Biomod/2012/TU Dresden/Nanosaurs/Project/Vesicles: Difference between revisions
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{{Biomod/2012/TU Dresden/Nanosaurs/Header}} | {{Biomod/2012/TU Dresden/Nanosaurs/Header}} | ||
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$( "#tabs" ).tabs(); | $( "#tabs" ).tabs(); | ||
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<div id="tabs" class="tabs-bottom"> | <div id="tabs" class="tabs-bottom"> | ||
<ul> | <ul> | ||
<li><a href="#tabs-1">Lipid Vesicles</a></li> | <li><a href="#tabs-1">Lipid Vesicles</a></li> | ||
<li><a href="#tabs-2"> | <li><a href="#tabs-2">Experiments with single stranded oligonucleotides</a></li> | ||
<li><a href="#tabs-3">Gel Analysis</a></li> | <li><a href="#tabs-3">Gel Analysis</a></li> | ||
<li><a href="#tabs-4">DNA origami | <li><a href="#tabs-4">Experiments with DNA origami</a></li> | ||
</ul> | </ul> | ||
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<h2>Overview</h2> | <h2>Overview</h2> | ||
<p>In the very early stages of our project, we decided that our central “tethering species” would be Giant Unilamellar Vesicles (GUVs). We would then attach several DNA origami structures to GUVs via cholesterol modified DNA oligonucleotides. The structure encloses oligonucleotic “catcher strands” and is initially locked by means of an aptamer lock. When a ligand specific to the aptamer is introduced into the system the origami structure would open to reveal these catcher strands. Target species in the solution, which have “receiver strands” complementary to the catcher strands, can then get tethered to the GUVs when the catcher and receiver strands hybridize. We decided that our ideal system would contain Large Unilamellar Vesicles (LUVs) as the “tethered target species”.</p> | <p>In the very early stages of our project, we decided that our central “tethering species” would be Giant Unilamellar Vesicles (GUVs). We would then attach several DNA origami structures to GUVs via cholesterol modified DNA oligonucleotides. The structure encloses oligonucleotic “catcher strands” and is initially locked by means of an aptamer lock. When a ligand specific to the aptamer is introduced into the system the origami structure would open to reveal these catcher strands. Target species in the solution, which have “receiver strands” complementary to the catcher strands, can then get tethered to the GUVs when the catcher and receiver strands hybridize. We decided that our ideal system would contain Large Unilamellar Vesicles (LUVs) as the “tethered target species”.</p> | ||
<div style="margin:auto; width:598px; box-shadow: 0 0 10px #888888; border: 5px solid white;"> | |||
<a href="http://openwetware.org/images/9/9b/BM12_nanosaurs_DeconV1_final.gif" title="DiD labeled GUV (red) tethered by open DNA origami structure to Fast-DiO GUV (green)" rel="lightbox[group1]"> | |||
<img src="http://openwetware.org/images/0/07/BM12_nanosaurs_400x_deconV1_final.gif"> | |||
</a> | |||
</div> | |||
<h3>Introduction</h3> | <h3>Introduction</h3> | ||
<p>We decided to start with a simple system consisting of single stranded DNA oligonucleotides on both the “tethering” and “target” species until the DNA origami structures were fabricated .Based on previous research work | <p>We decided to start with a simple system consisting of single stranded DNA oligonucleotides on both the “tethering” and “target” species until the DNA origami structures were fabricated .Based on previous research work [1], we assumed that the optimal number of anchored oligonucleotide strands per lipid molecule in the vesicles’ membrane was of, 5* 10-3 for GUVs and 4*10-4 for LUVs.</p> | ||
<h3>Materials</h3> | <h3>Materials</h3> | ||
<p>The giant unilamellar vesicles (GUVs) were prepared using electroformation | <p>The giant unilamellar vesicles (GUVs) were prepared using electroformation and the large unilamellar vesicles (LUVs) were prepared using rehydration and extrusion <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#protocols">(Lab Book -> Protocols)</a>. The composition of both of the phospholipid vesicles was the same and consists of 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). The charged vesicles had in addition varying volume amounts (0%-10%) of 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). For some experiments the lipids were labeled with fluorescent dyes, Fast-DiO with an emission at 488 nm and DiD with an emission at 647 nm. There were two different versions of the SLB buffer <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a> depending on the experiments. The single stranded oligonucleotides experiments were with SLB; meanwhile for the origami experiments the SLB buffer included magnesium.</p> | ||
<p>Our cholesterol-modified oligonucleotides were three: catcher A (tethering oligonucleotide used only for the experiments with single stranded oligonucleotides | <p>Our cholesterol-modified oligonucleotides were three: catcher A (tethering oligonucleotide used only for the experiments with single stranded oligonucleotides), anchor-complementary (part of the double-stranded tethering oligonucleotide for the origami structure, catcher-complementary (oligonucleotide for the LUVs as target species).The only non-cholesterol-modified oligonuclotide used was named as “receiver A” since it consisted in the complementary strand for catcher A. Receiver A was modified depending on the target molecule used for the experiments with single stranded oligonucleotides. The target species were Streptavidin-conjugated Quantum dots 625, Alexa 488, Alexa 488-conjugated Streptavidin and finally LUVs.</p> | ||
<p>The experiments were carried out in multiwell plates, each well having a total volume of 40µl. Before using the imaging wells, we incubated for at least 30 minutes with a solution of bovine serum albumin (BSA) ( | <p>The experiments were carried out in multiwell plates, each well having a total volume of 40µl. Before using the imaging wells, we incubated for at least 30 minutes with a solution of bovine serum albumin (BSA) <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a> which was removed previously to setting the experiments. The imaging was done using Zeiss LSM 780 CC3 and the pictures were taken at the equatorial plane of the vesicles.</p> | ||
<h3>Experimental procedure</h3> | <h3>Experimental procedure</h3> | ||
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<ol> | <ol> | ||
<li>GUVs electroformation</li> | <li>GUVs electroformation</li> | ||
<li>Well pasivation ( | <li>Well pasivation with BSA (prevent vesicles disruption)</li> | ||
<li>GUVs observation under the light microscope (checking the stability)</li> | <li>GUVs observation under the light microscope (checking the stability)</li> | ||
<li>Target species preparation:</li> | <li>Target species preparation:</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li>LUVs formation and calibration | <li>LUVs formation and calibration.</li> | ||
<li>Streptavidin-biotin interaction: the biotinylated receiver A oligonuclotide were incubated for 10 min with Quantum dot 625-Streptavidin or Alexa 488-Streptavidin | <li>Streptavidin-biotin interaction: the biotinylated receiver A oligonuclotide were incubated for 10 min with Quantum dot 625-Streptavidin or Alexa 488-Streptavidin.</li> | ||
</ol> | </ol> | ||
<li>Anchoring of cholesterol-modified DNA oligonucleotides:</li> | <li>Anchoring of cholesterol-modified DNA oligonucleotides:</li> | ||
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<li>The well was then imaged using Zeiss LSM 780 CC3 inverse confocal microscope.</li> | <li>The well was then imaged using Zeiss LSM 780 CC3 inverse confocal microscope.</li> | ||
</ol> | </ol> | ||
<div class="clear"></div> | |||
<h2>References</h2> | |||
<p>1. Beales P A, Vanderlick T K (2007), Specific Binding of Different Vesicle Populations by the Hybridization of Membrane-Anchored DNA. J Phys Chem A 111, 12372-12380</p> | |||
</div> | </div> | ||
<div id="tabs-2"> | <div id="tabs-2"> | ||
<h2>Experiments with single stranded oligonucleotides</h2> | <h2>Experiments with single stranded oligonucleotides</h2> | ||
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<h3>Alexa labeled DNA oligonucleoides as target species</h3> | <h3>Alexa labeled DNA oligonucleoides as target species</h3> | ||
<p>In the first set of experiments, receiver A strands labeled with Alexa 488 were used as the target species. | <p>In the first set of experiments, receiver A strands labeled with Alexa 488 were used as the target species <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a>. In the control experiments, no catcher A strands were used. On imaging along the focal plane, clear distinguishable fluorescent rings were observed around the GUVs. Such rings were not observed in the control wells. This clearly indicated that the receiver A strands were hybridizing with the catcher A strands, resulting in such rings.</p> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<div class="img_gal | <div class="img_gal"> | ||
<h4>Schemes</h4> | |||
<div class="img_gbox" style="height: | |||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species.png"><img src="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_s.png" /></a> | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species.png"><img src="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_s.png" /></a> | |||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_CONTROL.png | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_CONTROL.png"><img src="http://www.openwetware.org/images/1/12/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_CONTROL_s.png" /></a> | ||
<div class="descr">Control set up</div> | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with Alexa 488-receiver A</h4> | <h4>Experiment with Alexa 488-receiver A</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/b/b8/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen.jpg"><img src="http://www.openwetware.org/images/7/7b/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen_s.jpg" /></a> | <a rel="lightbox[group2]" title="Alexa 488=receiver A excitation" href="http://www.openwetware.org/images/b/b8/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen.jpg"><img src="http://www.openwetware.org/images/7/7b/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen_s.jpg" /></a> | ||
<div class="descr">Alexa 488 excitation</div> | <div class="descr">Alexa 488 excitation</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/9/9d/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans.jpg"><img src="http://www.openwetware.org/images/7/7c/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans_s.jpg" /></a> | <a rel="lightbox[group2]" title="Transmitted light" href="http://www.openwetware.org/images/9/9d/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans.jpg"><img src="http://www.openwetware.org/images/7/7c/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans_s.jpg" /></a> | ||
<div class="descr">Transmitted light</div> | <div class="descr">Transmitted light</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control experiment w/o catcher A</h4> | <h4>Control experiment w/o catcher A</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen_s.jpg" /></a> | <a rel="lightbox[group3]" title="Control-Alexa 488 excitation" href="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen_s.jpg" /></a> | ||
<div class="descr">Alexa 488 excitation</div></a> | <div class="descr">Alexa 488 excitation</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/1/10/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans_s.jpg" /></a> | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/1/10/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans_s.jpg" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
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<div class="clear"></div> | <div class="clear"></div> | ||
<div class="img_gal | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/5/5d/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species.png"><img src="http://www.openwetware.org/images/8/8e/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species_s.png" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/5/5d/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species.png"><img src="http://www.openwetware.org/images/8/8e/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species_s.png" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="http://www.openwetware.org/images/f/f8/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_speciesCONTROL.png | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/f/f8/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/2c/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_speciesCONTROL_s.png" /></a> | ||
<div class="descr">Control set up</div> | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with Strept-Alexa 488-receiver A</h4> | <h4>Experiment with Strept-Alexa 488-receiver A</h4> | ||
<div class="img_gbox" style="height:180px;"> | |||
