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Biomod/2014/ASU

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< <a href="/wiki/Biomod" title="Biomod">Biomod</a> | <a href="/wiki/Biomod/2014" title="Biomod/2014">2014</a>
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  • /
.project_box > img { margin-left: 90px; }
  1. protocols, #polymers_protocols, #origami_protocols, #reaction_protocols, #nanocontainer_protocols, #imaging_protocols {
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margin-bottom: 30px; margin-left: 30px; margin-top: 20px; } li > ul { margin-left: 10px; } /*achievement*/ .achievement_box{ background: none repeat scroll 0 0 #FFFFFF; color: #000000; display: block; float: left; font-size: 18px; font-weight: 300; line-height: 1.6; margin-left: 180px; margin-right: auto; overflow: hidden; padding: 10px; width: 50%; }
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<p class = "serif" style = "color: #000000; font-size: 25px;">Overall Design</p>


<p style = "color: #000000; font-size: 18px;">The goal of this project was to develop a method to make a DNA structure with the same capabilities for protein binding as a protein antibody. We aimed to design a DNA structure with the ability to specifically bind to a target by exploiting the 3 dimensional surface of the protein. </p>

<p style = "color: #000000; font-size: 18px;">Traditional antibodies are comprised of several domains comprising light chains and heavy chains (Figure D1). The antibody consists of a variable domain and a constant domain (Figure D2) in which the variable domain allows the antibody to bind to foreign antigens.</p>

<img src = "http://openwetware.org/images/2/22/Screen_Shot_2014-10-25_at_8.32.26_PM.png">

<p style = "color: #000000; font-size: 18px;">One of the key design strategies for our project was to create a wide variety of DNA ‘antibodies’ by adding random sequences into oligonucleotides which are then incorporated into a DNA tile. These oligonucleotides will then form into a diverse range of secondary structures. The aim is that tiles with a correct combination of oligonucleotides will coordinate among themselves to bind a target protein. Using these DNA tiles and oligonucleotides, we hope to mimic the antibody-antigen model as depicted in Figure D3:</p>
<img src = "http://openwetware.org/images/3/35/Screen_Shot_2014-10-25_at_9.03.08_PM.png">
<p class = "serif" style = "color: #000000; font-size: 25px;">DNA Tile Design</p>


<p style = "color: #000000; font-size: 18px;">Previous studies have shown that multiple aptamers for alpha-thrombin on the edge of a DNA tile could bind a protein multivalently at specific distances from one another2. However, we have focused on multivalency to the geometric face of a DNA nanostructure, rather than the edge, because (1) this limits the number of possible conformations that a set of strands can take, simplifying the selective process, (2) it allows for two-dimensional rather than solely one-dimensional spacing of aptamer elements, and (3) planar spaces on larger origami structures generally have a large amount of functionalizable space, while edges are frequently nonexistent or busy.</p> <p style = "color: #000000; font-size: 18px;">The DNA tile structure was designed to be 28 nm x 9 nm. The tile consisted of 4 DNA bundles (helices) consisting of a total of 84 base pairs. The 4 oligonucleotides are incorporated in the 2nd and 3rd bundles as shown below in Figure D4: </p>
<img src = "http://openwetware.org/images/1/11/Screen_Shot_2014-10-25_at_9.05.40_PM.png">
<p style = "color: #000000; font-size: 18px;">Alpha-thrombin was chosen as the target protein for this project as it was well characterized and had been used previously in studies investigating aptamer binding. The size of the DNA tile was chosen so that oligonucleotides could be inserted at a spacing relative to the dimensions of alpha-thrombin shown below. </p>
<img src = "http://openwetware.org/images/8/81/Screen_Shot_2014-10-25_at_9.07.16_PM.png">

<p class = "serif" style = "color: #000000; font-size: 25px;">Oligonucleotide Design</p>


