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

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     </tr>
     </tr>
     <tr style="font-family: Arial; font-weight: bold;">
     <tr style="font-family: Arial; font-weight: bold;">
       <td style="text-align: justify;">Figure X. AFM image
       <td style="text-align: justify;">Figure 30. AFM image
of GST-AZPA4 bound to DNA origami rectangles <span
of GST-AZPA4 bound to selected positions on DNA origami rectangles <span
  style="font-weight: normal;"> (tapping mode imaging in air).
  style="font-weight: normal;"> (AFM tapping mode imaging in air).
A yellow rectangle marks the origami with all of the three binding
A yellow rectangle marks the origami with all of the three binding
sites occupied. Red arrows indicate unbound protein adsorbed to mica.</span></td>
sites occupied. Red arrows indicate unbound protein adsorbed to mica.</span></td>
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finger proteins to attachment staple strands integrated into DNA
finger proteins to attachment staple strands integrated into DNA
origami rectangle. We used a DNA origami rectangle design inspired by
origami rectangle. We used a DNA origami rectangle design inspired by
the original DNA origami paper (Rothemund, <span
the original DNA origami paper (Rothemund, 2006, see
style="font-style: italic;">Nature</span> 2006, see
Methods / <a
Methods / <a
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methgeneraldesignstrategy">DNA
  href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methgeneraldesignstrategy">DNA
Line 515: Line 514:
correct folding
correct folding
of zinc finger chimeras and characterization of their ability to bind
of zinc finger chimeras and characterization of their ability to bind
to the designed oligonucleotide hairpins determined by the Alpha-screen
to the designed oligonucleotide hairpins determined by the AlphaScreen
and EMSA assay, we proceeded with optimizing conditions for binding to
and EMSA assay, we proceeded with optimization of conditions for binding to
DNA origami rectangle. To achieve successful binding, several factors
DNA origami rectangle. To achieve successful binding, several factors
had to be taken into account:
had to be taken into account:
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DNA origami rectangles had to be removed to minimize the number of
DNA origami rectangles had to be removed to minimize the number of
potential binding sites for protein chimeras,</li>
potential binding sites for protein chimeras,</li>
   <li>the time of incubation with DNA origami had to be optimized
   <li>the time of incubation with DNA origami had to be optimized to allow reaching
to allow for obtaining equilibrium of binding and prevent potential
equilibrium and prevent potential protein inactivation due to air oxidation of cysteine
protein inactivation due to air oxidation (this might be also avoided
residues of ZFPs (this might be also avoided by incubation in an inert atmosphere).</li>
by incubation in an inert atmosphere).</li>
</ul>
</ul>
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<span style="font-family: Arial;">
<span style="font-family: Arial;">
Successful binding was demonstrated for GST-AZPA4 using the following
Successful binding was demonstrated for GST-AZPA4 using the following protocol: DNA origami rectangles were prepared in 5 nM concentration with 5x staple strand excess in 1x TB/Mg<sup>2+</sup> buffer. Standard staple strands at positions Bb1, ES3, Be1 were replaced with attachment staples for AZPA4. 1x TB/Mg<sup>2+</sup> buffer was removed by filtration and replaced with 35 mM Hepes, pH = 7.0, 50 mM KCl, 12.5 mM MgCl<sub>2</sub>, 100 uM ZnCl<sub>2</sub>, 5 mM TCEP and 5 % (v/v) glycerol. DNA origami was incubated with 200x protein excess for 2 h, i.e. 70x excess with respect to the number of available attachment staples per origami, which is also consistent with the results from EMSA and AlphaScreen. Samples for AFM imaging were prepared fresh and imaged in a tapping mode in air. Control was the same DNA origami without added protein.  
protocol: DNA origami rectangles were prepared in 5 nM concentration
with 5x staple strand excess in 1x TB/Mg2+ buffer. Standard staple
strands at positions Bb1, ES3, Be1 were replaced with attachment
staples for AZPA4. 1x TB/Mg2+ buffer was removed by filtration and
replaced with 35 mM Hepes, pH = 7.0, 50 mM KCl, 12.5 mM MgCl2, 100 uM
ZnCl2, 5 mM TCEP and 5 % (v/v) glycerol. DNA origami was incubated with
200x protein excess for 2h, i.e. 70x excess with respect to the number
of available attachment staples per origami, which is also consistent
with the results from electrophoretic mobility shift assay and
Alpha-screen. Samples for AFM imaging were prepared fresh and imaged in
tapping mode under air. The control was the same origami without the
added protein.
</span><br style="font-family: Arial;">
</span><br style="font-family: Arial;">
<br style="font-family: Arial;">
<br style="font-family: Arial;">
<table
<table
  style="margin-top: 3px; margin-bottom: 5px; float: left; width: 100%; height: 50px;"
  style="margin-top: 3px; margin-bottom: 20px; float: left; width: 100%; height: 50px;"
  border="0" cellpadding="0" cellspacing="0">
  border="0" cellpadding="0" cellspacing="0">
   <tbody>
   <tbody>
     <tr>
     <tr>
       <td style="text-align: center;"><img
       <td style="text-align: center;"><img
  style="font-family: Arial; width: 890px; height: 293px;" alt=""
  style="font-family: Arial; width: 890px; height: 293px; padding: 0 0 10px 0;" alt=""
  src="http://openwetware.org/images/4/45/Slika2.png"></td>
  src="http://openwetware.org/images/4/45/Slika2.png"></td>
     </tr>
     </tr>
     <tr style="font-family: Arial;">
     <tr style="font-family: Arial;">
       <td style="text-align: justify;"><span
       <td style="text-align: justify;"><span
  style="font-weight: bold;">Figure Y: Specific binding of ZFP
  style="font-weight: bold;">Figure 31: Specific binding of ZFP
chimeras to DNA origami in comparison to control </span> a) DNA
chimeras to DNA origami in comparison to control </span> a) DNA
origami sample prepared as described. b) DNA origami control without
origami sample prepared as described. b) DNA origami control without
the protein.<br><br></td>
protein.</td>
     </tr>
     </tr>
   </tbody>
   </tbody>
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<span style="font-family: Arial;">
<span style="font-family: Arial;">
Results demonstrate that we can bind protein domains to the specific
Results demonstrate that we can bind protein domains to the specific
position on DNA origami based on protein DNA interactions. Further
position on DNA origami based on ZFP-DNA interactions. Further
experiments are planned to demonstrate simultaneous binding of several
experiments are planned to demonstrate simultaneous binding of several
different ZFPs to different binding sites on the same DNA origami
different ZFPs to different binding sites on the same DNA origami
board.<br><br>
board.
</span>
</span>


