Biomod/2013/Todai/Result

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           <img src="http://openwetware.org/images/3/30/Figure11_12-Todai.png" width="360px" height="240px">
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           <img src="http://openwetware.org/images/2/24/Koyama_131027_1-Todai.JPG" width="320px" height="192px">
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           <img src="http://openwetware.org/images/2/24/Koyama_131027_1-Todai.JPG" width="240px" height="240px">
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       <p class ="paragraph">
       <p class ="paragraph">
       Product of click reaction appeared over 375 uM CuSO<sub>4</sub> concentration. Combining with the stability data, we decided to use 625 uM CuSO<sub>4</sub> condition.
       Product of click reaction appeared over 375 uM CuSO<sub>4</sub> concentration. Combining with the stability data, we decided to use 625 uM CuSO<sub>4</sub> condition.
 +
      </p>
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              <div class="res-conclusion">
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              --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
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    </div>
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    </article>
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    <!--◆◆4.4 Cupper-free click chemistry◆◆-->
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    <article>
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    <h3><a name="Cupper-free_click_reaction"></a>4) Cupper-free click reaction</h3>
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    <!--Method-->
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    <div class="zairyou-heading">[Method]</div>
 +
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    <div id ="step4_3)">
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      <p class="paragraph">
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Click reaction demands cupper catalyst, which works as a toxine in human body. Therefore, we studied about cupper-free click reaction for the application to human body.
 +
        (<a target="_blank" href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols" style="color:#e00000;">
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        protocols
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        </a>
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        )
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        </p>
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    </div>
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 +
      <div class="zairyou-heading">[Result & Discussion]</div>
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      <div class="res-conclusion">
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      a) Optimum concentration of CuSO<sub>4</sub> to OCK
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    </div>
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          <figure>
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        <center>
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        <img src="http://openwetware.org/images/2/25/Gelphoto1-Todai.png" width=480px height=270px>
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 +
        </center>
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      </figure>
 +
      <p class="paragraph>
 +
We first measured the Cu-free click reaction in solution (without no
 +
catalyst nor accelerator). The association time at 2 uM oligonucleotide
 +
condition was 17.1 h, and appearent association time was estimated as
 +
8.1 [1/M/s].
 +
</p>
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    <br>
 +
    <br>
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    </div>
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      <div class="res-conclusion">
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      b) Optimum concentration of CuSO<sub>4</sub> to click reaction
 +
    </div>
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          <figure>
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        <center>
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        <img src="http://openwetware.org/images/9/94/ClickResult-Todai.png" width=600px height=450px>
 +
 +
        </center>
 +
      </figure>
 +
      <p class ="paragraph">
 +
We first measured the Cu-free click reaction in solution (without no
 +
catalyst nor accelerator). The association time at 2 uM oligonucleotide
 +
condition was 17.1 h, and appearent association time was estimated as
 +
8.1 [1/M/s].
 +
</p>
               <div class="res-conclusion">
               <div class="res-conclusion">
               --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
               --> Optimum concentration of CuSO<sub>4</sub>: 625 uM
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         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
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         [1] CanDo(<a href="http://cando-dna-origami.org/usersguide)">http://cando-dna-origami.org/usersguide</a>)
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         [1] CanDo(<a href="http://cando-dna-origami.org/usersguide">http://cando-dna-origami.org/usersguide</a>)
         </a>
         </a>
         </div>
         </div>

Revision as of 23:52, 26 October 2013


Result-Todai nanORFEVRE-

 Result

 Contents

  • STEP 1: DNA strands assemble to form designed structures

  • STEP 2: Penetration into the membrane

  • STEP 3: Recognition of cancer-specific proteins

  • STEP 4: Oligomerization in solution

  •  Oligomeric Cell Killer (OCK)

     STEP 1: DNA strands assemble to form designed structures

    1)Optimize the condition to assemble OCK

    [Method]

    The DNA nanostructure, "Oligomeric Cell Killer" , was designed to achieve our goal(-->Project).

    The result of simulation by "CanDo[1]" showed the shape and flexibility of OCK. To know the optimum condition of the structure assembly, we did experiments in three conditions as follows.

    • At different concentration of MgCl2
    • At different incubate temperature
    • At different length of incubate time
    ( protocols )







    [Result & Discussion]
    Optimum concentration of MgCl2
    Agarose-gel electrophoresis to research the optimum concentration of MgCl2

    Fast-migrating species upon agarose-gel electrophoresis was yielded at 10~20mM MgCl2 condition. At higher MgCl2 concentration, a sub-band, which might be a dimer, appeared.

    --->> Optimum concentration of MgCl2: 10mM


    Optimum incubate temparature
    Agarose-gel electrophoresis to research the optimum temperature

    Fast-migrating species upon agarose-gel electrophoresis was yielded at 52.0 °C.

    --->> Optimum temparature : 52.0 °C


    To decide optimum length of incubate time
    Agarose-gel electrophoresis to research the optimum time

    The band for 3h is fast migrated and sharp.

    --->> Optimum incubate time : 3h

    2) TEM imaging of the 3D structure of OCK

    [Method]

    Gel electrophoresis cannot visualize the 3D structure of OCK, so it was confirmed by Transmission electron microscopy (TEM). ( protocols )




    [Result & Discussion]
    TEM imaging of OCK
    TEM image of OCK Three monomers of OCK were observed in this figure.

    TEM images confirm that our OCK has two domains. Comparing the observed structure to our design, one domain match the shape and size to plane-like domain. And the other domain matches to stick-like domain. Furthermore, in close watching the images, DNA well, which exists one side of plane-like domain, could be detected.

