Biomod/2011/Slovenia/BioNanoWizards/ideaverticalstacks

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</ul> <!-- End PureCSSMenu.com MENU --> </div> </div> </div> <!-- end #header --> <div id="page" class="container"> <div id="content"> <div class="post"> <div class="entry"><big><big><big><big><span 

style="color: black; font-weight: bold;">Vertical stacks</span></big></big></big></big><br>

<br> <span style="font-family: Arial;"> DNA origami rectangles based on a single-stranded M13 DNA have dimensions around 100 nm (Rothemund, 2006). Extension into the third dimension created variable shapes such as different geometrical objects (Shih, 2004) and a box (Andersen, 2009), weaving the compact 3D objects from several connected layers (Douglas, 2009). 3D shells were also produced by bending dsDNA (Han, 2011). Two dimensional tiling by stitching DNA origami rectangles was reported (Endo, 2010). In general 3D DNA origami design has so far relied mostly on shaping a single M13 DNA strand, which restricted to some extent more complex functional applications of bionanomolecular structures. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> The main motivation for the idea of creating vertical stacks of DNA origami is the possibility to combine two or more differently-modified DNA origami breadboards, (e.g. metalized or covered with bound conductive carbon nanotubes), which is not possible using single continuous thread. Precise positioning of DNA origami layers could be achieved using tethers connecting two neighboring DNA origami layers. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> Vertical stacking of DNA origami layers could be accomplished using either DNA or proteins as tethers. The vertical order of DNA origami layers in the stack could be designed at will using interacting tethers on each face of the DNA origami layers. The common approach of DNA and protein tethers is that they both include replacement of conventional staples with their extended versions, resulting in either single-stranded or double-stranded DNA extensions which point perpendicularly to the plane of DNA origami to directly interact or serve as a protein docking site, respectively. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> This type of vertical stacks may lead to <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appnanoelectronics">nanoscale

electronic applications</a>, elaborated in the Discussion. Stacking could additionally be used to create masks for the formation of 3D patterns similar as in photolithography. Vertical stacking can increase the density of integration, which is an approach that is actively pursued in the development of 3D integrated circuits.</span><br> <br style="font-family: Arial;"> <br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">DNA tethers</span></big></big><br>

<br style="font-family: Arial;"> <span style="font-family: Arial;"> The idea of DNA tethers is to use unique complementary single stranded DNA sequences protruding perpendicularly from the DNA origami. Their positions on the opposing face of the other DNA origami stacking partner are placed at the mirrored positions (Figure 1). Implementation of this idea requires rational selection of complementary staple extensions and some additional modifications (described in Methods) as the tethering segments protrude from different faces of DNA origami rectangles. By introducing such staples so that they project out of the DNA origami perpendicularly in both directions, multiple (in theory infinite) DNA origami stacks could be formed. Precise positioning of two DNA origami layers above each other requires introduction of several tethers and their asymmetric arrangement to prevent formation of staggered overlays. </span><br style="font-family: Arial;"> <span style="font-family: Arial;"></span><br> <table 

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     <td style="text-align: center;"><img
style="font-family: Arial; width: 800px; height: 385px;" alt=""
src="http://openwetware.org/images/e/e9/VerticalstackDNAtethers.png"></td>
   </tr>
   <tr style="font-family: Arial;">
     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 1. Vertical DNA origami

stacks based on single-stranded DNA tethers. </span>DNA origami planes are modified independently using staple strands with ssDNA extensions protruding perpendicularly into solution.<br> 

     </td>
   </tr>
 </tbody>

</table><br><br><br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">

Protein tethers</span></big></big><br> <span style="font-family: Arial;"><br> DNA tethers have some potential drawbacks, in the first place the lack of rigidity in comparison to globular protein domains. We reasoned that we could use the same idea of ZFP-based DNA origami add-ons also for creation of vertical stacks. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;"> For this purpose DNA origami is modified at selected sites with the same type of double stranded ZFP binding sites perpendicular to the plane of DNA origami as in protein add-ons. We developed two approaches to protein tethers based on twin ZFP binding domains and ZFP-protein heterodimeric tethering domain. </span><br style="font-family: Arial;"> <br> <br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">Bifunctional - twin

ZFP based approach</span></big></big><br> <br style="font-family: Arial;"> <span style="font-family: Arial;"> Here, the tethering molecule is composed of two distinct ZFPs each recognizing origami attachment site on a different DNA origami layer. This chimeric protein fusion, bridged with a solubility domain (MBP), is prepared in recombinant form in bacteria. Similarly to DNA tethering approach one could in theory expect formation of a long vertical stack by the addition of two different ZFP attachment sites on the opposing faces of DNA origami </span><br style="font-family: Arial;"> <br> <table 

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     <td style="text-align: center;"><img
style="font-family: Arial; width: 890px; height: 298px;" alt=""
src="http://openwetware.org/images/d/d8/ProteintetheringFINAL.png"></td>
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   <tr style="font-family: Arial;">
     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 2: Protein-based tethering

of DNA origami planes with twin ZFP chimeras. </span>(a) Basic element for such protein mediated tethering comprises a solubility enhancing domain (MBP) flanked by two distinct ZFPs. (b) Numerous recombinantly produced twin ZFP chimeras stabilize DNA origami planes at defined distance locking down the stack.<br> 

