Biomod/2012/TU Dresden/Nanosaurs/Project/DNA origami

From OpenWetWare

< Biomod | 2012 | TU Dresden/Nanosaurs | Project(Difference between revisions)
Jump to: navigation, search
Current revision (01:39, 28 October 2012) (view source)
 
(32 intermediate revisions not shown.)
Line 1: Line 1:
-
{{Biomod/2012/TU Dresden/Nanosaurs/Header}}
+
{{Biomod/2012/TU Dresden/Nanosaurs/Header}}  
-
<html>
+
 +
<html>
<script>
<script>
$(function() {
$(function() {
Line 6: Line 7:
});
});
</script>
</script>
 +
 +
<body>
<div id="tabs" class="tabs-bottom">
<div id="tabs" class="tabs-bottom">
<ul>
<ul>
-
<li><a href="#tabs-1">students</a></li>
+
<li><a href="#tabs-1">Design</a></li>
-
<li><a href="#tabs-2">supervisors</a></li>
+
<li><a href="#tabs-2">Assembly</a></li>
-
<li><a href="#tabs-3">supervisors</a></li>
+
<li><a href="#tabs-3">Cargo Attachment</a></li>
-
<li><a href="#tabs-4">supervisors</a></li>
+
</ul>
</ul>
<div class="tabs-spacer"></div>
<div class="tabs-spacer"></div>
<div id="tabs-1">
<div id="tabs-1">
-
<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>
+
<h2>What is DNA origami?</h2>
-
<div id="guv_vid">
+
-
<iframe width="480" height="360" src="http://www.youtube.com/embed/uJj4Ul6Q9OA" frameborder="0" allowfullscreen></iframe>
+
<p>DNA origami is a well-established technique in nanotechnology which involves the folding of DNA
-
</div>
+
to create various 2D or 3D patterns and shapes at the nanoscale. More in detail, it makes use of a
-
<h3>Introduction</h3>
+
long single strand of DNA known as scaffold strand, which acts like a backbone or support for a
 +
particular structure to be made. In order to shape the construct in a certain way, several shorter
 +
strands of different lengths, called staple strands, are hybridized (bound) to specific parts of
 +
the scaffold profiting from the specificity of interactions between complementary base pairs in DNA.
 +
The way these binding sites are chosen, determines how the structure is going to fold and in which
 +
shape it ends up. </p>
 +
 +
<h2>Our DNA origami structure</h2>
 +
 +
<h3>Requirements</h3>
 +
 +
<p>In order to accomplish the purpose of our project, the DNA origami shell must fulfill the
 +
following requirements:</p>
 +
 +
<ul>
 +
<li>Comprise a tethering platform for the attachment to lipid bilayers.</li>
 +
<li>Include a variable catching platform which allows the structure to bind specific target species. </li>
 +
<li>Avoid unspecific binding of non-wanted targets.</li>
 +
<li>Provide a trigger mechanism which enables binding upon signal.</li>
 +
<li>Contain one or more parts with fluorescent labels for testing and imaging purposes.</li>
 +
</ul>
 +
 +
<h2>Design</h2>
 +
 +
<p>DNA origami is a well-established technique in nanotechnology which involves the folding of DNA
 +
to create various 2D or 3D patterns and shapes at the nanoscale. More in detail, it makes use of a
 +
long single strand of DNA known as scaffold strand, which acts like a backbone or support for a
 +
particular structure to be made. In order to shape the construct in a certain way, several shorter
 +
strands of different lengths, called staple strands, are hybridized (bound) to specific parts of
 +
the scaffold profiting from the specificity of interactions between complementary base pairs in DNA.
 +
The way these binding sites are chosen, determines how the structure is going to fold and in which
 +
shape it ends up. </p>
 +
 +
<p>Our structure was inspired by the DNA origami Logic Nanorobot by Douglas et al.
 +
(Science 335, 831-8XX (2012)). We chose the model proposed in this paper because its shell-like
 +
shape provides binding specificity while its lock mechanism offered a triggering platform.<br>  
 +
In our design, the height of the structure was lowered to 20nm while increasing its width to 45nm.
 +
This is intended to decrease the chances of molecules diffusing into the origami in its closed
 +
configuration while making it lay flatter on the vesicle surface to enhance binding. Most notably,
 +
the structure was adjusted to have anchor and catcher strands which allow binding to the carrier
 +
vesicle and to the target species respectively.<br>  
 +
The functional principle of the DNA origami shell is that in its closed state (i.e its lock strands
 +
are hybridized to each other), the single stranded catcher oligonucleotides inside are shielded.
 +
This means they are not accessible for anything from the outside to hybridize to them.
 +
The locks can be triggered to open once a certain protein is around. When the lock strands are not
 +
hybridized anymore, the DNA origami shell opens up due to thermal fluctuations. The catcher strands
 +
inside are then freely accessible.
 +
</p>
-
<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 (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), 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>
+
 +
<div class="img_set">
 +
<a  rel="lightbox[origami]" title="DNA origami - front view" href="http://openwetware.org/images/b/b1/BM12_nanosaurs_Overview_Front_view.jpg"><img src="http://openwetware.org/images/f/ff/BM12_nanosaurs_Overview_Front_view_s.jpg"></a>
 +
<a  rel="lightbox[origami]" title="DNA origami - side view" href="http://openwetware.org/images/a/a4/BM12_nanosaurs_Overview_Side_view.jpg"><img src="http://openwetware.org/images/4/4f/BM12_nanosaurs_Overview_Side_view_s.jpg"></a>
 +
<a  rel="lightbox[origami]" title="DNA origami - top view" href="http://openwetware.org/images/4/4e/BM12_nanosaurs_Overview_Top_view.jpg"><img src="http://openwetware.org/images/7/71/BM12_nanosaurs_Overview_Top_view_s.jpg"></a>
 +
</div>
 +
<div class="clear"></div>
 +
<div class="img_set">
 +
<a  rel="lightbox[origami]" title="CanDo simulation - front view" href="http://openwetware.org/images/d/de/BM12_nanosaurs_Shell_guidestrands_fluctuations_2_front.gif"><img src="http://openwetware.org/images/9/99/BM12_nanosaurs_Shell_guidestrands_fluctuations_2_front_s.gif"></a>
 +
<a  rel="lightbox[origami]" title="CanDo simulation - side view" href="http://openwetware.org/images/8/8d/BM12_nanosaurs_Shell_guidestrands_fluctuations_3_side.gif"><img src="http://openwetware.org/images/f/f0/BM12_nanosaurs_Shell_guidestrands_fluctuations_3_side_s.gif"></a>
 +
<a  rel="lightbox[origami]" title="CanDo simulation - top view" href="http://openwetware.org/images/f/fc/BM12_nanosaurs_Shell_guidestrands_fluctuations_1_top.gif"><img src="http://openwetware.org/images/5/55/BM12_nanosaurs_Shell_guidestrands_fluctuations_1_top_s.gif"></a>
 +
</div>
 +
