User:Avinash Parida/test project: Difference between revisions

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<ul class="active-founders secondary-nav"> <li class="founders tree-8" > <a href="threed.html" style="color:red;">3D DNA Origami</a> </li> <li class="2D tree-8"> <a href="twod.html">2D DNA Origami</a> </li> <li class="jobs tree-8"> <a href="gallery.html">Gallery</a> </li> <li class="home-agency tree-8"> <a href="brainstorming.html">Brainstorming</a> </li> <li class="taj tree-8"> <a href="tajmahal.html" >About Taj Mahal</a> </li>

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<p class="description rte"> <div class="outer-mag"> <div class="mag-article grid grid-w180-g10"> <div class="header-chapter"> <h1 class="heading-1" style="font-size:34px;text-align:center">DNA Origami of Taj Mahal</h1>  <div class="box box-gray" style="padding:30px;"> <h2 style="padding:0px 0 10px 0;">Abstract</h2> <p>The recent worldwide research on constructions using 3D DNA origami technique is attracting many researchers, to explore newer and diverse possibilities of making useful tools as well as novel structures. In particular, Castro et al demonstrated how to design and produce an object shaped like a robot using 3D DNA origami. Also, in April 2011, Hao Yan et al had demonstrated hollow curved surfaces using 3D DNA origami. Motivated by these researches, we aim to develop a highly complex non-trivial 3D structure of the Taj Mahal using two different construction techniques. We also propose an automation algorithm for creating 3D structures similar to ours using the first technique.</p> </div> </div>

<li> <article> <header> <h1><a name="introduction">Introduction</a></h1> <p class="infos"></p> </header> <p>The first designing technique is similar to that proposed by Castro et al, and uses caDNAno for CAD and a honeycomb lattice. We tried to maintain various kinds of stability in the structure using available designing principles proposed through various different researches worldwide. We also present an algorithm aimed at automating the development of similar 3D structures using caDNAno.</p><br/>

<p>The second technique we worked on is quite challenging and tries to combine various different 3D structures into one big 3D structure, with a solid 3D base holding all the parts together. We have identified and designed the basic building blocks of Taj Mahal namely Decagonal Dome (29nm radius, 10 crossovers, 8 rings), Cylindrical Tubes (10nm radius tubular), Pyramidal Tops (13nm radius, 5 crossovers, 5 rings), Cubical Body (85nm width) and square base. The designing was done using Nano Engineers and Autodesk Maya. Combining these basic shapes is still a challenge and experimental validation for the same is needed. </p><br/> <p>Prior to this work, we have also created the 2D structures of the map of India and Gujarat state.</p> </article> </li>

<li> <article> <header> <h1><a name="background">Background</a></h1> <p class="infos"></p> </header> <p>Till date, 3D DNA Origami has been achieved using various different construction techniques. There are techniques for creating hollow container-like objects by folding up single layers of helices (11,12,16). Hao Yan et al have also recently presented a strategy to design and construct self-assembling DNA Nanostructures, which form 3D curved shapes.</p> <br/> <p>Also, it is possible to create space-filled structures using a multi-layered approach (13,14,15,17), though the yield is less and takes longer. One can either use a square lattice or a honeycomb lattice for building space-filled multi-layered structures.</p><br/> <p>There also exist a few techniques to join two specific kinds of 3D structures. For space-filled 3D structures using honeycomb lattice, we have two ‘Lock and Fit’ techniques to combine two individual 3D pieces. One is using the ‘Slotted cross’ shape technique and the other is using ‘Squared nut’ shape technique.</p> <br/>

<p>For joining space-filled 3D structures using a square lattice, there is no such ‘Lock and Fit’ technique in place currently.</p><br/>

<p>We also didn’t come across research, which reveals a definite technique for combining two individual 3D container structures.</p><br/>

<p>Another challenge for us while creating a large 3D shape was the size of the scaffold. Currently, most of the DNA origami is done using the scaffold of the single stranded m13 virus. m13 scaffold has a large scaffold length of over 7000 bps and it has low secondary structure formation, which makes it the best option for DNA Origami. To create larger structures, we would need multiple scaffolds since a length of 7000 base pairs would not be sufficient for the purpose. Research has been done to scale up 2D DNA Origami using tiled staplers, but no corresponding research work for scaling 3D structures using some kind of 3D tiles has been released yet.

