We then asked if TFO-directed crosslinking improves the resistance of functionalised origami to DNA-processing enzymes. We chose phage T7 RNA polymerase (T7 RNAP) as a suitable test since it has been shown to induce disassembly of DNA nanostructures by promoterindependent transcription. We reasoned that formation of the "super-staples" might provide "self-healing" properties to the origami and protect the structures from RNAP-dependent strand displacement. To test this, we first incubated purified TFO-free 258HP origami with a 2.5 uM T7 RNAP (500-fold molar excess), conditions that mimicked the estimated in vivo concentrations of commonly studied RNAPs. The origami was incubated with RNAP at 37 °C for 30 minutes and then treated with proteinase K before analysing the samples by AGE (Fig 5A, top panel). Analysis showed that, in the absence of nucleotide triphosphates (NTPs), most of the origami became distorted, but not disassembled by RNAP, as evidenced by the appearance of a step/smear pattern from the origami band to the well in the gel and by the absence of the released Cy5-labelled reporter staple (compare lanes 2 and 3). In contrast, incubation with RNAP in the presence of 0.5 mM NTPs resulted in complete destruction of the TFO-free origami, as seen from the full transition of the Cy5 signal from the origami band into the released Cy5-staple (lane 4). In addition, a strong smear detectable by ethidium bromide, migrating between the Cy5-labelled staple and the 0.5 kb dsDNA marker, appeared after incubation. Further incubation with RNase removed the smear, indicating that it represented the RNA non-specifically transcribed by RNAP from the nanostructures (lane 5). We conclude that TFO-free 258HP origami are bound by RNAP in the absence of NTPs and are disassembled under conditions permitting transcription. We next investigated if TFO-crosslinked origami became resistant to RNAP transcription (Fig