<div class="clear"></div>
-
<p>The giant unilamellar vesicles (GUVs) were prepared using electroformation (Hyperlink) and the large unilamellar vesicles (LUVs) were prepared using rehydration and extrusion (Hyperlink). 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 (Hyperlink) depending on the experiments. The single stranded oligonucleotides experiments were with SLB; meanwhile for the origami experiments the SLB buffer included magnesium.</p>
+
<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>Our cholesterol-modified oligonucleotides consisted were three: catcher A (tethering oligonucleotide used only for the experiments with single stranded oligonucleotides (Hyperlink)), anchor-complementary (part of the double-stranded tethering oligonucleotide for the origami structure (Hyperlink)), catcher-complementary (oligonucleotide for the LUVs as target species (Hyperlink)).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 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 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 mechanical properties and assumptions about the behavior of DNA (see <a href="http://cando-dna-origami.org/">CanDo</a>).  
 +
</p>
 +
 +
 +
<h2>Functional parts</h2>
 +
 +
<h3>Scaffold</h3>
 +
 +
<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>
 +
</div>
 +
<div class="img_right img_link">
 +
<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>
 +
<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>
 +
<h3>Core</h3>
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Core - Front view" href="http://openwetware.org/images/1/14/BM12_nanosaurs_2_core_front.jpg"><img src="http://openwetware.org/images/4/47/BM12_nanosaurs_2_core_front_s.jpg"></a>
 +
</div>
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Core - Side view" href="http://openwetware.org/images/6/65/BM12_nanosaurs_2_core_side.jpg"><img src="http://openwetware.org/images/d/d2/BM12_nanosaurs_2_core_side_s.jpg"></a>
 +
</div>
 +
<p>There are 171 core oligos which give the structure its stability, its basic shape and hold all the functional parts together. To avoid that several structures stack together, the turning points of the scaffold are left single stranded for about 10 to 30 nucleotides. This means that in those regions no staple strands hybridize thus comprising highly flexible single strand DNA.
 +
</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) (Hyperlink) which was removed previously to setting the experiments. The imaging was done using Zeiss LSM 780 CC3 (Hyperlink) and the pictures were taken at the equatorial plane of the vesicles.</p>
+
<div class="clear"></div>
 +
<h3>Anchors</h3>
 +
<div class="img_right img_link">
 +
<a rel="lightbox[origami]" title="Anchors - Front view" href="http://openwetware.org/images/2/23/BM12_nanosaurs_4_anchors.jpg"><img src="http://openwetware.org/images/2/27/BM12_nanosaurs_4_anchors_s.jpg"></a>
 +
</div>
 +
<p>The anchor oligomers can hybridize to cholesterol labeled single strands that provide the attachment to the lipid membrane of the giant unilamellar vesicles and therefore connect the DNA origami structure permanently to them. There are 9 anchor strands to increase the probability of binding. They are 35nt long, containing a 5nt spacer at the DNA origami and a 30bp binding site for cholesterol oligomers on the vesicles.
 +
</p>
-
<h3>Experimental procedure</h3>
+
<div class="clear"></div>
 +
<h3>Catchers</h3>
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Catchers - Front view" href="http://openwetware.org/images/5/57/BM12_nanosaurs_3_catchers.jpg"><img src="http://openwetware.org/images/a/ae/BM12_nanosaurs_3_catchers_s.jpg"></a>
 +
</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.<br/>For our experiments the catcher strands were also labeled, for example with biotin to bind to streptavidin coated quantum dots.
 +
</p>
 +
 +
<div class="clear"></div>
 +
<h3>Locks</h3>
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Locks - Front view" href="http://openwetware.org/images/5/50/BM12_nanosaurs_5_locks_front.jpg"><img src="http://openwetware.org/images/b/bf/BM12_nanosaurs_5_locks_front_s.jpg"></a>
 +
</div>
 +
<div class="img_right img_link">
 +
<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>
 +
<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/
 +
  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>
 +
 +
<div class="clear"></div>
 +
 +
<h3>Edge staple</h3>
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Edge staple - Front view" href="http://openwetware.org/images/d/d5/BM12_nanosaurs_6_edgestaple.jpg"><img src="http://openwetware.org/images/b/bc/BM12_nanosaurs_6_edgestaple_s.jpg"></a>
 +
</div>
 +
<p>The edge staple is a single strand that is on the top side of the structure. It can be labeled with fluorophores such as Alexa 647 to add a fluorescent signal to the structure.
 +
</p>
-
<ol>
+
<div class="clear"></div>
-
<li>GUVs electroformation</li>
+
<h3>Guide staples</h3>
-
<li>Well pasivation (Hyperlink)</li>
+
<div class="img_right img_link">
-
<li>GUVs observation under the light microscope (checking the stability)</li>
+
<a  rel="lightbox[origami]" title="Guide staples - Side view" href="http://openwetware.org/images/5/51/BM12_nanosaurs_7_guide_staple_side.jpg"><img src="http://openwetware.org/images/a/a8/BM12_nanosaurs_7_guide_staple_side_s.jpg"></a>
-
<li>Target species preparation:</li>
+
-
<ol type="a">
+
-
<li>LUVs formation and calibration (Hyperlink).</li>
+
-
<li>Streptavidin-biotin interaction: the biotinylated receiver A oligonuclotide were incubated for 10 min with Quantum dot 625-Streptavidin or Alexa 488-Streptavidin (Hyperlink).</li>
+
-
</ol>
+
-
<li>Anchoring of cholesterol-modified DNA oligonucleotides:</li>
+
-
<ol type="a">
+
-
<li>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</li>
+
-
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.
+
-
</ol>
+
-
<li>Hybridization: the target species were mixed with the GUVs and incubated overnight.</li>
+
-
<li>The well was then imaged using Zeiss LSM 780 CC3 inverse confocal microscope.</li>
+
-
</ol>
+
</div>
</div>
-
<div id="tabs-2">
+
<div class="img_right img_link">
-
<h2>Experiments with single stranded oligonucleotides</h2>
+
<a  rel="lightbox[origami]" title="Guide staples - Top view" href="http://openwetware.org/images/6/69/BM12_nanosaurs_7_guide_staple_top.jpg"><img src="http://openwetware.org/images/1/16/BM12_nanosaurs_7_guide_staple_top_s.jpg"></a>
-
<p>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.</p>
+
</div>
-
 
