We first determined the effect of a 50 °C temperature challenge on 258HP origami that had been irradiated for 10 seconds in the presence and absence of a pso-TFO carrying a PEG4-NH2 group (pso-TFO-PEG4-NH2). AGE analysis of the TFO-free origami revealed that the nanostructure underwent rapid unfolding between the 20-and 30-sec timepoints, with no intermediates detectable within the time resolution of this assay (Fig 4A, top panel). This agrees with previously reported studies that show that the melting of staples within origami occurs cooperatively. TEM imaging also confirmed that after a 50-second incubation there were no intact origami remaining in the sample (Fig ). We next investigated if saturating and crosslinking the purified 258HP origami in the presence of a ~70-fold excess of pso-TFO over hairpins led to an enhancement in the stability of the complexes. Strikingly, we found that irradiated pso-TFO-loaded 258HP remained intact for at least 6250 seconds (~1.7 hours), consistent with at least a 250-fold increase in the lifetime (Fig 4A, middle panel). Finally, we asked if complexes prepared and crosslinked using our one-pot assembly method exhibited the same level of stabilisation. AGE analysis revealed that the structures appeared as stable as the TFO-saturated origami, and no unfolding products were again detectable for at least and) Experiments were undertaken on origami folded in the absence (top panel) or presence of pso-TFO-PEG4-NH2 under saturating (middle panel) or one-pot annealing (bottom panel) conditions. Origami containing pso-TFOs were subjected to irradiation at 365 nm for 10 seconds. Samples of 2 nM folded origami were then subjected to heat challenge at either 50 °C (A) or 60 °C (B) for the timepoints indicated. Samples were run on a 1% agarose gel in a pH 4.8 running buffer at room temperature. Bands for the origami and "super-staples" were visualised by scanning for Cy5-reporter sstaple fluorescence, whilst the scaffold strand, free TFO and dsDNA markers were visualised by scanning for ethidium bromide fluorescence; (C) Representative TEM images of 258HP origami prepared in the absence of TFO, irradiated and subjected to heat challenge for 0 and 50 seconds; (D) Representative TEM images of 258HP origami prepared in the presence of TFO, irradiated and subjected to heat challenge for 0 and 1250 seconds.