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<h2>Background</h2>
<h2>Aim</h2>
The core of our project is to structurally link several molecular beacons to form a single cooperative biosensor. We decided a circular structure would be more elegant and easier to model without lonely subunits on the ends. We've broken down our final design concept into two components: the "Core Barrel" which the individual sub-units are attached, and the "Co-operative linkers" which mechanically link all the sub-units together.
To design the two key components of our cooperative molecular biosensor: a barrel composed of 24 helices and the molecular beacons mounted upon it.  
<h2>Considerations</h2>
<h2>Considerations</h2>
We wanted maximal flexibility possible from a single design so we wouldn't need to redesign or reorder parts, saving us time and money. To achieve this we opted for a modular design where the 'parts' (oligos) could be easily interchanged independently without changing the rest of the design, in particular we needed the following design parameters to be modular:
We wanted maximal flexibility possible from a single design so we wouldn't need to re-design or re-order parts, saving us time and money. To achieve this we opted for a modular design where the components could be interchanged easily and independently without changing the rest of the design. In particular we needed the following features to be modular:<br><br>
<ul>
<ul>
<li>Signal specificity</li>
<li>The sequence targeted by our biosensor</li>
<li>Strength of clip which the signal must compete against</li>
<li>The strength of the clip that holds the molecular beacons closed</li>
<li>The number of sub-units in the whole sensor</li>
<li>The number of molecular beacons in the cooperative biosensor</li>
<li>The strength of cooperative linkage</li>
<li>The degree of cooperativity between molecular beacons</li>
</ul>
</ul>  
The first two design parameters were addressed in the <a target="_blank"href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single>single switch design</a>, the third would be determined by the design of the barrel at the core of our biosensor, and the last by the design of mounted molecular beacons.
The first two design parameters were addressed in the <a href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single>single switch design</a>, the third would be determined by the core barrel design, and the last but the cooperative linker design.
 
<h4>Core Barrel</h4>
For flexibly in the number subunits in a sensor we chose a 24 mer design so that we could alter the number of sub-units by including or excluding sub-units on various helices (factors of 24 are 1, 2 , 4, 6, 8, 12, and 24). The height of the barrel structure needs to be similar to the length of the molecular beacon sensor length (18-30nt) so we included only 2 staple + 1 scaffold linkage between any two helicies. This minimal size requires a custom scaffold strand: M13mp18, the standard scaffold in DNA origami, is far too long (7249nt) compared to custom scaffold length required (900nt). We developed a way to <a href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp3>create ssDNA scaffolds of any length</a>. This scaffold could be of any arbitrary sequence that is long enough. We decided to pay homage to our inspiration in the BFM and chose a section of the coding region of FliG, the protein involved in the switching mechanism of the BFM.
<br>
To design the 24mer structure we took two approaches: one theoretical and one spatial.
 
<h2>Theoretical Design</h2>
<div class="image-right">
<div><img src=http://openwetware.org/images/4/48/AJTBarrelcloser.png></div>
Fig. 1. CanDo output of the theoretically designed Core Barrel.
</div>
We found the closest cross-over periodicity using this <a href=http://openwetware.org/images/e/e8/Angles_per_bp_10.5.xlsx>handy table</a> of crossover periodicity and angle. The external angle for a 24mer is 180+/-15˚ and this would be the angle required between the crossovers to neighbouring helices. The 15˚ change in angle between needed is unattainable as a  +/-1bp change in periodicity equates to approx. +/-36˚change in angle, so we approximated the barrel as a 12mer which has and external angle of 180+/-30˚. We generated a bunch of CaDNAno models with different skips and/or inserts on different helices and modelled them with CanDo and selected the one with minimal twisting. <a href=http://openwetware.org/images/0/06/DesignOpt2AJT.json>Theoretical Design CaDNAno File</a>.
 
<h2>Spatial Modelling</h2>
Our super modeller, Jon, created a 3D representation of B-DNA. Unlike DNA origami's 'rules of thumb' it pays no heed to approximations such as axial symmetry of DNA and includes fine structural details such as major and minor grooves. Placing helices in the arrangement we desired for a 24mer barrel he manually altered the positions and orientations of each helix to minimise the length of crossover links and created a sequence model based on this 3D model (Fig. 2) and transcribed it into a <a href=http://openwetware.org/images/4/49/DesignOpt1.1Jon.json>CaDNAno File</a> to thread the scaffold sequence.  
 