+
-
<h3>Alexa labeled DNA oligonucleoides as target species</h3>
+
<p>There are two guide staples that should help to close the structure during the assembly. It increases the amount of structures that are closed after the assembly. The structure cannot open while the guide strands are there. Therefore they have an 8 base toehold and if fully complementary oligos are applied later, they would hybridize and the structure would still be closed, but now has the chance to be opened (if the locks are opened).
-
<p>In the first set of experiments, receiver A strands labeled with Alexa 488 were used as the target species. The protocol for the labeling procedure can be found here (Hyperlink). 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>
+
</p>
-
 
+
-
<div class="img_right img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a></div>
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - front view" href="big pic"><img src="small pic" /></a>
+
-
<div class="descr">Control set up</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - side view" href="Overview-Side-500.jpg"><img src="Overview-Side-100.jpg" /></a>
+
-
<div class="descr">GUV with Alexa 488 labeled receiver A</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - top view" href="Overview-Top-500.jpg"><img src="Overview-top-100.jpg" /></a>
+
-
<div class="descr">Transmitted light image of GUV</div>
+
-
</div>
+
-
</div>
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" /></a>
+
-
<div class="descr">GUV w/o catcher A </div></a>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" /></a>
+
-
<div class="descr">Transmited light image of GUV  </div></a>
+
-
</div>
+
-
</div>
+
-
 
+
-
<h3>Alexa 488 labeled Streptavidin molecules as target species</h3>
+
-
<p>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.</p>
+
-
<div class="img_left img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a></div>
+
<div class="clear"></div>
 +
<h2>cadnano</h2>
-
<div class="img_gal">
+
<p>In order to turn the idea of our sketch into an actual 3D DNA nanostructure, we used a software called caDNAno(learn more at http://cadnano.org/); a computational tool for DNA origami design. Using this tool one define the shape of the desired structure within a graphic environment by providing the hybridization sites for the staple strands and the scaffold length as input parameters. The program will then display the necessary sequences of the staple strands which can be ordered from a suitable company.
-
<div class="img_gbox">
+
</p>
-
<a  rel="lightbox[group1]" title="DNA origami - front view" href="big pic"><img src="small pic" /></a>
+
-
<div class="descr">Control set up</div>
+
<div class="img_set">
-
</div>
+
<a  rel="lightbox[origami]" title="Cadnano - not guided version" href="http://openwetware.org/images/f/fd/BM12_nanosaus_cadnano432.jpg"><img src="http://openwetware.org/images/8/8f/BM12_nanosaurs_cadnano162.jpg"></a>
-
<div class="img_gbox">
+
<a  rel="lightbox[origami]" title="Cadnano - guided version" href="http://openwetware.org/images/2/2b/BM12_nanosaurs_cadnano_guided400.jpg"><img src="http://openwetware.org/images/6/6d/BM12_nanosaurs_cadnano_guided150.jpg"></a>
-
<a  rel="lightbox[group2]" title="DNA origami - side view" href="Overview-Side-500.jpg"><img src="Overview-Side-100.jpg" /></a>
+
</div>
-
<div class="descr">GUV with Streptavidin labeled with Alexa-488 receiver A</div>
+
-
</div>
+
</div>
-
<div class="img_gbox">
+
<div class="tabs-spacer"></div>
-
<a  rel="lightbox[group2]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" /></a>
+
<div id="tabs-2">
-
<div class="descr">Transmitted light image of GUV</div>
+
<h2>Assembly</h2>
-
</div>
+
       
-
</div>
+
<p>To assemble the desired structure the following things need to be pipetted together:
 +
</p>
 +
 +
<ul>
 +
<li>Scaffold</li>
 +
<li>Set of staple oligos defining the features the assembled structure should have </li>
 +
<li>Folding buffer</li>
 +
<li>Water</li>
 +
</ul>
 +
 +
<p>The correct ratios and the recipe of the folding buffer can be found in the recipe section.
 +
          <br>Following a detailed protocol the mixture is heated up to 85°C and then cooled down very
 +
  slowly using a given temperature ramp. Especially in the area of 55°C the cooling process
 +
  is extremely slow since most of the assembly process happens in that temperature region.
 +
  The whole cooling process takes about 15 hours.<br>After the assembly the structures remain
 +
  stable at room temperature.
 +
        </p>
-
<div class="img_gal">
+
<h3>Purification</h3>
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" /></a>
+
<p>To have a greater yield of assembled structures, the ratio of staple strands to scaffold
-
<div class="descr">GUV w/o catcher A</div>
+
  strands is 7.5 to 1. To get rid of the leftover single strands after assembly, the samples
-
</div>
+
  are typically dialyzed for 1 to 2 hours using a 0.025µm filter.
-
<div class="img_gbox">
+
</p>
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" /></a>
+
-
<div class="descr">Transmited light image of GUV  </div>
+
<h2>Results</h2>
-
</div>
+
-
</div>
+
<p>In order to examine the shape of the structure, the samples were imaged using transmission
-
 
+
  electron (TEM) and atomic force microscopy (AFM).
-
<h3>Quantum dots 625-Streptavidin as target species</h3>
+
</p>
-
<p>Then we decided to use Quantum dots (QD) 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>
+
 
