Biomod/2013/Todai/Design: Difference between revisions

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<div class="rightbar-des">
<div class="rightbar-des">
   <ul>
   <ul>
     <li><a href="#1.Oligomeric Cell Killer">1. Oligomeric Cell Killer</a>
     <li><a href="#Oligomeric_Cell_Killer"> Oligomeric Cell Killer</a>
         <ul>
         <ul>
           <li><a href="#1-1.General design">1-1.<br>General design</a>
           <li><a href="#1._Structure">1. Structure</a>
          </li>
          <li><a href="#2._Function">2. Function</a>
<ul>
          <li><a href="#2.1_Sites for_penetration">2.1 Sites for penetration</a>
</li>
          <li><a href="#2.2_Recognition_system">2.2 Recognition system</a>
          </li>
          <li><a href="#2.3_Sites_for_oligomerization">2.3 Sites for oligomerization</a>
 
          </li>
<li><a href="#2.4_Pore_formation">2.4 Pore formation</a>
 
          </li>
 
</ul>
</li>
<li><a href="#References">References</a>
           </li>
           </li>
         </ul>
         </ul>
     </li>
     </li>
     <li><a href="#2.Cylinder in barrel by DNA origami">2. Cylinder in barrel</a>
      
        <ul>
          <li><a href="#2-0.Purpose">2-0. <br>Purpose</a>
          </li>
          <li><a href="#2-1.Geometrical features">2-1. <br>Geometrical features</a>
          </li>
          <li><a href="#2-2.Functional features">2-2. <br>Functional features</a>
          </li>
        </ul>
    </li>
   </ul>
   </ul>
</div>
</div>
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<!--1. Oligomeric Cell Killer-->
<!--1. Oligomeric Cell Killer-->


   <h1 class="heading"><a name="1.Oligomeric Cell Killer">&nbsp;1. Oligomeric Cell Killer</a></h1>
   <h1 class="heading"><a name="Oligomeric_Cell_Killer">&nbsp;Oligomeric Cell Killer</a></h1>


<!--1-1. Design as an approach to our goal-->
<!--1-1. Design as an approach to our goal-->


   <article>
   <article>
     <h1 class="title"><a name="1-1.General design">&nbsp;1-1. General design</a></h1>
 
 
    <p class="paragraph" id="par1">
Inspired by immune system, our goal is to fabricate pore forming DNA nanostructure killing the cancer cell (termed Oligomeric Cell killer: OCK). The designed
 
structure is shown below. Our design is characterized with four features: a broad plane-like domain to anchor on the cell surface, stick-domain to invade into
 
the cell membrane, safety lock system for cell recognition, and connectable sites for oligomerization and pore formation.
    </p>
    <br>
 
 
<center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/7/73/OCK_all-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/e/e1/Todai_OCK_flow.png" width="300px" height="300px" style="border:solid 1.5px black;">
        </figure>
 
        </td>
        </tr>
      </table>
    </center>
 
      <article>
        <ul class="Contents-list">
 
        <li>
        <b>Anchoring and penetration</b>
        </a>
<br>
<p class="paragraph">
OCKs anchor and penetrate lipid bilayer by their cholesterols.
<br>
-->Cholesterol sticking site, structure
</p>
<br>
</li>
 
 
        <li>
<b>Recognition (safety lock system)</b>
        <br>
<p class="paragraph">
The DNA aptamer of OCK recognizes cancer cell specific membrane protein and release the biotin-modified strand locked by the DNA aptamer.
<br>
-->Aptamer lock system
</p>
<br>
</li>
        <li><b>Oligomerization</b>
<br>
<p class="paragraph">
        The activated biotins bind to the streptavidins of other monomers, resulting in the trimerization of OCKs
<br>
-->Trimerization by streptavidin-biotin complex
</p>
        <br>
</li>
        <li>
        <b>Pore formation</b>
<br>
<p class="paragraph">
The physical constraint induced by oligomerization activates the azides and alkynes reactive groups on OCK, covalently bonding the stick-domain of
 
each subunits of trimeric OCKs by click chemistry. Lipids are excluded from the gap made by stick-domain of OCKs. As a result, a part of membrane is punched,
 
and pore formation is achieved
<br>
-->Click chemistry
</p>
<br>
</li>
</ul>
    <br>
 
<!--1. Structure-->
 
  <article>
     <h1 class="title"><a name="1._Structure">&nbsp;1. Structure</a></h1>
     <br>
     <br>


     <p class="paragraph">
     <p class="paragraph">
Inspired by immune system, our goal is to fablicate pore forming DNA nanostructure killing the cancer cell. The designed structure is shown below. Our design is characterized with three features: a broad plane part to anchor on the cell surface, bend of side edges to invade into the cell membrane, and connectable sites for oligomization.
The detailed structure and scale of OCK are shown below. OCK is composed of two main domains. The plane-like domain (red) sticks to lipid bilayer, and
 
the stick-domain (blue) penetrates into the lipid bilayer.
     </p>
     </p>
     <br>
     <br>
Line 101: Line 214:
     <figure>
     <figure>
       <center>
       <center>
       <img src="http://openwetware.org/images/a/ac/Des1-Todai.png" width="300" height="300">
       <img src="http://openwetware.org/images/1/1e/OCKnaname-Todai.png" width="300" height="300">
      <figcaption style="font-size:110%;position:relative;left:-20px;">
      General Design
      </figcaption>
       </center>
       </center>
     </figure>
     </figure>
     <br>
     <br>


