Thursday, September 1, 2016
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
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 13 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 3). The origami that may have visible streptavidin are circled in
blue. Figure 3b shows the same area imaged after Figure 3a, 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.
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.