Further increase in crosslinking efficiency might also be possible using TFOs containing multiple psoralen molecules (e.g. "triplex staples" 53 ) or using TFOs that contain stabilizing nucleotide analogues. We also demonstrated that, through the appropriate positioning of hairpins, such TFOfunctionalised, crosslinked structures become less susceptible to thermal denaturation and completely resistant to disassembly by concentrated phage T7 RNAP. Our RNAP sensitivity assay was inspired by the motivation to employ DNA origami as platforms for in vitro and in vivo single-molecule analyses of transcription initiation. We anticipated that un-crosslinked origami would be highly vulnerable to RNAPs, since origami, by definition, have numerous nicks and often have ssDNA modules, whereas RNAPs are known to non-specifically initiate from nicks and ssDNA. Sensitivity of nanostructures to viral RNAPs has been recently documented. We showed that TFO-driven crosslinking protected origamis from destruction by the transcribing T7 RNAP, consistent with the notion that the super-staples formed by crosslinking could not be irreversibly displaced by the transcribing enzyme. The finding that the crosslinked origami were still transcribed by RNAP showed that the nanostructure remained accessible to RNAP despite the presence of TFO, or the functionalization of the TFO 3′ end with the bulky PEG20K group. Optimization of the hairpin placement and/or the TFO functionalities will be needed to fully control non-specific origami binding by RNAP and other DNA-processing enzymes. Overall, the RNAP sensitivity assay offers more nuanced metrics of origami functionality in biological media compared to nuclease sensitivity assays, since it provides three separate read-outs -non-specific RNAP binding/distortion of origami, staple displacement, and RNA production.