    <p class="paragraph">
    <p class="paragraph" id="par3">
It requires enough free energy to penetrate membranes. This is compensated by the free energy gained from biding of the DNA nanostructure bound cholresterols to the lipid bilayer. The more cholesterols are equipped to the structure, the more stabilized it stays near the membrane. A broad plane gives much cholesterol binding sites, so this feature might be suitable to penetrate membranes. Although previous reseach (Danilo D. Lasic, et al.)<span class="ref-sup"><a href="#desref-1">[1]</a></span> reported the non-specific binding of DNA to the liposome, which is usually an undesiable feature, we thought that we could utilized this feature positively by means of the broad plane. Integrating broad plane into our DNA nanostructure, we expect some free energy gain by the non-specific binding of the broad plane to the bilayer, which may stabilize the binding of our DNA structure to the bilayer.
Recognizing specific trigger signal, OCK can trimerize, making a small hole (inner and outer diameter is 4 nm and 12 nm, respectively) to punch the
    </p>
 
    <br>
cell.
    <p class="paragraph">
</p>
To achieve an efficient pore forming system, one should balance between the cost of penetration of the nanostructure into the bilayer and the binding stability of the nanostructure to the bilayer. To overcome this dilemma, we introduced the "bend of side edges", which allow us to minimizing the penetration part, but also maximizing the anchoring part.
 
    </p>
 
    <br>
<center>
    <p class="paragraph">
      <table cellpadding="0">
To oligomerize, the DNA nanostructure must have some binding site to each other. Therefore, we introduced "connectable sites" into our nanostructure. It should be noted that hybridization is used for oligomerization method in the figure, we examine other method as well.
        <tr>
    </p>
        <td>
    <br>
        <figure>
    <br>
          <img src="http://openwetware.org/images/7/76/OCKmigipng-Todai.png" width="300px" height="300px" >
    <p class="paragraph">
        </figure>
Although we thought carefully, above design are mainly derived from speculation. We, therefore, need more information (e.g. interaction between DNA and liposome) to design our nanostructure more specifically. Thus, ss a first step toward our goal, we started with simple DNA origami structure: Cylinder in Barrel.
        </td>
    </p>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/0/02/OCKshoumen-Todai.png" width="300px" height="300px" >
        </figure>
        </td>
        </tr>
      </table>
    </center>
<br>
<center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/4/4d/Assemblyup-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/3/37/Assembly1-Todai.png" width="300px" height="300px" >
        </figure>
 
        </td>
        </tr>
      </table>
    </center>
 
 
<!--2. Function-->


  <article>
    <h1 class="title"><a name="2._Function">&nbsp;2. Function</a></h1>
<p class="paragraph" id="par4">
OCK is equipped with functional sites for penetration and oligomerization.
</p>
   </article>
   </article>
   <br>
   <br>
  <br>
  <br>


<!--2. Cylinder in barrel by DNA origami-->
<!--2.1 Sites for penetration-->
 
<article>
<h2 class="title"><a name ="2.1_Sites for penetration"></a>2.1 Sites for penetration</h2>
<h3 class="title">&nbsp;1)Cholesterol sticking sites</h3>
<center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/f/f6/OCK_anchoring-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/2/28/OCK_penetrate-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
        </tr>
      </table>
    </center>
 
<p class="paragraph" id="par5">It requires enough free energy to penetrate membranes. This is compensated by the free energy gained from biding of the
 
DNA nanostructure bound cholesterols to the lipid bilayer. The more cholesterols are equipped to the structure, the more stabilized it stays near
 
the membrane. We attached cholesterol to our OCK by two methods; hybridization and direct incorporation. Hybridization method is accomplished by hybridizing
 
the scaffold strand and cholesterol-modified ssDNA strands. Regarding direct incorporation method, we supplemented cholesterol modified
 
staples at the formation of OCK. For both methods, we design four cholesterol sites shown below.
</p>
<br>
          <center>
          <figure>
          <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width="300px" height="300px" >
          </figure>
          </center>
 
<h3 class="title">&nbsp;2)Plane-domain and stick-domain</h3>
<p class="paragraph id="par6">
To ensure the penetration of DNA nanostructure (six double-helical DNA domains. diameter of 6 nm), previous research used 26 cholesterol
 
binding sites (Martin Langecker, et al.<span class="ref-sup"><a href="#desref-1">[1]</a></span>) However, too much sites may cause the heterogeneity of the
 
sample and lower yields. To resolve this points, we paid attention to character of DNA : non-specific binding to the liposome (Danilo D. Lasic, et al.<span
 
class="ref-sup"><a href="#desref-2">[2]</a></span>), which we confirmed using Rectangle type DNA origami and POPC (see \Experiment\\Pilot study section).
 
Although the non-specific binding is usually an undesirable feature, we thought that we could utilize this feature positively by means of the broad plane-
 
domain. Integrating broad plane into our DNA nanostructure, we expect some free energy gain by the non-specific binding of the broad plane to the bilayer,
 
which may stabilize the binding of our DNA structure to the bilayer. Combining the assistant of cholesterols and plane-domain, we thought that it is easy for the stick domain to penetrate into membrane.
</p>
 
</article>
<br>
<!--2.2 Recognition system (safely lock system)-->
<h2 class="title"><a name="2.2_Recognition_system"></a>2.2 Recognition system (safely lock system)</h2>
<p class="paragraph" id="par7">
The key feature of OCK is keeping monomeric state in solution and on normal cell, while oligomerization on cancer cell. To ensure this feature,
 
recognition system (safety lock system) is required to prevent the non-specific oligomerization, while having ability to oligomerization.
</p>
 
    <center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/d/d1/OCK_AptamerLockBiotin-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/d/d1/OCK_AptamerUnlockBiotin-Todai.png" width="300px" height="300px"
        </figure>
        </td>
        </tr>
      </table>
    </center>
 
 
<p class="paragraph" id="par8">
As a recognition system, we use DNA aptamer. In general, DNA aptamer recognize specific ligand. And if some part of DNA aptamer is hybridized with
 
complementary strand, the complementary strand is released from the DNA aptamer on the binding of ligands, because ligands take over the DNA strands of DNA
 
aptamer from the complementally strands. We used this scheme as followings. Shown in the figure, the biotin modified aptamer complementary strands are
 
adhering to OCK, therefore the streptavidins cannot access to the biotin. On recognition of cancer specific membrane protein, biotin modified strands are
 
released and can bind to streptavidin. Therefore, the cancer cell recognition and OCK oligomerization are achieved simultaneously.
</p>
<p class="paragraph" id="par9"
In this project, PDGF was modified with cholesterols and used as model mimic membrane protein, as DNA aptamer system for PDGF was established.(S. M.