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

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

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style="padding: 0pt 0pt 5px; 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 30. AFM image

of GST-AZPA4 bound to selected positions on DNA origami rectangles <span 

style="font-weight: normal;"> (AFM 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> The central goal of our project was to confirm binding of chimeric zinc finger proteins to attachment staple strands integrated into DNA origami rectangle. We used a DNA origami rectangle design inspired by the original DNA origami paper (Rothemund, 2006, see Methods / <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/methgeneraldesignstrategy">DNA

origami design</a>). After initial characterization of the correct folding of zinc finger chimeras and characterization of their ability to bind to the designed oligonucleotide hairpins determined by the AlphaScreen and EMSA assay, we proceeded with optimization of conditions for binding to DNA origami rectangle. To achieve successful binding, several factors had to be taken into account: <br> <br> </span>

<ul> 

 <li>the concentration of protein chimeras added to DNA origami

should be in excess, but at the same time low enough to avoid formation of large protein aggregates, which cannot be removed by filtration, since they might interfere with atomic force microscope measurements,</li> 

 <li>excess of attachment staples which are not integrated into

DNA origami rectangles had to be removed to minimize the number of potential binding sites for protein chimeras,</li> 

 <li>the time of incubation with DNA origami had to be optimized to allow reaching

equilibrium and prevent potential protein inactivation due to air oxidation of cysteine residues of ZFPs (this might be also avoided by incubation in an inert atmosphere).</li> </ul> <br style="font-family: Arial;"> <span style="font-family: Arial;"> Successful binding was demonstrated for GST-AZPA4 using the following protocol: DNA origami rectangles were prepared in 5 nM concentration with 5x staple strand excess in 1x TB/Mg<sup>2+</sup> buffer. Standard staple strands at positions Bb1, ES3, Be1 were replaced with attachment staples for AZPA4. 1x TB/Mg<sup>2+</sup> buffer was removed by filtration and replaced with 35 mM Hepes, pH = 7.0, 50 mM KCl, 12.5 mM MgCl<sub>2</sub>, 100 uM ZnCl<sub>2</sub>, 5 mM TCEP and 5 % (v/v) glycerol. DNA origami was incubated with 200x protein excess for 2 h, i.e. 70x excess with respect to the number of available attachment staples per origami, which is also consistent with the results from EMSA and AlphaScreen. Samples for AFM imaging were prepared fresh and imaged in a tapping mode in air. Control was the same DNA origami without added protein. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <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="font-family: Arial; width: 890px; height: 293px; padding: 0 0 10px 0;" alt=""
src="http://openwetware.org/images/4/45/Slika2.png"></td>
   </tr>
   <tr style="font-family: Arial;">
     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 31: Specific binding of ZFP

chimeras to DNA origami in comparison to control </span> a) DNA origami sample prepared as described. b) DNA origami control without protein.</td> 

   </tr>
 </tbody>

</table> <span style="font-family: Arial;"> Results demonstrate that we can bind protein domains to the specific position on DNA origami based on ZFP-DNA interactions. Further experiments are planned to demonstrate simultaneous binding of several different ZFPs to different binding sites on the same DNA origami board. </span>

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