Biomod/2011/Slovenia/BioNanoWizards/resultssummary: Difference between revisions

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applications</a><br>
applications</a><br>
</span>
</span>
<br>
<br>
<br>
<big><big><span style="color: black; font-weight: bold;">1.
<big><big><span style="color: black; font-weight: bold;">1.
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<br>
<br>
<span style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-weight: bold;">We enhanced the
<span style="font-weight: bold;">We <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">enhanced the
solubility of zinc fingers so that they can be produced in large amount
solubility</a> of zinc fingers so that they can be produced in large amount
and used for <em>in vitro</em> applications</span>,
and used for <em>in vitro</em> applications</span>,
rather than only within living cells. Their solubility was increased by
rather than only within living cells. Their solubility was increased by genetic
fusing them with maltose-binding protein (MBP) or
fusing them with maltose-binding protein (MBP) or
glutathion-S-transferase (GST) domains, which can also facilitate
glutathione-S-transferase (GST) domains, which also facilitate
purification of protein fusions.</span><br>
purification.</span><br>
<br>
<table style="text-align: left; width: 100%;" border="0"
cellpadding="0" cellspacing="0">
  <tbody>
    <tr align="left">
      <td><img
style="margin-top: 10px; margin-bottom: 10px; width: 761px; height: 332px;"
alt=""
src="http://openwetware.org/images/e/ec/MBPsolublezadnji.png"></td>
    </tr>
    <tr align="justify">
      <td><span style="font-family: Arial;"><span
style="font-weight: bold;">
Figure 1: Production and isolation of MBP-fused ZFPs containing
six-finger domains.</span> Left to right: Coomassie stained
SDS-PAGE gels of MBP-6F6 (Mw of approximately 64.3 kDa) and MBP-2C7 (Mw
of approximately 64.9 kDa) soluble and insoluble fractions; Western
blot analysis of MBP-6F6 and MBP-2C7 soluble and insoluble fractions
using anti-His4 antibodies; Coomassie stained SDS-PAGE of isolated
MBP-6F6 (Mw of approximately 64.3 kDa); Coomassie stained SDS-PAGE of
isolated MBP-6F6 (Mw of approximately 64.9 kDa).
      </span></td>
    </tr>
  </tbody>
</table>
<br>
<br>
<span style="font-family: Arial;"><span
<span style="font-family: Arial;"><span
  style="font-weight: bold;">We tested DNA binding of
  style="font-weight: bold;">We demonstrated DNA binding of
zinc finger domains with increased specificity and affinity</span>
zinc finger domains with <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultstightbindingzfp">increased specificity and affinity</a></span>
based on the recognition of 18 bp binding sequence, which increases
based on the recognition of 18 bp binding sequence, which increases
their affinity for DNA towards the picomolar range of Kd, making their
their affinity for DNA towards the picomolar range of Kd, making their
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attachment staples was demonstrated by AlphaScreen and EMSA
attachment staples was demonstrated by AlphaScreen and EMSA
experiments.
experiments.
</span><span style="font-family: Arial; font-weight: bold;">We
</span>
demonstarted positional attachment of ZFPs to specific sites on DNA
<br>
<br><span style="font-family: Arial; font-weight: bold;">We
demonstrated <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/protdnahybrid">positional attachment</a> of ZFPs to specific sites on DNA
origami.<br>
origami.<br>
<br>
</span>
</span>
<table style="text-align: left; width: 100%;" border="0"
cellpadding="5" cellspacing="2">
  <tbody>
    <tr>
      <td style="text-align: left; vertical-align: bottom;"><img
style="padding-top: 0pt; padding-bottom: 0px; width: 389px; height: 277px;"
alt="" src="http://openwetware.org/images/6/6b/MBPZFP.png"></td>
      <td style="text-align: left;" valign="undefined"><span
style="font-family: Arial; font-weight: bold;"><img
style="padding: 0pt 0pt 5px; width: 457px; height: 279px;"
alt=""
src="http://openwetware.org/images/a/af/AlphaScreen_vsi.png"></span></td>
    </tr>
    <tr>
      <td style="vertical-align: top; text-align: justify;"><span
style="font-weight: bold;">Figure
2:
Threedimensional representation of chimeric ZFP</span>. <span
style="font-weight: normal;">MBP is shown in green and ZFP
(Zif268) is shown in cyan bound to the target DNA.&nbsp;</span></td>
      <td style="vertical-align: top; text-align: justify;"><span
style="font-weight: bold;">Figure 3: AlphaScreen binding
assay of GST-2C7, GST-AZPA4, GST-Zif268 and MBP-6F6 to their
corresponding DNA sequences.&nbsp;</span>Non-target
biotinylated
oligonucleotides were used as negative controls.</td>
    </tr>
  </tbody>
</table>
<table
style="margin: 3px 0px 30px 30px; width: 550px; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
  <tbody>
    <tr>
      <td style="text-align: center;"><img
style="padding-top: 0pt; padding-bottom: 5px; margin-top: 15px; width: 570px; height: 281px;"
alt="" src="http://openwetware.org/images/5/5c/Slika1prot.png"></td>
    </tr>
    <tr style="font-family: Arial; font-weight: bold;">
      <td style="text-align: justify;">Figure 4. AFM image
of GST-AZPA4 bound to DNA origami rectangles <span
style="font-weight: normal;"> (tapping mode imaging in air).
A yellow rectangle marks the origami with all of the three binding
sites occupied. Red arrows indicate unbound protein adsorbed to mica.</span></td>
    </tr>
  </tbody>
</table>
<br>
<br>
<big><big><span style="color: black; font-weight: bold;">2.
<big><big><span style="color: black; font-weight: bold;">2.
Vertical stacks of DNA origami</span></big></big>
Vertical stacks of DNA origami rectangles</span></big></big>
<br>
<br>
<br>
<br>
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combinations of more than one type of DNA origami derivatized with
combinations of more than one type of DNA origami derivatized with
different molecules (e.g. conductive carbon nanotubes or metals). For
different molecules (e.g. conductive carbon nanotubes or metals). For
this purpose combinations of two or more DNA origami breadboards into
this purpose combinations of two or more DNA origami layers arranged into
vertical stacks may produce some directly useful devices. Vertical
vertical stacks can be used for fabrication of useful nanoscale devices. Vertical
stacking could be accomplished using either DNA or proteins as tethers.
stacking could be accomplished using either DNA or proteins as tethers.
The vertical order of DNA origami plates and their number in stack
The vertical order of DNA origami layers and their number in a stack
could be designed at will using different tethers on each side of the
could be designed at will using different tethers on each side of the
DNA breadboard.</span>
DNA origami plates.</span>
<br>
<br>
<br>
<br>
<span style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-weight: bold;">We prepared perfectly
<span style="font-weight: bold;">We prepared perfectly
aligned DNA stack using DNA tethers.</span></span>
<a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsdnatethers">aligned DNA stack</a> using DNA tethers.</span></span>
<br>
<br>
<br>
<br>
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relatively easy to implement and ensure their uniqueness, the advantage
relatively easy to implement and ensure their uniqueness, the advantage
of protein tethers is that they can provide a fixed distance between
of protein tethers is that they can provide a fixed distance between
stacks, they are not modified with the same modifiers at the DNA
stacks due to the discrete size of used protein folds, they are not modified with the same modifiers at the DNA surface and can remain functional. <br>
surface and remain functional. <br>
<br>
<br>
<span style="font-weight: bold;">We designed two different
<span style="font-weight: bold;">We designed two different
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<ul>
<ul>
   <li><span style="font-family: Arial;">twin ZFP
   <li><span style="font-family: Arial;">twin ZFP
chimeras, having two different DNA binding domains that
chimeras, comprising two different DNA binding domains that
ensure anchoring to two different sides or faces of DNA origami and
ensure anchoring to two different sides or faces of DNA origami and
additional solubilizing and purification domains (MBP and His-tag,
additional solubilizing and purification domains (MBP and His-tag,
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   </li>
   </li>
</ul>
</ul>
<br>
<span style="font-family: Arial;"><span
<span style="font-family: Arial;"><span
  style="font-weight: bold;">We designed twin ZFP
  style="font-weight: bold;">We designed <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsproteintethers">twin ZFP
protein tethers,</span> produced them in recombinant form and
protein tethers</a>,</span> produced them in recombinant form and
purified them.</span><br>
purified them.</span><br>
<table
 
