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<p class="paragraph">4. Forming the polymerizing ability enough to deform the liposome</p>
<p class="paragraph">4. Forming the polymerizing ability enough to deform the liposome</p>
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<p class="paragraph">
As described in Structure, the Motor-Monomer has the finger-pointing shape and the hole to embed streptavidin in. These features enable the Motor-Monomers to connect with others like Lego blocks, which makes the Motor-Polymer stiffer. There are two types of structures Motor-Polymer would take as shown in figureX; both are supposed to be stiff enough.
As described in Structure, the Motor-Monomer has the finger-pointing shape and the hole to embed streptavidin. These features enable the Motor-Monomers to connect with others like Lego blocks, which makes the Motor-Polymer stiffer. There are two types of structures Motor-Polymer would take as shown in figureX; both are supposed to be stiff enough.
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<body> <font face="Futura,Arial,Frutiger" font size="24px">DESIGN</font> <br> <br> <p class="paragraph"> In order to achieve the project goal, we designed two constructs; the Receptor for the sensing system and the Motor-Monomer for the moving system. For design, we took advantage of our early trial experiences, which were done with the DNA origami structure used in the past (see Early Trial section). Especially we focused to design the structure to work effectively. <a name="2">&nbsp;</a></p>

<h1 class="title"><a name="receptor">&nbsp;The Receptor</a></h1>

<p class="paragraph"> The Receptor was developed to be inserted in the liposome with two functions: recognize an outside signal and activate the Motor-Monomers inside to start polymerization. </p> <p class="paragraph"> For this, we designed the Receptor that consists of the Activator and the Wall. The Activator has the ability to activate the Motor-Monomers to polymerize. Before the recognition of the outside signal, however, the Wall, which is connected with the Activator with linker, physically blocks the Activator to associate with Motor-Monomers. Upon outside signal recognition, the Wall separates from the Activator and the Activator is released into the liposome, resulting the activation of Motor-Monomers (the activation mechanism is described in the Motor-Monomer part). </p>

<br> <h1 class="big">Requirements</h1> <p class="paragraph">To develop the Receptor, the following three mechanisms have to be considered:</p>

<p class="paragraph"> <p class="menu">1. Embedding the Wall in the liposome <p class="menu">2. Linking the Activator to the liposome</p> <p class="menu">3. Connecting the Activator and the Wall</p> <p class="menu">4. Separating the Wall from the Activator</p> <p class="menu">5. Releasing the Activator into the liposome</p> </p>

<br> <h1 class="big">Structure (The Wall)</h1> <img src="http://openwetware.org/images/1/15/WallKashiwa.gif" width="325px" height="309px" align="right"> <br> <p class="paragraph"> The Wall is constructed using DNA origami method; made of 18 honeycomb structures. It is composed with the body and the extension bar. The body part is U-shaped and the extension bar part is 16 nm long to penetrate into liposome membrane. </p>

<p class="paragraph"> The upper ends of the body that attach to the liposome membrane are smooth, while the other downside ends are rough so that they prevent the Wall from self-aggregation. </p> <br clear="right">

<br> <h1 class="big" 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 the Wall in the liposome</p> <p class="paragraph"> The whole Wall structure is so big it cannot penetrate into the liposome. We therefore penetrated one honeycomb structure extended from the body part of the Wall into inner membrane of liposome. Moreover, the upper side of the Wall is modified with cholesterol to support the embedment into the liposome. </p>

<br> <p class="paragraph">2. Linking the Activator to the liposome</p> <p class="paragraph"> The Activator is a restriction enzyme named Hind&#8546;. We linked the Activator to the liposome by embedding the following Anchor into the liposome membrane. </p> <p class="paragraph"> The Anchor is composed of a double-stranded DNA chain and MISTIC protein (Membrane Integrating Sequence for Translation of Integral Membrane Protein Constructs). The reason we chose MISTIC was that it folds into the liposome membrane easily compared to other membrane proteins*. The double-stranded DNA chain is for linking the MISTIC and the Activator. </p>

<br> <p class="paragraph">3. Connecting the Activator and the Wall</p> <p class="paragraph"> To prevent the Motor-Monomers from associating the Activator before signal induction, the Activator has to be covered by the Wall, therefore, we connected the Activator and the Wall. </p> <p class="paragraph"> For this, we bound biotin-modified staple strands to the Activator and the Wall. When divalent streptavidin (mutant of wild type tetravalent streptavidin) is added, the Activator and the Wall are combined by the biotin-streptavidin binding. </p>

<br> <p class="paragraph">4. Separating the Wall from the Activator</p> <p class="paragraph"> To activate the Motor-Monomers and start the polymerization, the Wall has to separate from the Activator upon recognition of the outside signal. Our ultimate goal is to start patrolling of PoLICe when it recognizes the cancer markers. However it is much complicated and difficult to use native cancer marker as the signal. We therefore used much simple model signal: the restriction enzyme. </p> <p class="paragraph"> When the restriction enzyme is added, it cuts the recognition site on the DNA staple strand that connects the Wall and the Anchor. The Wall consequently separate from the Anchor and the Motor-Monomers are able to associate with the Activator. </p>