  <h1 class="heading"><a name="2.Cylinder in barrel by DNA origami">&nbsp;2. Cylinder in barrel by DNA origami</a></h1>
Douglas, et al.<span class="ref-sup"><a href="#desref-3">[3]</a></span>)
  <br>
</p>


  <center>
</article>
  <iframe width="420" height="315" src="http://www.youtube.com/embed/2I802ed96t0?rel=0" frameborder="0" allowfullscreen>
<br>
  </iframe>
<!--2.3 Sites for oligomerization-->
  </center>
<article>
  <br>
<h2 class="title"><a name="2.3_Sites_for_oligomerization"></a>2.3 Sites for oligomerization</h2>
<h3 class="title">&nbsp;1)Necessity of precise control</h3>
<p class="paragraph" id="par10">
When OCKs recognize cancer cell on the membrane, they are oligomerized (get trimer) and make pore. To achieve these steps, we designed the structure


of prototype, which was simpler than the current design.
</p>


<!--2-0. Purpose-->
    <center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/3/35/OCK_prototype-Todai.png" width="300px" height="300px" >


  <article>
        </figure>
    <h1 class="title"><a name="2-0.Purpose">&nbsp;2-0. Purpose</a></h1>
        </td>
    <br>


    <p class="paragraph">
    This barrel structure was designed to get some feedback for our final design of “Origomeric Cell Killer (hereafter “OCK")”. CaDNAno (version 2.2) was used to design the structure, and M13mp18 was chosen as the scaffold strand.
    </p>
    <br>
   
    <p class="paragraph">
    OCK needs to be oligomerized and be formed pore on the membrane, so we check following things by Cylinder.
    </p>


      <center>
        <td>
         <figure>
         <figure>
           <img src="http://openwetware.org/images/4/42/Des2_0purpose-Todai.png" width="420" height="420" style="border:solid 1.5px black;">
           <img src="http://openwetware.org/images/1/1d/OCK_current-Todai.png" width="300px" height="300px"  
        <br>
        <br>
        <figcaption style="position:relative;left:-10px;">
        What is intended to confirm by "Cylinder".
        </figcaption>
         </figure>
         </figure>
       </center>
        </td>
    <br>
        </tr>
       </table>
    </center>
 
<p class="paragraph" id="par11">
We choose biotin-streptavidin interaction as the oligomerization method, as the preliminary experiments using barrel structure (see \Experiment\\Pilot
 
study section) showed that the hybridization method was not a suitable way to oligomerization because of the difficulty to prevent the non-specific
 
interaction between monomers. We designed our prototype OCK as shown above, which has tow biotins on the left and right sides. However this simple design
 
produce not only the desired trimer structure, but also the undesired oligomer shown below.
</p>


    <p class="item">1) Can DNA nanostructures penetrate lipid membranes?</p>
    <p class="item">2) Can DNA nanostructures bind each other and make dimer(or more complex structure )in solution?</p>
    <p class="item">3) Can DNA nanostructures dimerize on membrane?</p>
    <p class="item">4) Can the direction of connection be controllable?</p>
    <br>
    <p class="paragraph">
    Therefore, the structure was equipped with following features.
    </p>
    
    
  </article>
    <center>
  <br>
      <table cellpadding="0">
  <br>
        <tr>
  <br>
        <td>
 
        <figure>
 
          <img src="http://openwetware.org/images/d/d2/OCK_goodTrimer.png" width="300px" height="300px" >
<!--2-1. Geometrical features-->
 
        </figure>
        </td>
 


  <article>
        <td>
    <h1 class="title"><a name="2-1.Geometrical features">&nbsp;2-1. Geometrical features</a></h1>
        <figure>
   
          <img src="http://openwetware.org/images/d/dc/OCK_badTrimer.png" width="300px" height="300px">  
    <br>
        </figure>
    <figure>
        </td>
      <center>
        </tr>
      <img src="http://openwetware.org/images/7/79/BarrelSize-Todai.png" width="300px" height="300px" >
      </table>
      <figcaption style="position:relative;left:-25px;">
    </center>
      The dimentions of a cylinder in barrel
    <div>
      </figcaption>
            <figure>
      </center>
          <img src="http://openwetware.org/images/b/b4/OCK_Biotin-Todai.png" width="300" height="300" style="float:right; margin:0;margin-left:10px;margin-
    </figure>
    <br>


    <p class="paragraph">
bottom:10px; margin-top:10px; position:relative;
    To get reliable information, the design of cylinder needs to be simple and realistic. We designed our Cylinder in Barrel according to Martin Langecker et al.<span class="ref-sup"><a href="#desref-2">[2]</a></span>
  left:35px;">
    </p>
        </figure>
        <br>
        <br>
<p class="paragraph" id="par12">
As a solution, we introduce the asymmetry into OCK, because symmetry of prototype produces the alternative form of oligomer. To do this, we built in  


