Biomod/2012/Tianjin/Project/YDNA

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<ul>

  	    <li class='active '><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin'><span>Home</span></a></li>
  	    <li class='active '><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Team'><span>Team</span></a>
       </li>
  	    <li class='has-sub'><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Project/Background'><span>Designs</span></a>
  	        <ul>
  	            <li><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Project/Background'><span>Background</span></a></li>
               <li><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Project/LogicGate'><span>The Logic Gate</span></a></li>
               <li><a href="http://openwetware.org/wiki/Biomod/2012/Tianjin/Project/YDNA"><span>Y-DNA</span></a></li>
               <li><a href="http://openwetware.org/wiki/Biomod/2012/Tianjin/Project/Origami"><span>The Origami Amplifier</span></a></li>
 	        </ul>

</li>

<li class='has-sub'><a href='#'><span>Experiments and Results</span></a>

  	        <ul>
  	            <li><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Result/LogicGate'><span>The Logic Gate</span></a></li>
               <li><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Result/YDNA'><span>Y-DNA</span></a></li>
               <li><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Result/Origami'><span>The Origami Amplifier</span></a></li>

</ul>

       </li>   	
  	    <li class='active'><a href="http://openwetware.org/wiki/Biomod/2012/Tianjin/Protocol"><span>Protocol</span></a>

</li>

           <li class='active'><a href='http://openwetware.org/wiki/Biomod/2012/Tianjin/Attributions'><span>Attributions</span></a></li>

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<html>

<div id="tianjin-right-content"> <ul><li> <a href="#" title="#">Background</a> </li><li> <a href="#" title="#">The Logic Gate</a><div class="tianjin-description">1xxx</div> </li><li> <strong class="selflink">Y-DNA</strong><div class="tianjin-description">2xxx</div> </li><li> <a href="#" title="#">The Origami Amplifier</a><div class="tianjin-description">3xxx</div> </li></ul> </div>

<div id="text-content"> </html>

Y-DNA

The Y-DNA was synthesized from three ssDNAs, each of which has partial complementary sequences to the other two ssDNAs. From last part, we know that our logic gate have a high selectivity and is a precise logic gate. It occurred to us that we can utilize this logic gate to control the formation of Y-DNA by making the logic gate one of the ssDNAs. Without adding any ions, the logic gate remain in its original structure, which leave no sequence available to form Y-DNA with other two ssDNAs. If we add Cu²⁺, the Cuzyme will self-cleave and reveal a long sequence, making it feasible to form a Y-DNA. Moreover, we knew how many logic gate have self-cleave according to our research in the previous part, so we can control the cut logic gate generated. In this way, we can control when and how many Y-DNA be produced.

Because we want to use the logic gate to form Y-DNA, we need to specifically investigate its behavior in Y-DNA formation. Using NuPack (Figure X), we predicted that our original logic gate may fail. First, we simulated the scenario when there is only the logic gate and two ssDNAs in the solution. The result showed that the three DNA strands formed Y-DNA, because the XXX region is too long, so that the two ssDNA structure can open the logic gate through strand replacement even without the activation of Cu²⁺. Therefore, we add a stem-loop structure by altering the sequence and adding several basepairs. In this way, Y-DNA can only form after adding Cu²⁺.

Subsequently, we simulated whether the improved logic gate would form a stable Y-DNA after EcoRI digestion. Again, the result didn’t give us a positive feedback. According to Figure (X), the sequence after digestion would be too short and too close to 8-17’s catalytic core to form a stable duplex. Therefore, we lengthened the sequence to ensure a stable Y-DNA after digestion. The function of this optimized new logic gate were investigated in the wet lab.

In conclusion, we not only repeated the conventional method of producing Y-DNA, but also introduced the logic gate to control when and how many Y-DNA are produced in the system. Next we’ll investigate how to make the Y-DNA polymerize in a controlled way.

Polymerization

The Y-DNAs we produced only contains a blunt end, which makes it unable to polymerize. In order to polymerize, we need to create a sticky end in each branch, then use T4 ligase to link these ends. In this way, the Y-DNAs are able to connect with each other, and form a large polymer. The Y-DNA is unable to form a linear structure due to its own structure. We thought that AFM can be used to determine its property.

There are two ways to have the sticky end: 1. designing it in the ssDNA from the beginning; 2. Using digestion to create the sticky end from blunt end after the formation of Y-DNA. In our experiment, we used the second way. Why? Because polymerization requires ligase to link Y-DNA together. Ordinarily, the sequences we ordered from companies contains a hydroxyl group at 5’ instead of a phosphate group that is essential for T4 ligation. While the sticky end created using enzyme digestion contains the phosphate group. Therefore, we chose to create the sticky end after the formation of Y-DNA.

EcoRI is used to digest Y-DNA, because EcoRI leaves a sticky end of AATT, which makes all sticky ends complimentary with each other. This eliminates the need to use multiple enzymes.

We know that 8-17 can only cleave substrate at a RNA site, so if the corresponding cleavage site of 8-17 on the Y-DNA is replaced with RNA, we are able to use the sensor to control the decomposition of Y-DNA. By adding Pb²⁺ into the Y-DNA polymer, 8-17 is activated to cut one branch in every Y-DNAs. As a result, the whole polymer structure will disintegrate. In this way, sensor shows its outstanding role in controlling the formation, polymerization and decomposition of Y-DNA.

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Biomod/2012/Tianjin/Project/YDNA

Y-DNA

Polymerization

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