Experiments were undertaken on origami folded in the absence (top panel) or presence of a pso-TFO (middle) or its PEG20K conjugate (bottom panel). These were prepared under onepot annealing conditions and the origami containing pso-TFOs were subjected to irradiation at 365 nm for 10 seconds. Samples of 2-5 nM folded origami were then incubated with 2.5 μM T7 RNAP in the presence or absence of 1 mM NTPs at 37 °C for 30 minutes and then subjected to proteinase K treatment for 15 minutes to degrade the protein. Additional samples that were subjected to RNase A treatment before proteinase K digestion were also prepared. Samples were run on a 1% agarose gel in a pH 4.8 running buffer at room temperature. Bands for the origami and the Cy5-labelled staple 1 were visualised by scanning for Cy5 fluorescence, whilst RNA transcripts and dsDNA markers were visualised by scanning for ethidium bromide fluorescence; (B) Representative TEM images of non-irradiated (left image) and irradiated (right image) 258HP-pso-TFO-PEG20K origami that had been subjected to RNAP challenge in the presence of NTPs.