Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM Experiments

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Tuesday, November 21, 2017








AFM Experiments

Figure 1. The Random Walking Playground used for AFM experiments. The dark blue dots represent track 1 strands. The light blue dots represent track 2 strands. The light blue dots in the upper left corner are hairpin markers used to tell the orientation of origami in AFM. The green dot is a biotinylated control staple that was used in experiments after the walker could not be observed to see what streptavidin looks like when it is on origami.

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 (Figure 4) 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. Figure 5 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) as drawn in Figure 2a. 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 (Figure 6). Various hypotheses were formed and tested as to why the walker could not be seen.

Figure 2. (a) 3'-end-biotinylated walker on track; (b) 5'-end-biotinylated walker on track.

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

Figure 3. We started by trying to form a regular rectangle to make sure our protocols worked as expected.

Origami with Probes

Figure 4. We reformed the regular rectangle using our modified staple strands (at the locations we would put the tracks in Figure 1) to show that the probes don't interfere with the binding in the rectangle.

Origami with Tracks

Figure 5. We showed that our probes bind in the proper location by adding track strands and noting where they bind on the origami.

Origami with Unobservable Walker

Figure 6. We attempted to insert our walker at the beginning of the track for the first time, but we can't see it on the AFM images. This could be due to any one of a number of issues.

Origami with Streptavidin Control

Figure 7. We first see what our walker should look like by adding another control to our origami. The new bright dot is the protein streptavidin.

Origami with Observable Annealed Walkers

Figure 8. After some work, we can now see walkers on the surface of our origami.

Origami with Observable Walkers on Tracks