<p style = "color: #000000; font-size: 18px;">We had to consider several factors in the design of the oligonucleotide strands to maximize the chances for the binding of the DNA antibody to alpha-thrombin. In order to achieve this we created 46 base pair oligonucleotides containing a 20 base pair random DNA sequence in the middle of the strand. Two known DNA sequences flank the random oligonucleotide sequence: a conserved 15 base pair region at the 5’ end of each sequence allows for amplification by PCR, and another 11 base pair region at the 3’ end is responsible for correct insertion into the DNA tile and also acts as a primer during PCR.</p>
<img src = "http://openwetware.org/images/8/8f/Screen_Shot_2014-10-25_at_9.09.28_PM.png">
<p class = "serif" style = "color: #000000; font-size: 25px;">Selection Process</p>


<p style = "color: #000000; font-size: 18px;">One of the key challenges in this process is the characterization of binding between proteins and DNA antibodies. A pool of DNA antibodies were created using random sequences which result in many thousands of different oligonucleotide combinations and sequences on each DNA tile. Each of these combinations of random oligonucleotides will create unique and diverse secondary and quaternary structures on the DNA tile as the oligonucleotides interact with themselves and one another. The protein will selectively bind DNA antibodies with a complementary structure. This will allow for selection of single DNA antibodies from a pool of many thousands of variations. Electrophoretic mobility shift assay (EMSA) and affinity chromatography was used to characterize this process and to identify the DNA antibodies that have bound to the target protein. Randomized oligonucleotides which compose the selected antibodies can then be amplified by PCR and then reutilised in a new generation of tiles composed of oligonucleotides from the enriched pool. This process of selection can then be repeated in a similar method to systematic evolution of ligands by exponential enrichment (SELEX). Multiple cycles of this procedure can be performed to narrow the pool and enhance specific binding.</p>
<img src = "http://openwetware.org/images/f/fe/Screen_Shot_2014-10-25_at_9.11.35_PM.png">
<p class = "serif" style = "color: #000000; font-size: 25px;">DNA Antibody Potential Applications</p>


<p style = "color: #000000; font-size: 18px;">DNA antibodies due to their versatility in structure and function can be used for a variety of applications such as:</p> <p style = "color: #000000; font-size: 18px;">     1) Immunology and drug targeting/delivery.</p> <p style = "color: #000000; font-size: 18px;">     2) Low cost high stability antibody substitute</p> <p style = "color: #000000; font-size: 18px;">     3) Enhanced programmable antibodies</p> <p style = "color: #000000; font-size: 18px;">     4) Precipitation/tagging of proteins</p> <p style = "color: #000000; font-size: 18px;">     5) Incorporation into larger DNA nanostructures </p>
<p class = "serif" style = "color: #000000; font-size: 25px;">Conclusion of Project Design</p>


<p style = "color: #000000; font-size: 18px;">As shown above, our project design not only characterizes the DNA antibody structure, but also the binding properties relative to the target protein. In our experiment, we utilize several conformations of oligonucleotides at the insertion site of the DNA tile in order to explore different pathways of constructing DNA antibodies.</p>

<a class="fancybox.iframe yt" href="//www.youtube.com/embed/6olQTUOQjCk?list=UUwmG88y6SZpJSxGwicxZEdw"><img src="http://openwetware.org/images/thumb/4/4e/Simulation_vid.jpg/800px-Simulation_vid.jpg"></a>


<p class = "serif" style = "color: #000000; font-size: 25px;">References:</p>


<p style = "color: #000000; font-size: 18px;">     1. Barclay A (2003). "Membrane proteins with immunoglobulin-like domains – a master superfamily of interaction </p> <p style = "color: #000000; font-size: 18px;">     molecules". Semin Immunol 15 (4): 215–223</p> <p style = "color: #000000; font-size: 18px;">     2. Rinker, S., Ke, Y., Liu, Y. Chhabra, R., Yan, H. Self-assembled DNA nanostructures for distance-dependent</p> <p style = "color: #000000; font-size: 18px;">     multivalent ligand-protein binding. Nature Nanotechnology 3, 418-422 (2008).</p> <p style = "color: #000000; font-size: 18px;">     3. https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/t7009pis.pdf</p>



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