-
 
+
<h3>TEM</h3>
-
<div class="img_right img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a></div>
+
 +
<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/Lab_book">protocols</a>).
 +
</p>
 +
 +
<div class="img_set">
 +
<a  rel="lightbox[origami]" title="TEM overview & close-up of open structures" href="http://openwetware.org/images/a/a8/BM12_nanosaurs_TEMOpen.jpg"><img src="http://openwetware.org/images/4/40/BM12_nanosaurs_TEMOpen_s.jpg"></a>
 +
<a  rel="lightbox[origami]" title="TEM overview & close-up of closed lying structures" href="http://openwetware.org/images/7/7b/BM12_nanosaurs_TEMClosed.jpg"><img src="http://openwetware.org/images/d/d2/BM12_nanosaurs_TEMClosed_s.jpg"></a>
 +
<a  rel="lightbox[origami]" title="TEM overview & close-up of closed upright standing structures" href="http://openwetware.org/images/f/ff/BM12_nanosaurs_TEMUpright.jpg"><img src="http://openwetware.org/images/b/b3/BM12_nanosaurs_TEMUpright_s.jpg"></a>
 +
</div>
 +
 +
<p>The TEM images demonstrate a successful assembly of both types of structures. In particular
 +
  they show a significant difference in shape between the open and the closed structures. Open
 +
  structures were typically twice as long as closed structures (see below). For these images
 +
  the closed structures were assembled including the guide strands, but also the not guided
 +
  constructs showed a conformational change with a high percentage of the structures being closed.
 +
          <br/>Producing negatively stained samples (using short staining times) it was possible to image the
 +
  closed structures standing upright. The pictures show that the shape of the cross section is
 +
  rather variable. However, most of the structures show a high degree of integrity, i.e. a closed
 +
  circumference supporting that the structures are really closed.<br/>Evaluating several individual
 +
  structures the following average lateral dimensions of both types of DNA origami were obtained:
 +
        </p>
 +
 +
<table>
 +
<tr>
 +
<th rowspan="2">&#91;nm&#93;</th>
 +
<th colspan="2">closed</th>
 +
<th>open</th>
 +
</tr>
 +
<tr>
 +
<th>width</th>
 +
<th>length</th>
 +
<th>full length</th>
 +
</tr>
 +
<tr>
 +
<td><b># of measurements</b></td>
 +
<td>26</td>
 +
<td>29</td>
 +
<td>40</td>
 +
</tr>
 +
<tr>
 +
<td><b>result (95&#37; STD)</b></td>
 +
<td>48,9 &#177; 5,9</td>
 +
<td>39,6 &#177; 3,4</td>
 +
<td>71,4 &#177; 3,8</td>
 +
</tr>
 +
<tr>
 +
<td><b>relative error &#91;&#37;&#93; </b></td>
 +
<td>12,0</td>
 +
<td>8,6</td>
 +
<td>5,3</td>
 +
</tr>
 +
<tr>
 +
<td><b>Expected</b></td>
 +
<td>45</td>
 +
<td>40-44</td>
 +
<td>80-88</td>
 +
</tr>
 +
<tr>
 +
<td><b>Possible reason</b> <br/> <b>for deviation</b></td>
 +
<td></td>
 +
<td></td>
 +
<td>Hinges and edges <br/> floppy single strands</td>
 +
</tr>
 +
</table>
 +
 +
<div class="img_set">
 +
<a  rel="lightbox[origami]" title="Distribution of width for closed structures" href="http://openwetware.org/images/c/c4/BM12_nanosaurs_histograms_Width_%28closed%29800.jpg"><img src="http://openwetware.org/images/b/b6/BM12_nanosaurs_histogram_Width_%28closed%29250.jpg"></a>
 +
<a  rel="lightbox[origami]" title="Distribution of length for closed structures" href="http://openwetware.org/images/6/6e/BM12_nanosaurs_histograms_Length_%28closed%29800.jpg"><img src="http://openwetware.org/images/8/8d/BM12_nanosaurs_histogram_Length_%28closed%29250.jpg"></a>
 +
<a  rel="lightbox[origami]" title="Distribution of full length for open structures" href="http://openwetware.org/images/4/4e/BM12_nanosaurs_histograms_Length_%28open%29800.jpg"><img src="http://openwetware.org/images/4/43/BM12_nanosaurs_histogram_Length_%28open%29250.jpg"></a>
 +
</div>
 +
 +
<p>For the closed structure the length, as well as the width, match nicely the expected values.
 +
  The slightly higher width can be explained by assuming that the structures laying down flat which
 +
  increases the lateral dimension due to the bending down of the side walls.<br>
 +
          The open structure however appeared to be shorter than one would expect if one doubles the length
 +
  of a closed structure. This can be explained due to the fact that the turning points, as well as
 +
  the hinges, were left as single strands making them more flexible. Therefore they do not necessarily
 +
  have to be stretched to their full lengths. In general the open structure shows increased flexibility
 +
  and degrees of freedom compared to the closed constructs.
 +
        </p>
 +
 +
<h3>AFM</h3>
 +
<p>To further proof of the correct assembly, the open and closed structures were sent to the Spanish
 +
  National Center for Biotechnology in Madrid. There Dr. Fernando Moreno-Herrero and Maria Eugenia
 +
  Fuentes obtained a series of magnificent AFM images.The following pictures show the different samples
 +
  in an overview (left) as well as an enlarged view of a single structure (right).</p>
-
<div class="img_gal">
+
<div style="width: 400px;" class="img_set">
-
<div class="img_gbox">
+
<a  rel="lightbox[origami]" title="AFM overview of open structures" href="http://openwetware.org/images/2/2a/BM12_nanosaurs_AFM_Open_overview.jpg"><img src="http://openwetware.org/images/b/ba/BM12_nanosaurs_AFM_Open_overview_s.jpg""></a>
-
<a  rel="lightbox[group1]" title="DNA origami - front view" href="big pic"><img src="small pic" /></a>
+
<a  rel="lightbox[origami]" title="AFM close-up of an open structure" href="http://openwetware.org/images/6/63/BM12_nanosaurs_AFM_Open_single.jpg"><img src="http://openwetware.org/images/c/c5/BM12_nanosaurs_AFM_Open_single_s.jpg"></a>
-
<div class="descr">Control set up</div>
+
<a  rel="lightbox[origami]" title="AFM overview of closed structures" href="http://openwetware.org/images/5/54/BM12_nanosaurs_AFM_Closed_overview.jpg"><img src="http://openwetware.org/images/a/aa/BM12_nanosaurs_AFM_Closed_overview_s.jpg"></a>
-
</div>
+
<a  rel="lightbox[origami]" title="AFM close-up of a closed structure" href="http://openwetware.org/images/6/67/BM12_nanosaurs_AFM_Closed_single.jpg"><img src="http://openwetware.org/images/d/d4/BM12_nanosaurs_AFM_Closed_single_s.jpg"></a>
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - side view" href="Overview-Side-500.jpg"><img src="Overview-Side-100.jpg" /></a>
+
-
<div class="descr">GUVs with QD-Strep-receiver A</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - top view" href="Overview-Top-500.jpg"><img src="Overview-top-100.jpg" /></a>
+
-
<div class="descr">Transmitted light image of GUV</div>
+
-
</div>
+
-
</div>
+
</div>
</div>
 +
<div class="img_right img_link">
 +
  <a  rel="lightbox[origami]" title="Developed blob model" href="http://openwetware.org/images/3/3e/BM12_nanosaurs_AFM_Blob_model800.jpg"><img src="http://openwetware.org/images/1/19/BM12_nanosaurs_AFM_Blob_model800_s.jpg"></a>
 +
</div>
 +
 
