Biomod/2011/Slovenia/BioNanoWizards/protdnahybrid

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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

style="margin: 3px 0px 30px 30px; width: 550px; height: 50px; float: right;"
border="0" cellpadding="0" cellspacing="0">
 <tbody>
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     <td style="text-align: center;"><img
style="padding: 0pt 0pt 5px; width: 570px; height: 281px;"
alt="" src="http://openwetware.org/images/5/5c/Slika1prot.png"></td>
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     <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

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border="0" cellpadding="0" cellspacing="0">
 <tbody>
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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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