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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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 <tbody>
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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 X. 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> 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, <span 

style="font-style: italic;">Nature</span> 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 Alpha-screen and EMSA assay, we proceeded with optimizing 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 for obtaining equilibrium of binding and prevent potential protein inactivation due to air oxidation (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/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;"> <br style="font-family: Arial;"> <table 

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     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure Y: 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 the 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 protein 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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