 +
<p>The open structures appear very homogeneous in shape, whereas a rather large degree of heterogeneity
 +
  was found in the AFM images for the closed structures. A possible explanation for the less defined
 +
  shape of the closed structures could be that those samples have been purified via Freeze ‘N Squeeze
 +
  DNA Gel Extraction whereas the open samples have just been dialyzed. However, the Freeze’N squeeze
 +
  purification gives less background, which means the sample is purer. Since the structures appear to
 +
  be very fragile, the dialysis is a more suitable purification method to leave the structures intact.
 +
          <br>Further measurements on seemingly intact closed structures provided three major classes of different
 +
  shapes. These can be interpreted by the following model developed by Dr. Fernando Moreno-Herrero and
 +
  Maria Eugenia Fuentes:
 +
        </p>
 +
 
 +
  <p>The evaluations of the lateral dimensions of the origami structures in the AFM images are depicted
 +
      in the table below:
 +
  </p>
 +
 
 +
<table>
 +
<tr>
 +
<th colspan="2">&#91;nm&#93;</th>
 +
<th> width</th>
 +
<th> length</th>
 +
<th> height <br/> peaks</th>
 +
<th> height <br/> valleys</th>
 +
</tr>
 +
<tr>
 +
<td><b>open</b></td>
 +
<td><b>one half</b></td>
 +
<td>54,7 &#177; 3,9</td>
 +
<td>41,5 &#177; 3,2</td>
 +
<td>3,8 &#177; 0,4</td>
 +
<td>1,2 &#177; 0,3</td>
 +
</tr>
 +
<tr>
 +
<td rowspan="3"><b>closed</b></td>
 +
<td><b>2 blobs</b></td>
 +
<td>79,2 &#177; 3,4</td>
 +
<td>57,3 &#177; 5,3</td>
 +
<td>8,9 &#177; 1,7</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td><b>3 blobs</b></td>
 +
<td>85,8 &#177; 6,7</td>
 +
<td>69,2 &#177; 5,6</td>
 +
<td>8,5 &#177; 1,8</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td><b>4 blobs</b></td>
 +
<td>90,5 &#177; 1,2</td>
 +
<td>59,9 &#177; 6,0</td>
 +
<td>7,3 &#177; 1,6</td>
 +
<td>---</td>
 +
</tr>
 +
<tr>
 +
<td><b>Expected</b></td>
 +
<td></td>
 +
<td>45</td>
 +
<td>40-44</td>
 +
<td>10 / 20</td>
 +
<td></td>
 +
</tr>
 +
</table>
 +
 
 +
  <p>The length of the open structure matches very well with the expectations. The width is slightly
 +
  too large and the height is too low. This can be explained by the fact that the fragile structure
 +
  preferably lays down flat on the surface and also gets pushed down by the AFM tip.<br>The increase
 +
  of width and length for the closed structure can be explained by an increased tip convolution due to the
 +
  increased height of the structure. However the height matches very well, since it is twice the height of
 +
  the open structure indicating that the desired conformational change has been successfully achieved.<br>
 +
  The various heights of the close structure also go along very well with the model of the different positions
 +
  of the hexagonal DNA multifilaments.<br>In total the AFM and the TEM images confirm a successful DNA origami
 +
  assembly and the expected change in conformation between open and closed structures for the majority of the
 +
  objects. Also the dimensions are well in agreement with the expectations taking into account some explainable
 +
  deviations due to the flexibility of the structure and the limitations of the method that was applied.
 +
          </p>
 +
 
 +
   
 +
</div>
 +
<div class="tabs-spacer"></div>
<div id="tabs-3">
<div id="tabs-3">
-
<h2>Gel Analysis</h2>  
+
<h2>Gel shift assays</h2>
-
<p>After an unsuccessful approach of visualizing the quantum dots, we decided to check the efficiency of oligo-oligo hybridization and binding to streptavidin coated quantum dots on PAGE. The entire set of the experiments below was performed in 12% PA gel (protocol 1-hyperlink).</p>
+
 +
<p>In order to test the specific binding of cargo to our structures and calibrate the sample conditions, several
 +
gel shift assays were performed. The most relevant ones are highlighted in this section.<br>For internal controls
 +
two different schemes for cargo attachment were followed: Loading the cargo based on streptavidin-biotin interaction
 +
and employing DNA strand hybridization. In these experiments we used streptavidin coated quantum dots which can be
 +
attached to the origami in two ways:
 +
        </p>
 +
 +
 +
               
 +
<ul>
 +
<li>Directly binding to internal 5’ biotinylated strands.</li>
 +
<li>Binding of the quantum dots to 3’ biotinylated oligonucleotides which can then hybridize to the internal catcher strands of the origami.</li>
 +
</ul>
 +
 +
<p> To make the gels easy to understand, we use the following conventions for defining which components were loaded in each lane:
 +
</p>
-
<h3>Biotinylation and Streptavidin binding</h3>
+
<div style="margin:auto; width:250px; box-shadow: 0 0 10px #888888; border: 5px solid white;">
-
<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>
+
<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>
 +
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Buffer calibration" href="http://openwetware.org/images/1/1a/BM12_Nanosaurs_ImageGel2.jpg"><img src="http://openwetware.org/images/2/27/BM12_Nanosaurs_ImageGel2_s.jpg"><div>Fig.2 Buffer Calibration</div></a>
 +
</div>
 +
 +
<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)
 +
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
 +
the structure becomes less homogeneous and possibly the structures are also more prone to dimerization. Based on this,
 +
we took 8mM as our standard buffer for further experiments.
 +
</p>
 +
 +
 +
<div class="clear"></div>
 +
<h3>Structure overview</h3>
 +
 +
<div class="img_left img_link">
 +
<a  rel="lightbox[origami]" title="Structure overview" href="http://openwetware.org/images/f/fd/BM12_Nanosaurs_ImageGel1.jpg"><img src="http://openwetware.org/images/1/10/BM12_nanosaurs_ImageGel1_100_s.jpg"><div>Fig.3 Structure overview</div></a>
 +
</div>
 +
 +
<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
 +
be seen that in lane number 2 there is a second band above the expected band for the structure. This likely shows that the
 +
open structures tend to aggregate more than the closed structures, which can be attributed to two main factors; MgCl2 induced
 +
stacking interactions and hybridization between the free locks of adjacent structures.
 +
</p>
 +
 +
 +
<div class="clear"></div>
 +
<h3>Quantum dot binding</h3>
 +
 +
<div class="img_right img_link">
 +
<a  rel="lightbox[origami]" title="Quantum dot binding" href="http://openwetware.org/images/6/61/BM12_Nanosaurs_ImageGel3.jpg"><img src="http://openwetware.org/images/9/98/BM12_Nanosaurs_ImageGel3_s.jpg"><div>Fig.4 Quantum dot binding</div></a>
 +
</div>
 +
 +
<p>After confirming the assembly quality of our structures, cargo attachment tests were performed. In particular, we employed
 +
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.<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>
-
<div class="img_left img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a>
+
-
<div class="descr">Efficiency of biotinylation reaction. From left: oligo, biotynylated oligo, oligo+streptavidin, biotynylated-oligo+streptavidin</div>
+
<p>
-
</div>
+
</p>
-
 