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 </tbody>

</table> <br> <br style="font-family: Arial;"> <span style="font-family: Arial;">Introduction of several protein tethers is expected to significantly increase the stability of such stacks due to the cooperative interactions between several neighboring binding sites, therefore even ZFPs with weaker affinities could be used for the stacking purpose. The expected affinity of six neighboring ZFP interactions should exceed the biotin/streptavidin affinity. Formation of stacks requires the precise stoichiometry of each component in the reaction mixture as the excess of ZFP protein tethers could be inhibitory to the interaction, while the lower ration would result in missing tethers. One solution would be to first functionalize one DNA origami plane with excessive amounts of twin ZFP chimeras, remove the surplus of proteins and then titrate the system with the second DNA origami plane. </span><br 

style="font-family: Arial;">

<br> <br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">Protein heterodimer

approach</span></big></big><br> <br style="font-family: Arial;"> <span style="font-family: Arial;">Tethering two faces of DNA origami could be also achieved by the incorporation of different protein heterodimerization domains. In contrast to twin ZFP protein tethers two different proteins have to be prapared. However the advantage of the heterodimer approach is that interactions between heterodimers could be regulated and derivatization of DNA origami with bound ZFP-interacting domains can be performed separately.</span><br 

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     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 3: Protein tethers of DNA

origami based on protein heterodimerization. </span>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas eu massa velit. Donec sed risus lacus, ac sollicitudin enim. Proin iaculis sollicitudin felis non congue. Nulla massa odio, facilisis at dictum sed, pretium ut elit.</td> 

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</table> <br> <br><br><br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">Coiled-coil-dimer based

approach</span></big></big><br> <span style="font-family: Arial;"><br> Coiled-coils are naturally occurring protein domains comprising intertwined alpha helices in parallel or antiparallel orientation. The idea here is to genetically fuse each of the two coiled-coil forming segments to one of the two MBP-ZFP chimeras. Coiled-coil interactions therefore mediate stacking of DNA origami layers decorated at the defined positions through the specific ZFP binding domain. Preferably the interactions between coiled-coil partners are antiparallel (McClain, 2001), with coiled-coil segment serving as a rigid rod, separating the two layers.</span><br 

style="font-family: Arial;">

<br> <br> <big style="font-family: Arial;"><big><span 

style="color: black; font-weight: bold;">SH3-SH3 ligand

based approach</span></big></big><br> <br style="font-family: Arial;"> <span style="font-family: Arial;">SH3-SH3 ligand pair forms tight and specific interaction and has been used to assemble protein scaffold in a biosynthetic reaction (Dueber, 2009). We considered this interaction, even though it is in the sub μM range, for the formation of a vertical stack. Addition of a soluble SH3 ligand peptide could be used to regulate the disassembly of the stack. Similarly to the strategy using coiled-coils, two DNA origami planes would need to be separately functionalized with the two chimeras and mixed at equimolar ratios to form a stack.</span><br> <br> <hr style="width: 100%; height: 2px;"> <small></small> <ul> 

 <li><small>Andersen ES, Dong

M, Nielsen MM, Jahn K, Subramani R, Mamdouh W, Golas MM, Sander B, Stark H, Oliveira CL, Pedersen JS, Birkedal V, Besenbacher F, Gothelf KV, Kjems J (2009) Self-assembly of a nanoscale DNA box with a controllable lid. <span style="font-style: italic;">Nature</span> 459: 73-76.</small></li> 

 <li><small>Douglas SM, Dietz

H, Liedl T, Högberg B, Graf F, Shih WM (2009) Self-assembly of DNA into nanoscale three-dimensional shapes. <span 

style="font-style: italic;">Nature</span> 459: 414-418.</small></li>
 <li><small>Dueber JE, Wu GC,

Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD (2009) Synthetic protein scaffolds provide modular control over metabolic flux. <span style="font-style: italic;">Nature Biotechnol </span>28: 753-9.</small></li> 

 <li><small>Endo M, Sugita T,

Katsuda Y, Hidaka K, Sugiyama H (2010) Programmed-assembly system using DNA jigsaw pieces. <span style="font-style: italic;">Chemistry</span> 18: 5362-5368.</small></li> 

 <li><small>Han D, Pal S,

Nangreave J, Deng Z, Liu Y, Yan H (2011) DNA origami with complex curvatures in three-dimensional space. <span 

style="font-style: italic;">Science</span> 332:342-6.</small></li>
 <li><small>McClain DL, Woods

HL, Oakley MG (2001) Design and Characterization of a Heterodimeric Coiled Coil that Forms Exclusively with an Antiparallel Relative Helix Orientation. <span style="font-style: italic;">J. Am. Chem. Soc.</span> 123: 3151-3152.</small></li> 

 <li><small>Rothemund PWK

(2006) Folding DNA to create nanoscale shapes and patterns. <span 

style="font-style: italic;">Nature</span> 440: 297-302.</small></li>
 <li><small>Shih WM, Quispe JD,

Joyce GF (2004) A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. <span style="font-style: italic;">Nature</span> 427: 618-621.</small></li> </ul> <!-- TU SE KONEA GLAVNO BESEDILO NA STRANI --> </div> </div> </div> <!-- end #content --> <div style="clear: both;">&nbsp;</div> </div> <!-- end #page --></div> <div id="footer-content" class="container"> <div id="footer-bg"> <table 

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