To probe the limits of pso-TFO-driven stabilization, we repeated the panel of thermal challenge experiments at a higher temperature of 60°C, using the same three types of irradiated origami. This time, AGE analysis showed that the TFO-free origami unfolded completely in 10-20 seconds, i.e., at an expectedly faster rate than at 50 °C (Fig 4B, top panel). In contrast, the TFO-crosslinked origami remained intact for at least 50 seconds, irrespective of whether the complexes had been saturated with TFO or had been prepared by one-pot assembly. Further incubation at 60 °C caused partial unfolding of the crosslinked origami which was detected as (i) a ~3-fold and 8-fold reduction in fluorescence in the Cy5-labeled origami at 250 and 1250 seconds, respectively, and (ii) a progressive downward shift in the Cy5-labeled origami at 250 and 1250 seconds (Fig 4B, middle and bottom panels). Despite partial unfolding, the migration of both types of crosslinked origami never reached the level of the Cy5-free ssDNA scaffold, indicating that they might have retained some structural elements despite prolonged incubation at 60°C. In addition, a Cy5-labeled species appeared below the origami band which migrated between the 0.5 and 1 kb dsDNA markers. This species is likely to be similar in nature to the "super-staples" observed in the 2HP and 3HP origami crosslinking experiments (Fig ). Overall, these temperature challenge experiments indicate that pso-TFO mediated crosslinking leads to a dramatic stabilization of the HP-origami and is not affected by the method of their assembly. These experiments also confirmed that irradiated origami undergo incomplete crosslinking, as the fully crosslinked super-staple (comprised of 258 hairpins) would be expected to migrate at the rate of the ssDNA scaffold (migrates at ~1.7 kb), or a heavier species.