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The band of M13+staple ran longer than the band of M13, it showed that the structure which had big molecular weight was created. The lower fuzzy band was the band of excess staple DNA. When we put stapleDNA both 100 and 4 times of M13mp18, the annealing went well.  
The band of M13+staple ran longer than the band of M13, it showed that the structure which had big molecular weight was created. The lower fuzzy band was the band of excess staple DNA. When we put stapleDNA both 100 and 4 times of M13mp18, the annealing went well.  


[[Image:BIOMOD-2012-UTokyo-UTHongo-origamielectrophoresis160nM.png|border|bottom|500px|Fig.1 1.6 nM of M13mp18 and 160 nM of staple DNA]]
[[Image:BIOMOD-2012-UTokyo-UTHongo-origamielectrophoresis160nM.png|border|bottom|x400px|Fig.1 1.6 nM of M13mp18 and 160 nM of staple DNA]]
[[Image:BIOMOD-2012-UTokyo-UTHongo-origamielectrophoresis.png|border|bottom|500px|Fig.2 4 nM of M13mp18 and 16 nM of staple DNA]]
[[Image:BIOMOD-2012-UTokyo-UTHongo-origamielectrophoresis.png|border|bottom|x400px|Fig.2 4 nM of M13mp18 and 16 nM of staple DNA]]
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In this section, we write about our experiment.

Assembly of the DNA Shell

First, we mixed scaffold (used base sequence of M13) and staples, and ascertained whether DNA Shell was hybridized as we had designed by agarose gel electrophoresis and atomic force microscope.


The staple DNA was mixed with M13mp18 (160 or 16 nM of each staple DNA, 1.6 or 4 nM of M13mp18, a 100 or 4 –fold excess of staple DNA) in 1x TAE/Mg buffer. When we added DNA with molecules such as the fluorescence, the quencher and the biotin, those density became all the density same as staple DNA. Samples were annealed two hours from 95℃ to 20℃ in a thermal cycler at a rate of 6.25℃/10minutes 12 steps.

Agarose Gel Electrophoresis

We confirmed that we had the structure which had the bigger molecular weight than original M13(scaffold) by electrophoresis. Samples were electrophoresed in a 0.6 % agarose gel containing 1X TAE/Mg buffer. The agarose gel was run at 4℃ for 70 minutes (Fig.1 & 2).

The band of M13+staple ran longer than the band of M13, it showed that the structure which had big molecular weight was created. The lower fuzzy band was the band of excess staple DNA. When we put stapleDNA both 100 and 4 times of M13mp18, the annealing went well.

Fig.1 1.6 nM of M13mp18 and 160 nM of staple DNA Fig.2 4 nM of M13mp18 and 16 nM of staple DNA

AFM

These DNA origami (M13 + all staples, M13 + all staples except ones which combine with M13 between the squares (except LB and RB), M13 + all staples except one which combine with M13 between the center square and the right square (except RB)) were observed by using atomic force microscope (AFM) to confirm that these DNA origami were formed correctly. 1xTAE/Mg solution was utilized as buffer.

M13+staple (represent 500nm at one side)


M13+staple (without LB,RB)


Outlook

First picture shows that the origami with all staples was formed as designed. Also, these pictures may show that the first origami differ from the second one in the structure between two squares. There was the point where it seemed that origami connected with each other aside. This is probably because each Shell was attracted each other by π-π stacking interaction. We designed Shell as base pair lined lengthways, so it was appropriate for Shell to connect sideways by π-π stacking interaction.

Capturing ability

Next, we ascertained whether DNA Shell ccaptured target molecules by agarose gel electrophoresis, fluorometry, and AFM.

Agarose gel electrophoresis

We ascertained whether the migration distance changed after target DNA was added in the solution with DNA Shell. After having annealed the sample which added control1 and control2 to M13 and staple, we electrophoresed and confirmed a change of the phoresis distance. Samples were electrophoresed in a 0.6 % agarose gel containing 1X TAE/Mg buffer. The agarose gel was run at 4℃ for 70 minutes.