<br> <p class="paragraph"> 5. Releasing the Activator in the liposome </p> <p class="paragraph"> For efficient interaction between the Activator and the Motor-Monomers, the Activator had to be released into the liposome because the Motor-Monomers are less likely to access the membrane anchored Activator because of steric hindrance. </p> <p class="paragraph"> To release the Activator, we put another restriction enzyme in the liposome. The restriction enzyme cuts the double-stranded DNA chain that links the Activator and the Anchor, and the Activator is therefore released. <a name="1">&nbsp;</a> </p>

<br> <br>

<h1 class="title"><a name="motormonomer">&nbsp;The Motor-Monomer</a></h1>

<p class="paragraph"> The Motor-Monomers are developed to start polymerization to form the Motor-Polymer when the Receptor recognizes the outside signal, and consequently deform the liposome. </p> <p class="paragraph"> For this, we designed the Motor-Monomer equipped with biotin and streptavidin, biotin at the top part and streptavidin at the middle part. The streptavidin is inactivated before being activated by the Activator; biotin binding capacity is lost. The Activator of the Receptor restores the binding capacity and the Motor-Monomers therefore start polymerizing after activation by the Activator. </p> <br> <h1 class="big">Requirements</h1> <p class="paragraph">To develop the Motor-Monomer, the following mechanisms have to be considered:</p>

<p class="paragraph"> <p class="menu">1. Encapsulating the Motor-Monomers into the liposome</p> <p class="menu">2. Inactivating the polymerization ability of the Motor-Monomer</p> <p class="menu">3. Restoring the polymerizing ability of the Motor-Monomer with the Activator</p> <p class="menu">4. Forming the Motor-Polymer stiff enough to deform the liposome</p> </p>

<br> <h1 class="big">Structure</h1> <p class="paragraph"> The Motor-Monomer is constructed using DNA origami; made up of 6 honeycomb structures. It is shaped like finger-pointing and 2 honeycomb structures are longer than the others for 65 nm. There is a hole in the middle part of the Motor-Monomer to embed streptavidin. The length of each part is shown in the picture. </p> <p class="paragraph">The ends of the honeycomb structures are rough to prevent from cohesion.</p>

<br> <h1 class="big">Strategy</h1> <p class="paragraph">The details of our strategy to fulfill the requirements are described below.</p> <br> <p class="paragraph">1. Encapsulating the Motor-Monomers into the liposome</p> <p class="paragraph"> If the Motor-Monomers and the Activator are put into the liposome at the same time, the Motor-Monomers are activated before the signal induction. We therefore planned to connect the Activator and the Wall in the liposome first, and then fuse that liposome to another liposome containing the Motor-Monomers. </p>

<br> <p class="paragraph"> 2. Inactivating the polymerization ability of the Motor-Monomer </p> <img src="http://openwetware.org/images/5/50/Biotinanddesthiobiotin.png" width="400px" height="142px" align="right"> <p class="paragraph"> To prevent the Motor-Monomers from polymerizing in inactivated state, despite allowing polymerization in activated state, we used the following approach. </p> <p class="paragraph"> We used the difference of the binding strength between the biotin and desthiobiotin to streptavidin. Desthiobiotin is a biotin analogue that binds less tightly to biotin-binding proteins and is easily displaced by biotin*. </p> <p class="paragraph"> For the inactivation process, two complementally oligonucleotides having biotin or desthiobiotin were hybridized and the resulting duplex were bound with streptavidin. Supported by biotin strand, the desthiobiotin are stably tethered to the streptavidin, giving resistance to biotin displacement. </p>

<p class="paragraph"> Finally, this inactivated streptavidins were equipped to Motor-Monomer preventing the polymerization before activation. </p> <br clear="right">

<br> <p class="paragraph">3. Restoring the polymerizing ability of streptavidin with the Activator</p> <p class="paragraph"> As mentioned above, desthiobiotin-streptavidin binding is supported by stable biotin-avidin binding through the hybridization of DNA duplex. We supposed that by cutting the DNA duplex, desthiobiotin loses its supporter and is easily dissociated from the streptavidn, allowing the binding of biotin on the different Motor-Monomer. </p> <p class="paragraph"> Hind&#8546;, a restriction enzyme cleaves the specific DNA sequence, was chosen as the Activator to cut the DNA duplex. After cleavage by Hind&#8546;, the length of the remaining sequence is short, therefore dehybridization of the duplex are supposed to be occurred easily. </p>

<br> <p class="paragraph">4. Forming the polymerizing ability enough to deform the liposome</p> <p class="paragraph"> As described in Structure, the Motor-Monomer has the finger-pointing shape and the hole to embed streptavidin. These features enable the Motor-Monomers to connect with others like Lego blocks, which makes the Motor-Polymer stiffer. There are two types of structures Motor-Polymer would take as shown in figureX; both are supposed to be stiff enough. </p> <p class="paragraph"> Moreover, two single-stranded DNA staples complementary to each other are bound to different parts on the Motor-Monomer (figureX). Those staples form duplexes with the staples on others, which strengthen the connection of the Motor-Monomers. </p>

<br> <br> <h2 class="reference">Reference</h2> <p class="reference">1.M. Douglas et al, “A logic-gated nanorobot for targeted transport of molecular payloads”, Science, 2012 Feb 17; 335(6070): 831-4. <br>2.Science. 2014;344(6179):65-9.Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT.Iinuma R, Ke Y, Jungmann R, Schlichthaerle T, Woehrstein JB, Yin P.</p> </body>

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