80-104, respectively. The assembly conditions and folding yields are summarized in Supplementary Table . In general, a 10-fold excess of TFO staples was mixed with the linearized scaffold in acetate buffer (pH 5) and annealed from 80 °C to 50 °C overnight. Even though tsDNA packs a high density of negative phosphate charges, triplex origami structures do not require divalent cation concentrations higher than what is common for DNA origami (15-20 mM Mg 2+ ). The assembled structures were analysed by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The length and width of the structures are defined as the distance along and perpendicular to the helical axes, respectively. Rough yield estimates are calculated as the percentage of well-folded structures observed on full-size microscopy images (Supplementary Figs. 15-79). Structures whose features are partially obscured (e.g. by other piled-on structures or AFM imaging artefacts) are omitted from the calculations. A structure is qualitatively considered well-formed if the scaffold has been compacted successfully and approximates the designed dimensions without significant structural defects.