Biomod/2012/TU Dresden/Nanosaurs/Project/DNA origami - Revision history

Ali Ghaemi at 05:39, 28 October 2012

2012-10-28T05:39:58Z

←Older revision		Revision as of 05:39, 28 October 2012
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	<p>To image the structures via transmission electron microscopy the samples were stained with		<p>To image the structures via transmission electron microscopy the samples were stained with
-	uranyl acetate (see <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/	+	uranyl acetate (see <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/Lab_book">protocols</a>).
-	Lab_book">protocols</a>).	+
	</p>		</p>

Ali Ghaemi at 02:56, 28 October 2012

2012-10-28T02:56:57Z

←Older revision		Revision as of 02:56, 28 October 2012
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	<p>The middle row of the pictures above shows simulations of the possible fluctuations of the closed DNA origami structure including		<p>The middle row of the pictures above shows simulations of the possible fluctuations of the closed DNA origami structure including
-	the guide strands. These short movies were obtained by using ~~a service~~ called CanDo that calculates a ~~mechanical~~ prediction	+	the guide strands. These short movies were obtained by using an online resource called CanDo (Computer-aided engineering for DNA origami) that calculates a computational prediction
-	of the fluctuations based on the cadnano file of the structure and ~~general~~ properties and assumptions about the behavior of DNA (see <a href="http://cando-dna-origami.org/">CanDo</a>).	+	of the fluctuations based on the cadnano file of the structure and mechanical properties and assumptions about the behavior of DNA (see <a href="http://cando-dna-origami.org/">CanDo</a>).
	</p>		</p>

Ali Ghaemi at 02:53, 28 October 2012

2012-10-28T02:53:24Z

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	inside are then freely accessible.		inside are then freely accessible.
	</p>		</p>
		+
		+

	<div class="img_set">		<div class="img_set">
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	<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;"><a rel="lightbox[origami]" title="3D image created with Maya" href="http://openwetware.org/images/4/43/BM12_nanosaurs_MayaOrigami.jpg"><img src="http://openwetware.org/images/a/a6/BM12_nanosaurs_MayaOrigami_s.jpg"></a></div>		<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;"><a rel="lightbox[origami]" title="3D image created with Maya" href="http://openwetware.org/images/4/43/BM12_nanosaurs_MayaOrigami.jpg"><img src="http://openwetware.org/images/a/a6/BM12_nanosaurs_MayaOrigami_s.jpg"></a></div>

		+	<p>The middle row of the pictures above shows simulations of the possible fluctuations of the closed DNA origami structure including
		+	the guide strands. These short movies were obtained by using a service called CanDo that calculates a mechanical prediction
		+	of the fluctuations based on the cadnano file of the structure and general properties and assumptions about the behavior of DNA (see <a href="http://cando-dna-origami.org/">CanDo</a>).
		+	</p>

Gilbert S. Canon at 01:52, 28 October 2012

2012-10-28T01:52:10Z

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	</p>		</p>

-	~~<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;">~~	+
-	<a rel="lightbox[origami]" title="Convention chart for gel images" href="http://openwetware.org/images/a/aa/BM12_Nanosaurs_Convention_Chart.jpg"><img src="http://openwetware.org/images/4/46/BM12_Nanosaurs_Convention_Chart_s.jpg"><div>Fig.1 Convention chart for gel images</div> </a>	+
-	~~</div>~~	+
-	~~<div class="clear"></div>~~	+

	<ul>		<ul>
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	<p> To make the gels easy to understand, we use the following conventions for defining which components were loaded in each lane:		<p> To make the gels easy to understand, we use the following conventions for defining which components were loaded in each lane:
	</p>		</p>
-	~~<div class="clear"></div>~~

