Biomod/2014/VCCRI/LabBook/Coop

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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">

<h2>Background</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.

<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:

<ul> <li>Signal specificity</li> <li>Strength of clip which the signal must compete against</li> <li>The number of sub-units in the whole sensor</li> <li>The strength of cooperative linkage</li> </ul>

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 by 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. After 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 converted this 3D model (Fig. 2) 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><img src="http://openwetware.org/images/2/25/2014-EchiDNA-EXP3-BARREL-SCAFFOLD.png" /></div> Fig. 2. 3D model of spatially designed Core Barrel </div>

<h4>Cooperative Linker</h4> 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. <br>

<div class="image-center"> <div><img src="http://openwetware.org/images/e/ec/Subunit_specs.png" /></div> Fig. 3. Design and specifications for cooperatively-linked subunit </div>

<br> 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 href=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. <br> With 12 sub-units included, the whole sensor should look like this: <br> <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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Biomod/2014/VCCRI/LabBook/Coop

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