A single-fold triplex origami structure self-assembled from a 651 bp dsDNA scaffold and four TFO staples. The tsDNA appears to be wider than dsDNA and the two are therefore discernible by atomic force microscopy, which evidences that all four TFO staples bind efficiently (Fig. ). We progressively decreased the length of the 180° looped dsDNA region from 84 nm (255 bp) to 2 nm (6 bp) and retained a near-perfect folding yield of >90% (Fig. , Supplementary Figs. ). This was rather surprising, considering the 40-50 nm persistence length of dsDNA, and evidences the ability of TFO staples to fold duplex DNA despite its inherent rigidity. It is likely that the doublehelical base stacking geometry breaks in small loops, as evidenced by their end-to-end aggregation at high cation concentrations (Supplementary Figs. 16-17). Moreover, because of the inherent rigidity of dsDNA, the structural integrity of the assembled structure does not require the scaffold to be fully saturated by staple strands. Regions in which the scaffold is left bare can therefore be inserted at will as dsDNA spacers between triplex target domains, resulting in "patterned" triplex origami structures that alternate between double-and triple-stranded regions. It must, however, be acknowledged that the bending of a stiff scaffold demands an energy penalty that is paid by staple binding; too few or too weakly binding staples leave that penalty unpaid (Supplementary Fig. ). Still, using only 9-18 staples, a 2434 bp dsDNA scaffold assembled into five unique simple shapes, all with near-perfect yields (Fig. , Supplementary Figs. ).