		+	<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;">
		+	<a rel="lightbox[origami]" title="Convention chart for gel images" href="http://openwetware.org/images/a/aa/BM12_Nanosaurs_Convention_Chart.jpg"><img src="http://openwetware.org/images/4/46/BM12_Nanosaurs_Convention_Chart_s.jpg"><div>Fig.1 Convention chart for gel images</div> </a>
		+	</div>
		+	<div class="clear"></div>
		+

	<h3>Buffer calibration</h3>		<h3>Buffer calibration</h3>

Gilbert S. Canon at 01:47, 28 October 2012

2012-10-28T01:47:59Z

←Older revision		Revision as of 01:47, 28 October 2012
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	</p>		</p>

-	<div ~~class~~="~~img_left img_link~~">	+	<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;">
	<a rel="lightbox[origami]" title="Convention chart for gel images" href="http://openwetware.org/images/a/aa/BM12_Nanosaurs_Convention_Chart.jpg"><img src="http://openwetware.org/images/4/46/BM12_Nanosaurs_Convention_Chart_s.jpg"><div>Fig.1 Convention chart for gel images</div> </a>		<a rel="lightbox[origami]" title="Convention chart for gel images" href="http://openwetware.org/images/a/aa/BM12_Nanosaurs_Convention_Chart.jpg"><img src="http://openwetware.org/images/4/46/BM12_Nanosaurs_Convention_Chart_s.jpg"><div>Fig.1 Convention chart for gel images</div> </a>
	</div>		</div>

Gilbert S. Canon at 01:41, 28 October 2012

2012-10-28T01:41:25Z

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	<p>In order to enhance the quality of the assemblies, the effect of the folding buffer on the yield and structural		<p>In order to enhance the quality of the assemblies, the effect of the folding buffer on the yield and structural
	integrity of the origami was examined. Four different buffers with various MgCl2 concentrations (8mM, 10mM, 12mM, 14mM)		integrity of the origami was examined. Four different buffers with various MgCl2 concentrations (8mM, 10mM, 12mM, 14mM)
-	were used for assembling open and closed structures, as can be seen in Fig.1. From the pictures obtained, one can see	+	were used for assembling open and closed structures, as can be seen in Fig.2. From the pictures obtained, one can see
	that by increasing the MgCl2 concentration, the band for the closed structure blurs and shifts up. This indicates that		that by increasing the MgCl2 concentration, the band for the closed structure blurs and shifts up. This indicates that
	the structure becomes less homogeneous and possibly the structures are also more prone to dimerization. Based on this,		the structure becomes less homogeneous and possibly the structures are also more prone to dimerization. Based on this,
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	</div>		</div>

-	<p>At first, the quality of the basic open and closed assemblies was tested. As shown in lanes 2 and 3, both assembled	+	<p>At first, the quality of the basic open and closed assemblies was tested. As shown in lanes 2 and 3 (Fig.3), both assembled
	structures have a different structure and therefore run differently on the gel compared to the scaffold. Moreover, it can		structures have a different structure and therefore run differently on the gel compared to the scaffold. Moreover, it can
	be seen that in lane number 2 there is a second band above the expected band for the structure. This likely shows that the		be seen that in lane number 2 there is a second band above the expected band for the structure. This likely shows that the
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	<p>After confirming the assembly quality of our structures, cargo attachment tests were performed. In particular, we employed		<p>After confirming the assembly quality of our structures, cargo attachment tests were performed. In particular, we employed
-	attachment through hybridization (Fig.3). Quantum dot cargos that carried oligomers complementary to the catcher strands of the	+	attachment through hybridization (Fig.4). Quantum dot cargos that carried oligomers complementary to the catcher strands of the
-	origamis were added to the open and closed structures. Subsequently binding preferences were determined.	+	origamis were added to the open and closed structures. Subsequently binding preferences were determined.<br>
		+	From the results obtained (Fig.4) one can identify a clear gel shift due to quantum dots binding in lanes 2 and 5. However,
		+	there’s not a noticeable difference between the open and closed configurations as the ratio of bound vs. unbound structures cannot
		+	be determined straight forward. In order to have a better idea about binding preference and to discard problems with the structure,
		+	a further experiment involving the catchers of the system was proposed.
	</p>		</p>