All the samples which were added control to come to have a shorter phoresis distance than the sample which wasn’t add. It is thought that it became hard to go through the mesh of the gel by Shell having been closed.

Fluorometry

After having annealed the sample which contained M13, staple DNA, DNA with a fluorescence molecule and DNA with quencher molecule, we took the sample 100μl and added 1xTAE/Mg buffer 400μl and measured the fluorescence intensity. We recorded a fluorescence change when we put control DNA.

Fluorescence change (1 equal amount of control1)


After having added control, intensity of fluorescence decreased. It is thought that the fluorescence decreased because Shell closed by having put control and fluorescence molecules approached quencher molecules.

The next graph is the change of the peak numerical value when we changed the density of control1. We calculated peak numerical value after having added control for 100 at the intensity of fluorescence of the peak before adding control.


We changed the number of fluorescence molecules and the quencher molecules which spread from the Shell surface (12, 4 or 1) and, in the case of each, checked how became a fluorescence change. In the lower graph, peak numerical value before adding control is 100.

In the case of 12 and 4 fluorescence molecules, there is not the big difference, but there is a bigger fluorescence change than in case of 1 fluorescence molecule. It may be said that this becomes easy to detect a fluorescence change because plural fluorescence molecules approach with quencher molecules at a time when Shell was closed when density of controlDNA is small.


Following target DNA, we checked whether we could capture streptavidin with Shell.

After having annealed the sample which contained M13, staple DNA, DNA with a fluorescence molecule, DNA with quencher molecule and DNA with biotin, we took the sample 100μl and added 1xTAE/Mg buffer 400μl and measured the fluorescence intensity. We recorded a fluorescence change when we put SA.

Fluorescence change (1 equal amount of SA)



After having added SA, intensity of fluorescence decreased. It is thought that the fluorescence decreased because Shell closed by having put SA and fluorescence molecules approached quencher molecules.

The next graph is the change of the peak numerical value when we changed the density of SA. We calculated peak numerical value after having added SA for 100 at the intensity of fluorescence of the peak before adding SA.


A fluorescence change of 1/8 equal amount of SA is the biggest. It is thought that a fluorescence change becomes small, when SA density is big, the SA of 2 molecules is connected to biotin spread from two sides of Shell, and Shell does not close and when SA density is small, a ratio of Shell to close is small.

In case of high concentration
In case of low concentration
zero concentration

AFM

First, we tried the version of target DNA.

This picture was captured by AFM. In the picture, some square-like structures could be seen. They are exactly DNA Shell. And it is observed that almost central part of the Shell is white. This shows that central part is thicker than the other part of it.

File:Biomod-2012-UToky​o-UT-Hongo-AFMclosecontrol analysis.jpg

In this picture, upper graph display the horizontal height of the line in the bottom picture and each down-pointing triangles are corresponded between upper graph and bottom picture.


Following target DNA, we checked whether we could capture streptavidin with Shell.

At the upper left of the above picture, we could see the square structure which has two white points in the center. This structure is the Shell. The following pictures show the sectional pitch.

These pictures show:

  • Shell is 91.797nm (almost 90nm) width
  • Shell is 2.075nm (almost 2nm) thick
  • The white points of Shell are 3.882nm and 4.096nm(almost 4nm) thick
  • There is a part with almost 0nm thick at around 21nm from the side of Shell

Shell is constructed from double helix, so it is reasonable to think that the value 2nm is Shell's thickness. The thickness at the white points in the Shell are equivalent to the thickness for approximately two folds of double helix. In addition, width of Shell at the design stage is approximately 120nm and the structure seen this time, width is almost 2/3. It is reasonable to suppose that this is because one outer sheet of the Shell is on top of the other sheet. That's why we estimated that Shell captured streptavidin. The white points of the Shell are small compared with the Shell itself because streptavidin is small (almost 60 Å)

Immobilizing on microfluidic device

We confirmed if DNA origami is immobilized upon the surface of flowing channel of the microfluidic device.