style="margin-top: 3px; margin-bottom: 20px; float: left; width: 100%; height: 50px;"
 
border="0" cellpadding="0" cellspacing="0">
  <tbody>
    <tr>
      <td style="text-align: center;"><img
style="padding-top: 0pt; padding-bottom: 10px; font-family: Arial; margin-top: 15px; width: 890px; height: 512px;"
alt="" src="http://openwetware.org/images/f/f5/Slika5.png"></td>
    </tr>
    <tr style="font-family: Arial;">
      <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 5: Formation of DNA
origami stacks with the expected height visualized by the AFM.</span>
The upper left sample was annealed from 70 °C and the lower from 60 °C.
Both samples contained perfectly arranged double layer stacks. Red
arrows indicate the zigzag path of the profile extraction made with
PicoImage software (Agilent). Profile clearly depicts that the height
of stacks is double in size of the non-stacked DNA origami rectangles.</td>
    </tr>
  </tbody>
</table>
<br>
<br>
<span style="color: white;">.</span>
<br>
<table style="text-align: left; width: 50%; height: 370px;"
border="0" cellpadding="0" cellspacing="0">
  <tbody>
    <tr>
      <td style="text-align: right;"><img
src="http://openwetware.org/images/6/64/Twin_ZFPs_isolation.png"
alt=""
style="padding: 0pt 0pt 10px; font-family: Arial; width: 260px; height: 362px;"></td>
      <td style="text-align: justify; vertical-align: top;"><span
style="font-weight: bold;">Figure 7: Coomassie Brilliant
Blue stain of the twin ZFP proteins purified using chelating
chromatography.</span> Grey arrow depicts the position of
2C7-MBP-6F6 (Mw of 86.03 kDa) and the black arrow the position of
AZPA4-MBP-6F6 (Mw of 83.93 kDa). Proteins were produced under the same
conditions as BRET triple fusions: 2x YT medium supplemented with 10
g/L glucose, 100 mg/L antibiotic kanamycin and 0.5 mM ZnCl2 / 30 °C or
37 °C / 160rpm / ~5 or 7 h induction with 1 mM IPTG.</td>
    </tr>
  </tbody>
</table>
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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/applabonchip">Lab-on-a-nanochip</a></li>
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class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosynthteticcompartments">Biosynthetic

