Targetable hairpins are introduced into origami by the attachment of nucleotide sequences to the 5′ and 3′ ends of any two adjacent staples (i.e., staple N and N+1). The extension of staple N+1 (purple) encodes for a stem-loop hairpin and contains the TFO binding sequence (shown in yellow) and a 4-nt linker that contains the TpA sequence destined for psoralen crosslinking (in bold). Staple N (red) encodes the 4-nt compliment to the linker so that, upon origami assembly, a crosslinkable duplex between adjacent hairpins is formed. Targeting by a pso-TFO leads to psoralen intercalation across the TpA step (shown as a black bar). Irradiation with UVA (365 nm) light triggers mono-and bis-adduct formation between psoralen and the thymidine bases in the TpA step (shown by the boxes), covalently attaching the TFO to the origami. Bis-adduct formation between staples in proximal hairpins further tethers the TFO and leads to formation of "super-staples" that enhance the structural integrity of the origami. Functionalization is achieved using pso-TFOs that carry additional moieties (X) at their 3′ end; (B) Design of the prototypical DNA origami triangle used in this study. Scaffold routing for 0HP and 258HP origami are shown in blue and the 258 hairpins in 258HP are shown as black semi-circles. Routing of the staples ensured that staple-staple junctions and TFO-binding hairpins alternated between opposite faces of the triangle to allow functionalization of origami on both sides. The folding, crosslinking and degradation of these complexes was monitored using a Cy5-labelled reporter staple 1 (shown in red). Adjacent to staple 1 are reporter staples 0, 2, and 3 (orange, purple, and green, respectively) which are used to illustrate how TFO-binding hairpins are formed by adjacent staples.