We found that, in the absence of NTPs, the crosslinked complexes appeared intact after the RNAP challenge, indicating that, under NTP-free conditions, TFO loading and/or crosslinking reduces the affinity of RNAP for the origami (compare lanes 1 and 2). Most importantly, in the presence of NTPs, the crosslinked complexes appeared resistant to RNAP disassembly (lane 4). Interestingly, the RNA smear was still detectable after the transcription challenge, indicating that RNAP could still transcribe the crosslinked origami, but presumably could not displace the "super-staples" from the scaffold (lane 3). We thus investigated whether we could eliminate this residual transcription activity by attaching the bulky PEG20K to the 5′ end of the TFO (Fig 5A, bottom panel). We found that the crosslinked 258HP-TFO-PEG20K complexes were indistinguishable from the PEG-free 258HP-TFO complexes, i.e., they remained intact after transcription and still supported RNAP transcriptional activity. This finding was confirmed by TEM imaging which showed mostly intact 258HP-pso-TFO-PEG20K complexes after transcription, with minor damage in the triangle centres where the 3 central staples lacked hairpins due to design constraints (see Supplementary Table ) and at the sector joints (perhaps due to the low density of TFO-binding hairpins at these positions) (Fig ). We conclude that internal crosslinking by pso-TFOs and their conjugates protects HP origami from destruction by transcribing RNAP.