In a fully occupied honeycomb lattice, each staple helix has three nearest neighbors (e.g. helices 1, 3, 7, 8, 9, 10, 14, 16 in ). Our default rules allow antiparallel crossovers between adjacent staple helices only where the strand backbones arrive at points of closest proximity, which repeat every 21 base pairs if the helical twist is fixed at 10.5 base pairs per turn. Thus for a given staple helix, potential staple-crossover positions occur every seven base pairs, or two-thirds of a turn. Our default rules allow antiparallel crossovers between adjacent scaffold helices to occur five base pairs, or half a turn, upstream or downstream of allowed crossover positions for the associated staple helices. However, caDNAno permits the user to force crossovers between any two staple bases or between any two scaffold bases. Users should take care when forcing crossovers, as departure from the default rules may lead to folding failure if too much deviation from canonical DNA geometry is implied.
Figure 1. caDNAno Interface and design pipeline. (a) Screenshot of caDNAno interface. Left, Slice panel displays a cross-sectional view of the honeycomb lattice where helices can be added to the design. Middle, Path panel provides an interface to edit an unrolled (more ...)
The design process has four main steps. First, a target shape is approximated by selecting a raster-style scaffold path that passes between neighboring helices along antiparallel crossovers at allowed positions. Second, staple paths complementary to scaffold are assigned. By default, all permitted staple crossovers are included, except for those that would be five base pairs away from a scaffold crossover between the same two helices. Third, the staple paths are broken into shorter segments 18 to 49 bases long, usually with a mean length of 30 to 35 bases. Finally, the scaffold path is populated with the DNA sequence of the desired template (e.g. 7–8 kb M13-genome-based vector), and the complementary staple sequences are determined.
This design pipeline is integrated from start to finish in the caDNAno three-panel interface (a). The z
-axis is defined as parallel to the helical axes. The Slice panel (orange border) provides an x
cross-section view of the honeycomb helix lattice for any z
-depth, with helices represented as circles. When the user clicks on an empty circle, that helix position is made available for routing of scaffold and staple strands by adding a schematic side view of the same helix to the Path panel (blue border). The Path panel is used for nucleotide-level editing of scaffold- and staple-path connectivity, assigning DNA sequences to scaffold paths, and reading out of staple DNA sequences. The Render panel (grey border) provides a real-time, 3D cylinder model for visualizing the shape as it is constructed. In each panel, pan and zoom tool buttons allow the user to view or edit the shape at different positions and magnifications. The Slice and Path panels have specialized tools for making additions, edits, rearrangements or deletions to a design (detailed descriptions of the tool buttons are found in the Supplementary Note 1
). Completion of the design pipeline results in a list of staple DNA sequences corresponding to the schematics shown in each panel; the result also can be represented as a detailed SVG schematic (b).
The process of approximating a 3D shape with a scaffold path begins with selection of helices in the Slice panel to approximate a 2D projection of that shape. When a helix is added to the design in the Slice panel, the same helix also is made active in the Path panel and is populated with a three-base-long scaffold path by default. Thus, once the desired helices are added to the design via the Slice panel (a, orange panel), several short, disconnected scaffold paths are visible in the Path panel (c). The Path-panel editing tools are used to extend the scaffold paths in the z-direction and to connect neighboring helices with Holliday-junction crossovers. The goal is to complete a continuous raster-style traversal of the target shape using a scaffold path (d).
Once the scaffold path is complete, complementary staple paths are assigned by clicking the ‘Auto-staple’ tool button beneath the Path panel. Staple paths are created wherever scaffold is present, according to an algorithm that follows the aforementioned rules for crossover spacing (e). Staple paths that fall outside the preferred length range (18–49 bases) are highlighted, and the user is responsible for using the editing tools to break the staple paths into shorter segments. After all staples are edited into a satisfactory arrangement, the scaffold path is populated with a DNA sequence using the ‘Add Sequence’ tool. Several default sequences are provided, or the user can input his or her own. Additionally, a 3D model can be exported in X3D format, with double helices represented as cylinders of 2 nm diameter and 0.34 nm per base-pair length (f).
