Biomod/2014/VCCRI/LabBook/Single

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

<h2>Background</h2>

Molecular Beacons are a simple DNA-based probe for detecting the presence of a specific sequence of DNA or RNA in a sample. The mechanism of detection is via a conformational change in the beacon that '''occurs''' upon hybridising with the target DNA or RNA. This change in confirmation alters the distance between two fluorescent moieties changing the strength of fluorescent resonance energy transfer (FRET) resulting in a fluorescent readout. <br> Structurally, Molecular Beacons contain four domains.

</div> <ul> <li><b>1. Sensor domain:</b> A domain that is he reverse complement of the target DNA or RNA. usually between 20 and 30 nucleotides long.</li> <li><b>2. Stem domains:</b> Two short domains on either side of the sensor domain that are complementary with each other such that both ends bind to each other to form a hairpin structure. <li><b>3. Fluorophore domain:</b> A usually 5' end nucleotide modified with fluorescent dye molecule. <li><b>4. Quencher domain:</b> A usually 3' end nucleotide modified with a molecule that quenches fluorescent emission from the fluorophore when in close proximity. </ul> <h2>Considerations</h2> We identified the following parameters to be important in MB function: <br> <ul> <li> Sensor sequence - defines the target signal the MB is sensitive to. Ideally it should have not self-complimentary regions.</li>

 				<li> Sensor length - defines the stringency of detection as well as providing the free energy required to cause the conformational change and produce a signal.</li>
 				<li> Stem length  -  defines the free energy cost needed to overcome to activate the sensor by target strand hybridisation.</li>

</ul>

We needed to be able to explore the parameter space to optimise our co-operative switch - most importantly we will need to vary the free energy cost needed to overcome to produce a signal. To do this we need the stem domain to be different lengths.

Modifying oligos with both a Quencher and Fluorophore is expensive, has a low yield, and a slow turn around in synthesis. We tried to avoid needing to modify every version of a molecular beacon by deconstructing the four domains of normal single-strand MB and reconstructing it as an assembly of four oligos, each representing a domain (See Diagram below). This Modular design will allow us to quickly, easily, and cheaply generate different molecular beacon-like sensors by swapping out the different versions of each domain.

<h2>Single Switch Design 1</h2> We have already in the lab a 20nt alexour488 modified oligo - so we used that. For the remanding domains we generated random sequences (because we're rookies) and plugged them into NuPACK for analysis, and repeated until we achieved <a href= http://www.nupack.org/partition/show/497703?time_refresh=1.0&token=cRFkrceE6x>theoretical yield of assembly</a>. We then truncated one end of the clip strand to vary the strength of binding in the 'stem' domain <br>

Check out how this design performed in the lab <a href=http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp2>Here</a>, or for the nitty gritty sequences and protocols check the rough lab book LINK TO ROUGH LABBOOK

<div class="image-center"> <div><img src="http://openwetware.org/images/e/e8/2014-EchiDNA-SINGLE-DOMAINS.png" /></div> Fig 1. The domains of a conventional molecular beacon (left) and their corresponding strands in our modular switch (right). </div>

<h2>Single Switch Design 2</h2> The initial <a href=http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp2>experimental data </a> was a little hard to interpret and unpromising. To explain this behaviour it occurred to us that our first design may allow both the stem (clip) and target (signal) strands to be hybridised at the same tie forming a triangle confirmation (rather than a linear one) such that the target may bind but the fluorophore and quencher are still in close proximity and the signal is begin quenched. A strand by strand analysis of potential homo-/ hetreo-dimers suggested that the region of the sensor strand backbone left unbound when using shorter clip could bind to other regions in the sensor strand forming unwanted secondary structures.

<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>

To overcome this triangular confirmation we flipped the positions of the fluorophore/quencher strands with the clip strands so that the 20nt lengths of the fluorophore and quencher strands cannot form lengths of the triangle. Additionally, we used the clip sequences are the same as the signal sequences so that the clip holding the quencher and fluorophore together would be displaced by longer signal strand upon binding. Finally, because our design is modular, we altered the specificity of our sensor strand for a conserved sequence en the Ebola virus genome that has been used in a <a href=http://jvi.asm.org/content/78/8/4330.ful>Reverse Transcriptase PCR detection method </a>.

<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> </div> </div>

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