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<div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Design of Single Molecule Beacon</div>
<div id="LAB-BOOK-TITLE" style="padding-left:60px; text-align: justify;">Re-designing the Molecular Beacon</div>
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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>
<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.
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><br>
<br>
Structurally, molecular beacons contain four domains:
Structurally, Molecular Beacons contain four domains.


<div class="image-left">
<div class="image-left">
<div class="image-centre">
<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>
<div style="height:auto"><img src="http://openwetware.org/images/b/b9/2014-EchiDNA-LAB-BOOK-PROJECT_BACKGROUND-ANIMATION-BEACON.gif" /></div>
Fig.1 Cartoon animation of the mechanism of a molecular beacon.
Fig.1 The basic principles of a molecular beacon.
</div>
</div>
&nbsp;
&nbsp;
</div>
</div><br>
<ul>
<br>
<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>
<ul>
<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><orange>Sensor:</orange> A domain that is the reverse complement of the target DNA or RNA; usually around 25 nucleotides in length.</li>
<li><b>3. Fluorophore domain:</b> A usually 5' end nucleotide modified with fluorescent dye molecule.
<li><orange>Stem:</orange> Two complementary domains (around 5bp) on either side of the sensor domain, forming a hairpin structure.
<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.
<li><orange>Fluorophore:</orange> Typically a 5' nucleotide modified with fluorescent dye.
</ul>  
<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.
<h2>Considerations</h2>
</ul> <br><br>
We identified the following parameters to be important in MB function:
<h2>Considerations</h2>
We identified the following parameters to be important in molecular beacon:
<br>
<br>
<ul>
<ul>
<li> Sensor sequence - defines the target signal the MB is sensitive to. Ideally it should have not self-complimentary regions.</li>
<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> 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> <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> Stem length -  defines the free energy cost needed to overcome to activate the sensor by target strand hybridisation.</li>
   <li> <orange>Stem length</orange> defines the free energy cost that must be overcome to activate the fluorescent moiety.</li>
</ul>
</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.
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.
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.
<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>
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


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<h2>Single Switch Design #1</h2>
<div class="image-center">
<div class="image-center">
<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. The domains of a conventional molecular beacon (left) and their corresponding strands in our modular switch (right).
Fig 2. A conventional molecular beacon (left) and their <br>corresponding strands in our redesigned switch (right).
</div>
</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>
<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.  
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>
<br>
<div class="image-center">
<div class="image-center">
<div><img src="http://openwetware.org/images/0/06/2014-EchiDNA-SINGLE-TRIANGULATION.png" /></div>
<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.
Fig 3. A potential triangular confirmation of single switch #1. <br> The signal strand binds without triggering a change in fluorescence.
</div>
</div>
<br>
<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>.
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 class="image-center">
<div><img src="http://openwetware.org/images/b/b6/2014-EchiDNA-SINGLE-2ND-DESIGN.png" /></div>
<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).
Fig. 4. Single switch #2
</div>
</div>
<br>
<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
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.


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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> 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 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.

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