Experiments were undertaken on 1, 2, 3HP origami containing 1, 2, or 3 TFO-binding hairpins, respectively. HP1 was assembled using 3′-hairpin-modified staples 0 and 1, HP2 -using 3′-hairpin-modified staples 0, 1 and 2, and HP3 -using 3′-hairpin-modified staples 0, 1, 2, and 3. As a control, a 1HP minimal junction (top) was assembled from staples 0 and 1 but used a minimal "pseudoscaffold" oligo in place of origami scaffold. Each of these complexes contained staple 0 (orange) which lacked the 3′-hairpin extension. They also contained Cy5-labelled staple 1 (red) which allowed the products of the crosslinking reaction to be monitored; (B-C) Experiments undertaken on 1HP-minimal junction and 1HP origami that were first folded and then saturated with either the pso-TFO or its PEG5K conjugate. Samples were subjected to irradiation at 365 nm for the time points shown, and the products of the reaction were denatured and then separated on an 8% denaturing PAGE gel. Bands were visualized by scanning for Cy5-labeled staple 1 fluorescence. (D) Experiments undertaken on 1HP, 2HP and 3HP origami that were prepared by one-pot annealing in the presence of either pso-TFO or its PEG5K conjugate. They were subjected to irradiation at 365 nm for the time points shown, and the products of the reaction were denatured and then separated on an 8% denaturing PAGE gel. Lane M contains Cy5-labeled amplified PCR products of indicated lengths, denatured and separated on the same gel.