    <p class="paragraph">
biotin in one side (with recognition system to control the oligomerization), and streptavidin in other side. However, we found that simply attaching
    The cylinder domain is about 65 nm long (195 bp) and consists of six dsDNA helixes, so its diameter is 6 nm. The barrel domain is approximately 44 nm long (128 bp) and 48 helixes build this domain The thickness of bilayer is 2 nm, therefore, our cylinder (about 20 nm penetration part) is enough long to stick into the bilayer. By connecting the cylinder with barrel, our cylinder in barrel structure can be integrated more cholesterols than that without barrels.
    </p>


    <p class="paragraph">
streptavidin in one side is not enough for our OCK, especially in the oligomerization process of OCK (see next section). We solved this problem by introducing
    (Cholesterol is necessary to penetrate membranes, about which is written in next section.)
    </p>


  </article>
a well in OCK and embedding the streptavidin in the well.
  <br>
</p>
</div>


<!--2-2. Functional features-->


  <article>
<br>
    <h1 class="title"><a name="2-2.Functional features">&nbsp;2-2. Functional features</a></h1>
<br>
   
<br>
     <br>
     <br>
    <figure>
      <center>
      <img src="http://openwetware.org/images/a/a1/BarrelCholesterol-Todai.png" width="300px" height="300px" >
      <br>
      <br>
      <figcaption style="position:relative;left:-10px;">
      How "Cylinder" penetrates membranes
      </figcaption>
      <figcaption style="position:relative;left:-10px;">
      (This arrangement of lipids is refered to previous research. <span class="ref-sup"><a href="#desref-2">[2]</a></span>)
      </figcaption>
      </center>
    </figure>
     <br>
     <br>


    <p class="paragraph">
<h3 class="title">&nbsp;2)DNA well</h3>
Because DNA has negative charge, the DNA nanorobots have to gain some energy to penetrate lipid membrane, which is composed of amphiphilic molecules. This problem is solved by binding cholesterols to the structures. The barrel domain has 26 staple strands complementary to cholesterol modified DNA oligos, and modified oligos are hybridized to these staples. The DNA structure anchors itself to membrane by cholesterols, and it gives stability for the structure to stay near membranes. Therefore, the structure can pierce lipid bilayer.
    </p>
        <figure>
    <br>
          <img src="http://openwetware.org/images/3/34/OCK_SAWell.png" width="300" height="300" style="float:left; margin:0;margin-left:10px;margin-
 
bottom:10px;  position:relative;
  left:-35px;">
        </figure>
<p class="paragraph" id="par13">
Precise arrangement of OCK subunit is necessary for proper oligomerization, especially for the tight connection of stick-domain, as the size of lipids
 
are much small (-1 nm) compare to the size of OCK (-100nm). In this point of view, the flexibility of the position of streptavidin makes the situation


    <center>
difficult. To solve this problem easily, DNA well is equipped to OCK and the streptavidins were stored.
</p>
<br>
<br>
<br>
<br>
       <table cellpadding="0">
       <table cellpadding="0">
         <tr>
         <tr>
         <td>
         <td>
         <figure>
         <figure>
           <img src="http://openwetware.org/images/e/e3/Des2_2_3rdparagraph1-Todai.png" width="200px" height="200px" >
           <img src="http://openwetware.org/images/7/7f/OCK_SAjammer.png" width="300px" height="300px" >
          <figcaption>[Mechanism of binding-1]</figcaption>
 
          <figcaption>
          dimerized by the strands <br>
          sticking out from the top
          </figcaption>
         </figure>
         </figure>
         </td>
         </td>


         <td>
         <td>
         <figure>
         <figure>
           <img src="http://openwetware.org/images/7/7a/Des_2_2_3rdparagraph2-Todai.png" width="200px" height="200px" >
           <img src="http://openwetware.org/images/8/8c/OCK_SAinWell-Todai.png" width="300px" height="300px" >
              <figcaption>[Mechanism of binding-2]</figcaption>
 
              <figcaption>dimerized by the strands <br>
              sticking out from the side
              </figcaption>
         </figure>
         </figure>
         </td>
         </td>
         </tr>
         </tr>
       </table>
       </table>
    </center>
</article>
<br>
<!--2.4 Pore formation-->
<h2 class="title"><a name ="2.4_Pore_formation"></a>2.4 Pore formation</h2>
<h3 class="title">&nbsp;1)Click chemistry</h3>
        <figure>
          <img src="http://openwetware.org/images/d/d1/OCK_Clickgroup-Todai.png" width="300" height="300" style="  position:relative;
  left:-35px; float:left; margin:0;margin-right:-10px;margin-bottom:10px;">
 
        </figure>
<p class="paragraph" id="par14">
Thus far, the devices for regioselective oligomerization have been explained. However, there still remains a task; exclusion of lipids out from the


    <p class="paragraph">
gab made by trimer of OCKs. To do this, the linkers connecting each OCK subunits must be short enough to ensure the inter-subunit adhesion of OCKs's stick-
    To hybridize cholesterol modified oligos, three different sequences of staples were prepared.
    </p>


    <p class="item">
domain. In that view of point, streptavidin is not suitable glue, because the diameter of streptavidins is approximately 5 nm. On the other hand, 1, 2, 3 -
1:CCTCTCACCCACCATTCATC (Alexander johnson-Buck et al.<span class="ref-sup"><a href="#desref-3">[3]</a></span>)
    </p>
    <p class="item">
2:TAACAGGATTAGCAGAGCGAGG (Martin Langecker et al.<span class="ref-sup"><a href="#desref-2">[2]</a></span>)
    </p>
    <p class="item">
3:GGAACTTCAGCCCAACTAACATTTT
    </p>


