Biomod/2013/Todai/DesignOCK
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<li><a href="http://openwetware.org/wiki/Biomod/2013/Todai">Home</a>
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<li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Project">Project</a>
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<li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Design">Design</a>
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<li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">Result</a>
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<li><a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment">Experiment</a>
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<a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Contents">
Contents</a></li>
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<a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#PilotStudy">
Pilot Study</a></li>
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<a href="http://openwetware.org/wiki/Biomod/2013/Todai/Experiment#Protocols">
Protocols</a></li>
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<ul>
<li><a href="#1.Oligomeric Cell Killer">1. Oligomeric Cell Killer</a>
<ul>
<li><a href="#1-1.General design">1-1.<br>General design</a>
</li>
</ul>
</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>
</div>
<!--Design-->
<h1 class="big-title"><a name="Design"> Design</a></h1> <br>
<!--1. Oligomeric Cell Killer-->
<h1 class="heading"><a name="1.Oligomeric Cell Killer"> 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>
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<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/8/88/OCK_flow-Todai.png" width="300px" height="300px" style="border:solid 1.5px black;">
</figure>
</td>
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<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"> 1. Structure</a></h1> <br>
<p class="paragraph">
The detailed structure and scale of OCK are shown below. OCK 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>
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<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" >
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<img src="http://openwetware.org/images/4/4d/Assemblyup-Todai.png" width="300px" height="300px" >
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<img src="http://openwetware.org/images/3/37/Assembly1-Todai.png" width="300px" height="300px" >
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<!--2. Function-->
<article> <h1 class="title"><a name="2. Function"> 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">2.1 Sites for penetration</h2> <h3 class="title"> 1)Cholesterol sticking sites</h3>
<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 equipp ed 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>
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<figure>
<img src="http://openwetware.org/images/f/f6/OCK_anchoring-Todai.png" width="300px" height="300px" >
<figcaption>[Mechanism of binding-1]</figcaption>
<figcaption>
dimerized by the strands <br>
sticking out from the top
</figcaption>
</figure>
</td>
<td>
<figure>
<img src="http://openwetware.org/images/2/28/OCK_penetrate-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>
</td>
</tr>
</table>
</center>
<h3 class="title"> 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 [citation]. 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), 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 the stick-domain is easy to penetrate into membrane.
</p>
</article> <br> <!--2.2 Recognition system (safely lock system)--> <h2 class="title">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 [[citation]]. 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.
</p>
</article> <br> <!--2.3 Sites for oligomerization--> <article> <h2 class="title">2.3 Sites for oligomerization</h2> <h3 class="title"> 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> <!--necessary to change graphics-->
<center>
<table cellpadding="0">
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<figure>
<img src="http://openwetware.org/images/3/35/OCK_prototype-Todai.png" width="300px" height="300px" >
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<figure>
<img src="http://openwetware.org/images/1/1d/OCK_current-Todai.png" width="300px" height="300px"
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<!--necessary to change graphics-->
<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">
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<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>
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</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; position:relative;
left:35px;">
</figure>
<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>
<h3 class="title"> 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-right:-10px;margin-bottom:10px; position:relative;
left:-35px;">
</figure>
<br>
<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>
<center>
<table cellpadding="0">
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<td>
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<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>
</center>
</article> <br> <!--2.4 Pore formation--> <h2 class="title">2.4 Pore formation</h2> <h3 class="title"> 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 devises 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 窶� 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 (-C竕｡C) and azide (-N=N=N) 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 ~ 16 /M/s = 17h@0.1uM 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>
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<figure>
<img src="http://openwetware.org/images/a/a2/OCK_Cy3fluorescent-Todai.png" width="300px" height="300px" >
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<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"> References</a></h1>
<br>
<div>
<div class="reference-title">
<a name="desref-1">
[1] The Structure of DNA竏鱈iposome 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>
<div class="reference-title">
<a name="desref-2">
[2] 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-3">
[3] Multifactorial Modulation of Binding and Dissociation Kinetics on Two-Dimensional DNA Nanostructures
</a>
</div>
<div class="reference-author">
Alexander Johnson-Buck, Jeanette Nangreave, Shuoxing Jiang, Hao Yan, and Nils G. Walter
</div>
<div class="reference-journal" style="font-style:italic;">
Nano Letters 2013 13 (6), 2754-2759
</div>
</div>
<br>
<div>
<div class="reference-title">
<a name="desref-4">
[4] 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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