-	<p>~~From the results obtained (Fig.3) one can identify a clear gel shift due to quantum dots binding in lanes 2 and 5. However,~~	+	<p>
-	~~there’s not a noticeable difference between the open and closed configurations as the ratio of bound vs. unbound structures cannot~~	+
-	~~be determined straight forward. In order to have a better idea about binding preference and to discard problems with the structure,~~	+
-	~~a further experiment involving the catchers of the system was proposed.~~	+
	</p>		</p>
	<div class="clear"></div>		<div class="clear"></div>
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	</p>		</p>

-	<p>The results shown in fig.4 suggest a preference for the binding to the open structures compared to the closed structures when only a single catcher strand was present. If six catchers are used this difference was greatly reduced.	+	<p>The results shown in fig.5 suggest a preference for the binding to the open structures compared to the closed structures when only a single catcher strand was present. If six catchers are used this difference was greatly reduced.
	</p>		</p>

Gilbert S. Canon at 01:12, 28 October 2012

2012-10-28T01:12:38Z

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	<li>The quantum dots have a binding preference for the open structures over the closed ones.</li>		<li>The quantum dots have a binding preference for the open structures over the closed ones.</li>
	<li>This preference decreases if the number of catchers is increased.</li>		<li>This preference decreases if the number of catchers is increased.</li>
-	<li>? The attachment performance through hybridization or biotin-streptavidin interaction is comparable.</li>	+	<li>The attachment performance through hybridization or biotin-streptavidin interaction is comparable.</li>
	</ul>		</ul>

Gilbert S. Canon at 00:54, 28 October 2012

2012-10-28T00:54:06Z

←Older revision		Revision as of 00:54, 28 October 2012
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	<a rel="lightbox[origami]" title="Locks - Side view" href="http://openwetware.org/images/a/a4/BM12_nanosaurs_5_locks_side.jpg"><img src="http://openwetware.org/images/5/58/BM12_nanosaurs_5_locks_side_s.jpg"></a>		<a rel="lightbox[origami]" title="Locks - Side view" href="http://openwetware.org/images/a/a4/BM12_nanosaurs_5_locks_side.jpg"><img src="http://openwetware.org/images/5/58/BM12_nanosaurs_5_locks_side_s.jpg"></a>
	</div>		</div>
-	<p>The DNA origami has two locks, one on each side. Each lock consists out of two oligomers: an aptamer (blue) and its complementary oligo (green). The total length of each lock half extruding from the origami is 44 nt. 24nt of them are complementary to each other. The remaining 20nt that are not complementary, are between origami and the complementary region.	+	<p>The DNA origami has two locks, one on each side. Each lock consists out of two oligomers: an aptamer (blue) and its complementary oligo (green). The total length of each lock half extruding from the origami is 44 nt. 24nt of them are complementary to each other. The remaining 20nt that are not complementary, are between origami and the complementary region.<br>The aptamer is triggered by the protein PDGF. It preferably binds to a specific site on PDGF binding, which can thus open the lock by dissociating it from the complementary oligo (see (see <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/
-	~~</p>~~	+	Project/Aptamer">Aptamer</a>).Locks are only present in closed structures. If a strictly open structure was needed for the experiments the lock staples without any overhang were applied (“locks_nohang”).
-	<p>The aptamer is triggered by the protein PDGF. It preferably binds to a specific site on PDGF binding, which can thus open the lock by dissociating it from the complementary oligo (see (see <a href="http://openwetware.org/wiki/Biomod/2012/TU_Dresden/Nanosaurs/	+
-	Project/Aptamer">Aptamer</a>).	+
-	~~</p>~~	+
-	~~<p>~~Locks are only present in closed structures. If a strictly open structure was needed for the experiments the lock staples without any overhang were applied (“locks_nohang”).	+
	</p>		</p>
		+
	<div class="clear"></div>		<div class="clear"></div>

