    Fig. S2. Schematic representation of the interstrand proximity assay with measured pairs.

    Fig. S3. Data obtained from the intrastrand proximity assay and double-exponential fits.

    Fig. S4. Calculation of mean folding time and binding probabilities.

    Fig. S5. Data obtained from the interstrand proximity assay and double-exponential fits.

    Fig. S6. Binding probabilities derived from the interstrand proximity assay and double-exponential fits.

    Fig. S7. Laser-scanned images of 2% agarose gels placed in a water bath.

    Fig. S8. Design diagram of the brick-like test object variant 1, prepared using cadnano.

    Fig. S9. Laser-scanned image of 2% agarose gel placed in a water bath.

    Fig. S10. Ring graph representation of the scaffold strand and distance calculation.

    Table S1. Averaged raw data of the intrastrand proximity assay.

    Table S2. Averaged raw data of the interstrand proximity assay.

    Table S3. Reaction progress data of the intrastrand proximity assay.

    Table S4. Reaction progress data of the interstrand proximity assay.

    Table S5. Staple strand sequences of the used DNA origami test objects.

    References (32, 33)

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Design diagram of the brick-like test object variant 0, prepared using cadnano.
    • Fig. S2. Schematic representation of the interstrand proximity assay with measured pairs.
    • Fig. S3. Data obtained from the intrastrand proximity assay and double-exponential fits.
    • Fig. S4. Calculation of mean folding time and binding probabilities.
    • Fig. S5. Data obtained from the interstrand proximity assay and double-exponential fits.
    • Fig. S6. Binding probabilities derived from the interstrand proximity assay and double-exponential fits.
    • Fig. S7. Laser-scanned images of 2% agarose gels placed in a water bath.
    • Fig. S8. Design diagram of the brick-like test object variant 1, prepared using cadnano.
    • Fig. S9. Laser-scanned image of 2% agarose gel placed in a water bath.
    • Fig. S10. Ring graph representation of the scaffold strand and distance calculation.
    • References (32, 33)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (.txt format). Averaged raw data of the intrastrand proximity assay.
    • Table S2 (.txt format). Averaged raw data of the interstrand proximity assay.
    • Table S3 (.txt format). Reaction progress data of the intrastrand proximity assay.
    • Table S4 (.txt format). Reaction progress data of the interstrand proximity assay.
    • Table S5 (Microsoft Excel format). Staple strand sequences of the used DNA origami test objects.

    Files in this Data Supplement:

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