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<p class="paragraph">In order to simplify the condition of ring structures, we designed the Motor-Monomer with a ring structure at first (fig??). Strand a, a’, b, and b’are ssDNAs embedded on the ends of the Motor-Monomer X and Y, like figXX. Strand a-b pair and a'-b' pair are partially complementary. In contrast, strand a-a' pair and b-b' pair are completely complementary. By using these staples, Motor-Monomers becomes hetero-polymer. It helps us prevent the Motor-Monomers from polymerizing before building ring structures, and increase the stiffness of the Motor-Polymer because it enables the Motor-Monomers to get close to each other.</p> | <p class="paragraph">In order to simplify the condition of ring structures, we designed the Motor-Monomer with a ring structure at first (fig??). Strand a, a’, b, and b’are ssDNAs embedded on the ends of the Motor-Monomer X and Y, like figXX. Strand a-b pair and a'-b' pair are partially complementary. In contrast, strand a-a' pair and b-b' pair are completely complementary. By using these staples, Motor-Monomers becomes hetero-polymer. It helps us prevent the Motor-Monomers from polymerizing before building ring structures, and increase the stiffness of the Motor-Polymer because it enables the Motor-Monomers to get close to each other.</p> | ||
<p class="paragraph"><img src="http://openwetware.org/images/3/3f/Design1kashiwa.PNG" width=" | <img src="http://openwetware.org/images/8/8d/Design7kashiwa.PNG" width="500px"> | ||
<p class="paragraph">However, the persistence length of the ssDNA is only a few nanometers while the length of Flip-Tile is 34 nm. Therefore, it is difficult to make ring structures after we embed ring staples into the ends of the Motor-Monomers. To resolve this problem, we introduced short ssDNAs (named cover staples), which were partly complementary to the ring staples to form double strand DNA, which are much more rigid structure (the persistence length of dsDNA is about 50 nm). Take everything into consideration, we mixed the ring staples and the cover staples at first. Then, we mixed this complex with the Flip-tile structure, and could embed the ring structure into the Flip-Tile structure. Subsequently, we dissociated the cover staples from ring structure by raising the temperature to the melting temperature of the cover staples. In this way, we could get the Motor-Monomer which had a closed staple ring structure.</p> | |||
<p class="paragraph"><img src="http://openwetware.org/images/3/3f/Design1kashiwa.PNG" width="300px" align="left">Next, when the Initiator strands are released, the Monomer opens its ring via strand displacement. Then, the free end of the Monomer forms a DNA duplex by a displacement with one end of another Monomer and the other end becomes free. In this way, the Monomers polymerize one by one.</p> | |||
<br> | <br> | ||
<p class="paragraph">2.Bending stiffness</p> | <p class="paragraph">2.Bending stiffness</p> | ||
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<head> <style> h1.sub {
font-size:24px;
} </style> </head> <font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font><body> <br> <br> <a name="2"></a> <p class="paragraph">In order to achieve the <a href=Project#3>project goal</a>, we designed two constructs using DNA origami; the Receptor for the sensing system and the Motor-Monomer for the moving system.</p> <br> <h1 class="title"><a name="receptor"> The Receptor</a></h1>
<img src="http://openwetware.org/images/6/67/Designreceptor.jpg" width="200px" margin="-10px 0px" align="right">
<p class="paragraph">The Receptor was developed to recognize an external signal on the liposome surface upon which it releases the Polymerization Initiator inside. For this, we designed a pair of heterounits for the Receptor, the Receptor E and the Receptor A. They dimerize when an external signal is recognized and cause the release of the Polymerization Initiator (In this case, the single-stranded DNA).</p>
<h1 class="sub">Requirements</h1> <p class="paragraph">To develop the Receptor, the following three mechanisms have to be considered :</p>
<p class="paragraph">1. Embedding of Receptor on the liposome <br>2. Recognization of the external signal <br>3. Release of the Polymerization Initiator internally</p>
<h1 class="sub">Structure</h1>
<figure> <img src="http://openwetware.org/images/c/c1/Receptorimage.png" width="400px"> <figcaption>fig.1</figcaption>
<p class="paragraph">Provisory structure of Receptor for experiments. We attached aptamer sequence(red) and Initiater(orange) to OCK Structure; composed of floater domain(blue) and membrane integrating domain(yellow). Green staple has connecting role between Receptor and Initiater.</p>
<h1 class="sub" clear="both">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> <br> <p class="paragraph">1. Embedding of Receptor on the liposome</p>
<p class="paragraph">The natural Receptor often ends up sticking on the outside of the liposome and rarely penetrates it. Therefore, we modified the Receptor with cholesterol so that it penetrates the liposome and can access the interior.</p> <br> <p class="paragraph">2. Recognization of the external signal. </p>
