Experiments were undertaken using a TFO concentration of 100 μM, i.e., ~7-fold excess of We then repeated the folding at pH 8.0 (i.e., under conditions that favour origami annealing) and characterised complex formation by AGE analysis at pH 4. We then asked if one-pot assembly can be used to prepare origami targeted with TFOs modified with bigger functional groups. To test this, we used pso-TFOs modified at their 3′ ends with poly (ethylene glycol) moieties of molecular weights 5,000 and 20,000 (pso-TFO-PEG5K and pso-TFO-PEG20K, respectively). Such PEG modifications might protect origami from non-specific protein binding. As before, we annealed the complexes at pH 8.0 in the presence of an excess of pso-TFO-PEG5K/20K and assayed the formation of the origami-TFO complex by AGE at pH 4.8. Analysis of the gels revealed that the attachment of multiple PEG groups to the nanostructures severely reduced the mobility of the origami complexes