Representative TEM images of purified 258HP (top image) and 0HP (bottom image) origami triangles; (B) AGE analysis of the raw folding mix of 258HP origami loaded at different concentrations of TFOs. Origami were first prepared by annealing 50 nM scaffold with 150 nM staples. The TFOs were then supplied at the stated concentrations and the reaction incubated at pH 4.8 for 1 hr at room temperature. Bands corresponding to the origami and the free staples were visualized by scanning for Cy5-labeled staple 1 fluorescence. A DNA ladder was visualized by scanning for ethidium bromide fluorescence and positions of the 1, 3 and 10 kb bands are as indicated. Lanes 2-4 contained samples incubated with the unmodified TFO, whilst lanes 6-8 contained the dual-labelled pso-TFO-C6-NH2. "O" -origami monomer; "A"origami aggregate/oligomer (expected to form during origami foldingd 43 ); "O-TFO" -origami loaded with TFO, "S" -Cy5-labeled staple 1, "S-TFO" -staple 1 loaded with TFO; (C) Functionalization and crosslinking of 258HP origamis: effects of pso-TFO-PEG5K and 365 nm irradiation on the mobility of 258HP in AGE at different pH. One-pot assembly conditions were used to prepare 258HP loaded with pso-TFO-PEG5K. The samples were then irradiated with 365 nm light and analysed by AGE at pH 4.8 (left gel, annotated in red) and pH 8.0 (right gel, annotated in blue). Bands corresponding to the origami and the free staples were visualized by scanning for Cy5-labeled staple 1 fluorescence. Positions of the wells in the gel and the 1-, 3-, and 10-kb DNA ladder bands are as indicated. Labels of nucleic acid species are as in B; (D) Representative TEM images of one-pot crosslinked and purified 258HP origami loaded with pso-TFO-PEG20K; (E) Site-specific targeting of 27HP origamis with pso-TFO-PEG4biotin. Left: scaffold routing for 27HP origami which contains 27 TFO-binding hairpins in three clusters. TFO-bound streptavidin molecules are shown as orange circles. Right: One-pot assembly conditions were used to prepare 27HP origami loaded and crosslinked with pso-TFO-PEG4-biotin. The complexes were then incubated with 100 μM streptavidin for 30 min, dried and imaged by AFM.