<div class="img_gbox" style="height: | <a rel="lightbox[group2]" title="Strept-Alexa 488-receiver A excitation" href="http://www.openwetware.org/images/f/f2/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen.jpg"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen_s.jpg" /></a> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/f/f2/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen.jpg"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen_s.jpg" /></a> | |||
<div class="descr">Alexa 488 excitation</div> | <div class="descr">Alexa 488 excitation</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans.jpg"><img src="http://www.openwetware.org/images/9/9a/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans_s.jpg" /></a> | <a rel="lightbox[group2]" title="Strept-Alexa 488-receiver A Transmitted light" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans.jpg"><img src="http://www.openwetware.org/images/9/9a/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans_s.jpg" /></a> | ||
<div class="descr">Transmitted light</div> | <div class="descr">Transmitted light</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control experiment w/o catcher A</h4> | <h4>Control experiment w/o catcher A</h4> | ||
<div class="img_gbox" style="height: | <div class="clear"></div> | ||
<a rel="lightbox[group3]" title=" | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="Control-Strept-Alexa 488-receiver A excitation" href="http://www.openwetware.org/images/9/95/BM12_nanosaurs_Guv%2Cstrep-alexa _488%2Ccontrol-1SBgreen.jpg"><img src="http://www.openwetware.org/images/0/0d/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBgreen_s.jpg" /></a> | |||
<div class="descr">Alexa 488 excitation</div> | <div class="descr">Alexa 488 excitation</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans_s.jpg" /></a> | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans_s.jpg" /></a> | ||
<div class="descr">Transmited light</div> | <div class="descr">Transmited light</div> | ||
</div> | </div> | ||
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<h3>Quantum dot 625-Streptavidin as target species</h3> | <h3>Quantum dot 625-Streptavidin as target species</h3> | ||
<p>Then we decided to use Quantum dots ( | <p>Then we decided to use Quantum dots (QDs) since they not only have a high quantum yield but also do not bleach and could provide better quality of images. The QD-625 has an emission maximum at 625nm. It has a size of around 25nm. Biotinylated receiver A was first hybridized with QD-Strep, QD-Strep-receiver A, and then later this complex was added to the GUVs bearing chol-catcher A oligos. The controls did not contain the chol-catcher A oligos on the GUVs. The molar ratio of chol-catcher A : Biotinylated-receiver A : QD-Strep = 1:1:0.5 was used. As opposed to the previous results, a fluoresdent ring around the vesicles was not observed. Even more puzzling was the fact that we could not see the QD in solution.</p> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<div class="img_gal | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/3/30/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species.png"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species_s.png" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/3/30/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species.png"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species_s.png" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/e/e8/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/f/f5/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL_s.png" /></a> | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/e/e8/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/f/f5/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL_s.png" /></a> | ||
<div class="descr">Control set up</div> | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with QD-Strep-receiver A</h4> | <h4>Experiment with QD-Strep-receiver A</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/5/53/BM12_nanosaurs_NegativeQDred.jpg"><img src="http://www.openwetware.org/images/6/6f/BM12_nanosaurs_NegativeQDred_s.jpg" /></a> | <a rel="lightbox[group2]" title="QD-Strep-receiver A excitation" href="http://www.openwetware.org/images/5/53/BM12_nanosaurs_NegativeQDred.jpg"><img src="http://www.openwetware.org/images/6/6f/BM12_nanosaurs_NegativeQDred_s.jpg" /></a> | ||
<div class="descr">QD excitation</div> | <div class="descr">QD excitation</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/c/c7/BM12_nanosaurs_NegativeQDtrans.jpg"><img src="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_NegativeQDtrans_s.jpg" /></a> | <a rel="lightbox[group2]" title="QD-Strep-receiver A Transmitted light" href="http://www.openwetware.org/images/c/c7/BM12_nanosaurs_NegativeQDtrans.jpg"><img src="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_NegativeQDtrans_s.jpg" /></a> | ||
<div class="descr">Transmitted light</div> | <div class="descr">Transmitted light</div> | ||
</div> | </div> | ||
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<div id="tabs-3"> | <div id="tabs-3"> | ||
<h2>Gel Analysis</h2> | <h2>Gel Analysis</h2> | ||
<p>After an unsuccessful approach of visualizing the | <p>After an unsuccessful approach of visualizing the QDs, we decided to check the efficiency of oligo-oligo hybridization and binding to Streptavidin-coated QDs on PAGE. The entire set of the experiments below was performed in 12% PA gel <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a>.</p> | ||
<div class="img_right img_link"><a rel="lightbox" href="http://www.openwetware.org/images/a/a2/BM12_nanosaurs_1stgel.jpg"><img src="http://www.openwetware.org/images/e/e0/1stgel.jpg"></a></div> | <div class="img_right img_link"><a rel="lightbox" href="http://www.openwetware.org/images/a/a2/BM12_nanosaurs_1stgel.jpg"><img src="http://www.openwetware.org/images/e/e0/1stgel.jpg"></a></div> | ||
<h3>Biotinylation and Streptavidin binding</h3> | <h3>Biotinylation and Streptavidin binding</h3> | ||
<p>First we checked an efficiency of biotinylation of oligos complementary to cholesterol-coupled oligos. For the gel experiments, oligos without cholesterol were used. There was a shift in the bands between the control oligos and the biotinylated oligo. Also the efficient binding of biotinylated oligo to streptavidin (in lane 4) was observed.</p> | <p>First we checked an efficiency of biotinylation of oligos complementary to cholesterol-coupled oligos. For the gel experiments, oligos without cholesterol were used. There was a shift in the bands between the control oligos and the biotinylated oligo. Also the efficient binding of biotinylated oligo to streptavidin (in lane 4) was observed.</p> | ||
< | <h5>Efficiency of biotinylation reaction. From left: oligo, biotynylated oligo, oligo+streptavidin, biotynylated-oligo+streptavidin</h5> | ||
<h3>Optimal QD-Oligo ratio</h3> | <h3>Optimal QD-Oligo ratio</h3> | ||
<p>In order to optimize the ratio of | <p>In order to optimize the ratio of QDs to the oligonucleotides, different ratios of QDs were applied to constant amount of oligonucleotides (70ng). The optimal molar ratio of oligonucleotides to QDs was found to be 2:1 since the amount of oligonucleotides not bound to QDs was less and also to have a high probability that a single quantum dot is bound by just two oligonucleotides. The other lanes have more free oligonucleotides and therefore would lead to more unspecific binding in solution.</p> | ||
<div class="img_left img_link"><a rel="lightbox" href="http://www.openwetware.org/images/5/5f/BM12_nanosaurs_2ndgel.jpg"><img src="http://www.openwetware.org/images/f/fa/2ndgel.jpg"></a> | <div class="img_left img_link"><a rel="lightbox" href="http://www.openwetware.org/images/5/5f/BM12_nanosaurs_2ndgel.jpg"><img src="http://www.openwetware.org/images/f/fa/2ndgel.jpg"></a> | ||
</div> | </div> | ||
< | <h5>From left: oligonucleotide w/o biotin+QD; Ratio biotinylated-oligonucleotides:QDs 1:1,2:1, 3:1, 5:1,7:1, 10:1.</h5> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h3>Oligo and Quantum dot hybridization</h3> | <h3>Oligo and Quantum dot hybridization</h3> | ||
<div class="img_right img_link"><a rel="lightbox" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_3rdgel.jpg"><img src="http://www.openwetware.org/images/d/d7/3rdgel.jpg"></a></div> | <div class="img_right img_link"><a rel="lightbox" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_3rdgel.jpg"><img src="http://www.openwetware.org/images/d/d7/3rdgel.jpg"></a></div> | ||
<p>Finally, the complete system used for | <p>Finally, the complete system used for QDs experiment was checked on the gel. The results show that catcher A hybridizes with receiver A with high efficiency. However when QD was added, efficiency drops down significantly.</p> | ||
< | <h5>From left: catcher A, biotinylated catcher A-complementary and catcher A + Qds added 30 min later, biotinylated catcher A-complementary and Qds incubated separately + catcher A added 30 min later, biotinylated catcher A-complementary + catcher A | ||
</ | </h5> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h3>Spectral analysis of Quantum dots</h3> | <h3>Spectral analysis of Quantum dots</h3> | ||
<p>We were facing some difficulties in observing the | <p>We were facing some difficulties in observing the QDs' fluorescent both in solution and on the lipid membranes. A poor signal was observed even at high laser powers in confocal microscopy. The PA gels proved that there was no problem with the hybridization of QDs to biotinylated-oligos. Therefore, pure QDt samples of different concentrations were prepared and directly observed on cover slips and bright QDs at relatively low laser power could be observed. Then, we obtained the spectra of these QDs by doing a fluorescence emission scan (excited at 458nm). A peak signal was observed at 615-625nm which is consistent to the QD manufacturer specifications used. When the same analysis was done with our vesicles samples mentioned before, we found that the spectra was not the same as of the QDs due to the background fluorescence of the contaminated lipids samples at high laser powers. This prompted us to increase the concentration of the QDs from 0.1 nM to 10 nM. Subsequently, the concentration of the cholesterol-modified oligonucleotides was increased 50-fold.</p> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<h4>Fluorescent emission scan</h4> | <h4>Fluorescent emission scan</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title=" | <a rel="lightbox[group1]" title="False colour" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LIVECOLOUR.jpg"><img src="http://www.openwetware.org/images/c/ce/BM12_nanosaurs_LIVECOLOUR_s.jpg" alt="Caption" /></a> | ||
<div class="descr">False | <div class="descr">False color</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title=" | <a rel="lightbox[group1]" title="Bright field" href="http://www.openwetware.org/images/f/ff/BM12_nanosaurs_BRGHTFIELD.jpg"><img src="http://www.openwetware.org/images/c/c0/BM12_nanosaurs_BRGHTFIELD_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Bright field</div> | <div class="descr">Bright field</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_MAXEM625.jpg"><img src="http://www.openwetware.org/images/c/cf/BM12_nanosaurs_MAXEM625_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Maximum emission at 625nm" href="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_MAXEM625.jpg"><img src="http://www.openwetware.org/images/c/cf/BM12_nanosaurs_MAXEM625_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">Max em at 625nm</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Vesicles sample</h4> | <h4>Vesicles sample</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/c/c6/BM12_nanosaurs_BACKGROUNDNOISE.jpg"><img src="http://www.openwetware.org/images/8/85/BM12_nanosaurs_BACKGROUNDNOISE_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Lipids background signal" href="http://www.openwetware.org/images/c/c6/BM12_nanosaurs_BACKGROUNDNOISE.jpg"><img src="http://www.openwetware.org/images/8/85/BM12_nanosaurs_BACKGROUNDNOISE_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Background | <div class="descr">Background signal</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/e/e5/BM12_nanosaurs_BACKGROUNDNOISEtrans.jpg"><img src="http://www.openwetware.org/images/b/b0/BM12_nanosaurs_BACKGROUNDNOISEtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Bright field" href="http://www.openwetware.org/images/e/e5/BM12_nanosaurs_BACKGROUNDNOISEtrans.jpg"><img src="http://www.openwetware.org/images/b/b0/BM12_nanosaurs_BACKGROUNDNOISEtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Bright field</div></a> | <div class="descr">Bright field</div></a> | ||
</div> | </div> | ||
Line 216: | Line 223: | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h3>Quantum | <h3>Quantum dots 625-Streptavidin as target species (Higher concentration)</h3> | ||
<p>With the concentration mentioned before, bright fluorescent rings were observed around the GUVs and none in the controls. Thus, we were able to target a large species like | <p>With the concentration mentioned before, bright fluorescent rings were observed around the GUVs and none in the controls. Thus, we were able to target a large species like QDs on the vesicles.</p> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