<div class="image-center">
<div class="image-center">
<div><img src="http://openwetware.org/images/2/25/2014-EchiDNA-EXP3-BARREL-SCAFFOLD.png" /></div>
<div><img src="http://openwetware.org/images/d/d9/2014-EchiDNA-COOP-MODULARITY.png" /></div>
Fig. 2. 3D model of spatially designed Core Barrel
Fig. 1. Modular aspects of our design
</div>
</div>


<h4>Cooperative Linker</h4>
<h2>Barrel</h2>
Heres the tricky part: We needed to be able to mount our molecular beacon sub-units on the core barrel structure and mechanically link them together. To achieve this we  used two four-way junctions for each sensor, one at the each end, so that every junction has a branch that forms one end of the molecular beacon, one branch that staples into the core barre,l and two branches that link to other junctions on either side.  
Our <a target="_blank" href= "http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp5">model</a> demonstrated that the number of switches in our cooperative biosensor would affect its overall behaviour. Therefore we wanted to design a barrel that could support a flexible number of switches, allowing us to explore and optimise the behaviour of our cooperative biosensor. We chose to create a barrel with 24 helices so that we could alter the number of sub-units by systematically including or excluding switches on the various helices (factors of 24 include 1, 2 , 4, 6, 8, 12, and 24). <br><br>


 
The height of the barrel needed to be similar to the length of the molecular beacon (18-30nt). Combined with the number of helices, this constrained the size of the entire barrel. This necessitated a custom scaffold strand as the standard scaffold in DNA origami, M13, is far too long (around 7000nt). Therefore we developed a method for <a href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp3>creating ssDNA scaffolds of any length</a>. Since this scaffold could be of any arbitrary sequence, we decided to pay homage to the <a target="_blank" href="http://openwetware.org/wiki/Biomod/2014/VCCRI/Project/Problem">Bacterial Flagella Motor</a>, by choosing as the scaffold for our barrel a section of the coding region of FliG, the protein involved in the switching mechanism of the BFM.
<!-- Centred image with caption -->
<br><br>
We took two different approaches to designing the staples necessary to fold our DNA scaffold into the barrel at the core of our biosensor.
<br>
<div class="image-right">
<div><img src="http://openwetware.org/images/2/25/2014-EchiDNA-EXP3-BARREL-SCAFFOLD.png" /></div>
Fig. 2. 3D model of spatially designed barrel
</div>
<h3>Spatial Approach</h3>
We created a 3D representation of DNA from first principles, which unlike CaDNAno's 'rules of thumb' pays no heed to approximations such as axial symmetry of DNA and includes fine structural details such as major and minor grooves. After placing helices in the arrangement we desired for a 24mer barrel we manually altered the positions and orientations of each helix to minimise the length of crossover links and converted this 3D spatial model into a <a href=http://openwetware.org/images/4/49/DesignOpt1.1Jon.json>CaDNAno File</a> for computation of the scaffold and staple sequences. <br>
<h3>Theoretical Approach</h3>
<div class="image-right">
<div><img src="http://openwetware.org/images/9/9a/2014-EchiDNA-DESIGN-COOP_BEACON_DNA_PERIODICITY.png" /></div>
Fig. 3. Angles generated between adjacent helices by staples [from Castro, et al. (2011)]
</div>
We wanted to make a barrel, which meant that neighbouring helices had to align with specific angles between them. Neighbouring helices are connected by staple strands that move around a curve before connecting neighbouring helices. This means that the number of nucleotides in a staple is directly related to the angle between neighbouring helices, and thus to whether or not the helices fold naturally into a barrel.
<div class="image-left">
<div><img src=http://openwetware.org/images/4/48/AJTBarrelcloser.png></div>
Fig. 4. CanDo output of the theoretically designed Core Barrel.
</div> <br>
<br>We calculated that a 15˚ change in angle was required between neighbours, which allowed us to approximate the barrel as a 12mer with angles of 30˚. Based on this we generated a bunch of CaDNAno models with skips and inserts on different helices and modelled them with CanDo to select <a href=http://openwetware.org/images/0/06/DesignOpt2AJT.json>the design with minimal twisting</a>.<br><br>
We anticipate that both of these designs would form an adequate barrel to mount our molecular beacons, however, we chose the proceed with the spatial design as we wanted to test this novel method of generating staples.
<br>
<h2>Mounted Molecular Beacons</h2>
Now HERE'S the tricky part: we wanted to arrange our molecular beacons on the barrel and mechanically link them together. Importantly, we wanted the link between neighbours to be variable so that we could completely characterise the effect of cooperativity on the behaviour of the biosensor. To achieve this we used two symmetrical four-way junctions for each molecular beacon. The four-way junctions allowed us to connect each molecular beacon with its neighbours and with the barrel.
<br>
<div class="image-center">
<div class="image-center">
<div><http://openwetware.org/images/e/ec/Subunit_specs.png" /></div>
<div><img src="http://openwetware.org/images/6/64/2014-EchiDNA-COOP-STRAND-DESIGN-UNBOUND.png" /></div>
Fig. 3. Design and specifications for cooperatively-linked subunit
Fig. 4. Design and specifications for cooperatively-linked subunit
</div>
 