     STEP 2: Penetration into the membrane

    1) Flotation assay

    [Method]

    OCK was designed to penetrate lipid bilayer. However, it is difficult to conclude the penetration of OCK. Therefore, we first did flotation assay to detect the interaction of OCK with lipid. ( protocols )




    [Result & Discussion]
    The fluorescence intensity of NIL (in liposome) in each fraction
    The result of fluorescence spectrophotometer (JASCO, FP-6500) showed that liposome distributed mostly in fraction 3(lower layer).
    1% Agarose gel electrophoresis of each fraction in sample 1, 2
    1% Agarose gel electrophoresis of each fraction in sample 3, 4

    With the condition of cholesterol +/ liposome+, the peak fraction was No.3, which coinced with peak fraction of liposome. In contrast, lacking of cholesterol or liposome, OCK exist mainly in fraction No.2. As the peak fraction of OCK shifted from fraction No.2 to No.3, with the attachment of cholesterol and existence of liposome, we concluded that OCK stack in liposome.

    --> OCK stack in liposome.

    2) Preparation of GUVs

    [Method]

    GUV, Giant Unilamellar Vesicle, was prepared to visualize the sticking of OCK in membrane. The comparation between the fluorescence of OCK (Cy5) and GUV(NIL, Nile Red) was expected to suggest the sticking. ( protocols )




    [Result & Discussion]

    GUVs were observed with confocal laser scanning microscope (Carl Zeiss, LSM 5 Exciter). As the tracer of GUVs, 0.1 mol% Nile Red (Ex 553 nm, Em 637 nm) was used. About 10 um of GUVs were observed.


    [Result & Discussion]
    Integration of aptamer strands into DNA origami tile

    We confirmed the integration of aptamer attached strands (aptamer strands) into rectangle DNA origami tile (rect-tile).


    Responsibility of aptamer

    We confirmed the responsibility of aptamer sequence embedded in rect-tile (shown above). The position of Cy5-PDGF band coincided with that of DNA tile, showing that the aptamers work also on rect-tile. Furthermore, the linker length between aptamer sequence and staple sequence, the latter staple sequence is embedded into rect-tile, does not affect the binding ability of aptamer to PDGF.


    Blocking capabbility of lock system by aptamer

    We confirmed the blocking capability of our lock system for streptavidin binding (left figure, the image of gel electrophoresis). Our lock system consists of two strands: biotin attached strands (biotin strands) and aptamer attached strands (aptamer strands). These two strands hybridize each other in inactive form and hide biotin moiety from the streptavidin by steric hindrance effect. We confirmed this blocking capability by mixing Cy3 labeled streptavidin with lock system embedded rect-tile. Data indicates that the slight blocking capability upon shorten the polyT linker between aptamer sequence and staple sequence. Recently, we tried other sequence and have better results, which may be presented in the Jamboree in Boston.

    Optimum embedding condition of our lock system into rect-tile
    Next, we optimize the embedding condition of our lock system into rect-tile. This time full length of biotin strands were used instead of truncated ones used in above figure. Data indicate that the integrate efficiency of both biotin strands and aptamer strands into rect-tile is independent on the incubate temperature. We improve our lock system everyday. Don't miss our presentation in Jaboree in Boston !

    2) Embedding of recognition system to OCK

    [Method]

    To embed recognition system to OCK, we equiped PDGF aptamer used in rect-tile to OCK and confirmed the association of aptamer and PDGF . ( protocols )

    [Result & Discussion]
    Recognition of PDGF by DNA aptamer on OCK
    -->PDGF was recognized by the aptamer of OCK

    [Result & Discussion]
    Streptavidins induced oligomerization
    The mixing ratio of streptavidin to OCK was equal to 5:3, which means the mixing ratio of streptavidin to biotin was equal to 5:6 in the condition (L+R).

    2) TEM imaging of OCK dimers connected by streptavidin-biotin interaction

    [Method]

    Dimers of OCKs were also imaged by TEM to confirm the bands observed in the experiment 3.1) originated from the dimers. ( protocols )




    [Result & Discussion]

    Dimers of OCKs were observed in this experiment and two of them were shown above.

    -->The dimerization by streptavidin-biotin complex was confirmed.

    3) Oligomerization by Click reaction

    [Method]

    Azide and alkyne, which function as a reactive group of click reaction, are also equiped to OCK. It demands Cu+ as catalyst, but too high concentration of Cu+ (cation) might denaturate OCK like Mg2+. Therefore, we optimized the concentration of Cu+ to OCK first, and then the optimum Cu+ concentration to click reaction was investigated. ( protocols )

    [Result & Discussion]
    a) Optimum concentration of CuSO4 to OCK
    -->Optimum concentration of CuSO4: 625 uM or less


b) Optimum concentration of CuSO4 to click reaction

Product of click reaction appeared over 375 uM CuSO4 concentration. Combining with the stability data, we decided to use 625 uM CuSO4 condition.

--> Optimum concentration of CuSO4: 625 uM

4) Cupper-free click reaction

[Method]

Click reaction demands cupper catalyst, which works as a toxine in human body. Therefore, we studied about cupper-free click reaction for the application to human body. ( protocols )

[Result & Discussion]
a) Optimum concentration of CuSO4 to OCK

b) Optimum concentration of CuSO4 to click reaction

We first measured the Cu-free click reaction in solution (without no catalyst nor accelerator). The association time at 2 uM oligonucleotide condition was 17.1 h, and appearent association time was estimated as 8.1 [1/M/s].

--> Optimum concentration of CuSO4: 625 uM









 Reference

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