We used caDNAno to design seven different honeycomb-pleated-origami rectangular blocks (a, top row), creating a simple scaffold-path trajectory that followed the same approximate path through each structure: as viewed down the helical axes, close-packing rows of helices were arrayed within the honeycomb framework in an x
-raster pattern (i.e. left to right, then down, then right to left, then down, etc.); the connectivity of neighboring scaffold helices is more apparent in partially folded cylinder models (b, top row). The x
-raster rows within the honeycomb framework are corrugated; they stagger up and down and encompass helices that are actually at two different y
-positions. Similarly, virtual y
-oriented layers can be defined that stagger left and right and encompass helices that are at two different x
-positions. The shapes were folded either from a 7560-base scaffold into 60 parallel helices or from an 8064-base scaffold into 64 parallel helices to create number-of-rows versus number-of-helices-per-x
-raster-row combinations of 15 × 4, 10 × 6 (analyzed independently in ref. 14), 8 × 8, 6 × 10, 4 × 16, 3 × 20, 2 × 30. Each helix was allotted 126 bases of scaffold. Of those 126 bases, 98 were paired with complementary staples, and the remaining 28 bases were divided into front and rear unpaired loop fragments at the ends of each helix (detailed schematics and staple lists are included in Supplementary Notes 2 and 3
Figure 2. Transmission electron microscopy (TEM) and agarose-gel analysis of DNA-origami blocks. The nomenclature of the designs is m × n, where m is the number of x-raster rows, and n is the number of helices per x-raster row. (i), 15 × 4 motif; (more ...)
Each of the shapes was folded in separate chambers by heat denaturation followed by cooling for renaturation, and analyzed by agarose-gel electrophoresis (c). The seven shapes varied significantly in leading band yield, mobility, and sharpness, as well as amount of undesired formation of higher-order aggregates. We estimated folding yields as integrated intensity of material that migrated as a leading band divided by total intensity of material in the lane up to and including the well (d). Material in each of the leading bands was isolated by physical extraction and analyzed by negative-stain transmission electron microscopy (a). For each shape, 100 randomly selected individual-particle images were collected, and folding yields were estimated (e). A particle was judged to be well-folded if its outline could be aligned with a semi-transparent projection model of the corresponding design and it exhibited no obvious defects such as missing, broken, disrupted, or smeared out sections more than 3 nm away from the unpaired scaffold loops at the front and rear interfaces. For example, of the five particle images shown for the 4 × 16 design in a(v), only the topmost particle was counted as well-folded.
Only folding with three of the seven designs—four-helix-per-x
-raster or 15 × 4 (two y
-raster or 10 × 6 (three y
-raster or 2 × 30 (two x
-layers)—produced sharp leading monomer bands by agarose-gel electrophoresis (c). Thus designs with a smaller number of x
-layers or y
-layers may have a folding advantage due to fewer numbers of highly embedded helices, which may be more difficult to assemble, and perhaps also due to the lower crossover densities. Consistent with this trend, single-layer shapes fold much faster and to higher yield (9
). Folding with the six-helix-per-x
-raster (10 × 6) design produced the leading band with the greatest mobility, while folding with the four-helix-per-x
-raster (15 × 4) design produced the leading band with the greatest intensity, indicating the best yield. Our previous results suggested that faster gel mobility of the same design under different folding conditions correlates with fewer defects (14
), although it is more difficult to interpret mobility differences across designs with inherently different shapes.
-raster (10 × 6) shape appeared the most robust of the seven designs in terms of yielding particles that are intact after folding, staining, and drying (e). We also have found that this six-helix-x
-raster design performs well when used to construct shapes with as few as three x
-raster rows (i.e. 18 helices total) and longer lengths of helices (data not shown). Interestingly, the 15 × 4 and 2 × 30 designs produced particles that appeared bent when adhering to the grid surface with a perpendicular orientation of the helical axes; it is possible that the positively charged stain is deforming these particles, but that the other designs produce particles that are sufficiently thick to resist such deformation. Thinner objects such as the 15 × 4 and 2 × 30 designs might be suitable for some applications if a staining artifact is the cause of the observed deformations. Further studies will be necessary for optimizing design parameters that might affect folding yield, such as staple-break-point distribution, scaffold routing, and scaffold- versus staple-crossover densities (19
The construction of complex, 3D DNA nanostructures will increase the range of applications that can be addressed, but also will add complexity to the design process. By restricting design space to the honeycomb-lattice framework, we reduce the number of choices that need to be made when implementing a 3D DNA-origami shape while retaining a significant amount of flexibility. Our caDNAno software package relieves the user from completing the tedious conversion of a creative design to oligonucleotide sequences. We have found that caDNAno performs favorably when compared to ad hoc methods for generating staple sequences for a new shape design, typically reducing the time required for monotonous sequence assignment from days or weeks down to a few hours.
In addition to supporting the design of basic shapes such as rectangular blocks, caDNAno provides tools to introduce deviations from the basic honeycomb architecture, such as forced crossovers, to create very complicated designs. Additional software development will be required to make designs of these non-standard motifs more natural, for example for caDNAno to predict the structural consequences of these changes. More work is also needed to see what design rules lead to stable structures; for examples of designs that folded successfully, although with varying yields, see the gallery section at http://cadnano.org/