Biomod/2012/UTokyo/UT-Hongo/Assembly

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And we showed the intensity of fluorescence of each image to the graph.
And we showed the intensity of fluorescence of each image to the graph.
[[Image:Biomod-2012-UTokyo-UT-Hongo Fixing on microfluidics (image-2) ver1.jpg| thumb | 700px |Fig.4 Fluorescence images of DNA shells in a microchannel ]]
[[Image:Biomod-2012-UTokyo-UT-Hongo Fixing on microfluidics (image-2) ver1.jpg| thumb | 700px |Fig.4 Fluorescence images of DNA shells in a microchannel ]]
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After water was introduced, fluorescence intensity greatly fell. It because
 
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After water was introduced, fluorescence intensity greatly fell. It because
=Supporting enzyme=
=Supporting enzyme=

Revision as of 11:53, 26 October 2012

The University of Tokyo


In this section, we write about our experiment. Our experiment consists of four themes;

  • Assembly of the DNA Shell
  • Capturing Ability
  • Immobilizing on microfluidic device
  • Supporting enzyme

In each theme, we experimented this summer and can get various valuable data. we wrote our data and results in order.

Contents

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

Fig.1 4 nM of M13mp18 and 16 nM of staple DNA
Fig.1 4 nM of M13mp18 and 16 nM of staple DNA

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.

AFM

These DNA origami (M13 + all staples) were observed by using atomic force microscope (AFM) to confirm that these DNA Shell were formed correctly. 1xTAE/Mg solution was utilized as buffer.

Image:Biomod-2012-UTokyo-UT-Hongo-ununited.png



This AFM picture clearly shows that the origami with all staples was formed as designed. 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

fig.1
fig.1

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 target DNA (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 at 4℃ for 70 minutes.

All the samples which were added target DNA 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 target DNA.

Fluorescence change (1 equal amount of target DNA)

step1

fig.1
fig.1

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

step2

fig.2
fig.2

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

step3

fig.3
fig.3

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 target DNA is 100.

step4

fig.4
fig.4

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 target DNA 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)

Adding SA

fig.1
fig.1

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.

Changing the density of SA

fig.2
fig.2

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.

Fluorimetry

fig.1
fig.1
fig.2
fig.2
fig.3 dimer
fig.3 dimer

We performed fluorimetry in three kinds of Shell ([1] Shell where biotin grew on both sides (Normal Shell) [2] Shell where biotin grew only in the fluorescence side [3] Shell where biotin grew only in the quencher side). There was not the big difference between [3] and [1]. However, only [2] came to have a particularly big fluorescence change. The 2 molecules of biotin grow from one Shell in [2] and [3]. Because 4 molecules of biotin are connected for 1 molecule of SA, it is thought that two Shell catched one SA, and a dimer was formed. In this case a large number of fluorescence molecules would approach at a time in [2], and it is thought that concentration quenching happened, and the fluorescence decreased.


AFM

fig.1 Closing DNA Shell with target DNA
fig.1 Closing DNA Shell with target DNA

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.

fig.2 Analysis image of closing DNA Shell
fig.2 Analysis image of closing DNA Shell

We found that the thickness of the white point in the Shell (Fig.1) was about 4.9nm(Fig.2). In this picture, the left upper graph shows the sectional pitch that cut Shell perpendicularly. Target DNA is 26bp or about 5.8 nm, so we estimate that target DNA hybridized with staple DNA with a little curve.

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

fig.3 Closing DNA Shell with Streptavidin
fig.3 Closing DNA Shell with Streptavidin

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

Fig.1 Relative fluorescence intensities of DNA shells in a microchannel
Fig.1 Relative fluorescence intensities of DNA shells in a microchannel
  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 was 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.

Fig.2 Fluorescence images of DNA shells in a microchannel
Fig.2 Fluorescence images of DNA shells in a microchannel

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

Streptavidin is introduced into microfluidic device

Fig.3 Relative fluorescence intensities of DNA shells in a microchannel
Fig.3 Relative fluorescence intensities of DNA shells in a microchannel
  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 image to the graph.

Fig.4 Fluorescence images of DNA shells in a microchannel
Fig.4 Fluorescence images of DNA shells in a microchannel


After water was introduced, fluorescence intensity greatly fell. It because

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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