|Line 66:||Line 66:|
DNA origami is needed to make the DNA tablet because the main body of the tablet is based on DNA origami. However, the best buffer conditions for a DNA origami and the bistable system are not same, so we searched the optimal working conditions to combine them. We also tested if we can observe DNA origami in mixed buffers because the bistable system contains three kinds of enzymes and some proteins that may damage the origami. The purpose of the experiment is to find conditions compatible with both the origami and the bistable system and to make sure that the enzymes of the bistable system do not destroy the DNA origami. We also wanted to check that we can observe by AFM the origami while in a mix that contains proteins. The DNA origami we used consists of m13 and a set of 216 staples, which was designed by Shelley F. J. Wickham et al. The merit of this origami is that it is as flat as possible, and it is thus easier to observe its surface by AFM.
Compatibility with the Bistable System
In order to combine DNA origami with the bistable system, we needed to overcome the difference in their best conditions.
- The bistable system uses some enzymes (a polymerase -Bst large Fragment-, an exonuclease -ttRecJ- and a nicking enzyme -NBI) which may destroy DNA origami. The polymerase could extend the 3'end of the staples strands along the scaffold, starting form the nicks; the exonuclease could slowly degrade the staple strands.
- The bistable works at higher temperature, so this may also impact the stability of the structure.
- The bistable system also uses BSA. It can prevent origami to stick to mica, and we may not get good images by AFM.
In experiments below, we tried to find out the compromise of conditions between DNA origami and the bistable system which enables both the bistable system and DNA origami to work properly.
The direct link to the result of the experiments about compability with the bistable system is here.
We made a solution of the DNA origami. We put M13 and the 216 kinds of staple strands in tubes and made them anneal for about an hour in a PCR machine. We made three tubes of origami solution whose difference is the concentration of staple strands.
We observed DNA origami which we made on September 12th by AFM. We used the AFM at Suyama Lab in Komaba I Campus. We could not observe the middle-concentrated solution of origami.
- AFM Image of Low-Concentrated Solution of Origami
- AFM Image of High-Concentrated Solution of Origami
As you can see from the picture above, in both solutions, no origami was well structured. Therefore, we decided to do the same experiment again without keeping them in the freezer for a day.
We made the DNA origami solution again. We steeply lowered the concentration of staple strands. Also, in the experiment, the origami was not kept in the freezer but observed by AFM just after it was annealed .
As you can see the picture above, the origami was well structured.