Line 223: | Line 230: | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height:190px;"> | <div class="img_gbox" style="height:190px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/4/48/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x.png"><img src="http://www.openwetware.org/images/9/99/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/4/48/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x.png"><img src="http://www.openwetware.org/images/9/99/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height:190px;"> | <div class="img_gbox" style="height:190px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/5/5b/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL.png"><img src="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/5/5b/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL.png"><img src="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up</div> | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with 50x concentration of QD</h4> | <h4>Experiment with 50x concentration of QD</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_QDexcited.jpg"><img src="http://www.openwetware.org/images/5/5a/BM12_nanosaurs_QDexcited_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="QDs excitation 50x concentration" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_QDexcited.jpg"><img src="http://www.openwetware.org/images/5/5a/BM12_nanosaurs_QDexcited_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">QDs excitation</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_QDtransmlight.jpg"><img src="http://www.openwetware.org/images/f/f9/BM12_nanosaurs_QDtransmlight_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="QDs 50x concentrationTransmited light" href="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_QDtransmlight.jpg"><img src="http://www.openwetware.org/images/f/f9/BM12_nanosaurs_QDtransmlight_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control experiment with 50x concentration of | <h4>Control experiment with 50x concentration of QDs w/o catcher A</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/6/67/BM12_nanosaurs_QDcont.jpg"><img src="http://www.openwetware.org/images/7/73/BM12_nanosaurs_QDcont_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="QDs excitation w/o catcher A" href="http://www.openwetware.org/images/6/67/BM12_nanosaurs_QDcont.jpg"><img src="http://www.openwetware.org/images/7/73/BM12_nanosaurs_QDcont_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">QDs excitation</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/c/c8/BM12_nanosaurs_QDconttrans.jpg"><img src="http://www.openwetware.org/images/8/83/BM12_nanosaurs_QDconttrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/c/c8/BM12_nanosaurs_QDconttrans.jpg"><img src="http://www.openwetware.org/images/8/83/BM12_nanosaurs_QDconttrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
Line 260: | Line 267: | ||
<h3>Vesicles with different amounts of origami</h3> | <h3>Vesicles with different amounts of origami</h3> | ||
<p>Various amounts of origami were used to find the optimum concentration required to visualize them on vesicles. We observed fluorescent rings around the GUVs with | <p>Various amounts of origami were used to find the optimum concentration required to visualize them on vesicles. We observed fluorescent rings around the GUVs with 1.25nM, 2.5nM and 5nM of the assembled origami. It was decided to use 2.5nM of origami for further experiments due to the bright ring observed with a moderate DNA origami consumption. The controls did not have any anchor-complementary strands.</p> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:170px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/f/f4/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami.png"><img src="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/f/f4/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami.png"><img src="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:170px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/4/44/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL.png"><img src="http://www.openwetware.org/images/5/52/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/4/44/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL.png"><img src="http://www.openwetware.org/images/5/52/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up</div> | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with | <h4>Experiment with 1.25nM of DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_4microlSB.jpg"><img src="http://www.openwetware.org/images/d/d2/BM12_nanosaurs_4microlSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Alexa 647 DNA origami excitation (1.25nM)" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_4microlSB.jpg"><img src="http://www.openwetware.org/images/d/d2/BM12_nanosaurs_4microlSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/6/61/BM12_nanosaurs_4microltransSB.jpg" | <a rel="lightbox[group2]" title="Transmited light (1.25nM)" href="http://www.openwetware.org/images/6/61/BM12_nanosaurs_4microltransSB.jpg"><img src="http://www.openwetware.org/images/c/c4/BM12_nanosaurs_4microltransSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with | <h4>Experiment with 2.5nM of DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_8microl_single2SB.jpg" | <a rel="lightbox[group3]" title="Alexa 647 DNA origami excitation (2.5nM)" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_8microl_single2SB.jpg"><img src="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_8microl_single2SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/5/50/BM12_nanosaurs_8microl_single2transSB.jpg" | <a rel="lightbox[group3]" title="Transmited light (2.5nM)" href="http://www.openwetware.org/images/5/50/BM12_nanosaurs_8microl_single2transSB.jpg"><img src="http://www.openwetware.org/images/8/84/BM12_nanosaurs_8microl_single2transSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with | <h4>Experiment with 5nM of DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/c/c4/BM12_nanosaurs_18microl_group2SB.jpg" | <a rel="lightbox[group4]" title="Alexa 647 DNA origami excitation (5nM)" href="http://www.openwetware.org/images/c/c4/BM12_nanosaurs_18microl_group2SB.jpg"><img src="http://www.openwetware.org/images/7/73/BM12_nanosaurs_18microl_group2SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/9/9e/BM12_nanosaurs_18microl_group2transSB.jpg" | <a rel="lightbox[group4]" title="Transmited light (5nM)" href="http://www.openwetware.org/images/9/9e/BM12_nanosaurs_18microl_group2transSB.jpg"><img src="http://www.openwetware.org/images/d/d3/BM12_nanosaurs_18microl_group2transSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control experiment w/o anchor-complementary oligos | <h4>Control experiment w/o anchor-complementary oligos (2.5nM)</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group5]" title="" href="http://www.openwetware.org/images/d/d8/BM12_nanosaurs_8microl_single_controlSB.jpg" | <a rel="lightbox[group5]" title="Alexa 647 DNA origami excitation w/o anchor-complementary oligos 2.5nM)" href="http://www.openwetware.org/images/d/d8/BM12_nanosaurs_8microl_single_controlSB.jpg"><img src="http://www.openwetware.org/images/3/3a/BM12_nanosaurs_8microl_single_controlSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group5]" title="" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_8microl_single_controltransSB.jpg" | <a rel="lightbox[group5]" title="Control-Transmited ligh" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_8microl_single_controltransSB.jpg"><img src="http://www.openwetware.org/images/7/76/BM12_nanosaurs_8microl_single_controltransSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
Line 316: | Line 323: | ||
<h3>Alexa 647 labeled catcher complementary DNA oligo strands as target species for the DNA origami structure</h3> | <h3>Alexa 647 labeled catcher complementary DNA oligo strands as target species for the DNA origami structure</h3> | ||
<p>After obtaining very promising results with the origami anchorage into the GUVs, we wanted to find out whether the catcher strands on the origami are able to hybridize with the catcher-complementary strands properly. We used Alexa 647-labeled | <p>After obtaining very promising results with the origami anchorage into the GUVs, we wanted to find out whether the catcher strands on the origami are able to hybridize with the catcher-complementary strands properly. We used Alexa 647-labeled catcher-complementary strands to hybridize with the origami catcher strands and the GUVs were labeled with the green fluorophore DiO. The control had the closed origami. We observed a strong ring around the GUVs with the open origami. In the control we observed no ring around the GUVs. With this positive result for the origami, the following step was to repeat the experiment using the LUVs targeting system.</p> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title=" | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structure.png"><img src="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structure_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title=" | <a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/5/5f/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structureCONTROLclosed.png"><img src="http://www.openwetware.org/images/2/22/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structureCONTROLclosed_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with open DNA origami structure</h4> | <h4>Experiment with open DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/1/19/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB.jpg"><img src="http://www.openwetware.org/images/3/35/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Alexa 647-catcher-complementary oligos excitation" href="http://www.openwetware.org/images/1/19/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB.jpg"><img src="http://www.openwetware.org/images/3/35/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Alexa 647 | <div class="descr">Alexa 647</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/1/18/BM12_nanosaurs_OpenCy3origamiSB.jpg"><img src="http://www.openwetware.org/images/0/01/BM12_nanosaurs_OpenCy3origamiSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Cy3 DNA origami excitation" href="http://www.openwetware.org/images/1/18/BM12_nanosaurs_OpenCy3origamiSB.jpg"><img src="http://www.openwetware.org/images/0/01/BM12_nanosaurs_OpenCy3origamiSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_GuvDiDSB.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_GuvDiDSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_GuvDiDSB.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_GuvDiDSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUVs</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with closed DNA origami structure</h4> | <h4>Experiment with closed DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/7/7d/BM12_nanosaurs_CONTROLCatcher-compalexa647SB.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_CONTROLCatcher-compalexa647SB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Alexa 647-catcher-complementary oligos excitation" href="http://www.openwetware.org/images/7/7d/BM12_nanosaurs_CONTROLCatcher-compalexa647SB.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_CONTROLCatcher-compalexa647SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Alexa 647 | <div class="descr">Alexa 647</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_CONTROLguvsDiDSB.jpg"><img src="http://www.openwetware.org/images/7/74/BM12_nanosaurs_CONTROLguvsDiDSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_CONTROLguvsDiDSB.jpg"><img src="http://www.openwetware.org/images/7/74/BM12_nanosaurs_CONTROLguvsDiDSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUVs</div></a> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="img_gal"> | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with open DNA origami structure</h4> | <h4>Experiment with open DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen.jpg"><img src="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen.jpg"><img src="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/7/73/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred.jpg"><img src="http://www.openwetware.org/images/b/be/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Alexa 647 DNA origami excitation" href="http://www.openwetware.org/images/7/73/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred.jpg"><img src="http://www.openwetware.org/images/b/be/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/6/62/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp.jpg"><img src="http://www.openwetware.org/images/c/ce/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/6/62/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp.jpg"><img src="http://www.openwetware.org/images/c/ce/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with closed DNA origami structure</h4> | <h4>Experiment with closed DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBred.jpg" | <a rel="lightbox[group3]" title="Alexa 647 DNA origami excitation" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBred.jpg"><img src="http://www.openwetware.org/images/d/d3/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/b/ba/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans.jpg"><img src="http://www.openwetware.org/images/0/02/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/b/ba/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans.jpg"><img src="http://www.openwetware.org/images/0/02/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment w/o DNA origami structure</h4> | <h4>Experiment w/o DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/8/81/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred.jpg"><img src="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group4]" title="Alexa 647 excitation of DNA origami" href="http://www.openwetware.org/images/8/81/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred.jpg"><img src="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="http://www.openwetware.org/images/6/69/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBtrans.jpg | <a rel="lightbox[group4]" title="Control-Transmited light" href="http://www.openwetware.org/images/6/69/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBtrans.jpg"><img src="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