    <p class="noindent-paragraph">
triazole (the product of Click chemistry) is adequate, because the scale of the product is in the sub- to a few nanometer range (In this section, click
    These are different in the length and in sequence. The sequence of cholesterol modified oligos are perfectly complementary to the above three sequences, and their 5' ends are modified with a cholesterol.
    </p>


    <p class="paragraph">
chemistry means the Huisgen cycloaddition reaction between alkyne and azide). Therefore, OCKs are equipped with alkyne and azide reactive groups.  
To achieve oligomerization, the OCK has binding site by means of hybridization. Two pairs of sequences were used for hybridization. Both sequences are according to previous work (Shawn M. Douglas, et al. <span class="ref-sup"><a href="#desref-4">[4]</a></span>). These are derived from aptamer sequence, one is TE17, the other is sgc8c (and the complementary strands to these, so two pairs). These sequences were chosen because it is considered that these sequences don't prevent the folding of scaffold. The sequences are:
</p>
    </p>
<!--necessary to change graphics-->
    <p class="item">1:TCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGA TT(sgc8c aptamer + TT)
    </p>
    <p class="item">2:TT CAGCACCCAGTCAGAAGCAGGTGTTCGGAGTTTTGTATTGCGTAGCTG(TT+ TE17 aptamer )
    </p>


    <p class="noindent-paragraph">
    Designed structures have either the aptamer sequences (1, 2) or the two complementary strands (1, 2). When two structures with different pairs are mixed and hybridization happens, these structures hence bind each other through two binding sites. Two types of binding sites were designed to test whether the direction of connection can be controlled. One type of binding site uses staples sticking out from the ends of scaffolds, the other uses staples from side of OCK.
    </p>


    <figure>
     
       <center>
       <center>
      <img src="http://openwetware.org/images/5/5a/Des_2_2_4thparagraph-Todai.png" width="300px" height="300px" >
        <figure>
      <figcaption style="position:relative;left:-20px">
          <img src="http://openwetware.org/images/d/d1/600x240CLick-Todai.png" width="600" height="240" >
      Fluorescent materials are equipped
        <br>
      <br>by streptavidin-biotin interaction.
        <br>
      </figcaption>
 
        </figure>
       </center>
       </center>
    </figure>
     <br>
     <br>
<p clas="paragraph" id="par15">
Usually, this click reaction demands copper as catalyst. However, copper does not exist in human body. Therefore, copper free click chemistry was
studied in this project. As explained in result, the reaction rate of copper free click chemistry is very slow in solution (association rate constant ~ 8.1
/M/s = 17h@2uM reagents), but is accelerated when the reaction groups (azide and alkyne) are forced to be close. In other words, azide and alkyne reactive
groups do not react each other in solution, but easy to react each other after oligomerization. This character is very suitable to prevent non-specific


    <p class="paragraph">
oligomerization, while accelerating the specific oligomerization.
    To detect the cylinder piercing membrane, biotin modified staple strands (biotin-oligo) are attached at the bottom of OCK and hybridized with complementally strands labeled with fluorescence. Strept-avidins (SA) encapsulated in the liposome could bind to the OCK, and only the biotin-oligo penetrating liposome could bind to the SA, which can be detected by the gel shift assay.
</p>
    </p>
      <center>
        <figure>
          <img src="http://openwetware.org/images/c/c5/OCK_ConstraintClick-Todai.png" width="300" height="300" style="border:solid 1.5px black;">
        <br>
        <br>


        </figure>
      </center>
     <br>
     <br>
    <p class="paragraph">
<p class="paragraph">
    From the results of experiments with “Cylinder” we will decide our final design.
To detect whether click reaction happened, OCKs have two fluorescent dyes, Cy3 and Cy5. These fluorescent dyes are adhere to the azide-modified site
    </p>
 
  </article>
and the alkyne-modified site respectively (dye - dye distance is roughly 1 - 2 nm), so FRET signal between Cy3 and Cy5 is observed if click reaction occurred.
  <br>
</p>
  <br>
  <br>
    <center>
      <table cellpadding="0">
        <tr>
        <td>
        <figure>
          <img src="http://openwetware.org/images/a/a2/OCK_Cy3fluorescent-Todai.png" width="300px" height="300px" >
 
        </figure>
        </td>
 
 
        <td>
        <figure>
          <img src="http://openwetware.org/images/8/83/OCK_Cy5FRET-Todai.png" width="300px" height="300px" >


        </figure>
        </td>
        </tr>
      </table>
    </center>
<!--References-->
<!--References-->


Line 329: Line 598:
     <h1 class="title"><a name="References">&nbsp;References</a></h1>
     <h1 class="title"><a name="References">&nbsp;References</a></h1>
     <br>
     <br>


     <div>     
     <div>     
         <div class="reference-title">
         <div class="reference-title">
          <a name="desref-1">
        <a name="desref-1">
          [1] The Structure of DNA−Liposome Complexes
        [1] Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures  
          </a>
        </div>
          <div class="reference-author">
          Danilo D. Lasic,Helmut Strey, Mark C. A. Stuart, Rudolf Podgornik,  and Peter M. Frederik
          </div>
              <div class="reference-journal">
              Journal of the American Chemical Society 1997 119 (4), 832-833
              </div>
    </div>
    <br>
    <div>   
        <div class="reference-title">
        <a name="desref-2">
        [2] Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures  
         </a>
         </a>
         </div>
         </div>
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               </div>
               </div>
     </div>
     </div>
    <br>
<br>
     <div>     
     <div>     
         <div class="reference-title">
         <div class="reference-title">
        <a name="desref-3">
          <a name="desref-2">
        [3] Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;DNA Nanostructures
          [2] The Structure of DNA−Liposome Complexes
        </a>
          </a>
         </div>
         </div>
           <div class="reference-author">
           <div class="reference-author">
           Alexander Johnson-Buck, Jeanette Nangreave, Shuoxing Jiang, Hao Yan, and Nils G. Walter
           Danilo D. Lasic,Helmut Strey, Mark C. A. Stuart, Rudolf Podgornik, and Peter M. Frederik
           </div>
           </div>
               <div class="reference-journal" style="font-style:italic;">
               <div class="reference-journal">
               Nano Letters 2013 13 (6), 2754-2759
               Journal of the American Chemical Society 1997 119 (4), 832-833
               </div>
               </div>
     </div>
     </div>
     <br>
     <br>
    <div>   
 
