Biomod/2014/VCCRI/LabBook/Application

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

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We believe that our work is just the beginning of a fantastic project. We imagine many new directions which will continue to lead towards a deeper scientific understanding of cooperativity and to unique biosensing technologies that exploit this powerful design principle.

<h2>Bio-layer interferometry</h2>

It would be possible to empirically determine the rate constants underlying the mathematical model of our cooperative molecular biosensor. One method of approaching this would be bio-layer interferometry. Essentially, this would involve tethering one of our molecular beacons onto a surface using streptavidin-biotin affiliation, then washing solutions over the surface containing oligos of interest. As the tethered molecular beacon interacts with molecules in solution - for instance, the signal strand that opens the molecular beacon - we can observe the rate of this interaction by observing the rate of change in optical thickness of the surface. This technqiue would deepen our understanding of molecular beacons, leading directly to a more powerful predictive model, and perhaps offering an alternate method of detecting conformational changes in DNA nanotechnology.

<div class="image-center"> <div><img src="http://openwetware.org/images/5/5d/2014-EchiDNA-Bio-layer_interferometry_with_analyte_binding.gif" /></div> </div><br> <h2>Single molecule fluorescence</h2>

Some of the folks in <a href="http://www.victorchang.org.au/home/our-research/faculty-detail/?faculty_name=dr-lawrence-lee&division_name=structural-computational-biology"> our lab</a> have started building a single-molecule fluorescence microscope, which would essentially allow us to dilute a sample of our biosensor to the point where you can see single molecules if they are fluorescent. <br><br> Our switch is smaller than the wavelength of light. This means that although you couldn’t actually see the thing, it would be possible to tell, for instance, when two objects like a switch and a signal are co-localised in time, indicating binding of the switch. It would also be possible to use super-fast cameras and automated image analysis to generate statistical answers to questions like, ‘how often does a single switch open, causing a false positive fluorescent output, even in the absence of the target DNA?’, or, ‘to what degree does the opening of one switch make its neighbours more likely to open?’ <br><br> This technique would provide us with a lot of confidence in our design, and would also presents opportunities to inform our mathematical model in greater depth. <br> <h2>Hybridisation chain reaction</h2> 

				There are alternative outputs to our biosensor than fluorescence which may be purely DNA-based. Fluorescence is very powerful, but it is also quite expensive (a single fluorophore-modifed oligo cost us around $250), there seems to be background fluorescence caused by inefficient quenching of the light emitted by the fluorophore, and most importantly, it doesn’t amplify the signal. An alternative DNA-based output would be cheaper and with clever design might remove the need for the purify away staples from the assembly mix, which currently limits the life-span of our biosensor.

<br><br> 

				Most conventional technologies for sensing DNA employ PCR prior to detecting the target DNA, which does three things - 
				 	<ul>
				 	 	<li>amplifies the signal so that it can be detected above noise or background fluorescence,</li>
				 	 	<li>generates false positives through ‘mutations’ in PCR amplification,</li>
				 	 	<li>and makes quantification of the initial concentration of the target molecule difficult.</li>
				 	</ul>
				If our biosensor triggered an amplification cascade or a chemical chain reaction AFTER switching, then it might avoid generating false positives. Furthermore, if we had a characterised set of switches, each tuned to give a discrete output at a different threshold concentration, then by comparing the set we could estimate the concentration of the target.

<div class="image-center"> <div><img src="http://openwetware.org/images/0/0e/2014-EchiDNA-FUTURE-CHAIN.png" /></div> </div>


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</div> <br><br> <a id="next-link" href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook">Click here to go back to the Lab Book Overview<a> </div> </div> 

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Contact: <a href="mailto:BIOMODaustralia@gmail.com">BIOMODaustralia@gmail.com</a> | ©2014 Team EchiDNA. All rights reserved. This team is supported by the <a id="vccri" href="http://www.victorchang.edu.au">Victor Chang Cardiac Research Institute</a>. | <a id="edit-link" href="">Edit</a>

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