Line 424: | Line 431: | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with open DNA origami structure</h4> | <h4>Experiment with open DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/7/75/10%25LUVSPS_open.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Fast-DiO LUVs" href="http://www.openwetware.org/images/7/75/10%25LUVSPS_open.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/9/95/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSBtrans.jpg" | <a rel="lightbox[group1]" title="Transmited light" href="http://www.openwetware.org/images/9/95/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with closed DNA origami structure</h4> | <h4>Experiment with closed DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/4/40/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBgreen.jpg" | <a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/4/40/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBgreen.jpg"><img src="http://www.openwetware.org/images/b/bd/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/9/98/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBtrans.jpg" | <a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/9/98/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/6/6a/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment w/o DNA origami structure</h4> | <h4>Experiment w/o DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBgreen.jpg" | <a rel="lightbox[group3]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBgreen.jpg"><img src="http://www.openwetware.org/images/5/57/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBtransp.jpg" | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBtransp.jpg"><img src="http://www.openwetware.org/images/f/f2/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBtransp_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
Line 476: | Line 483: | ||
<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/5/51/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species.png"><img src="http://www.openwetware.org/images/d/de/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/5/51/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species.png"><img src="http://www.openwetware.org/images/d/de/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/5/54/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1.png"><img src="http://www.openwetware.org/images/b/b9/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up (w/o catcher-complementary)" href="http://www.openwetware.org/images/5/54/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1.png"><img src="http://www.openwetware.org/images/b/b9/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/2/23/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/2d/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control set up (closed origami structure)" href="http://www.openwetware.org/images/2/23/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/2d/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | ||
<div class="descr">Control set up | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Experiment with open DNA origami structure</h4> | <h4>Experiment with open DNA origami structure</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/8/87/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!.jpg"><img src="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="DiD GUVs excitation" href="http://www.openwetware.org/images/8/87/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!.jpg"><img src="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/e/e6/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans.jpg"><img src="http://openwetware.org/images/b/bf/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/e/e6/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans.jpg"><img src="http://openwetware.org/images/b/bf/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control | <h4>Control experiment w/o catcher-complementary oligos</h4> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/a/a8/BM12_nanosaurs_Control_to_the_huggingsSB.jpg"><img src="http://www.openwetware.org/images/2/2c/BM12_nanosaurs_Control_to_the_huggingsSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="DiD GUVs excitation" href="http://www.openwetware.org/images/a/a8/BM12_nanosaurs_Control_to_the_huggingsSB.jpg"><img src="http://www.openwetware.org/images/2/2c/BM12_nanosaurs_Control_to_the_huggingsSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUVs</div></a> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/8/88/BM12_nanosaurs_Control_to_the_huggingsSBtrans.jpg"><img src="http://www.openwetware.org/images/3/31/BM12_nanosaurs_Control_to_the_huggingsSBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/8/88/BM12_nanosaurs_Control_to_the_huggingsSBtrans.jpg"><img src="http://www.openwetware.org/images/3/31/BM12_nanosaurs_Control_to_the_huggingsSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | </div> | ||
Line 511: | Line 518: | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<p>Encouraged by the results with uniformly labeled vesicles we had prepared two classes of vesicles stained either with red fluorescent dye (DID) or with green (DIO). In the controls without the catcher-complementary strands, the GUVs do not cluster and do not seem to attract each other.</p> | <p>Encouraged by the results with uniformly labeled vesicles we had prepared two classes of vesicles stained either with red fluorescent dye (DID) or with green (DIO). In the controls without the catcher-complementary strands, the GUVs do not cluster and do not seem to attract each other.</p> | ||
<div class="clear"></div> | |||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <h4>Fluorescent GUVs</h4> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen.jpg"><img src="http://www.openwetware.org/images/9/93/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen_s.jpg" alt="Caption" /></a> | <div class="img_gbox" style="height:180px;"> | ||
<div class="descr"> | <a rel="lightbox[group1]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen.jpg"><img src="http://www.openwetware.org/images/9/93/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr">GUVs</div> | |||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred.jpg"><img src="http://www.openwetware.org/images/2/26/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="DiD LUVs excitation" href="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred.jpg"><img src="http://www.openwetware.org/images/2/26/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/e/ec/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans.jpg"><img src="http://www.openwetware.org/images/b/b2/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Transmited light" href="http://www.openwetware.org/images/e/ec/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans.jpg"><img src="http://www.openwetware.org/images/b/b2/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div></a> | <div class="descr">Transmited light</div></a> | ||
</div> | |||
<div class="clear"></div> | |||
<h4>Experiment with open DNA origami structure</h4> | |||
<div class="img_gbox" style="height:200px;"> | |||
<a rel="lightbox[group1]" title="Fast-DiO and DiD GUVs excitation 1" href="http://www.openwetware.org/images/e/e3/BM12_nanosaurs_Kissing_vesicles_%28guvs%29.lsm_%28RGB%29.jpg"><img src="http://www.openwetware.org/images/7/75/BM12_nanosaurs_Kissing_vesicles_%28guvs%29.lsm_%28RGB%29_s.jpg" alt="Caption" /></a> | |||
<div class="descr">Fast-DiO and DiD GUVs</div> | |||
</div> | |||
<div class="img_gbox" style="height:200px;"> | |||
<a rel="lightbox[group1]" title="Fast-DiO and DiD GUVs excitation 2" href="http://www.openwetware.org/images/f/ff/BM12_nanosaurs_Kissing_vesicles_%28guvs%292.lsm_%28RGB%29SB.jpg"><img src="http://www.openwetware.org/images/7/7d/BM12_nanosaurs_Kissing_vesicles_%28guvs%292.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a> | |||
<div class="descr">Fast-DiO and DiD GUVs</div> | |||
</div> | </div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<p>But, we could also notice vesicles being attracted to each other with the closed origami structure. This led us to conclude that the structures | |||
<p>But, we could also notice vesicles being attracted to each other with the closed origami structure. This led us to conclude that the DNA origami structures might not be totally closed.</p> | |||
<div class="clear"></div> | |||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <h4>Control experiment with closed DNA origami structure</h4> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/ | <div class="img_gbox" style="height:180px;"> | ||
<div class="descr">Control | <a rel="lightbox[group1]" title="Control 1(closed DNA origami)" href="http://www.openwetware.org/images/7/74/BM12_nanosaurs_Kissing_control.lsm_%28RGB%29SB-800.jpg"><img src="http://www.openwetware.org/images/f/f6/BM12_nanosaurs_Kissing_control.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Control 1</div> | |||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/ | <a rel="lightbox[group1]" title="Control 2(closed DNA origami)" href="http://www.openwetware.org/images/e/e8/BM12_nanosaurs_Kissing.control.amazing.lsm_%28RGB%29SB-800.jpg"><img src="http://www.openwetware.org/images/c/c1/BM12_nanosaurs_Kissing.control.amazing.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Control | <div class="descr">Control 2</div> | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h3>LUVs with variable (3 | <h3>LUVs with variable (3 and 7 mol%) negative charge (DOPS) with catcher complementary strands as target species</h3> | ||
<p>We needed to optimize the amount of negative charge such that Fast-DiO labeled (Green) LUVs could interact with DiD labeled (Red) GUVs in the presence of origami, but not cluster in its absence. We started by preparing LUVs with 3 mol% DOPS. There were controls with the closed origami, without any origami and one more without origami but with the catcher-complementary. Bright rings were present in all the samples including the controls.</p> | <p>We needed to optimize the amount of negative charge such that Fast-DiO labeled (Green) LUVs could interact with DiD labeled (Red) GUVs in the presence of origami, but not cluster in its absence. We started by preparing LUVs with 3 mol% DOPS. There were controls with the closed origami, without any origami and one more without origami but with the catcher-complementary. Bright rings were present in all the samples including the controls.</p> | ||
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<h4>Schemes</h4> | <h4>Schemes</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a> | ||
<div class="descr">Set up</div> | <div class="descr">Set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a> | ||
<div class="descr">Control | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group1]" title="" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | <a rel="lightbox[group1]" title="Control (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a> | ||
<div class="descr">Control | <div class="descr">Control set up</div> | ||
</div> | </div> | ||
</div> | </div> | ||
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<h4>Open origami 3%PS </h4> | <h4>Open origami 3%PS </h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/4/43/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green.jpg"><img src="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/4/43/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green.jpg"><img src="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox"style="height:180px;"> | ||