         <div class="reference-title">
         <div class="reference-title">
         <a name="desref-4">
         <a name="desref-3">
         [4] A logic-gated nanorobot for targeted transport of molecular payloads.  
         [3] A logic-gated nanorobot for targeted transport of molecular payloads.  
         </a>
         </a>
         </div>
         </div>
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   <br>
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 <ul>
    <li><a href="#Oligomeric_Cell_Killer"> Oligomeric Cell Killer</a>
       <ul>
         <li><a href="#1._Structure">1. Structure</a>
         </li>
         <li><a href="#2._Function">2. Function</a>

<ul> 

         <li><a href="#2.1_Sites for_penetration">2.1 Sites for penetration</a>

</li> 

         <li><a href="#2.2_Recognition_system">2.2 Recognition system</a>
         </li>
         <li><a href="#2.3_Sites_for_oligomerization">2.3 Sites for oligomerization</a>

         </li>

<li><a href="#2.4_Pore_formation">2.4 Pore formation</a> 

         </li>

</ul> </li> <li><a href="#References">References</a> 

         </li>
       </ul>
    </li>
    
 </ul>

</div>


<!--Design--> 

  <h1 class="big-title"><a name="Design">&nbsp;Design</a></h1>
  <br>

<!--1. Oligomeric Cell Killer--> 

  <h1 class="heading"><a name="Oligomeric_Cell_Killer">&nbsp;Oligomeric Cell Killer</a></h1>

<!--1-1. Design as an approach to our goal--> 

  <article>



    <p class="paragraph" id="par1">

Inspired by immune system, our goal is to fabricate pore forming DNA nanostructure killing the cancer cell (termed Oligomeric Cell killer: OCK). The designed

structure is shown below. Our design is characterized with four features: a broad plane-like domain to anchor on the cell surface, stick-domain to invade into

the cell membrane, safety lock system for cell recognition, and connectable sites for oligomerization and pore formation. 

    </p>
    <br>



<center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/7/73/OCK_all-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/e/e1/Todai_OCK_flow.png" width="300px" height="300px" style="border:solid 1.5px black;">
       </figure>

       </td>
       </tr>
     </table>
   </center>

      <article>
        <ul class="Contents-list">

        <li>
        <b>Anchoring and penetration</b>
        </a>

<br> <p class="paragraph"> OCKs anchor and penetrate lipid bilayer by their cholesterols. <br> -->Cholesterol sticking site, structure </p> <br> </li> 


       <li>

<b>Recognition (safety lock system)</b> 

       <br>

<p class="paragraph"> The DNA aptamer of OCK recognizes cancer cell specific membrane protein and release the biotin-modified strand locked by the DNA aptamer. <br> -->Aptamer lock system </p> <br> </li> 

       <li><b>Oligomerization</b>

<br> <p class="paragraph"> 

       The activated biotins bind to the streptavidins of other monomers, resulting in the trimerization of OCKs

<br> -->Trimerization by streptavidin-biotin complex </p> 

       <br>

</li> 

       <li>
       <b>Pore formation</b>

<br> <p class="paragraph"> The physical constraint induced by oligomerization activates the azides and alkynes reactive groups on OCK, covalently bonding the stick-domain of

each subunits of trimeric OCKs by click chemistry. Lipids are excluded from the gap made by stick-domain of OCKs. As a result, a part of membrane is punched,

and pore formation is achieved <br> -->Click chemistry </p> <br> </li> </ul> 

    <br>

<!--1. Structure--> 

  <article>
   <h1 class="title"><a name="1._Structure">&nbsp;1. Structure</a></h1>
   <br>

    <p class="paragraph">

The detailed structure and scale of OCK are shown below. OCK is composed of two main domains. The plane-like domain (red) sticks to lipid bilayer, and

the stick-domain (blue) penetrates into the lipid bilayer. 

    </p>
    <br>

    <figure>
     <center>
      <img src="http://openwetware.org/images/1/1e/OCKnaname-Todai.png" width="300" height="300">
     </center>
    </figure>
    <br>

    	<p class="paragraph" id="par3">
	Recognizing specific trigger signal, OCK can trimerize, making a small hole (inner and outer diameter is 4 nm and 12 nm, respectively) to punch the

cell. </p> 


<center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/7/76/OCKmigipng-Todai.png" width="300px" height="300px" >
       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/0/02/OCKshoumen-Todai.png" width="300px" height="300px" >
       </figure>
       </td>
       </tr>
     </table>
   </center>

<br> 

<center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/4/4d/Assemblyup-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/3/37/Assembly1-Todai.png" width="300px" height="300px" >
       </figure>

       </td>
       </tr>
     </table>
   </center>

<!--2. Function--> 

  <article>
   <h1 class="title"><a name="2._Function">&nbsp;2. Function</a></h1>

<p class="paragraph" id="par4"> OCK is equipped with functional sites for penetration and oligomerization. </p> 