+
<div class="clear"></div>
-
<h3>Optimal QD-Oligo ratio</h3>
+
<h3>Catcher influence on binding</h3>
-
<p>In order to optimize the ratio of quantum dots to the oligonucleotides, different ratios of quantum dots were applied to constant amount of oligonucleotides (70ng). The optimal molar ratio of oligonucleotides to quantum dots was found to be 2:1 since the amount of oligonucleotides not bound to quantum dots 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[origami]" title="Catcher influence on binding" href="http://openwetware.org/images/8/8a/BM12_Nanosaurs_ImageGel4.jpg"><img src="http://openwetware.org/images/a/af/BM12_Nanosaurs_ImageGel4_s.jpg"><div>Fig.5 Catcher influence on binding</div></a>
 +
</div>
 +
 +
<p>The previous results showed that there was still considerable binding to the closed structures. This might be due to the               catcher strands sticking out on the wrong side of the structure. Therefore, in addition to the construct with all 6 catchers to two other versions containing only one catcher were tested for cargo binding. One of them contained a single 5’ biotinylated oligo and the other contained only a single catcher for hybridization mediated binding.
 +
</p>
 +
 +
<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>To further support this, we quantified the binding preference of the structures from the gel image based on the relative intensities of the bands which showed a shift due to quantum dot binding and of the bands that contained the origami only. The obtained results are shown in the table below.
 +
</p>
 +
<div class="clear"></div>
 +
<table>
 +
<tr>
 +
  <th>Lane</th>
 +
  <th>Construct</th>
 +
  <th>Shifted<br/>band</th>
 +
  <th>Construct<br/>band</th>
 +
  <th>Ratio<br/>shifted/construct</th>
 +
  <th>QD affinity ratio<br/>open/closed</th>
 +
</tr>
 +
<tr>
 +
<td>4</td>
 +
<td>Open 1C</td>
 +
<td>1942</td>
 +
<td>5580</td>
 +
<td>0.35</td>
 +
<td rowspan="2">1.61</td>
 +
</tr>
 +
<tr>
 +
<td>11</td>
 +
<td>Closed 1C</td>
 +
<td>672</td>
 +
<td>3111</td>
 +
<td>0.22</td>
 +
</tr>
 +
<tr>
 +
<td>6</td>
 +
<td>Open 1C5'</td>
 +
<td>5216</td>
 +
<td>4025</td>
 +
<td>1.30</td>
 +
<td rowspan="2">1.70</td>
 +
</tr>
 +
<tr>
 +
<td>13</td>
 +
<td>Closed 1C5'</td>
 +
<td>2617</td>
 +
<td>3437</td>
 +
<td>0.76</td>
 +
</tr>
 +
<tr>
 +
<td>8</td>
 +
<td>Open 6C</td>
 +
<td>2937</td>
 +
<td>2944</td>
 +
<td>1.00</td>
 +
<td rowspan="2">1.24</td>
 +
</tr>
 +
<tr>
 +
<td>15</td>
 +
<td>Closed 6C</td>
 +
<td>4106</td>
 +
<td>5123</td>
 +
<td>0.80</td>
 +
</tr>
 +
</table>
-
<div class="img_right img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a>
+
<p> From these data, it can be seen that:  
-
<div class="descr">From left: oligonucleotide w/o biotin+QD; Ratio biotinylated-oligonucleotides:QDs 1:1,2:1, 3:1, 5:1,7:1, 10:1.</div>
+
</p>
-
</div>
+
-
 
+
<ul>
-
<h3>Oligo and Quantum dot hybridization</h3>
+
<li>The quantum dots have a binding preference for the open structures over the closed ones.</li>
-
<p>Finally, the complete system used for the experiments (in 3.1.) (hyperlink) 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>
+
<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>  
-
<div class="img_right img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a>
+
</ul>
-
<div class="descr">From left: catcher A, biotinylated catcher A-complementary and catcher A(separately hybridized)+ 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</div>
+
-
</div>
+
<p>However, in all cases closed structures still bind the cargos to a significant extend. The reason for this unexpected behavior still need to be explored. It may be that still to many misfolded closed origami structures are formed during assembly. This could be improved by a more careful adjustment of the annealing conditions.
-
 
+
</p>
-
<h3>Spectral analysis of Quantum dots</h3>
+
-
<p>We were facing some difficulties in observing the quantum dots both in solution and on the lipid membranes. A poor signal was observed even at high laser powers (70%) in confocal microscopy (?). The PA gels proved that there was no problem with the hybridization of quantum dots to biotinylated oligos. Therefore, pure quantum dot samples of different concentrations was prepared and directly observed on cover slips and bright quantum dots at relatively low laser power could be observed. We then obtained the spectra of these quantum dots by doing a fluorescence emission scan (excited at 458nm). A peak signal was observed at 615-625nm which is consistent to the quantum dot manufacturer specifications that we used. When the same  analysis was done with our vesicles containing samples mentioned before, we found that the spectra was not the same as of the quantum dots  due to the background fluorescence of the contaminated lipids at high laser powers. This prompted us to increase the concentration of the quantum dots from 0.1 nM to 10 nM. Subsequently, the concentration of the oligos was also increased 50-fold.</p>
+
-
 
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - front view" href="big pic"><img src="small pic" alt="Caption" /></a>
+
-
<div class="descr">False colour</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - side view" href="Overview-Side-500.jpg"><img src="Overview-Side-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Bright field</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Maximum emission at 625nm of 10nM Quantum dot samples on the cover slip</div></a>
+
-
</div>
+
-
</div>
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Background signal of the GUVs</div></a>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Bright field</div></a>
+
-
</div>
+
-
</div>
+
-
 
