The removal of TFO placement restrictions makes polyR scaffolds excellent tools for testing triplex origami design variables, such as the effects of crossover density and the minimum required length of recognition domains. We observed that in some cases, it is possible to use domains of only 9 bt, which are too short to form stable triplexes in non-scaffolded systems (Supplementary Fig. ). In order to study the importance of looped scaffold regions in structures of higher spatial complexity, the polyR-854bp scaffold was folded into various rectangles with thirteen staples each and varying dsDNA loop lengths (Fig. ). These tiny rectangles use a similar staple routing as depicted in Fig. , but skip the dsDNA spacers to achieve a higher overall crossover density. PolyR-854bp rectangles featuring dsDNA loops of 2 bp, 6 bp and 11 bp respectively folded with yields of 15%, 36% and 47%, thus revealing a clear preference for longer loops (Supplementary Figs. 47-49). Additionally, the larger polyR-3135bp scaffold was also folded into a densely triplexed rectangle with 11 bp loops (Fig. ). However, the yield of this polyR-3135bp rectangle was poor (<10%) because of splitting malformations. The polyR-3135bp scaffold assembled with nearperfect (>90%) yield into a densely triplexed five-fold bundle. AFM and TEM analysis revealed that these bundles twist extensively, in spite of the overall successful compaction of the scaffold as evidenced by electrophoretic migration (Supplementary Fig. ).