Biomod/2012/UTokyo/UT-Komaba/Experiment/Original Origami
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Concept
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[1].
The merit of this origami is that it is as flat as possible, and it is thus easier to observe its surface by AFM.
Mission
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 polyerase -Bst large Fragment-, an exonuclease -ttRecJ- and a nicking enzyme -NBI) which may destroy DNA origami.
- 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.
Experiment
September 12th
We made a solution of the DNA origami. We put M13 and 216 kinds of staple strands in tubes and kept them in PCR for about an hour. We made three tubes of origami solution whose difference is the concentration of staple strands.
September 13th
We observed DNA origami which we made September 12th by AFM. We used 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 fridge for a day.
September 14th
We made the DNA origami solution again. We steeply lowered the concentration of staple strands. Also, in the experiment, the origami was not be kept in the fridge but observed by AFM just after it was annealed .
As you can see the picture above, the origami was well structured.
September 19th
- Normal DNA Origami
We prepared two types of DNA origami solution. The purpose of the experiment was to compare the different result from the different concentration of the solutions. The both concentrations of staple strands were as high as we did in September 12th.
- DNA Origami with Enzymes
We use enzymes in the bistable system, therefore, we make sure that the enzymes do not destroy the DNA origami. We put nickase, polymerase and exnuclease with DNA origami and left them in the PCR. We put DNA origami solution made in September 19th, enzymes and some essential chemicals
- DNA Origami with BSA
We are also worried if BSA interrupt the AFM and we can not get clear images of DNA origami. Therefore, we prepare the solution for September 20th. We put DNA origami solution made in September 19th, BSA and some essential chemicals.
September 20th
We observed three types of DNA origami made in September 19th. The three types is normal DNA origami, DNA origami with BSA and DNA origami with enzymes.
- Normal DNA Origami
We observed the origami by AFM, but the structures of both DNA origami were destroyed as you can see from the picture below.
- DNA Origami with BSA
We need BSA for the bistable sytem so that we had to make sure that we can see the DNA structure when there are BSA in the liquid of the origami. Before we observed the origami with BSA by AFM, we rinsed 5μL of the origami liquid with 40μL of buffer for 4 times. Therefore, as you can see from the picture below, BSA did not disturb the observation of AFM, we can get the clear picture of DNA structure.
- DNA Origami with Enzymes
In the bistable system, we use three kinds of enzymes so that we had to make sure that the enzymes do not destroy the origami. As the result, we could see the DNA structure clearly, which proved that the enzymes do not destroy the DNA origami. Moreover, we observed the well-done origami structure in this experiment.