</p> <div class="img-div"> <a href="imgs/3d/technique_1/wiki/taj_2.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/taj_2.gif" alt="wiki-img" class="image-style"> <p class="img-text"><b>Figure 1 </b>| The Taj Mahal, Agra, India.</p> </a> </div>

</article> </li>

<li> <article> <header> <h1><a name="method">Method</a></h1> <p class="infos" style="font-size:14px;padding:20px 0 5px 0;">TECHNIQUE 1</p> </header>

<p>

<p class="MsoNormal"><span >Designing the layout, evaluating the design and determining the staple sequences of the Taj Mahal using 3D Multilayer DNA Origami in Taj Mahal, with a single long m13mp18 scaffold, on a honeycomb lattice.</span></p>

<br />

<p class="MsoNormal"><span >Technique 1 for designing the Taj Mahal using DNA Origami relies on principles already established through worldwide researches, and attempts to take it one step further by providing an enhanced 3D effect to the object. This design process is similar to the one used to design the 3D DNA Robot like structure, in the research published recently by Castro et al. The Taj Mahal was designed while keeping in mind various stability factors, to ensure that it maintains its structure when it is self-assembles under lab conditions.</span></p>

<br />

<p class="MsoNormal"><b><span >Method:</span></b></p>

<p class="MsoNormal"><span >A long single stranded scaffold is used for designing the structure. m13 scaffold was chosen for creating the Taj Mahal, because of two reasons. First is that the length of the scaffold, i.e. 7249 base pairs (bps), is sufficiently large to create the structure. Secondly, there is low secondary structure formation in the m13 Scaffold, so chances of occurrence of errors are low.</span></p>

<br />

<p class="MsoNormal"><span >As the DNA Origami principle stated long ago by Paul Rothemund goes:</span></p>

<p class="MsoNormal"><span><i>“Our goal is to choose a continuous route through the scaffold path and then generate a list of staples that would force the scaffold to adopt that configuration in the test-tube.”</i></span></p>

<br />

<p class="MsoNormal"><span >The Taj Mahal is designed as a space-filling multilayer origami object on a honeycomb lattice. As a consequence, each double-helical domain in the lattice has up to three neighbours arranged in threefold symmetry (fig 1). </span></p> <br /> <br />

<p class="MsoNormal"><b><span >Crossover spacing rule:</span></b></p>

<p class="MsoNormal"><span >A B-form DNA strand can be assumed to contain 10.5 bps per helix. Thus, this implies that the strands rotate by 240 degrees about the helical axis every 7 bps, by 480 degrees every 14 bps and two complete turns or 720 degrees every 21 bps. caDNAno also gives us the option to extend the length of the strands in the honeycomb lattice by multiples of 21 bps. In a honeycomb lattice, each strand has upto three neighbours in the honeycomb lattice. Hence, to ensure that DNA the DNA strands are confined to the honeycomb lattice, crossovers can be placed to each of the three neighbouring strands in constant intervals of 7 bps or every 240 degrees. <span>  </span></span></p>

<p class="MsoNormal"><span >It has been stated in previous research (6), that deviating from the constant 7-bp crossover spacing rulein the honeycomb-lattice packing causes local undertwist as well as axial strain (14).</span></p><br/><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/honeycomb-latice.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/honeycomb-latice.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 2 </b>| Spacing in the Honeycomb lattice. Each double-helical domain can have up to three neighbouring double-helical domains.</p> </a> </div>

<p class="MsoNormal"><span ><b>Some more previously established Designing constraints and principles:</b></span></p> <br/> <p>• According to a recent research (3), it has been established that the DNA Origami structure needs to have a crossover density to avoid the double helical domains from bowing out. It is known that a crossover density of one crossover per 7-8 base pairs, the inter-helical gap created by bowing reduces to 0.5 nm as compared to 1.5 nm for crossover density of 1 per 26 bps.</p> <br/> <p> • The staple strands can undergo multiple crossovers and connecting multiple neighbouring domains but they must obey the length constraints, i.e. the scaffold length must lie between 17 to 50 bps non-inclusive (6). Currently, caDNAno doesn’t obey this principle in its auto-stapling feature. Thus, the staples have to be manually broken after applying auto-staple.</p> <br/> <p>• According to previous research (1,2,16). unpaired single stranded scaffold segments can be used as entropic springs to support tensegrity structures, and they are useful in preventing unwanted base-stacking interactions at objet interface. Single stranded scaffold or staples can also serve as hybridization anchors for direct site attachments.</p> <br/> <p>• As stated by Castro et al in their research (6), the square lattice packing rule allows for creating densely packed objects with rectangular features but may require additional effort to eliminate potentially undesired global twist deformations. The honeycomb lattice packing rule by default create straight albeit more porous structures. Thus, we chose the honeycomb lattice for creating our structure.

