AFM is a useful tool, not only to be able to see the walker, but also to image origami and make sure it is well-formed when certain parameters are changed. It is arguably the best way to debug any origami-related problems the system may have, since you can see the origami, and potentially see problems it may have. Thus, we began by imaging origami (second image) with probes laid out as shown in the random walking playground. In a few of the rectangles, the probes can be seen as a faint line across the diagonal, but a more obvious feature is many distorted and/or destroyed origami. The third image shows origami that has tracks in addition to probes, where the tracks can be seen as a clear diagonal line on the origami, with much fewer distorted/destroyed origami. To image the walker, we ordered walkers with biotin attached at the 3’ end (the top of the walker). A streptavidin molecule can bind to up to four biotin molecules, and since streptavidin is fairly large, it can be seen as a bright spot under AFM, which will contrast it to the darker origami. Unfortunately, when we added the biotinylated walker start complex to the origami, nothing could be seen. Various hypothesis were formed and tested as to why the walker could not be seen.
One hypothesis is that the strepta- vidin 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 (orange dot in the random walking playground). 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 the fifth image 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 (see fifth image). 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.