<a rel="lightbox[group2]" title="" href="http://www.openwetware.org/images/0/0f/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans.jpg"><img src="http://www.openwetware.org/images/8/8a/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/0/0f/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans.jpg"><img src="http://www.openwetware.org/images/8/8a/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div> | <div class="descr">Transmited light</div> | ||
</div> | </div> | ||
Line 574: | Line 594: | ||
<h4>Control with closed DNA origami structure</h4> | <h4>Control with closed DNA origami structure</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/c/ca/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSB.jpg"><img src="http://www.openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Fast-DiO excitation" href="http://www.openwetware.org/images/c/ca/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSB.jpg"><img src="http://www.openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVS</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group3]" title="" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/d/d1/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group3]" title="Transmited light" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/d/d1/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div> | <div class="descr">Transmited light</div> | ||
</div> | </div> | ||
Line 586: | Line 606: | ||
<h4>Control with no DNA origami structure</h4> | <h4>Control with no DNA origami structure</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB.jpg"><img src="http://openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group4]" title="Fast-DiO excitation (w/o DNA origami)" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB.jpg"><img src="http://openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/d/dd/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans.jpg"><img src="http://www.openwetware.org/images/3/3e/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group4]" title="Control-Transmited light" href="http://www.openwetware.org/images/d/dd/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans.jpg"><img src="http://www.openwetware.org/images/3/3e/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div> | <div class="descr">Transmited light</div> | ||
</div> | </div> | ||
Line 597: | Line 617: | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<p>Hence, we needed to use a medial value between 3 and 10 mol% and therefore LUVs with 7 mol% DOPS were prepared and targeted by the Cy3-labeled origami on the GUV surface. The previous controls were repeated for this experiment | <p>Hence, we needed to use a medial value between 3 and 10 mol% and therefore LUVs with 7 mol% DOPS were prepared and targeted by the Cy3-labeled origami on the GUV surface. The previous controls were repeated for this experiment. Bright fluorescent rings were present in both the samples with open and closed origami structures, but it was not present in the control with catcher-complementary strands. Thus, we decided that the optimal negative charge of 7 mol% was required on the LUVs to screen the unspecific binding. The reason for a fluorescent ring with the closed DNA origami structure might be due to the fact that the porcentage of the open structure after the assembly of the closed structure could be high enough to allow the targeting hybridization <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Project/DNA_origami#tabs-2">(Project->DNAorigami->Assembly)</a>.</p> | ||
<h4>Open origami 7%PS</h4> | <h4>Open origami 7%PS</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group5]" title="" href="http://www.openwetware.org/images/9/93/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/2/20/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group5]" title="Fast-DiO LUVs excitation 7%PS" href="http://www.openwetware.org/images/9/93/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/2/20/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group5]" title="" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange.jpg"><img src="http://www.openwetware.org/images/1/14/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group5]" title="Cy3 DNA origami excitation 7%PS" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange.jpg"><img src="http://www.openwetware.org/images/1/14/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group5]" title="" href="http://www.openwetware.org/images/8/8f/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/d/dc/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group5]" title="DiD excitation of GUV 7%PS" href="http://www.openwetware.org/images/8/8f/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/d/dc/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUV</div> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 617: | Line 637: | ||
<h4>Control with closed DNA origami structure</h4> | <h4>Control with closed DNA origami structure</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group6]" title="" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/1/1e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group6]" title="Fast-DiO LUVs excitation (closed DNA origami)" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/1/1e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group6]" title="" href="http://openwetware.org/images/d/d6/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp.jpg"><img src="http://openwetware.org/images/5/58/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group6]" title="Cy3 DNA origami excitation(closed DNA origami)" href="http://openwetware.org/images/d/d6/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp.jpg"><img src="http://openwetware.org/images/5/58/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">DNA origami</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group6]" title="" href="http://www.openwetware.org/images/e/ef/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/4/41/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_%28Custom%29.jpg" alt="Caption" /></a> | <a rel="lightbox[group6]" title="DiD GUV excitation(closed DNA origami)" href="http://www.openwetware.org/images/e/ef/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/4/41/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_%28Custom%29.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUV</div> | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
<h4>Control | <h4>Control w/o DNA origami structure</h4> | ||
<div class="img_gal"> | <div class="img_gal"> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group7]" title="" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen.jpg"><img src="http://www.openwetware.org/images/d/d5/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group7]" title="Fast-DiO LUVs excitation (w/o DNA origami)" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen.jpg"><img src="http://www.openwetware.org/images/d/d5/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">LUVs</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group7]" title="" href="http://www.openwetware.org/images/c/c2/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred.jpg"><img src="http://www.openwetware.org/images/d/da/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group7]" title="DiD GUV excitation (w/o DNA origami)" href="http://www.openwetware.org/images/c/c2/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred.jpg"><img src="http://www.openwetware.org/images/d/da/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred_s.jpg" alt="Caption" /></a> | ||
<div class="descr"> | <div class="descr">GUV</div> | ||
</div> | </div> | ||
<div class="img_gbox" style="height: | <div class="img_gbox" style="height:180px;"> | ||
<a rel="lightbox[group7]" title="" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp.jpg"><img src="http://www.openwetware.org/images/1/12/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp_s.jpg" alt="Caption" /></a> | <a rel="lightbox[group7]" title="Transmited light (w/o DNA origami)" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp.jpg"><img src="http://www.openwetware.org/images/1/12/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp_s.jpg" alt="Caption" /></a> | ||
<div class="descr">Transmited light</div> | <div class="descr">Transmited light</div> | ||
</div> | </div> | ||
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 23:47, 27 October 2012
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- <a href="#tabs-1">Lipid Vesicles</a>
- <a href="#tabs-2">Experiments with single stranded oligonucleotides</a>
- <a href="#tabs-3">Gel Analysis</a>
- <a href="#tabs-4">Experiments with DNA origami</a>
Overview
In the very early stages of our project, we decided that our central “tethering species” would be Giant Unilamellar Vesicles (GUVs). We would then attach several DNA origami structures to GUVs via cholesterol modified DNA oligonucleotides. The structure encloses oligonucleotic “catcher strands” and is initially locked by means of an aptamer lock. When a ligand specific to the aptamer is introduced into the system the origami structure would open to reveal these catcher strands. Target species in the solution, which have “receiver strands” complementary to the catcher strands, can then get tethered to the GUVs when the catcher and receiver strands hybridize. We decided that our ideal system would contain Large Unilamellar Vesicles (LUVs) as the “tethered target species”.
<a href="http://openwetware.org/images/9/9b/BM12_nanosaurs_DeconV1_final.gif" title="DiD labeled GUV (red) tethered by open DNA origami structure to Fast-DiO GUV (green)" rel="lightbox[group1]"> <img src="http://openwetware.org/images/0/07/BM12_nanosaurs_400x_deconV1_final.gif"> </a>
Introduction
We decided to start with a simple system consisting of single stranded DNA oligonucleotides on both the “tethering” and “target” species until the DNA origami structures were fabricated .Based on previous research work [1], we assumed that the optimal number of anchored oligonucleotide strands per lipid molecule in the vesicles’ membrane was of, 5* 10-3 for GUVs and 4*10-4 for LUVs.
Materials
The giant unilamellar vesicles (GUVs) were prepared using electroformation and the large unilamellar vesicles (LUVs) were prepared using rehydration and extrusion <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#protocols">(Lab Book -> Protocols)</a>. The composition of both of the phospholipid vesicles was the same and consists of 1, 2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). The charged vesicles had in addition varying volume amounts (0%-10%) of 1, 2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). For some experiments the lipids were labeled with fluorescent dyes, Fast-DiO with an emission at 488 nm and DiD with an emission at 647 nm. There were two different versions of the SLB buffer <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a> depending on the experiments. The single stranded oligonucleotides experiments were with SLB; meanwhile for the origami experiments the SLB buffer included magnesium.
Our cholesterol-modified oligonucleotides were three: catcher A (tethering oligonucleotide used only for the experiments with single stranded oligonucleotides), anchor-complementary (part of the double-stranded tethering oligonucleotide for the origami structure, catcher-complementary (oligonucleotide for the LUVs as target species).The only non-cholesterol-modified oligonuclotide used was named as “receiver A” since it consisted in the complementary strand for catcher A. Receiver A was modified depending on the target molecule used for the experiments with single stranded oligonucleotides. The target species were Streptavidin-conjugated Quantum dots 625, Alexa 488, Alexa 488-conjugated Streptavidin and finally LUVs.
The experiments were carried out in multiwell plates, each well having a total volume of 40µl. Before using the imaging wells, we incubated for at least 30 minutes with a solution of bovine serum albumin (BSA) <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a> which was removed previously to setting the experiments. The imaging was done using Zeiss LSM 780 CC3 and the pictures were taken at the equatorial plane of the vesicles.
Experimental procedure
- GUVs electroformation
- Well pasivation with BSA (prevent vesicles disruption)
- GUVs observation under the light microscope (checking the stability)
- Target species preparation:
- LUVs formation and calibration.
- Streptavidin-biotin interaction: the biotinylated receiver A oligonuclotide were incubated for 10 min with Quantum dot 625-Streptavidin or Alexa 488-Streptavidin.
- Anchoring of cholesterol-modified DNA oligonucleotides:
- To GUVs and LUVs: the vesicles were incubated at room temperature for a period of two hours with the corresponding cholesterol oligos. At the end of this process, most strands were anchored to the lipid vesicles NOTE: for the experiments with origami previous to the anchoring, the cholesterol-modified anchor-complementary oligonucleotides were incubated for 30 min to hybridize with the corresponding origami structure.
- Hybridization: the target species were mixed with the GUVs and incubated overnight.
- The well was then imaged using Zeiss LSM 780 CC3 inverse confocal microscope.