<img src="http://openwetware.org/images/7/72/Thrombin.png" width="200px" align="right"> <p class="paragraph">We used Thrombin as an external signal in this experiment. Thrombin, a serine protease, has specific sites where different DNA aptamers bind. We inserted thrombin-binding DNA aptamer sequences into the Receptor heterounits. Furthermore, we bound a staple strand to each heterounit, two strands to be complementary to each other.</p> <p class="paragraph">Therefore, when the aptamers in the Receptor heterounits bind to the binding sites of thrombin, the heterounits join together to form a dimer. We should keep in mind that we have to optimize the thrombin concentration; an excess of thrombin causes errors because of the Receptor E and the Receptor A binding to the separate thrombin.</p> <br> <p class="paragraph">3. Release of the Polymerization Initiator.</p> <p class="paragraph">We designed the Polymerization Initiator as a single-stranded DNA which is partially complementary to a specific domain of the Receptor E. When the Receptor heterounits form a dimer, the strand displacement occurs to form a more stable DNA duplex and the initially-bound ssDNA is released, which works as the Polymerization Initiator.<a name="1"> </a></p><br> <br> <h1 class="title"><a name="motormonomer"> The Motor-Monomer</a></h1>
<p class="paragraph">The Motor-Monomer was developed to begin the polymerization when it caught the Polymerization Initiator. For this, we designed two types of the Motor-Monomers, Motor-Monomer X and Motor-Monomer Y, which have closed rings; when the Polymerization Initiator is caught, the Motor-Monomers join to form the Motor-Polymer by opening their ring structures one after another.</p> <center> <table><tr> <td><img src="http://openwetware.org/images/0/04/Designmonomer.png" width="350px"></td> <td><img src="http://openwetware.org/images/e/ef/Designpolymer.jpg" width="350px"></td> </tr></table> </center>
<h1 class="sub">Requirements</h1> <p class="paragraph">To develop the Motor-Monomer, the following two requirements have to be considered:</p>
<p class="paragraph">1. The Motor-Monomers should not polymerize without the Initiator. <br>2. The Motor-Polymer should be stiff enough to deform the liposome.</p>
<h1 class="sub">Structure</h1> <p class="paragraph">Provisory structure of Motor-Monomer for experiments. We attached six connecting staples(orange) to Flip-tile(blue). The length of connecting staple was determined for making ring formation.( Flip-tile_Science2012) </p> <center> <img src="http://openwetware.org/images/9/97/Monomerimage.png" width="350px"> <img src="http://openwetware.org/images/9/95/Design3kashiwa.png" width="350px"> </center> <h1 class="sub">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> <br> <p class="paragraph">1.Controllability</p> <center> <p><img src="http://openwetware.org/images/b/b0/Design8.png" width="350px"> <img src="http://openwetware.org/images/a/aa/Design2.png" width="350px"></p> </center>
<p class="paragraph">In order to simplify the condition of ring structures, we designed the Motor-Monomer with a ring structure at first (fig??). Strand a, a’, b, and b’are ssDNAs embedded on the ends of the Motor-Monomer X and Y, like figXX. Strand a-b pair and a'-b' pair are partially complementary. In contrast, strand a-a' pair and b-b' pair are completely complementary. By using these staples, Motor-Monomers becomes hetero-polymer. It helps us prevent the Motor-Monomers from polymerizing before building ring structures, and increase the stiffness of the Motor-Polymer because it enables the Motor-Monomers to get close to each other.</p>
<img src="http://openwetware.org/images/8/8d/Design7kashiwa.PNG" width="500px"> <p class="paragraph">However, the persistence length of the ssDNA is only a few nanometers while the length of Flip-Tile is 34 nm. Therefore, it is difficult to make ring structures after we embed ring staples into the ends of the Motor-Monomers. To resolve this problem, we introduced short ssDNAs (named cover staples), which were partly complementary to the ring staples to form double strand DNA, which are much more rigid structure (the persistence length of dsDNA is about 50 nm). Take everything into consideration, we mixed the ring staples and the cover staples at first. Then, we mixed this complex with the Flip-tile structure, and could embed the ring structure into the Flip-Tile structure. Subsequently, we dissociated the cover staples from ring structure by raising the temperature to the melting temperature of the cover staples. In this way, we could get the Motor-Monomer which had a closed staple ring structure.</p> <p class="paragraph"><img src="http://openwetware.org/images/3/3f/Design1kashiwa.PNG" width="300px" align="left">Next, when the Initiator strands are released, the Monomer opens its ring via strand displacement. Then, the free end of the Monomer forms a DNA duplex by a displacement with one end of another Monomer and the other end becomes free. In this way, the Monomers polymerize one by one.</p> <br> <p class="paragraph">2.Bending stiffness</p>
<p class="paragraph">Our initial approach was to design three closed rings in the Motor-Monomer to develop enough stiffness by limiting flexibility. Subsequently, we found that three-rings-Monomers are likely to form the branched Polymer, based on probability theory. Therefore, we try a new approach according to the reported reference (Iinuma et al)¹.</p>
<p class="paragraph"> <ul><li>Firstly, we planed to introduce concavo-convex surface to ensure the geometry specificity of the adherend. Secondly, additional short sticky strands (a few bases) were introduce to strengthen the binding stability.</li> </ul> </p></body> <img src="http://openwetware.org/images/8/81/Design4kashiwa.png" width="250px"> <br> <br> <h1 class="title">Reference</h1> <p>1.Science. 2014;344(6179):65-9.Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Iinuma R1, Ke Y, Jungmann R, Schlichthaerle T, Woehrstein JB, Yin P.</p>
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