 
We used a <a href=http://openwetware.org/images/a/a9/Coopband_nupack_script_x12.txt>script</a> for NuPACK design to generate the sequences of the strands not already defined in all 24 junctions. We then used NuPACK analysis to analyse the theoretical yield of each junction in isolation for equimolar rations of oligos. We re-iterated this design/analysis loop until we had a <a http://openwetware.org/images/1/15/Coop_Switch_adapter_and_band_sequences.xlsx> sequence set</a> that had high theoretical yields. We wanted to be able to vary the strength of the structural link between sub-units so we ordered a range of 'linker' strand sets with 2 and 8 single polyT in their middle, as well as a set of 'null' linkers that do not link with their neighbours.
 
 
 
<!-- Centred image with caption -->
<div class="image-center">
<div><img src="http://openwetware.org/images/f/f5/2014-EchiDNA-LAB-BOOK-EXP-4-BUTTON.png" /></div>
Fig 1. The quick brown fox jumps over the lazy dog.The quick brown fox jumps over the lazy dog.
</div>
</div>


<br>
We wanted to be able to vary the strength of the band connecting molecular beacons, so we ordered a range of bands with 2 and 8 single polyT in their middle, as well as a set of 'null' bands that should prevent any cooperativity between neighbours by removing the physical connection.
<br><br>
Given all these constraints, we used a <a href=http://openwetware.org/images/a/a9/Coopband_nupack_script_x12.txt>script</a> for NUPACK design to generate all the remaining undefined sequences in each four-way junction. We then used NUPACK analysis to determine the theoretical yield of each junction in isolation for equimolar rations of oligos. We re-iterated this design-analysis loop until we had a <a href=http://openwetware.org/images/1/15/Coop_Switch_adapter_and_band_sequences.xlsx> sequence set</a> that had high theoretical yields.
<br><br>
With 12 sub-units included, the whole sensor should look like this:
<br>
<div class="image-center">
<div style="height:auto;"><img src="http://openwetware.org/images/0/07/2014-EchiDNA-SOLUTION-COOP-UNBOUND-SPIN.gif" /></div>
</div>
<br>
<br>


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<div id="LAB-BOOK-TOP"> <div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Design of Cooperative Beacon</div> </div> <div id="LAB-BOOK-REPEAT"> <div id="LAB-BOOK-TEXT">

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<h2>Aim</h2> To design the two key components of our cooperative molecular biosensor: a barrel composed of 24 helices and the molecular beacons mounted upon it.

<h2>Considerations</h2> We wanted maximal flexibility possible from a single design so we wouldn't need to re-design or re-order parts, saving us time and money. To achieve this we opted for a modular design where the components could be interchanged easily and independently without changing the rest of the design. In particular we needed the following features to be modular:<br><br> <ul> <li>The sequence targeted by our biosensor</li> <li>The strength of the clip that holds the molecular beacons closed</li> <li>The number of molecular beacons in the cooperative biosensor</li> <li>The degree of cooperativity between molecular beacons</li> </ul> The first two design parameters were addressed in the <a target="_blank"href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Single>single switch design</a>, the third would be determined by the design of the barrel at the core of our biosensor, and the last by the design of mounted molecular beacons. <div class="image-center"> <div><img src="http://openwetware.org/images/d/d9/2014-EchiDNA-COOP-MODULARITY.png" /></div> Fig. 1. Modular aspects of our design </div>

<h2>Barrel</h2> Our <a target="_blank" href= "http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp5">model</a> demonstrated that the number of switches in our cooperative biosensor would affect its overall behaviour. Therefore we wanted to design a barrel that could support a flexible number of switches, allowing us to explore and optimise the behaviour of our cooperative biosensor. We chose to create a barrel with 24 helices so that we could alter the number of sub-units by systematically including or excluding switches on the various helices (factors of 24 include 1, 2 , 4, 6, 8, 12, and 24). <br><br>