</p>

<br />

<p class="MsoNormal"><span ><b>Basic Workflow:</b></span></p>

<p>The Taj Mahal was structurally divided into the four pillars, the central dome and the base. Each structure was created individually using five different single scaffolds and then the pillars and the central dome were connected to the base, using scaffold crossovers. Staplers were then applied keeping the stability constraints in mind, and then the m13 sequence was installed in the scaffold and staplers.</p><br/>

<p class="MsoNormal"><span ><b>Workflow:</b></span></p> <p>• A silhouette of the Taj Mahal was chosen to get an idea of the dimensions of The Taj Mahal. The structure was divided into six major components, i.e. the four pillars, central dome and the base. Then these structures were further divided into grids, so that it could be designed using available CAD software for DNA Origami.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/taj-shadow.jpg" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/taj-shadow.jpg" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 3 </b>| A silhouette of the Taj Mahal, chosen for reference and understanding the dimensions of the structure.</p> </a> </div>

<p>• Based on the dimensions of The Taj Mahal, there were two designing options we had to choose from, for designing the Taj Mahal. They are depicted in fig. x. If the entire length and width available in caDNAno for the structure was to be used, choosing the configuration in fig.x(a) made more sense if the length and breadth of The Taj Mahal structure were considered. </p><br/>

<p>• However, since we calculated that we would not be able to use the entire space of caDNAno due to limitation on the size of the scaffold we could use, we decided to go with configuration in fig. x(b) for the sake of simplicity.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/brainstorm-design.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/brainstorm-design.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 4</b> | The two possible configurations which could be used for designing the Taj Mahal. Configuration (a) made more sense if the entire length and width of caDNAno was to be used. However, since a the scaffold length was limited to 7249 bps, configuration (b) was chosen for the sake of simplicity.</p> </a> </div>

<p>• Next, 108 single stranded scaffolds in the honeycomb lattice of the Taj Mahal were chosen. The length of each of these scaffold was chosen as 84 bps. Next the tools provided in caDNAno were used to break the scaffold at desired points to bring out specific shapes. </p><br/>

<p>• After this, a continuous route through all the 108 strands was chosen. This was done by installing scaffold crossovers. The task was not trivial and multiple iterations had to be made to finally join the entire structure together. The shape was compromised a bit during this process, and a few strands had to be left out, leaving 104 strands in the structure finally.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/scaffold-path.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/scaffold-path.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 5</b> | Initially, 108 single strands were chosen for the structure. While choosing a continuous route through the scaffold, the structure had to be compromised a bit and finally a continuous route through 104 strands was chosen. The structure was made modular as it can be seen. The four pillars, and the central dome were connected to the base using scaffold crossovers.</p> </a> </div>

<p>After a continuous route was established, staples were installed in the structure, mostly using Auto-Staple feature provided in caDNAno, and some manually. Moreover, auto-staple feature doesn’t generate perfect staples. The staplers had to be modified at lot of places to meet the length constraints. This was all done manually.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/panel-2-strands.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/panel-2-strands.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 6 </b>| The staplers generated to keep the 3D Taj Mahal structure stable at various levels of zoom.</p> </a> </div>

<p>Next, the sequence of m13 was installed into the main scaffold, and the sequence of the staplers was generated simultaneously. The Staple Sequences are the extracted and copied onto a spreadsheet. The finished structure is depicted in fig.7.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/taj-mahal-3d.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/taj-mahal-3d.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 7</b> | The 3D view taken by caDNAno panel-3 for The Taj Mahal structure finally generated by technique 1.</p> </a> </div>

<p>The staple sequence, which is thus generated, can be used to generate the Taj Mahal in lab, by using the methods, which are already being used worldwide. (6, 13).</p><br/>

<p><b>General info about the structure:</b></p> <p>The scaffold sequence used by to create the Taj Mahal structure is that of m13mp18. However, the entire 7249 bps of the sequence were not required, and the length of scaffold used was 6508 bps. The number of staplers used in the structure are 215.</p><br/> <p> An xls file of the stapler sequence that we generated and the m13mp18 sequence that we installed are available for download in the <a href="result.html">results</a> section of the Wiki.</p><br/>

<br />

<p class="MsoNormal"><b><span >Calculating the dimensions of DNA Origami object:</span></b></p>

<p class="MsoNormal"><span >The following rules were used to calculate the dimensions of the structure:</span></p><br/>