  </article>
  <br>

<!--2.1 Sites for penetration-->

<article> <h2 class="title"><a name ="2.1_Sites for penetration"></a>2.1 Sites for penetration</h2> <h3 class="title">&nbsp;1)Cholesterol sticking sites</h3> <center> 

     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/f/f6/OCK_anchoring-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/2/28/OCK_penetrate-Todai.png" width="300px" height="300px" >

       </figure>
       </td>
       </tr>
     </table>
   </center>

<p class="paragraph" id="par5">It requires enough free energy to penetrate membranes. This is compensated by the free energy gained from biding of the

DNA nanostructure bound cholesterols to the lipid bilayer. The more cholesterols are equipped to the structure, the more stabilized it stays near

the membrane. We attached cholesterol to our OCK by two methods; hybridization and direct incorporation. Hybridization method is accomplished by hybridizing

the scaffold strand and cholesterol-modified ssDNA strands. Regarding direct incorporation method, we supplemented cholesterol modified

staples at the formation of OCK. For both methods, we design four cholesterol sites shown below. </p> <br> 

         <center>
         <figure>
         <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width="300px" height="300px" >
         </figure>
         </center>

<h3 class="title">&nbsp;2)Plane-domain and stick-domain</h3> <p class="paragraph id="par6"> To ensure the penetration of DNA nanostructure (six double-helical DNA domains. diameter of 6 nm), previous research used 26 cholesterol

binding sites (Martin Langecker, et al.<span class="ref-sup"><a href="#desref-1">[1]</a></span>) However, too much sites may cause the heterogeneity of the

sample and lower yields. To resolve this points, we paid attention to character of DNA : non-specific binding to the liposome (Danilo D. Lasic, et al.<span

class="ref-sup"><a href="#desref-2">[2]</a></span>), which we confirmed using Rectangle type DNA origami and POPC (see \Experiment\\Pilot study section).

Although the non-specific binding is usually an undesirable feature, we thought that we could utilize this feature positively by means of the broad plane-

domain. Integrating broad plane into our DNA nanostructure, we expect some free energy gain by the non-specific binding of the broad plane to the bilayer,

which may stabilize the binding of our DNA structure to the bilayer. Combining the assistant of cholesterols and plane-domain, we thought that it is easy for the stick domain to penetrate into membrane. </p>

</article> <br> <!--2.2 Recognition system (safely lock system)--> <h2 class="title"><a name="2.2_Recognition_system"></a>2.2 Recognition system (safely lock system)</h2> <p class="paragraph" id="par7"> The key feature of OCK is keeping monomeric state in solution and on normal cell, while oligomerization on cancer cell. To ensure this feature,

recognition system (safety lock system) is required to prevent the non-specific oligomerization, while having ability to oligomerization. </p> 


   <center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/d/d1/OCK_AptamerLockBiotin-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/d/d1/OCK_AptamerUnlockBiotin-Todai.png" width="300px" height="300px" 
       </figure>
       </td>
       </tr>
     </table>
   </center>


<p class="paragraph" id="par8"> As a recognition system, we use DNA aptamer. In general, DNA aptamer recognize specific ligand. And if some part of DNA aptamer is hybridized with

complementary strand, the complementary strand is released from the DNA aptamer on the binding of ligands, because ligands take over the DNA strands of DNA

aptamer from the complementally strands. We used this scheme as followings. Shown in the figure, the biotin modified aptamer complementary strands are

adhering to OCK, therefore the streptavidins cannot access to the biotin. On recognition of cancer specific membrane protein, biotin modified strands are

released and can bind to streptavidin. Therefore, the cancer cell recognition and OCK oligomerization are achieved simultaneously. </p> <p class="paragraph" id="par9" In this project, PDGF was modified with cholesterols and used as model mimic membrane protein, as DNA aptamer system for PDGF was established.(S. M.

Douglas, et al.<span class="ref-sup"><a href="#desref-3">[3]</a></span>) </p>

</article> <br> <!--2.3 Sites for oligomerization--> <article> <h2 class="title"><a name="2.3_Sites_for_oligomerization"></a>2.3 Sites for oligomerization</h2> <h3 class="title">&nbsp;1)Necessity of precise control</h3> <p class="paragraph" id="par10"> When OCKs recognize cancer cell on the membrane, they are oligomerized (get trimer) and make pore. To achieve these steps, we designed the structure

of prototype, which was simpler than the current design. </p> 

   <center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/3/35/OCK_prototype-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/1/1d/OCK_current-Todai.png" width="300px" height="300px" 
       </figure>
       </td>
       </tr>
     </table>
   </center>

<p class="paragraph" id="par11"> We choose biotin-streptavidin interaction as the oligomerization method, as the preliminary experiments using barrel structure (see \Experiment\\Pilot

study section) showed that the hybridization method was not a suitable way to oligomerization because of the difficulty to prevent the non-specific

interaction between monomers. We designed our prototype OCK as shown above, which has tow biotins on the left and right sides. However this simple design

produce not only the desired trimer structure, but also the undesired oligomer shown below. </p> 