Control is introduced into microfluidic device

1. A solution containing of fluorescent labeled-DNA origami(M13 + staples) was introduced into a microchannel on the microfluidic device. After rinsing to remove DNA origami not immobilized on the microchannel, observation of remaining fluorescence from immobilized DNA origami is performed (a). The scale bar is 100 μm.

2. The fluorescence was almost kept even if water is introduced into the microchannel (b). Decreasing of fluorescent intensity was observed due to destruction of immobilized DNA origami.

3. The fluorescence strength would be weak if the control solution is introduced into the microchannel (c).

And we showed the intensity of fluorescence of each iamge to the graph.

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Therefore, we could confirme that DNA origami was immobilized onto the surface of the microchannel.

Streptavidin is perfused into microfluidic device

1. A solution containing of fluorescent labeled-DNA origami(attached Biotin) was introduced into a microchannel on the microfluidic device. After rinsing to remove DNA origami not immobilized on the microchannel, observation of remaining fluorescence from immobilized DNA origami is performed (d). The scale bar is 100 μm.

2. The fluorescence was kept even if water is introduced into the microchannel (e).

3. The fluorescence strength would be weak if the streptavidin is introduced into the microchannel (f).

And we showed the intensity of fluorescence of each iamge to the graph.

Error creating thumbnail: File with dimensions greater than 12.5 MP　 Error creating thumbnail: File with dimensions greater than 12.5 MP　 Error creating thumbnail: File with dimensions greater than 12.5 MP　 　

Supporting enzyme

Based on the results mentioned above, we did further advanced experiment. We ascertained whether DNA Shell supported enzymes.

Background

We used tetramethylbenzidine (TMB), streptavidin with horseradish peroxidase (HRP) labeling and trypsin. TMB can be oxidized for the reduction of hydrogen peroxide to water by peroxidase enzymes such as HRP. When TMB is oxidized, the color of the solution takes on a blue color. If pH of the solution is low (for example, using sulfric acid), the color turns into yellow. The former blue color can be read at a wavelength of nm and the latter yellow color can be read at nm.

We made the use of this chemical reaction. Trypsin is protease. Now suppose the solution with HRP, hydrogen peroxide and TMB. In acid condition, TMB may be oxidized and change color of the solution. However, if trypsin exists with HRP solution, it can be imagined easily that trypsin decompose HRP and the chemical reaction mentioned above does not happen. Then, the DNA Shell is added. The DNA Shell protects HRP by combining streptavidin with biotin sticked to the end of spreading DNA from the Shell, so we can expect the oxidization of TMB and changing color.

Experiment condition

Extinction measurement

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   <h2 style="border-bottom: none;">BIOMOD 2012 Team UT-Hongo</h2>
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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo">Top</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#description">Abstract</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#youtube">YouTube</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo#navi">Links</a></li> </ul> 

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro">Motives</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Focus">Focus</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Idea_of_DNA_Shell">Idea</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Functionalities_Exhibited">Funcitonalities</a></li> 

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Intro#Conceptual_Blueprint_of_the_Structure">Blueprint</a></li>

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly">Design & Results</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Design">Design</a></li> 

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Assembly#Adding_functionality">Function</a></li>
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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method">Method</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#AFM">AFM</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Photometer">Photometer</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Electrophoresis">Electrophoresis</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Ultraviolet_Irradiation">Others</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Method#Reagent">Reagent</a></li> </ul> 

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork">Progress & Beyond</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#Variety_of_Target_Substances">Target Variety</a></li> 

         <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/FutureWork#DNA_Shell_with_Functionality">Functionalization</a></li>
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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Team">Team</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Team#Info">Info</a></li> 

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<h3><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement">Acknowledgement</a></h3> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Mentor">Mentor</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Professors">Professors</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Sponsors">Sponsors</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/UTokyo/UT-Hongo/Acknowledgement#Special_Thanks">Special Thanks</a></li> </ul> 

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