+
-
<h3>Quantum dot 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 quantum dots on the vesicles.</p>
+
-
 
+
-
<div class="img_right img_link"><a rel="lightbox" href="link to big image"><img src="link to small image"></a>
+
-
<div class="descr">Set up</div>
+
-
</div>
+
-
 
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group1]" title="DNA origami - front view" href="big pic"><img src="small pic" alt="Caption" /></a>
+
-
<div class="descr">Control set up</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - side view" href="Overview-Side-500.jpg"><img src="Overview-Side-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Fluorescent of QD with 50x concentration</div>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group2]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Transmited light image of GUV</div></a>
+
-
</div>
+
-
</div>
+
-
<div class="img_gal">
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">GUV w/o catcher A</div></a>
+
-
</div>
+
-
<div class="img_gbox">
+
-
<a  rel="lightbox[group3]" title="DNA origami - top view" href="Overview-Top-500.jpg"></a><img src="Overview-top-100.jpg" alt="Caption" /></a>
+
-
<div class="descr">Transmited light image of GUV</div></a>
+
-
</div>
+
-
</div>
+
-
 
+
-
<div id="tabs-4">
+
</div>
</div>
-
</div>
+
<div class="tabs-spacer"></div>
 +
 +
</body>
</html>
</html>

Current revision

What is DNA origami?

DNA origami is a well-established technique in nanotechnology which involves the folding of DNA to create various 2D or 3D patterns and shapes at the nanoscale. More in detail, it makes use of a long single strand of DNA known as scaffold strand, which acts like a backbone or support for a particular structure to be made. In order to shape the construct in a certain way, several shorter strands of different lengths, called staple strands, are hybridized (bound) to specific parts of the scaffold profiting from the specificity of interactions between complementary base pairs in DNA. The way these binding sites are chosen, determines how the structure is going to fold and in which shape it ends up.

Our DNA origami structure

Requirements

In order to accomplish the purpose of our project, the DNA origami shell must fulfill the following requirements:

  • Comprise a tethering platform for the attachment to lipid bilayers.
  • Include a variable catching platform which allows the structure to bind specific target species.
  • Avoid unspecific binding of non-wanted targets.
  • Provide a trigger mechanism which enables binding upon signal.
  • Contain one or more parts with fluorescent labels for testing and imaging purposes.

Design

DNA origami is a well-established technique in nanotechnology which involves the folding of DNA to create various 2D or 3D patterns and shapes at the nanoscale. More in detail, it makes use of a long single strand of DNA known as scaffold strand, which acts like a backbone or support for a particular structure to be made. In order to shape the construct in a certain way, several shorter strands of different lengths, called staple strands, are hybridized (bound) to specific parts of the scaffold profiting from the specificity of interactions between complementary base pairs in DNA. The way these binding sites are chosen, determines how the structure is going to fold and in which shape it ends up.

Our structure was inspired by the DNA origami Logic Nanorobot by Douglas et al. (Science 335, 831-8XX (2012)). We chose the model proposed in this paper because its shell-like shape provides binding specificity while its lock mechanism offered a triggering platform.
In our design, the height of the structure was lowered to 20nm while increasing its width to 45nm. This is intended to decrease the chances of molecules diffusing into the origami in its closed configuration while making it lay flatter on the vesicle surface to enhance binding. Most notably, the structure was adjusted to have anchor and catcher strands which allow binding to the carrier vesicle and to the target species respectively.
The functional principle of the DNA origami shell is that in its closed state (i.e its lock strands are hybridized to each other), the single stranded catcher oligonucleotides inside are shielded. This means they are not accessible for anything from the outside to hybridize to them. The locks can be triggered to open once a certain protein is around. When the lock strands are not hybridized anymore, the DNA origami shell opens up due to thermal fluctuations. The catcher strands inside are then freely accessible.

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 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 mechanical properties and assumptions about the behavior of DNA (see CanDo).

Functional parts

Scaffold

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.
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.

Core

There are 171 core oligos which give the structure its stability, its basic shape and hold all the functional parts together. To avoid that several structures stack together, the turning points of the scaffold are left single stranded for about 10 to 30 nucleotides. This means that in those regions no staple strands hybridize thus comprising highly flexible single strand DNA.

Anchors

The anchor oligomers can hybridize to cholesterol labeled single strands that provide the attachment to the lipid membrane of the giant unilamellar vesicles and therefore connect the DNA origami structure permanently to them. There are 9 anchor strands to increase the probability of binding. They are 35nt long, containing a 5nt spacer at the DNA origami and a 30bp binding site for cholesterol oligomers on the vesicles.

Catchers

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.
For our experiments the catcher strands were also labeled, for example with biotin to bind to streptavidin coated quantum dots.

Locks

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.
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 Aptamer).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”).

Edge staple

The edge staple is a single strand that is on the top side of the structure. It can be labeled with fluorophores such as Alexa 647 to add a fluorescent signal to the structure.

Guide staples

There are two guide staples that should help to close the structure during the assembly. It increases the amount of structures that are closed after the assembly. The structure cannot open while the guide strands are there. Therefore they have an 8 base toehold and if fully complementary oligos are applied later, they would hybridize and the structure would still be closed, but now has the chance to be opened (if the locks are opened).

cadnano

In order to turn the idea of our sketch into an actual 3D DNA nanostructure, we used a software called caDNAno(learn more at http://cadnano.org/); a computational tool for DNA origami design. Using this tool one define the shape of the desired structure within a graphic environment by providing the hybridization sites for the staple strands and the scaffold length as input parameters. The program will then display the necessary sequences of the staple strands which can be ordered from a suitable company.

Assembly

To assemble the desired structure the following things need to be pipetted together:

  • Scaffold
  • Set of staple oligos defining the features the assembled structure should have
  • Folding buffer
  • Water

The correct ratios and the recipe of the folding buffer can be found in the recipe section.
Following a detailed protocol the mixture is heated up to 85°C and then cooled down very slowly using a given temperature ramp. Especially in the area of 55°C the cooling process is extremely slow since most of the assembly process happens in that temperature region. The whole cooling process takes about 15 hours.
After the assembly the structures remain stable at room temperature.

Purification

To have a greater yield of assembled structures, the ratio of staple strands to scaffold strands is 7.5 to 1. To get rid of the leftover single strands after assembly, the samples are typically dialyzed for 1 to 2 hours using a 0.025µm filter.