References
1. Beales P A, Vanderlick T K (2007), Specific Binding of Different Vesicle Populations by the Hybridization of Membrane-Anchored DNA. J Phys Chem A 111, 12372-12380
Experiments with single stranded oligonucleotides
Our tethering species were always GUVs. Since the previous data on the optimal concentration of the oligos on the vesicles were available only for homogenous systems (consisting either GUVs or LUVs), we started with simple tethered target species (fluorophores) to find the optimal concentration of the components and moved on to our final target (LUVs). The tethering and target systems used in this first stage were the catcher A strands and receiver A strands respectively. The molar ratio between the tethering and target oligos was always kept as 1:1.
Alexa labeled DNA oligonucleoides as target species
In the first set of experiments, receiver A strands labeled with Alexa 488 were used as the target species <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a>. In the control experiments, no catcher A strands were used. On imaging along the focal plane, clear distinguishable fluorescent rings were observed around the GUVs. Such rings were not observed in the control wells. This clearly indicated that the receiver A strands were hybridizing with the catcher A strands, resulting in such rings.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species.png"><img src="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_s.png" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_CONTROL.png"><img src="http://www.openwetware.org/images/1/12/BM12_nanosaurs_Alexa_labeled_DNA_oligonucleoides_as_target_species_CONTROL_s.png" /></a>
Experiment with Alexa 488-receiver A
<a rel="lightbox[group2]" title="Alexa 488=receiver A excitation" href="http://www.openwetware.org/images/b/b8/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen.jpg"><img src="http://www.openwetware.org/images/7/7b/BM12_nanosaurs_Guv%2Calexa_488-1SBgreen_s.jpg" /></a>
<a rel="lightbox[group2]" title="Transmitted light" href="http://www.openwetware.org/images/9/9d/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans.jpg"><img src="http://www.openwetware.org/images/7/7c/BM12_nanosaurs_Guv%2Calexa_488-1SBtrans_s.jpg" /></a>
Control experiment w/o catcher A
<a rel="lightbox[group3]" title="Control-Alexa 488 excitation" href="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBgreen_s.jpg" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/1/10/BM12_nanosaurs_Guv%2Calexa_488%2Ccontrol-1SBtrans_s.jpg" /></a>
Alexa 488 labeled Streptavidin molecules as target species
Subsequently, Streptavidin labeled with Alexa-488 was used as the target species. In the control experiments, no catcher A strands were added. Fluorescent rings were also present around the GUVs.. The control wells didn’t present such rings. This confirmed that it was possible to hybridize more than just oligonucleotides.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/5/5d/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species.png"><img src="http://www.openwetware.org/images/8/8e/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_species_s.png" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/f/f8/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/2c/BM12_nanosaurs_Alexa_488_labeled_Streptavidin_molecules_as_target_speciesCONTROL_s.png" /></a>
Experiment with Strept-Alexa 488-receiver A
<a rel="lightbox[group2]" title="Strept-Alexa 488-receiver A excitation" href="http://www.openwetware.org/images/f/f2/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen.jpg"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBgreen_s.jpg" /></a>
<a rel="lightbox[group2]" title="Strept-Alexa 488-receiver A Transmitted light" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans.jpg"><img src="http://www.openwetware.org/images/9/9a/BM12_nanosaurs_Guv%2Cstrep-alexa_488-2SBtrans_s.jpg" /></a>
Control experiment w/o catcher A
<a rel="lightbox[group3]" title="Control-Strept-Alexa 488-receiver A excitation" href="http://www.openwetware.org/images/9/95/BM12_nanosaurs_Guv%2Cstrep-alexa _488%2Ccontrol-1SBgreen.jpg"><img src="http://www.openwetware.org/images/0/0d/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBgreen_s.jpg" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/9/92/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans.jpg"><img src="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guv%2Cstrep-alexa_488%2Ccontrol-1SBtrans_s.jpg" /></a>
Quantum dot 625-Streptavidin as target species
Then we decided to use Quantum dots (QDs) since they not only have a high quantum yield but also do not bleach and could provide better quality of images. The QD-625 has an emission maximum at 625nm. It has a size of around 25nm. Biotinylated receiver A was first hybridized with QD-Strep, QD-Strep-receiver A, and then later this complex was added to the GUVs bearing chol-catcher A oligos. The controls did not contain the chol-catcher A oligos on the GUVs. The molar ratio of chol-catcher A : Biotinylated-receiver A : QD-Strep = 1:1:0.5 was used. As opposed to the previous results, a fluoresdent ring around the vesicles was not observed. Even more puzzling was the fact that we could not see the QD in solution.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/3/30/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species.png"><img src="http://www.openwetware.org/images/0/0c/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species_s.png" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/e/e8/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/f/f5/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_speciesCONTROL_s.png" /></a>
Experiment with QD-Strep-receiver A
<a rel="lightbox[group2]" title="QD-Strep-receiver A excitation" href="http://www.openwetware.org/images/5/53/BM12_nanosaurs_NegativeQDred.jpg"><img src="http://www.openwetware.org/images/6/6f/BM12_nanosaurs_NegativeQDred_s.jpg" /></a>
<a rel="lightbox[group2]" title="QD-Strep-receiver A Transmitted light" href="http://www.openwetware.org/images/c/c7/BM12_nanosaurs_NegativeQDtrans.jpg"><img src="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_NegativeQDtrans_s.jpg" /></a>
Gel Analysis
After an unsuccessful approach of visualizing the QDs, we decided to check the efficiency of oligo-oligo hybridization and binding to Streptavidin-coated QDs on PAGE. The entire set of the experiments below was performed in 12% PA gel <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book#recipes">(Lab Book -> Recipes)</a>.
Biotinylation and Streptavidin binding
First we checked an efficiency of biotinylation of oligos complementary to cholesterol-coupled oligos. For the gel experiments, oligos without cholesterol were used. There was a shift in the bands between the control oligos and the biotinylated oligo. Also the efficient binding of biotinylated oligo to streptavidin (in lane 4) was observed.
Efficiency of biotinylation reaction. From left: oligo, biotynylated oligo, oligo+streptavidin, biotynylated-oligo+streptavidin
Optimal QD-Oligo ratio
In order to optimize the ratio of QDs to the oligonucleotides, different ratios of QDs were applied to constant amount of oligonucleotides (70ng). The optimal molar ratio of oligonucleotides to QDs was found to be 2:1 since the amount of oligonucleotides not bound to QDs was less and also to have a high probability that a single quantum dot is bound by just two oligonucleotides. The other lanes have more free oligonucleotides and therefore would lead to more unspecific binding in solution.
From left: oligonucleotide w/o biotin+QD; Ratio biotinylated-oligonucleotides:QDs 1:1,2:1, 3:1, 5:1,7:1, 10:1.
Oligo and Quantum dot hybridization
Finally, the complete system used for QDs experiment was checked on the gel. The results show that catcher A hybridizes with receiver A with high efficiency. However when QD was added, efficiency drops down significantly.
From left: catcher A, biotinylated catcher A-complementary and catcher A + Qds added 30 min later, biotinylated catcher A-complementary and Qds incubated separately + catcher A added 30 min later, biotinylated catcher A-complementary + catcher A
Spectral analysis of Quantum dots
We were facing some difficulties in observing the QDs' fluorescent both in solution and on the lipid membranes. A poor signal was observed even at high laser powers in confocal microscopy. The PA gels proved that there was no problem with the hybridization of QDs to biotinylated-oligos. Therefore, pure QDt samples of different concentrations were prepared and directly observed on cover slips and bright QDs at relatively low laser power could be observed. Then, we obtained the spectra of these QDs by doing a fluorescence emission scan (excited at 458nm). A peak signal was observed at 615-625nm which is consistent to the QD manufacturer specifications used. When the same analysis was done with our vesicles samples mentioned before, we found that the spectra was not the same as of the QDs due to the background fluorescence of the contaminated lipids samples at high laser powers. This prompted us to increase the concentration of the QDs from 0.1 nM to 10 nM. Subsequently, the concentration of the cholesterol-modified oligonucleotides was increased 50-fold.
Fluorescent emission scan
<a rel="lightbox[group1]" title="False colour" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LIVECOLOUR.jpg"><img src="http://www.openwetware.org/images/c/ce/BM12_nanosaurs_LIVECOLOUR_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Bright field" href="http://www.openwetware.org/images/f/ff/BM12_nanosaurs_BRGHTFIELD.jpg"><img src="http://www.openwetware.org/images/c/c0/BM12_nanosaurs_BRGHTFIELD_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Maximum emission at 625nm" href="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_MAXEM625.jpg"><img src="http://www.openwetware.org/images/c/cf/BM12_nanosaurs_MAXEM625_s.jpg" alt="Caption" /></a>
Vesicles sample
<a rel="lightbox[group1]" title="Lipids background signal" href="http://www.openwetware.org/images/c/c6/BM12_nanosaurs_BACKGROUNDNOISE.jpg"><img src="http://www.openwetware.org/images/8/85/BM12_nanosaurs_BACKGROUNDNOISE_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Bright field" href="http://www.openwetware.org/images/e/e5/BM12_nanosaurs_BACKGROUNDNOISEtrans.jpg"><img src="http://www.openwetware.org/images/b/b0/BM12_nanosaurs_BACKGROUNDNOISEtrans_s.jpg" alt="Caption" /></a>
Quantum dots 625-Streptavidin as target species (Higher concentration)
With the concentration mentioned before, bright fluorescent rings were observed around the GUVs and none in the controls. Thus, we were able to target a large species like QDs on the vesicles.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/4/48/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x.png"><img src="http://www.openwetware.org/images/9/99/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50x_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/5/5b/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL.png"><img src="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Quantum_dot_625-Streptavidin_as_target_species50xCONTROL_s.png" alt="Caption" /></a>
Experiment with 50x concentration of QD
<a rel="lightbox[group2]" title="QDs excitation 50x concentration" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_QDexcited.jpg"><img src="http://www.openwetware.org/images/5/5a/BM12_nanosaurs_QDexcited_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="QDs 50x concentrationTransmited light" href="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_QDtransmlight.jpg"><img src="http://www.openwetware.org/images/f/f9/BM12_nanosaurs_QDtransmlight_s.jpg" alt="Caption" /></a>
Control experiment with 50x concentration of QDs w/o catcher A
<a rel="lightbox[group3]" title="QDs excitation w/o catcher A" href="http://www.openwetware.org/images/6/67/BM12_nanosaurs_QDcont.jpg"><img src="http://www.openwetware.org/images/7/73/BM12_nanosaurs_QDcont_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/c/c8/BM12_nanosaurs_QDconttrans.jpg"><img src="http://www.openwetware.org/images/8/83/BM12_nanosaurs_QDconttrans_s.jpg" alt="Caption" /></a>
Experiments with DNA Origami
We then proceeded with the experiments using our DNA origami structure. To prevent unspecific binding, we used GUVs with 10 mol% negative lipid species of DOPS for all the experiments. The anchor-complementary and catcher-complementary oligonucleotides were used for these experiments.
