One hypothesis is that the streptavidin stock was bad or for some reason was not binding properly to DNA. To prove this was not the case, we ordered a biotinylated staple (green dot in Figure 1). The streptavidin was seen as a very bright spot on most origami. We decided to use the staple in all future AFM experiments as a control. A second hypothesis was that the insertion mechanism was not working, and that the walker was ﬂoating in solution. This seems unlikely, because free ﬂoating wakers could diﬀuse to the goal when triggered, but the ﬂuorescence experiments showed that in the absence of tracks, walkers did not reach the goal. In any case, this hypothesis was tested by inserting another biotinylated staple at SP10. The streptavidin can be seen but less frequently than the biotinylated control staple. This low frequency could simply be due to stoichiometric diﬀerences, since in Figure 7 we see that the control staple appears on almost all origami, whereas before it was absent in many. A third hypothesis was that the streptavidin is too high from the surface on DNA with multiple nicks that could easily “dodge” the AFM tip. A proposed solution for this was to order walkers biotinylated at the 3’ end instead, so it would only be 20 base pairs away from the surface. When this was attempted, the walker was still not seen. One possibility was that the streptavidin was there but since it was still far from the surface, its brightness would blend in with that of tracks. Thus, origami was formed without any probes except for SP10. Since this was the only probe, we could now anneal the walker (biotinylated at the 3’ end) onto the origami, rather than inserting it. Surprisingly, several bright spots were seen where the walker should be (Figure 8). The origami that may have visible streptavidin are circled in blue. Figure 8b shows the same area imaged after Figure 8a, and we see that two of the origami with potential visible walkers still had the same spot. This could suggest that the problem is not that the walker “dodges” the tip, but rather that for some other reason most origami do not have a walker start complex with streptavidin. However, since one of the circled origami (the top right one) does not seem to have the bright spot again, that is not necessarily the case. It could be that on the origami with visible walkers, the walkers somehow got stuck in some position, so the streptavidin can no longer move. The red circled origami also contains a bright spot on the side opposite the control staple and marker that appears in both images, but this spot is neither in the right position nor anywhere on the track. This could also be an instance of a walker that got stuck in some position on the origami (perhaps after being dismantled from its original position). To make matters more confusing, to the right of the bottommost blue circle are two origami that appear to have three bright spots all on the same side. In other images, more potential visible walkers were seen, as well as more unexplainable spots. The most likely hypothesis is still that the streptavidin moves too much to be caught by the tip. To counter this, we are currently trying to lock down the walker at a given position using the surrounding tracks, and the fact that multiple biotin molecules can be bound to one streptavidin.
Regular Rectangular Origami
Origami with Probes
Origami with Tracks
Origami with Unobservable Walker
Origami with Streptavidin Control
Origami with Observable Annealed Walkers
Origami with Observable Walkers on Tracks