<p>1. Since the space between two basepairs in a DNA strand is approximately 0.34 nm, the length of the scaffold can be calculated as 0.34 * number of (bps) in the longest strand used.</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/honeycomb.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/honeycomb.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 8</b> | Calculating various dimensions of the honeycomb lattice (a) The honeycomb structure of the lattice can be represented by a hexagon. (b),(c) We know that the diameter of a DNA helix is 2 nm. Using mathematical principals, we can find out various angles and distances in the hexagon. (d),(e) The interhellical distance between two double-hellical domains placed in (d) is 4nm whereas the distance in configuration (e) is 3.46 nm (f) The height of the hexagon was calculated as 4 nm and the width is 3.46 nm.</p> </a> </div>

<p>2. The diameter of DNA helix is 2 nm. Thus, in a honeycomb lattice, the distance various lengths and dimensions can be calculated mathematically (fig. x). Thus, it was calculated that the distance between two hexagonal lattices is 3.44 nm length-wise and 4 nm height-wise (fig. x).</p><br/>

<div class="img-div"> <a href="imgs/3d/technique_1/wiki/diagram-taj.gif" title="img" target="_blank"> <img src="imgs/3d/technique_1/wiki/diagram-taj.gif" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 9</b> | Calculating the dimensions of the Taj Mahal. The height of the pillars, h1, was calculated to be 10 nm. The height of the central dome, h2, was calculated as 12.5 nm. The base length, h4 was calculated as 28.56 nm, whereas the base width, h4 was calculated to be 39.56 nm.</p> </a> </div>

<p>According to the above calculations, the dimensions of our structure were calculated as following (fig.9):</p></br> <p>h1 = 2.5 * (honeycomb height) nm = (2.5 * 4) nm = 10 nm</p> <p>h2 = 3 * (honeycomb height) nm = (3 * 4.828) nm = 12.5 nm</p> <p> h3 = 84 * (length of 1 bp) nm = (84 * 0.34) nm = 28.56 nm</p> <p> h4 = 11.5 * (honeycomb width) nm = (11.5 * 3.44) nm = 39.56 nm</p> </p><br/>

<p>Thus, the structure of the Taj Mahal was successfully created and the staplers were generated using Technique 1.</p><br/>

<p class="infos" style="font-size:14px;padding:20px 0 5px 0;">TECHNIQUE 2</p>

<p>The Taj Mahal was successfully designed using Technique-1 and the staplers for the structure were generated. However, the resolution of the structure was not satisfactory for us. A higher resolution structure was desired and since no technique currently allowed us to do so satisfactorily for 3D structures, creating one would be a good contribution for the community. Thus, it was decided that existing techniques will be looked into and an attempt will be made to scale up 3D DNA Origami by using the combining various research done worldwide. A detailed documentation of our brainstorming can be found in the <a href="brainstorming.html">brainstorming section of the Wiki.</a>.</p><br/>

<p><b>Stability of the structure:</b></p> <p>We wanted a higher resolution 3D model of the Taj Mahal. Since it was not possible to have a single large scaffold, which would create the whole Taj Mahal, due to stability constraints and constraints on the size of the scaffold, it meant that individual pieces of the Taj Mahal which were around 7000 base pairs long would have to be created and then it was necessary to come up with a scaling technique to join these pieces to form the final structure. We also had to ensure that the technique ensured stability of the structure. There are various factors under stability that had to be considered:</p><br/>

<p><i><strong>Stability factor 1 - The base of the structure – Mechanical stability</strong></i></p> <p>We needed a firm base for the entire Taj Mahal structure, which could hold all our individual pieces together in place, and provide mechanical stability to the structure.</p><br/>

<p><i><strong> Stability factor 2 - Thermodynamic stability: </strong></i></p> We were also aware that the structures that we designed needed to be thermodynamically stable. We were initially not sure how to go about checking if structures are thermodynamically stable or not so we decided to do some research over this before making design decisions.</p><br/>

<p><i><strong>Stability factor 3 - Crossovers position stability:</strong></i></p> While designing the 3D Structures, we decided to follow the tried and tested principles for determining the positions of the scaffold and the stapler crossovers in the structure. </p><br/>

<p>1. Initially, efforts were made to visualize the 3D structures using Computer Aided Designing software. A hollow sphere was created and wrapped with material assumed to be DNA, according to principles stated in Hao Yan’s research (3), just for the purpose of understanding through modeling. Various models proposed in the research were created and a few of them at<a href="" target="_blank" style="color:red"> BIOMOD Experiments <img src="http://openwetware.org/skins/monobook/external.png"></a> </p><br/>

