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;">Re-designing the Molecular Beacon</div> </div> <div id="LAB-BOOK-REPEAT"> <div id="LAB-BOOK-TEXT">

<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> 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>  

</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 molecular beacon: <br> <ul> <li> <orange>Sensor sequence</orange> defines the target signal the molecular beacon 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 these parameters in our molecular beacon. 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 conventional molecular beacons and reconstructed them 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> <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><br> To generate sequences for our first molecular beacon, we used a mix of random and pre-defined sequences. Our lab already had a 20nt AlexaFluor488 modified oligo - we used this and in combination with random sequences for the remaining domains. We plugged these into NUPACK for analysis, and iterated analysis until we achieved<a href= "http://www.nupack.org/partition/show/528656?token=Ox6cCAHpk3&temperature=15.0"> high theoretical yields of assembly</a>. We then truncated the clip by increments, thus generating variations in 'clip strength' by altering the free energy of hybridisation with the loop of the molecular beacon. This allowed us to find the optimal clip strength for observing a clear change in fluorescence before we proceeded to construct our cooperative biosensor. <br><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 head to our <a href="http://biomodaustralia2014.postach.io/exp-2-2-titration-of-switch-against-signal" target="_blank">rough lab book</a>.

<h2>Single Switch Design #2</h2> The initial <a href=http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp2>experimental data</a> led to improvements in our designs. From our experiments we developed two hypotheses about our first single switch: <br> <ul> <li> It occurred to us that our first design may allow both the clip and signal strands to be hybridised at the same time, forming a trianglular rather than linear conformation. This means that even though signal binds the fluorophore may remain quenched. </li>

 				<li> We also observed unexpected results for shorter clips. In molecular beacons with shorter clips a region of the sensor was left unhybridised. Through a strand-by-strand analysis of homo- and hetero-dimers we found that this region resulted in an array of secondary structures that counfound our expectations even in this simple system. </li>

</ul> <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. <br> The signal strand binds without triggering a change in fluorescence. </div> <br> To overcome these problems we flipped the positions of the fluorophore and quencher strands. Additionally, we altered the clip so that it would compete with the signal to hybridise to the molecular beacon, removing the unhybrised nucleotides that caused secondary structures in our single switch #1. Finally, though our design is entirely modular and could detect any arbitrary sequence of DNA, we decided to make our molecular beacon relevant by altering the specificity of our sensor strand for a conserved sequence en the Ebola virus genome that has been used in a <a target="_blank" 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. Single switch #2 </div> <br> The new position of the clip removed the possibility of the alternate triangular conformation, while also removing unhybridised nucleotides and providing a simpler method to tether multiple switches together in our <a target="_blank"href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Coop">cooperative molecular biosensor</a>. Check out how this design performed in the lab compared to our first design <a target="_blank" href=http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Exp2>here</a>, or for the nitty gritty sequences and protocols head to the <a target="_blank" href="http://biomodaustralia2014.postach.io/">rough lab book</a>.

<h2>Conclusion</h2>

We have built on the proven biosensing technology of molecular beacons by teasing apart the different components of the system into separate, variable domains. This design has allowed our team to fully characterise the molecular beacon with a range of different clip strengths with different concentrations of the target strand of DNA. Furthermore, the redesigned molecular beacon is entirely compatible with the <a target="_blank"href="http://openwetware.org/wiki/Biomod/2014/VCCRI/LabBook/Coop">design of our cooperative molecular biosensor</a>, which will allow direct comparison of independently vs. cooperatively functioning molecular beacons.

</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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