Vesicles with different amounts of origami
Various amounts of origami were used to find the optimum concentration required to visualize them on vesicles. We observed fluorescent rings around the GUVs with 1.25nM, 2.5nM and 5nM of the assembled origami. It was decided to use 2.5nM of origami for further experiments due to the bright ring observed with a moderate DNA origami consumption. The controls did not have any anchor-complementary strands.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/f/f4/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami.png"><img src="http://www.openwetware.org/images/c/cd/BM12_nanosaurs_Vesicles_with_different_amounts_of_origami_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/4/44/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL.png"><img src="http://www.openwetware.org/images/5/52/BM12_nanosaurs_Vesicles_with_different_amounts_of_origamiCONTROL_s.png" alt="Caption" /></a>
Experiment with 1.25nM of DNA origami structure
<a rel="lightbox[group2]" title="Alexa 647 DNA origami excitation (1.25nM)" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_4microlSB.jpg"><img src="http://www.openwetware.org/images/d/d2/BM12_nanosaurs_4microlSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Transmited light (1.25nM)" href="http://www.openwetware.org/images/6/61/BM12_nanosaurs_4microltransSB.jpg"><img src="http://www.openwetware.org/images/c/c4/BM12_nanosaurs_4microltransSB_s.jpg" alt="Caption" /></a>
Experiment with 2.5nM of DNA origami structure
<a rel="lightbox[group3]" title="Alexa 647 DNA origami excitation (2.5nM)" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_8microl_single2SB.jpg"><img src="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_8microl_single2SB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Transmited light (2.5nM)" href="http://www.openwetware.org/images/5/50/BM12_nanosaurs_8microl_single2transSB.jpg"><img src="http://www.openwetware.org/images/8/84/BM12_nanosaurs_8microl_single2transSB_s.jpg" alt="Caption" /></a>
Experiment with 5nM of DNA origami structure
<a rel="lightbox[group4]" title="Alexa 647 DNA origami excitation (5nM)" href="http://www.openwetware.org/images/c/c4/BM12_nanosaurs_18microl_group2SB.jpg"><img src="http://www.openwetware.org/images/7/73/BM12_nanosaurs_18microl_group2SB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group4]" title="Transmited light (5nM)" href="http://www.openwetware.org/images/9/9e/BM12_nanosaurs_18microl_group2transSB.jpg"><img src="http://www.openwetware.org/images/d/d3/BM12_nanosaurs_18microl_group2transSB_s.jpg" alt="Caption" /></a>
Control experiment w/o anchor-complementary oligos (2.5nM)
<a rel="lightbox[group5]" title="Alexa 647 DNA origami excitation w/o anchor-complementary oligos 2.5nM)" href="http://www.openwetware.org/images/d/d8/BM12_nanosaurs_8microl_single_controlSB.jpg"><img src="http://www.openwetware.org/images/3/3a/BM12_nanosaurs_8microl_single_controlSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group5]" title="Control-Transmited ligh" href="http://www.openwetware.org/images/1/17/BM12_nanosaurs_8microl_single_controltransSB.jpg"><img src="http://www.openwetware.org/images/7/76/BM12_nanosaurs_8microl_single_controltransSB_s.jpg" alt="Caption" /></a>
Alexa 647 labeled catcher complementary DNA oligo strands as target species for the DNA origami structure
After obtaining very promising results with the origami anchorage into the GUVs, we wanted to find out whether the catcher strands on the origami are able to hybridize with the catcher-complementary strands properly. We used Alexa 647-labeled catcher-complementary strands to hybridize with the origami catcher strands and the GUVs were labeled with the green fluorophore DiO. The control had the closed origami. We observed a strong ring around the GUVs with the open origami. In the control we observed no ring around the GUVs. With this positive result for the origami, the following step was to repeat the experiment using the LUVs targeting system.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structure.png"><img src="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structure_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up" href="http://www.openwetware.org/images/5/5f/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structureCONTROLclosed.png"><img src="http://www.openwetware.org/images/2/22/BM12_nanosaurs_Alexa_647_labeled_catcher_complementary_DNA_oligo_strands_as_target_species_for_the_DNA_origami_structureCONTROLclosed_s.png" alt="Caption" /></a>
Experiment with open DNA origami structure
<a rel="lightbox[group2]" title="Alexa 647-catcher-complementary oligos excitation" href="http://www.openwetware.org/images/1/19/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB.jpg"><img src="http://www.openwetware.org/images/3/35/BM12_nanosaurs_Catcher-compAlexa647%28excited%29SB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Cy3 DNA origami excitation" href="http://www.openwetware.org/images/1/18/BM12_nanosaurs_OpenCy3origamiSB.jpg"><img src="http://www.openwetware.org/images/0/01/BM12_nanosaurs_OpenCy3origamiSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/2/28/BM12_nanosaurs_GuvDiDSB.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_GuvDiDSB_s.jpg" alt="Caption" /></a>
Experiment with closed DNA origami structure
<a rel="lightbox[group3]" title="Alexa 647-catcher-complementary oligos excitation" href="http://www.openwetware.org/images/7/7d/BM12_nanosaurs_CONTROLCatcher-compalexa647SB.jpg"><img src="http://www.openwetware.org/images/5/56/BM12_nanosaurs_CONTROLCatcher-compalexa647SB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_CONTROLguvsDiDSB.jpg"><img src="http://www.openwetware.org/images/7/74/BM12_nanosaurs_CONTROLguvsDiDSB_s.jpg" alt="Caption" /></a>
LUVs with catcher complementary strands as target species
After the positive results of the origami anchorage into the GUVs we proceeded to prove the correct hybridization of Fast-DiO fluorescent (Green) LUVs. The catcher-complementary strands anchored on the LUV surface were hybridized with the catcher strands on the Alexa 647-labeled origami structure in the open configuration. We included two controls, one with a closed origami and one without origami. We observed LUVs on the GUVs surface in both configurations of DNA origami and also in the control without the origami. The uncharged (without DOPS) LUVs tended to unspecifically bind with the GUVs.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a>
Experiment with open DNA origami structure
<a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen.jpg"><img src="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_Open_origami_and_luvs_%28DIOexcited%292SBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Alexa 647 DNA origami excitation" href="http://www.openwetware.org/images/7/73/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred.jpg"><img src="http://www.openwetware.org/images/b/be/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBred_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/6/62/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp.jpg"><img src="http://www.openwetware.org/images/c/ce/BM12_nanosaurs_Open_origami_and_luvs_%28alexa_647excited%292SBtransp_s.jpg" alt="Caption" /></a>
Experiment with closed DNA origami structure
<a rel="lightbox[group3]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Alexa 647 DNA origami excitation" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBred.jpg"><img src="http://www.openwetware.org/images/d/d3/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBred_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/b/ba/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans.jpg"><img src="http://www.openwetware.org/images/0/02/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBtrans_s.jpg" alt="Caption" /></a>
Experiment w/o DNA origami structure
<a rel="lightbox[group4]" title="" href="http://www.openwetware.org/images/a/a1/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen.jpg"><img src="http://www.openwetware.org/images/a/ad/BM12_nanosaurs_Closed_origamiAlexa647%2BLUVscontrolSBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group4]" title="Alexa 647 excitation of DNA origami" href="http://www.openwetware.org/images/8/81/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred.jpg"><img src="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBred_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group4]" title="Control-Transmited light" href="http://www.openwetware.org/images/6/69/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBtrans.jpg"><img src="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_Closed_origami_and_luv_%28alexa_647_and_Dio_exc%29_3CONTROL1SBtrans_s.jpg" alt="Caption" /></a>
LUVs with 10 mol% negative charge (DOPS) with catcher complementary strands as target species
When unspecific binding of uncharged LUVs was observed, we decided to use LUVs with 10 mol% negatively charged lipids (DOPS). In this case we did not observe unspecific binding neither in the samples with origami nor the control without origami.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a>
Experiment with open DNA origami structure
<a rel="lightbox[group1]" title="Fast-DiO LUVs" href="http://www.openwetware.org/images/7/75/10%25LUVSPS_open.jpg"><img src="http://www.openwetware.org/images/1/1c/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Transmited light" href="http://www.openwetware.org/images/9/95/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/e/ee/BM12_nanosaurs_Guvs_%2B_luvs_PS_open_origamiSBtrans_s.jpg" alt="Caption" /></a>
Experiment with closed DNA origami structure
<a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/4/40/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBgreen.jpg"><img src="http://www.openwetware.org/images/b/bd/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/9/98/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/6/6a/BM12_nanosaurs_Guvs_%2B_luvs_PS_closed_origamiSBtrans_s.jpg" alt="Caption" /></a>
Experiment w/o DNA origami structure
<a rel="lightbox[group3]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/d/d7/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBgreen.jpg"><img src="http://www.openwetware.org/images/5/57/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBtransp.jpg"><img src="http://www.openwetware.org/images/f/f2/BM12_nanosaurs_Control_guvs%2BluvsPS_no_ring2SBtransp_s.jpg" alt="Caption" /></a>
GUVs with 10 mol% negative charge (DOPS) with catcher-complement strands as target species
In parallel, we were trying to tether two classes of GUV. The LUVs in the previous experiment was replaced by GUVs. In the beginning GUVs only labeled in the same way with one dye were used. The control did not have the catcher-complement strands. The experiment brought surprising results. The vesicles seemed to be attracted to each other when the open origami structure was used. They seemed to change their shape in order to increase the contact area between them. We also noticed that the fluorescence in the interface between the tethered vesicles was much more intense than in the control hence suggesting that the interaction was mediated by the origami structure.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/5/51/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species.png"><img src="http://www.openwetware.org/images/d/de/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_species_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up (w/o catcher-complementary)" href="http://www.openwetware.org/images/5/54/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1.png"><img src="http://www.openwetware.org/images/b/b9/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROL1_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control set up (closed origami structure)" href="http://www.openwetware.org/images/2/23/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/2d/BM12_nanosaurs_GUVs_with_10_mol%25_negative_charge_%28DOPS%29_with_catcher-complement_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a>
Experiment with open DNA origami structure
<a rel="lightbox[group2]" title="DiD GUVs excitation" href="http://www.openwetware.org/images/8/87/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!.jpg"><img src="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBhugging!!_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/e/e6/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans.jpg"><img src="http://openwetware.org/images/b/bf/BM12_nanosaurs_Cy3openorigamicatchercomchol3SBtrans_s.jpg" alt="Caption" /></a>
Control experiment w/o catcher-complementary oligos
<a rel="lightbox[group3]" title="DiD GUVs excitation" href="http://www.openwetware.org/images/a/a8/BM12_nanosaurs_Control_to_the_huggingsSB.jpg"><img src="http://www.openwetware.org/images/2/2c/BM12_nanosaurs_Control_to_the_huggingsSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Control-Transmited light" href="http://www.openwetware.org/images/8/88/BM12_nanosaurs_Control_to_the_huggingsSBtrans.jpg"><img src="http://www.openwetware.org/images/3/31/BM12_nanosaurs_Control_to_the_huggingsSBtrans_s.jpg" alt="Caption" /></a>
Encouraged by the results with uniformly labeled vesicles we had prepared two classes of vesicles stained either with red fluorescent dye (DID) or with green (DIO). In the controls without the catcher-complementary strands, the GUVs do not cluster and do not seem to attract each other.
