Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM Experiments

Thursday, April 18, 2024 Home Members Project Protocols Progress Discussion References

AFM Experiments 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 2) 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 3 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 Images We started by trying to form a regular rectangle to make sure our protocols worked as expected. We reformed the regular rectangle using our modified staple strands to show that the probes don't interfere with the binding in the rectangle. We showed that our probes bind in the proper location by adding track strands and noting where they bind on the origami. 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. We first see what our walker should look like by adding another control to our origami. The new bright dot is the protein streptavidin. After some work, we can now see walkers on the surface of our origami.