In origami containing adjacent TFO-binding hairpins, a single staple can participate in the formation of two hairpins -one hairpin at the 5′ end (formed by the 4-nt staple extension) and another one at the 3′ end (formed by the 32-nt extension). We therefore asked if "superstaples" can form upon crosslinking of adjacent hairpins. Thus, we purified origami in which we added 1 (2HP) or 2 (3HP) additional hairpins adjacent to the hairpin formed by staple 0 and staple 1 (Fig and). This time experiments were performed on origami prepared using our one-pot assembly approach (Fig 3D). It was observed that, after irradiation for 1 and 9 seconds, both the 2HP and 3HP origami formed crosslinked species that migrated much slower than those observed for 1HP. This is consistent with formation of a three-staple adduct for 2HP, presumably mediated by two pso-TFOs, and a four-staple adduct for 3HP, presumably mediated by three pso-TFOs. Longer irradiation times did not significantly affect the pattern of crosslinking products, indicating that the formation of super-staples went to saturation (data not shown). We conclude that the targeting of proximal hairpins in origami leads to the formation of "super-staples" and that this site-specifically tethers the pso-TFO (and attached functionality) to precise locations on the origami. We also note that irradiation of the Cy5labeled DNA constructs for 3 seconds did not cause significant changes in Cy5 fluorescence in species detectable by PAGE, whilst irradiation for 9 seconds resulted in only a small ~20% decrease in Cy5 fluorescence (Fig ). This indicates that the power density of 365 nm irradiation used by our mini-LED is mostly benign to fluorophores attached to origami.