Fluorescent GUVs
<a rel="lightbox[group1]" title="Fast-DiO GUVs excitation" href="http://www.openwetware.org/images/b/b6/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen.jpg"><img src="http://www.openwetware.org/images/9/93/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="DiD LUVs excitation" href="http://www.openwetware.org/images/0/0b/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred.jpg"><img src="http://www.openwetware.org/images/2/26/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBred_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Transmited light" href="http://www.openwetware.org/images/e/ec/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans.jpg"><img src="http://www.openwetware.org/images/b/b2/BM12_nanosaurs_Control_red%2Bgreen_guvs2SBtrans_s.jpg" alt="Caption" /></a>
Experiment with open DNA origami structure
<a rel="lightbox[group1]" title="Fast-DiO and DiD GUVs excitation 1" href="http://www.openwetware.org/images/e/e3/BM12_nanosaurs_Kissing_vesicles_%28guvs%29.lsm_%28RGB%29.jpg"><img src="http://www.openwetware.org/images/7/75/BM12_nanosaurs_Kissing_vesicles_%28guvs%29.lsm_%28RGB%29_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Fast-DiO and DiD GUVs excitation 2" href="http://www.openwetware.org/images/f/ff/BM12_nanosaurs_Kissing_vesicles_%28guvs%292.lsm_%28RGB%29SB.jpg"><img src="http://www.openwetware.org/images/7/7d/BM12_nanosaurs_Kissing_vesicles_%28guvs%292.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a>
But, we could also notice vesicles being attracted to each other with the closed origami structure. This led us to conclude that the DNA origami structures might not be totally closed.
Control experiment with closed DNA origami structure
<a rel="lightbox[group1]" title="Control 1(closed DNA origami)" href="http://www.openwetware.org/images/7/74/BM12_nanosaurs_Kissing_control.lsm_%28RGB%29SB-800.jpg"><img src="http://www.openwetware.org/images/f/f6/BM12_nanosaurs_Kissing_control.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control 2(closed DNA origami)" href="http://www.openwetware.org/images/e/e8/BM12_nanosaurs_Kissing.control.amazing.lsm_%28RGB%29SB-800.jpg"><img src="http://www.openwetware.org/images/c/c1/BM12_nanosaurs_Kissing.control.amazing.lsm_%28RGB%29SB_s.jpg" alt="Caption" /></a>
LUVs with variable (3 and 7 mol%) negative charge (DOPS) with catcher complementary strands as target species
We needed to optimize the amount of negative charge such that Fast-DiO labeled (Green) LUVs could interact with DiD labeled (Red) GUVs in the presence of origami, but not cluster in its absence. We started by preparing LUVs with 3 mol% DOPS. There were controls with the closed origami, without any origami and one more without origami but with the catcher-complementary. Bright rings were present in all the samples including the controls.
Schemes
<a rel="lightbox[group1]" title="Set up" href="http://www.openwetware.org/images/2/2b/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species.png"><img src="http://www.openwetware.org/images/0/0e/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_species_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control (closed DNA origami)" href="http://www.openwetware.org/images/d/db/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROL_s.png" alt="Caption" /></a>
<a rel="lightbox[group1]" title="Control (w/o DNA origami)" href="http://www.openwetware.org/images/f/f3/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami.png"><img src="http://www.openwetware.org/images/2/25/BM12_nanosaurs_LUVs_with_catcher_complementary_strands_as_target_speciesCONTROLnoorigami_s.png" alt="Caption" /></a>
Open origami 3%PS
<a rel="lightbox[group2]" title="Fast-DiO LUVs excitation" href="http://www.openwetware.org/images/4/43/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green.jpg"><img src="http://www.openwetware.org/images/8/84/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origami_green_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group2]" title="Transmited light" href="http://www.openwetware.org/images/0/0f/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans.jpg"><img src="http://www.openwetware.org/images/8/8a/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_open_origamitrans_s.jpg" alt="Caption" /></a>
Control with closed DNA origami structure
<a rel="lightbox[group3]" title="Fast-DiO excitation" href="http://www.openwetware.org/images/c/ca/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSB.jpg"><img src="http://www.openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group3]" title="Transmited light" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans.jpg"><img src="http://www.openwetware.org/images/d/d1/BM12_nanosaurs_Guvs_%2B_luvs_3PS%2B_closed_origamiSBtrans_s.jpg" alt="Caption" /></a>
Control with no DNA origami structure
<a rel="lightbox[group4]" title="Fast-DiO excitation (w/o DNA origami)" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB.jpg"><img src="http://openwetware.org/images/e/e2/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSB_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group4]" title="Control-Transmited light" href="http://www.openwetware.org/images/d/dd/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans.jpg"><img src="http://www.openwetware.org/images/3/3e/BM12_nanosaurs_Guvs_%2B_luvs_3PS_controlSBtrans_s.jpg" alt="Caption" /></a>
Hence, we needed to use a medial value between 3 and 10 mol% and therefore LUVs with 7 mol% DOPS were prepared and targeted by the Cy3-labeled origami on the GUV surface. The previous controls were repeated for this experiment. Bright fluorescent rings were present in both the samples with open and closed origami structures, but it was not present in the control with catcher-complementary strands. Thus, we decided that the optimal negative charge of 7 mol% was required on the LUVs to screen the unspecific binding. The reason for a fluorescent ring with the closed DNA origami structure might be due to the fact that the porcentage of the open structure after the assembly of the closed structure could be high enough to allow the targeting hybridization <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Project/DNA_origami#tabs-2">(Project->DNAorigami->Assembly)</a>.
Open origami 7%PS
<a rel="lightbox[group5]" title="Fast-DiO LUVs excitation 7%PS" href="http://www.openwetware.org/images/9/93/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/2/20/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group5]" title="Cy3 DNA origami excitation 7%PS" href="http://www.openwetware.org/images/9/9b/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange.jpg"><img src="http://www.openwetware.org/images/1/14/BM12_nanosaurs_EXPGUVs%28DiD%29%2BOpenOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercompSBorange_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group5]" title="DiD excitation of GUV 7%PS" href="http://www.openwetware.org/images/8/8f/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/d/dc/BM12_nanosaurs_EXPGUVs%28DiD%29EXCITED%2BOpenOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_s.jpg" alt="Caption" /></a>
Control with closed DNA origami structure
<a rel="lightbox[group6]" title="Fast-DiO LUVs excitation (closed DNA origami)" href="http://www.openwetware.org/images/7/7e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen.jpg"><img src="http://www.openwetware.org/images/1/1e/BM12_nanosaurs_CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29EXCITED%2BCatchercompSBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group6]" title="Cy3 DNA origami excitation(closed DNA origami)" href="http://openwetware.org/images/d/d6/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp.jpg"><img src="http://openwetware.org/images/5/58/CONT1GUVs%28DiD%29%2BClosedOrigami%28Cy3%29_EXCITED%2BLUVs%28DiO%29%2BCatchercomp_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group6]" title="DiD GUV excitation(closed DNA origami)" href="http://www.openwetware.org/images/e/ef/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred.jpg"><img src="http://www.openwetware.org/images/4/41/BM12_nanosaurs_CONT1GUVs%28DiD%29EXCITED%2BClosedOrigami%28Cy3%29%2BLUVs%28DiO%29%2BCatchercompSBred_%28Custom%29.jpg" alt="Caption" /></a>
Control w/o DNA origami structure
<a rel="lightbox[group7]" title="Fast-DiO LUVs excitation (w/o DNA origami)" href="http://www.openwetware.org/images/9/9c/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen.jpg"><img src="http://www.openwetware.org/images/d/d5/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBgreen_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group7]" title="DiD GUV excitation (w/o DNA origami)" href="http://www.openwetware.org/images/c/c2/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred.jpg"><img src="http://www.openwetware.org/images/d/da/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBred_s.jpg" alt="Caption" /></a>
<a rel="lightbox[group7]" title="Transmited light (w/o DNA origami)" href="http://www.openwetware.org/images/2/2f/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp.jpg"><img src="http://www.openwetware.org/images/1/12/BM12_nanosaurs_CONT2GUVs%28DiD%29%2BLUVs%28DiO%29%2BCatchercomp2SBtransp_s.jpg" alt="Caption" /></a>