Results

In order to examine the shape of the structure, the samples were imaged using transmission electron (TEM) and atomic force microscopy (AFM).

TEM

To image the structures via transmission electron microscopy the samples were stained with uranyl acetate (see protocols).

The TEM images demonstrate a successful assembly of both types of structures. In particular they show a significant difference in shape between the open and the closed structures. Open structures were typically twice as long as closed structures (see below). For these images the closed structures were assembled including the guide strands, but also the not guided constructs showed a conformational change with a high percentage of the structures being closed.
Producing negatively stained samples (using short staining times) it was possible to image the closed structures standing upright. The pictures show that the shape of the cross section is rather variable. However, most of the structures show a high degree of integrity, i.e. a closed circumference supporting that the structures are really closed.
Evaluating several individual structures the following average lateral dimensions of both types of DNA origami were obtained:

[nm] closed open
width length full length
# of measurements 26 29 40
result (95% STD) 48,9 ± 5,9 39,6 ± 3,4 71,4 ± 3,8
relative error [%] 12,0 8,6 5,3
Expected 45 40-44 80-88
Possible reason
for deviation
Hinges and edges
floppy single strands

For the closed structure the length, as well as the width, match nicely the expected values. The slightly higher width can be explained by assuming that the structures laying down flat which increases the lateral dimension due to the bending down of the side walls.
The open structure however appeared to be shorter than one would expect if one doubles the length of a closed structure. This can be explained due to the fact that the turning points, as well as the hinges, were left as single strands making them more flexible. Therefore they do not necessarily have to be stretched to their full lengths. In general the open structure shows increased flexibility and degrees of freedom compared to the closed constructs.

AFM

To further proof of the correct assembly, the open and closed structures were sent to the Spanish National Center for Biotechnology in Madrid. There Dr. Fernando Moreno-Herrero and Maria Eugenia Fuentes obtained a series of magnificent AFM images.The following pictures show the different samples in an overview (left) as well as an enlarged view of a single structure (right).

The open structures appear very homogeneous in shape, whereas a rather large degree of heterogeneity was found in the AFM images for the closed structures. A possible explanation for the less defined shape of the closed structures could be that those samples have been purified via Freeze ‘N Squeeze DNA Gel Extraction whereas the open samples have just been dialyzed. However, the Freeze’N squeeze purification gives less background, which means the sample is purer. Since the structures appear to be very fragile, the dialysis is a more suitable purification method to leave the structures intact.
Further measurements on seemingly intact closed structures provided three major classes of different shapes. These can be interpreted by the following model developed by Dr. Fernando Moreno-Herrero and Maria Eugenia Fuentes:

The evaluations of the lateral dimensions of the origami structures in the AFM images are depicted in the table below:

[nm] width length height
peaks
height
valleys
open one half 54,7 ± 3,9 41,5 ± 3,2 3,8 ± 0,4 1,2 ± 0,3
closed 2 blobs 79,2 ± 3,4 57,3 ± 5,3 8,9 ± 1,7 ---
3 blobs 85,8 ± 6,7 69,2 ± 5,6 8,5 ± 1,8 ---
4 blobs 90,5 ± 1,2 59,9 ± 6,0 7,3 ± 1,6 ---
Expected 45 40-44 10 / 20

The length of the open structure matches very well with the expectations. The width is slightly too large and the height is too low. This can be explained by the fact that the fragile structure preferably lays down flat on the surface and also gets pushed down by the AFM tip.
The increase of width and length for the closed structure can be explained by an increased tip convolution due to the increased height of the structure. However the height matches very well, since it is twice the height of the open structure indicating that the desired conformational change has been successfully achieved.
The various heights of the close structure also go along very well with the model of the different positions of the hexagonal DNA multifilaments.
In total the AFM and the TEM images confirm a successful DNA origami assembly and the expected change in conformation between open and closed structures for the majority of the objects. Also the dimensions are well in agreement with the expectations taking into account some explainable deviations due to the flexibility of the structure and the limitations of the method that was applied.

Gel shift assays

In order to test the specific binding of cargo to our structures and calibrate the sample conditions, several gel shift assays were performed. The most relevant ones are highlighted in this section.
For internal controls two different schemes for cargo attachment were followed: Loading the cargo based on streptavidin-biotin interaction and employing DNA strand hybridization. In these experiments we used streptavidin coated quantum dots which can be attached to the origami in two ways:

  • Directly binding to internal 5’ biotinylated strands.
  • Binding of the quantum dots to 3’ biotinylated oligonucleotides which can then hybridize to the internal catcher strands of the origami.

To make the gels easy to understand, we use the following conventions for defining which components were loaded in each lane:

Buffer calibration

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) 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 the structure becomes less homogeneous and possibly the structures are also more prone to dimerization. Based on this, we took 8mM as our standard buffer for further experiments.

Structure overview

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 be seen that in lane number 2 there is a second band above the expected band for the structure. This likely shows that the open structures tend to aggregate more than the closed structures, which can be attributed to two main factors; MgCl2 induced stacking interactions and hybridization between the free locks of adjacent structures.

Quantum dot binding

After confirming the assembly quality of our structures, cargo attachment tests were performed. In particular, we employed 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.
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.

Catcher influence on binding

The previous results showed that there was still considerable binding to the closed structures. This might be due to the catcher strands sticking out on the wrong side of the structure. Therefore, in addition to the construct with all 6 catchers to two other versions containing only one catcher were tested for cargo binding. One of them contained a single 5’ biotinylated oligo and the other contained only a single catcher for hybridization mediated binding.

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.

To further support this, we quantified the binding preference of the structures from the gel image based on the relative intensities of the bands which showed a shift due to quantum dot binding and of the bands that contained the origami only. The obtained results are shown in the table below.

Lane Construct Shifted
band
Construct
band
Ratio
shifted/construct
QD affinity ratio
open/closed
4 Open 1C 1942 5580 0.35 1.61
11 Closed 1C 672 3111 0.22
6 Open 1C5' 5216 4025 1.30 1.70
13 Closed 1C5' 2617 3437 0.76
8 Open 6C 2937 2944 1.00 1.24
15 Closed 6C 4106 5123 0.80

From these data, it can be seen that:

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

However, in all cases closed structures still bind the cargos to a significant extend. The reason for this unexpected behavior still need to be explored. It may be that still to many misfolded closed origami structures are formed during assembly. This could be improved by a more careful adjustment of the annealing conditions.