<p>2. Next some time was spent in learning and researching NanoEngineer, which is a Molecular engineering tool for nanoscale design and simulation. All the tutorials given on the software’s website were studied and some simple 3D structures were created which could be used later in the Taj Mahal structure (fig. 1).</p><br/>

<div class="img-div"> <a href="name" title="img" target="_blank"> <img src="name" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 1</b> | (a) A Single Octagonal Ring (b) An Octagonal cylinder.</p> </a> </div>

<p>3. Developing these structures was good for a start but we needed to focus on the bigger goal. Thus we decided to use the design principles mentioned in some of the research papers related to 3D DNA Origami that we read.</p><br/> <p>We built a basic pyramid using the principles we studied.</p><br/>

<div class="img-div"> <a href="name" title="img" target="_blank"> <img src="name" alt="wiki-img" class="image-style"> <p class="img-text" style="padding:0 0 25px 0;"><b>Figure 2</b> | (a) A 3D DNA Origami Pyramid created using designing principles provided in Hao Yan’s research (3), from the Top view, (b) Lateral view, and (c) Top-Side view</p> </a> </div>

<p><b>Designing principles:</b></p><br/> <p>There were certain factors that had to be kept in mind while designing the structure. The designing principles that were followed are as following:</p><br/> <p>• The length of the single stranded scaffold represents the maximum number of base pairs that are available to form all of concentric layers (the number of rings and size of each can vary, however, the total number of base pairs is dictated by the scaffold). </p><br/> <p>• The actual size of the inner and outer rings must also conform to the design principles. The inner ring can not have such a small radius of curvature that the network of crossovers between it and the neighboring rings is not sufficient to constrain it.</p><br/> <p>• It may be possible for 3 crossovers between adjacent rings to create a stable, planar arrangement; however, the corresponding 30 bp ring would have too large a degree of curvature to accommodate the DNA itself.</p><br/> <p>• The circumference of each ring will also be affected by its relationship to the other rings, as reflected by the equation C = 2πr (ΔC = 2πΔr). Or C = 4a (ΔC = 4Δa) for square rings.</p><br/> <p>• Once the pattern and position of crossovers has been determined, the long single stranded scaffold strand (M13) is conceptually wound so that it comprises one of the two strands in every helical ring; each time the scaffold moves from one ring to the next a ‘scaffold crossover’ is created. Ideally scaffold crossovers should be staggered so that a single ‘seam’ is not created that could weaken the crossover network.</p><br/><br/>

<p>4. Brainstorming was done over how to join the individual pieces of 3D structures together. This proved to be the most challenging part of the project and the most important part as well. Detailed documentation of the brainstorming and discussions is provided in the <a href="brainstorming.html">brainstorming section of the Wiki.</a>.</p><br/>

<p>Finally, one of the proposed techniques was chosen to work on. It was proposed that first we would create individual 3D structures similar to those developed by Hao Yan et al in their research (3), using similar design principles. However, the proposed 3D structures would be a bit different in shape towards the base. Concentric rings would be provided around the base region by extending the same scaffold. </p><br/>

<p>Once all these structures with a flattened extension of base are created, then the concept of 2D tiling() would come into plan. 2D tiling is an established technique for scaling 2D DNA Origami structures. Since all the flattened bases of all our 3D structures would lie in the same plane, we could effectively use this technique to scale up the structures.</p><br/>

<p>The first step was to create a master plan of the entire structure</p><br/>

<p>Next We then came up with some basic designs, which used the principles that we would use later on in designing the Taj Mahal:</p><br/>

<p>Using our technique, all these structures can be joined using tiled staplers. </p><br/>

<p>Conclusion

Known issues: 1. Low mechanical stability 2. No experimental verification

Challenges: 1. A way to scale 3d dna origami

Possible Solutions: 1. Increasing the size of scaffold 2. Introducing 3d tiled staplers </p><br/>

</article> </li>

<div id="conclusion" class="box box-gray" style="padding:30px;"> <h2 style="padding:0px 0 10px 0;">Conclusion</h2> <p> The design of the desired structure of Taj Mahal using Technique-1 was successfully created. Keeping all the stability constraints in mind, the positioning of scaffold crossovers, stapler crossovers and the stapler sequence were determined. Designing using technique-2 is still a challenge. Possible proposed solutions are to find a way to create 3D DNA Tile staplers. Another way of scaling 3D DNA Origami is by increasing the size of the main scaffold either by joining multiple scaffolds using an enzyme such as ligase or finding an equally good but longer substitute for m13 scaffold which has low secondary structure formation.</p> </div>

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