We tested the conversion from nHS to rHS for structures folded from all three dsDNA scaffolds: the polyR-854bp, the polyR-3135bp, and a 4.9 kbp truncated fragment of the 60R21N-9kbp gene. These scaffolds were respectively assembled into a three-point star, six-point star, and rectangle with both nHS and rHS staple sets (Fig. ). The stars featured good to near-perfect folding yields for both binding motifs. The nHS rectangle also folded with a good yield (69%, n = 124), but the rHS rectangle did not assemble at all; presumably because the 15 bt target domains are too short to form stable rHS triplexes. A rectangle with an alternative staple routing utilizing 30 bt target domains did assemble, albeit with lower yield (27%, n = 62) than the nHS rectangle. In contrast, the rHS variants of the patterned and polyR five-fold hexagonal bundles in Fig. and Supplementary Fig. folded with good to high yields (patterned 84%, n = 59; polyR >90% on TEM and 72% on AFM, n = 67). Notably, a large majority of the rHS TFO staple recognition domains in the aforementioned high-yield structures contain too few guanines to form stable triplexes in isolation (Supplementary Fig. ). Reverse Hoogsteen triplex origami structures also fold orthogonally with conventional DNA origami structures in one reaction mixture (Fig. ).