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<div><img src="http://openwetware.org/images/e/e8/2014-EchiDNA-SINGLE-DOMAINS.png" /></div>
<div><img src="http://openwetware.org/images/e/e8/2014-EchiDNA-SINGLE-DOMAINS.png" /></div>
Fig 2. A conventional molecular beacon (left) and their corresponding strands in our redesigned switch (right).
Fig 2. A conventional molecular beacon (left) and their <br>corresponding strands in our redesigned switch (right).
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Revision as of 03:13, 25 October 2014

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<div id="LAB-BOOK-TOP"> <div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Re-designing the Molecular Beacon</div> </div> <div id="LAB-BOOK-REPEAT"> <div id="LAB-BOOK-TEXT">

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<h2>Aim</h2> To design a new kind of molecular beacon which is modular and capable of being tethered to its neighbours in our cooperative biosensor. <h2>Background</h2> <a href="http://onlinelibrary.wiley.com/doi/10.1002/anie.200800370/abstract?deniedAccessCustomisedMessage=&userIsAuthenticated=false" target="_blank"> Molecular beacons</a> (MBs) 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 conformation alters the distance between two fluorescent moieties, thus changing the strength of Förster resonance energy transfer (FRET) and resulting in a fluorescent readout. <br><br> Structurally, Molecular beacons contain four domains:

<div class="image-left"> <div class="image-centre"> <div style="height:auto"><img src="http://openwetware.org/images/b/b9/2014-EchiDNA-LAB-BOOK-PROJECT_BACKGROUND-ANIMATION-BEACON.gif" /></div> Fig.1 The basic principles of a molecular beacon. </div> &nbsp;

</div><br> <br> <ul> <li><orange>Sensor:</orange> A domain that is the reverse complement of the target DNA or RNA; usually around 25 nucleotides in length.</li> <li><orange>Stem:</orange> Two complementary domains (around 5bp) on either side of the sensor domain, forming a hairpin structure. <li><orange>Fluorophore:</orange> Typically a 5' nucleotide modified with fluorescent dye. <li><orange>Quencher:</orange> Typically a 3' nucleotide modified with a molecule that quenches fluorescent emission from the fluorophore. The efficiency of quenching is distance-dependent. </ul> <br><br> <h2>Considerations</h2> We identified the following parameters to be important in MB function: <br> <ul> <li> <orange>Sensor sequence</orange> defines the target signal the MB detects. Ideally it should have not self-complimentary regions that compete with the hairpin structure.</li> 

 				<li> <orange> Sensor length </orange>defines the accuracy of detection as well as providing the free energy required to cause the conformational change and produce a signal.</li>
 				<li> <orange>Stem length</orange> defines the free energy cost that must be overcome to activate the fluorescent moiety.</li>

</ul>

We knew we would have to optimise our cooperative biosensor by exploring the various possible designs of our MB. But synthesising DNA with quencher- and fluorophore-modifications is expensive, has a low yield, and a slow turn around in synthesis. Therefore we wanted to avoid synthesising a new molecular beacon every time we needed to vary any of the components of the system. <br><br> We deconstructed the four domains of normal single-strand MB and reconstructed it as an assembly of four oligos, each representing a separate domain. This modular design allowed 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> <br> <div class="image-center"> <div><img src="http://openwetware.org/images/e/e8/2014-EchiDNA-SINGLE-DOMAINS.png" /></div> Fig 2. A conventional molecular beacon (left) and their <br>corresponding strands in our redesigned switch (right). </div>

To generate sequences for our first MB, we used a mix of random and pre-defined sequences. Our lab already had a 20nt Alexafluor488 modified oligo - we used this and for the remanding domains generated random sequences 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


<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. <br> <div class="image-center"> <div><img src="http://openwetware.org/images/0/06/2014-EchiDNA-SINGLE-TRIANGULATION.png" /></div> Fig 3. A potential triangular confirmation of single switch 1 in which the signal strand is bound but no fluorescence occurs. </div> <br> 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/b/b6/2014-EchiDNA-SINGLE-2ND-DESIGN.png" /></div> Fig. 4. Second design of single switch 2: the location of the fluorophore and quencher strands as well as the displacement of the clip removes the possibility of the triangular confirmation that design 1 has (see Fig. 3). </div> <br> Check out how this design performed in the lab compared to our first design <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


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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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