The height of the barrel needed to be similar to the length of the molecular beacon (18-30nt). Combined with the number of helices, this constrained the size of the entire barrel. This necessitated a custom scaffold strand as the standard scaffold in DNA origami, M13, is far too long (around 7000nt). Therefore we developed a method for <a href= http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp3>creating ssDNA scaffolds of any length</a>. Since this scaffold could be of any arbitrary sequence, we decided to pay homage to the <a target="_blank" href="http://openwetware.org/wiki/Biomod/2014/VCCRI/Project/Problem">Bacterial Flagella Motor</a>, by choosing as the scaffold for our barrel a section of the coding region of FliG, the protein involved in the switching mechanism of the BFM. <br><br> We took two different approaches to designing the staples necessary to fold our DNA scaffold into the barrel at the core of our biosensor. <br> <div class="image-right"> <div><img src="http://openwetware.org/images/2/25/2014-EchiDNA-EXP3-BARREL-SCAFFOLD.png" /></div> Fig. 2. 3D model of spatially designed barrel </div> <h3>Spatial Approach</h3> We created a 3D representation of DNA from first principles, which unlike CaDNAno's 'rules of thumb' pays no heed to approximations such as axial symmetry of DNA and includes fine structural details such as major and minor grooves. After placing helices in the arrangement we desired for a 24mer barrel we manually altered the positions and orientations of each helix to minimise the length of crossover links and converted this 3D spatial model into a <a href=http://openwetware.org/images/4/49/DesignOpt1.1Jon.json>CaDNAno File</a> for computation of the scaffold and staple sequences. <br> <h3>Theoretical Approach</h3> <div class="image-right"> <div><img src="http://openwetware.org/images/9/9a/2014-EchiDNA-DESIGN-COOP_BEACON_DNA_PERIODICITY.png" /></div> Fig. 3. Angles generated between adjacent helices by staples [from Castro, et al. (2011)] </div> We wanted to make a barrel, which meant that neighbouring helices had to align with specific angles between them. Neighbouring helices are connected by staple strands that move around a curve before connecting neighbouring helices. This means that the number of nucleotides in a staple is directly related to the angle between neighbouring helices, and thus to whether or not the helices fold naturally into a barrel. <div class="image-left"> <div><img src=http://openwetware.org/images/4/48/AJTBarrelcloser.png></div> Fig. 4. CanDo output of the theoretically designed Core Barrel. </div> <br> <br>We calculated that a 15˚ change in angle was required between neighbours, which allowed us to approximate the barrel as a 12mer with angles of 30˚. Based on this we generated a bunch of CaDNAno models with skips and inserts on different helices and modelled them with CanDo to select <a href=http://openwetware.org/images/0/06/DesignOpt2AJT.json>the design with minimal twisting</a>.<br><br> We anticipate that both of these designs would form an adequate barrel to mount our molecular beacons, however, we chose the proceed with the spatial design as we wanted to test this novel method of generating staples. <br> <h2>Mounted Molecular Beacons</h2> Now HERE'S the tricky part: we wanted to arrange our molecular beacons on the barrel and mechanically link them together. Importantly, we wanted the link between neighbours to be variable so that we could completely characterise the effect of cooperativity on the behaviour of the biosensor. To achieve this we used two symmetrical four-way junctions for each molecular beacon. The four-way junctions allowed us to connect each molecular beacon with its neighbours and with the barrel. <br> <div class="image-center"> <div><img src="http://openwetware.org/images/6/64/2014-EchiDNA-COOP-STRAND-DESIGN-UNBOUND.png" /></div> Fig. 4. Design and specifications for cooperatively-linked subunit </div>


<br> We wanted to be able to vary the strength of the band connecting molecular beacons, so we ordered a range of bands with 2 and 8 single polyT in their middle, as well as a set of 'null' bands that should prevent any cooperativity between neighbours by removing the physical connection. <br><br> Given all these constraints, we used a <a href=http://openwetware.org/images/a/a9/Coopband_nupack_script_x12.txt>script</a> for NUPACK design to generate all the remaining undefined sequences in each four-way junction. We then used NUPACK analysis to determine the theoretical yield of each junction in isolation for equimolar rations of oligos. We re-iterated this design-analysis loop until we had a <a href=http://openwetware.org/images/1/15/Coop_Switch_adapter_and_band_sequences.xlsx> sequence set</a> that had high theoretical yields. <br><br> With 12 sub-units included, the whole sensor should look like this: <br> <div class="image-center"> <div style="height:auto;"><img src="http://openwetware.org/images/0/07/2014-EchiDNA-SOLUTION-COOP-UNBOUND-SPIN.gif" /></div> </div> <br> <br>


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