compartments</a></li> 

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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">Biosensors</a></li>
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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appnanoelectronics">Nanoelectronics</a></li>
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<!--[if lte IE 6]></td></tr></table></a><![endif]--></li> 

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construction</a></li> 

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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methprotprodandisol">Protein

production and isolation</a></li> 

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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methalphascreen">AlphaScreen

assay</a></li> 

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href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methbret">BRET

assay</a></li> 

       </ul>

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     <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methafm">AFM</a></li>
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 <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/team">Team</a></li>
 <li class="pureCssMenui"><a class="pureCssMenui"
href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/acknowledgments">Acknowledgments</a></li>

</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;">Results summary</span></big></big></big></big><br>

<br> <br> <span style="font-family: Arial;"> Results of our project experimentally demonstrate the <span 

style="font-weight: bold;">proof of the concept for DNA

origami-protein hybrids</span> that could be used in <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/applabonchip">advanced

applications</a><br> </span> <br> <big><big><span style="color: black; font-weight: bold;">1. Protein add-ons</span></big></big> <br> <br> <span style="font-family: Arial;"> <span style="font-weight: bold;">We <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultssolublezfp">enhanced the solubility</a> of zinc fingers so that they can be produced in large amount and used for <em>in vitro</em> applications</span>, rather than only within living cells. Their solubility was increased by genetic fusing them with maltose-binding protein (MBP) or glutathione-S-transferase (GST) domains, which also facilitate purification.</span><br> <br> <span style="font-family: Arial;"><span 

style="font-weight: bold;">We demonstrated DNA binding of

zinc finger domains with <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultstightbindingzfp">increased specificity and affinity</a></span> based on the recognition of 18 bp binding sequence, which increases their affinity for DNA towards the picomolar range of Kd, making their interaction with DNA almost irreversible. Specific binding to dsDNA attachment staples was demonstrated by AlphaScreen and EMSA experiments. </span> <br> <br><span style="font-family: Arial; font-weight: bold;">We demonstrated <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/protdnahybrid">positional attachment</a> of ZFPs to specific sites on DNA origami.<br> </span> <br> <big><big><span style="color: black; font-weight: bold;">2. Vertical stacks of DNA origami rectangles</span></big></big> <br> <br> <span style="font-family: Arial;"> Advanced technological applications of DNA origami may require combinations of more than one type of DNA origami derivatized with different molecules (e.g. conductive carbon nanotubes or metals). For this purpose combinations of two or more DNA origami layers arranged into vertical stacks can be used for fabrication of useful nanoscale devices. Vertical stacking could be accomplished using either DNA or proteins as tethers. The vertical order of DNA origami layers and their number in a stack could be designed at will using different tethers on each side of the DNA origami plates.</span> <br> <br> <span style="font-family: Arial;"> <span style="font-weight: bold;">We prepared perfectly <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsdnatethers">aligned DNA stack</a> using DNA tethers.</span></span> <br> <br> <span style="font-family: Arial;">While DNA tethers are relatively easy to implement and ensure their uniqueness, the advantage of protein tethers is that they can provide a fixed distance between stacks due to the discrete size of used protein folds, they are not modified with the same modifiers at the DNA surface and can remain functional. <br> <br> <span style="font-weight: bold;">We designed two different types of protein tethers:</span><br> <br> </span> <ul> 

 <li><span style="font-family: Arial;">twin ZFP

chimeras, comprising two different DNA binding domains that ensure anchoring to two different sides or faces of DNA origami and additional solubilizing and purification domains (MBP and His-tag, respectively).</span></li> 

 <li>heterodimeric ZFPs, comprising of DNA binding domain and a

heterodimerizing domain (SH3 peptide and SH3 domain or coiled-coil forming segments) that form a stable heterodimer.<br> 

 </li>

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

style="font-weight: bold;">We designed <a href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/resultsproteintethers">twin ZFP

protein tethers</a>,</span> produced them in recombinant form and purified them.</span><br>


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