Gilbert S. Canon at 00:51, 28 October 2012

2012-10-28T00:51:15Z

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	</div>		</div>

-	<p>The catcher strands are complementary to the cholesterol labeled single strands that are integrated in the lipid membrane of the large unilamellar vesicles. Once the DNA origami shell is open and the catcher strands are freely accessible, the LUVs can bind to the DNA origami due to the hybridization of the single strands. There are 6 catcher strands to increase the probability of binding. They are 35nt long, containing a 5nt spacer at the DNA origami and a 30nt binding site for the single strands on the vesicles.	+	<p>The catcher strands are complementary to the cholesterol labeled single strands that are integrated in the lipid membrane of the large unilamellar vesicles. Once the DNA origami shell is open and the catcher strands are freely accessible, the LUVs can bind to the DNA origami due to the hybridization of the single strands. There are 6 catcher strands to increase the probability of binding. They are 35nt long, containing a 5nt spacer at the DNA origami and a 30nt binding site for the single strands on the vesicles.<br/>For our experiments the catcher strands were also labeled, for example with biotin to bind to streptavidin coated quantum dots.
-	</p>	+
-		+
-	<p>For our experiments the catcher strands were also labeled, for example with biotin to bind to streptavidin coated quantum dots.	+
	</p>		</p>

Gilbert S. Canon at 00:48, 28 October 2012

2012-10-28T00:48:02Z

←Older revision		Revision as of 00:48, 28 October 2012
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	<h2>Functional parts</h2>		<h2>Functional parts</h2>

-	<h4>Scaffold</h4>	+	<h3>Scaffold</h3>
-	<p>The scaffold is a 7560 bases long single stranded circular DNA derived from the E.coli virus M13p18. It provides the basis to which the staple oligos can hybridize to form the structure. It winds its way through the whole DNA origami and ends exactly at the point where it started.<br/>It also connects the upper and lower half of the shell via two hinges. These are simply about 12 bases of single stranded DNA, meaning there are no staple strands hybridizing in this region.	+
-	~~</p>~~	+
	<div class="img_right img_link">		<div class="img_right img_link">
	<a rel="lightbox[origami]" title="Scaffold - Side view" href="http://openwetware.org/images/0/0a/BM12_nanosaurs_1_Scaffold_side.jpg"><img src="http://openwetware.org/images/a/a2/BM12_nanosaurs_1_Scaffold_side_s.jpg"></a>		<a rel="lightbox[origami]" title="Scaffold - Side view" href="http://openwetware.org/images/0/0a/BM12_nanosaurs_1_Scaffold_side.jpg"><img src="http://openwetware.org/images/a/a2/BM12_nanosaurs_1_Scaffold_side_s.jpg"></a>
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	<a rel="lightbox[origami]" title="Scaffold - Top view" href="http://openwetware.org/images/7/70/BM12_nanosaurs_1_Scaffold_top.jpg"><img src="http://openwetware.org/images/a/a6/BM12_nanosaurs_1_Scaffold_top_s.jpg"></a>		<a rel="lightbox[origami]" title="Scaffold - Top view" href="http://openwetware.org/images/7/70/BM12_nanosaurs_1_Scaffold_top.jpg"><img src="http://openwetware.org/images/a/a6/BM12_nanosaurs_1_Scaffold_top_s.jpg"></a>
	</div>		</div>
-		+	<p>The scaffold is a 7560 bases long single stranded circular DNA derived from the E.coli virus M13p18. It provides the basis to which the staple oligos can hybridize to form the structure. It winds its way through the whole DNA origami and ends exactly at the point where it started.<br/>It also connects the upper and lower half of the shell via two hinges. These are simply about 12 bases of single stranded DNA, meaning there are no staple strands hybridizing in this region.
		+	</p>

	<div class="clear"></div>		<div class="clear"></div>