Supporting Information: In situ structure and dynamics of DNA origami determined through molecular dynamics simulations

Design of the DNA origami structures. Using caDNAno, we designed the following three DNA origami structures: a straight 6by-3 honeycomb-pleated structure (HC), a straight 4-by-4 squarepleated structure (SQ), and a 6-by-3 honeycomb-pleated structure having a 90◦ programmable bend (HC-90◦). Below, we briefly describe our protocols for obtaining the caDNAno designs. Building a DNA origami structure in caDNAno begins with sketching the overall shape of the target structure using cylinders. Fig. S1A illustrates this step for the HC structure. The cylinders (numbered by index m) depict the volumes where fragments of duplex DNA will be placed; the blue and red tubes represent the backbone traces of the DNA strands. In the target structure, neighboring cylinders (m = i, i + 1) are connected to each other, forming a single sheet that includes all cylinders. The particular three-dimensional (3D) fold of the sheet is obtained by introducing additional connections between the cylinders, for example, between m = 2 and m = 11 in Fig. S1A. In the final structure, all cylinders are arranged parallel to one another and form either a honeycomb or square lattice pattern within the plane normal to the axes of the cylinders. For convenience, we define the z-axis to be parallel to the axes of the cylinders. Next, we determine the length, sequence and spatial arrangement of the DNA strands that can realize the cylinder model. At this stage of the process, it is convenient to represent the DNA origami object as a two-dimensional (2D) sheet. Fig. S1B, Fig. S2A, and Fig. S2B show 2D schematics of our HC, SQ and HC-90◦ objects, respectively. A long scaffold strand is manually designed to span the entire 2D sheet, crossing over between neighboring cylinders [1]. The caDNAno program generates a collection of short (usually 18 to 49 nucleotide-long) staple strands that, together with the scaffold strand, form duplex DNA. The nucleotide sequence of the staples uniquely will define their positions along the scaffold. In a 3D DNA origami structure, a staple strand can form a crossover between either two neighboring or two distant (within the 2D sheet) DNA helixes. Because duplex DNA has a helical pitch, staple crossovers can be realized only at specific locations in a periodic 3D origami structure. The caDNAno software places the staple crossovers at designated crossover planes. For the HC object, the crossover planes are depicted as semi-transparent rectangles in Fig. S1A and as gray columns in Fig. S1B. The location of the crossover planes depends on the type of the DNA lattice. In a honeycomb lattice, each cylinder has three nearest neighbors and the DNA helix rotates by 240◦between two consecutive crossover planes (by 270◦in a square lattice where each cylinder has four nearest neighbors). A 240◦or 270◦rotation of a B-form DNA helix is equivalent to an axial displacement by 7 or 8 base pairs, respectively. Thus, the crossover planes occur periodically and the structures repeat themselves every 3 or 4 crossover planes (21 or 32 base pairs) in the honeycomb or square lattice, respectively. Two neighboring crossover planes define an array cell. Each array cell contains 7 or 8 base pairs per cylinder in the honeycomb or square lattice, respectively [2], Fig. S1B. The scaffold crossovers can occur outside the staple crossover planes. For a large 3D DNA origami object, the majority of connections between the cylinders is realized by staple crossovers. The distributions of scaffold and staple crossovers in our designs of DNA origami objects are shown at the bottom of Fig. S1B, and Fig. S2A,B. In the final step, the scaffold strand is assigned a nucleotide sequence of a fragment of the M13 viral genome. The nucleotide sequence of the staple strands is chosen to complement the sequence of the scaffold strand. The sequence assignment is automated by the caDNAno program.