   <center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/d/d2/OCK_goodTrimer.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/d/dc/OCK_badTrimer.png" width="300px" height="300px"> 
       </figure>
       </td>
       </tr>
     </table>
   </center>
   <div>
           <figure>
         <img src="http://openwetware.org/images/b/b4/OCK_Biotin-Todai.png" width="300" height="300" style="float:right; margin:0;margin-left:10px;margin-

bottom:10px; margin-top:10px; position:relative; 

 left:35px;">
       </figure>
       <br>
       <br>

<p class="paragraph" id="par12"> As a solution, we introduce the asymmetry into OCK, because symmetry of prototype produces the alternative form of oligomer. To do this, we built in

biotin in one side (with recognition system to control the oligomerization), and streptavidin in other side. However, we found that simply attaching

streptavidin in one side is not enough for our OCK, especially in the oligomerization process of OCK (see next section). We solved this problem by introducing

a well in OCK and embedding the streptavidin in the well. </p> </div>


<br> <br> <br> 

    <br>
    <br>

<h3 class="title">&nbsp;2)DNA well</h3> 

       <figure>
         <img src="http://openwetware.org/images/3/34/OCK_SAWell.png" width="300" height="300" style="float:left; margin:0;margin-left:10px;margin-

bottom:10px; position:relative; 

 left:-35px;">
       </figure>

<p class="paragraph" id="par13"> Precise arrangement of OCK subunit is necessary for proper oligomerization, especially for the tight connection of stick-domain, as the size of lipids

are much small (-1 nm) compare to the size of OCK (-100nm). In this point of view, the flexibility of the position of streptavidin makes the situation

difficult. To solve this problem easily, DNA well is equipped to OCK and the streptavidins were stored. </p> <br> <br> <br> <br> 

     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/7/7f/OCK_SAjammer.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/8/8c/OCK_SAinWell-Todai.png" width="300px" height="300px" >

       </figure>
       </td>
       </tr>
     </table>

</article> <br> <!--2.4 Pore formation--> <h2 class="title"><a name ="2.4_Pore_formation"></a>2.4 Pore formation</h2> <h3 class="title">&nbsp;1)Click chemistry</h3> <figure> 

         <img src="http://openwetware.org/images/d/d1/OCK_Clickgroup-Todai.png" width="300" height="300" style="  position:relative;
 left:-35px; float:left; margin:0;margin-right:-10px;margin-bottom:10px;">

       </figure>

<p class="paragraph" id="par14"> Thus far, the devices for regioselective oligomerization have been explained. However, there still remains a task; exclusion of lipids out from the

gab made by trimer of OCKs. To do this, the linkers connecting each OCK subunits must be short enough to ensure the inter-subunit adhesion of OCKs's stick-

domain. In that view of point, streptavidin is not suitable glue, because the diameter of streptavidins is approximately 5 nm. On the other hand, 1, 2, 3 -

triazole (the product of Click chemistry) is adequate, because the scale of the product is in the sub- to a few nanometer range (In this section, click

chemistry means the Huisgen cycloaddition reaction between alkyne and azide). Therefore, OCKs are equipped with alkyne and azide reactive groups. </p> <!--necessary to change graphics--> 


     <center>
       <figure>
         <img src="http://openwetware.org/images/d/d1/600x240CLick-Todai.png" width="600" height="240" >
        <br>
        <br>

       </figure>
     </center>
    <br>

<p clas="paragraph" id="par15"> Usually, this click reaction demands copper as catalyst. However, copper does not exist in human body. Therefore, copper free click chemistry was

studied in this project. As explained in result, the reaction rate of copper free click chemistry is very slow in solution (association rate constant ~ 8.1

/M/s = 17h@2uM reagents), but is accelerated when the reaction groups (azide and alkyne) are forced to be close. In other words, azide and alkyne reactive

groups do not react each other in solution, but easy to react each other after oligomerization. This character is very suitable to prevent non-specific

oligomerization, while accelerating the specific oligomerization. </p> 

     <center>
       <figure>
         <img src="http://openwetware.org/images/c/c5/OCK_ConstraintClick-Todai.png" width="300" height="300" style="border:solid 1.5px black;">
        <br>
        <br>

       </figure>
     </center>
    <br>

<p class="paragraph"> To detect whether click reaction happened, OCKs have two fluorescent dyes, Cy3 and Cy5. These fluorescent dyes are adhere to the azide-modified site

and the alkyne-modified site respectively (dye - dye distance is roughly 1 - 2 nm), so FRET signal between Cy3 and Cy5 is observed if click reaction occurred. </p> 

   <center>
     <table cellpadding="0">
       <tr>
       <td>
       <figure>
         <img src="http://openwetware.org/images/a/a2/OCK_Cy3fluorescent-Todai.png" width="300px" height="300px" >

       </figure>
       </td>



       <td>
       <figure>
         <img src="http://openwetware.org/images/8/83/OCK_Cy5FRET-Todai.png" width="300px" height="300px" >

       </figure>
       </td>
       </tr>
     </table>
   </center>

<!--References--> 

  <article>
    <h1 class="title"><a name="References">&nbsp;References</a></h1>
    <br>

    <div>     
       <div class="reference-title">
       <a name="desref-1">
       [1] Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures 
       </a>
       </div>
          <div class="reference-author">
          Martin Langecker, Vera Arnaut, Thomas G. Martin, Jonathan List, Stephan Renner, Michael Mayer, Hendrik Dietz, and Friedrich C. Simmel
          </div>
             <div class="reference-journal">
             Science 16 November 2012: 338 (6109), 932-936. [DOI:10.1126/science.1225624] 
             </div>
    </div>

<br> 

    <div>     
       <div class="reference-title">
         <a name="desref-2">
         [2] The Structure of DNA−Liposome Complexes
         </a>
       </div>
          <div class="reference-author">
          Danilo D. Lasic,Helmut Strey, Mark C. A. Stuart, Rudolf Podgornik,  and Peter M. Frederik
          </div>
             <div class="reference-journal">
             Journal of the American Chemical Society 1997 119 (4), 832-833 
             </div>
    </div>
    <br>
  
       <div class="reference-title">
       <a name="desref-3">
       [3] A logic-gated nanorobot for targeted transport of molecular payloads. 
       </a>
       </div>
          <div class="reference-author">
          S. M. Douglas, I. Bachelet, G. M. Church
          </div>
             <div class="reference-journal">
             Science 335, 831 (2012)
             </div>
    </div>



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