Biomod/2011/Slovenia/BioNanoWizards/resultssummary

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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;">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> </span>

<br> <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 enhanced the solubility 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 fusing them with maltose-binding protein (MBP) or glutathion-S-transferase (GST) domains, which can also facilitate purification of protein fusions. </span> <table style="text-align: left; width: 100%;" border="0" 

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     <td valign="undefined"><span
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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). 

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     <td style="text-align: center;"><img
style="padding: 0pt 0pt 5px; width: 540px; height: 388px;"
alt="" src="http://openwetware.org/images/6/6b/MBPZFP.png"></td>
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   <tr style="font-family: Arial; font-weight: bold;">
     <td style="text-align: justify;">Figure 2:

Threedimensional representation of chimeric ZFP. <span 

style="font-weight: normal;">MBP is shown in green and ZFP

(Zif268) is shown in cyan bound to the target DNA.</span></td> 

   </tr>
 </tbody>

</table> <br> <br> <span style="font-family: Arial;"> <span style="font-weight: bold;">We tested DNA binding of zinc finger domains with increased specificity and affinity</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> <table 

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     <td style="text-align: center;"><img
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alt="" src="http://openwetware.org/images/a/af/AlphaScreen_vsi.png"></td>

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     <td style="text-align: justify;">Figure 3: AlphaScreen binding assay of GST-2C7, GST-AZPA4, GST-Zif268 and MBP-6F6 to their corresponding DNA sequences.<span
style="font-weight: normal;"> Higher output signal indicates that six-finger ZFPs GST-AZPA4 and GST-2C7 bind to their corresponding DNA sequences with higher affinity than three-finger ZFP GST-Zif268 (equal experimental conditions were used). Non-target biotinylated oligonucleotides were used as negative controls.</span></td>
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 </tbody>

</table> <span style="font-family: Arial; font-weight: bold;"> We demonstarted positional attachment of ZFPs to specific sites on DNA origami.</span> <br><br> <table 

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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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   <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> 

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 </tbody>

</table> <br><br> <big><big><span style="color: black; font-weight: bold;">2. Vertical stacks of DNA origami</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 breadboards into vertical stacks may produce some directly useful devices. Vertical stacking could be accomplished using either DNA or proteins as tethers. The vertical order of DNA origami plates and their number in stack could be designed at will using different tethers on each side of the DNA breadboard.</span> <br><br> <span style="font-family: Arial;"> <span style="font-weight: bold;">We prepared perfectly aligned DNA stack using DNA tethers.</span></span> <br><br> <table 

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     <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>
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</table> <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, they are not modified with the same modifiers at the DNA surface and remain functional. <br><br> <span style="font-weight: bold;">We designed two different types of protein tethers:</span><br><br> a. twin ZFP chimeras, having 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).<br><br> b. 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><br>

<span style="font-weight: bold;">We designed twin ZFP protein tethers,</span> produced them in recombinant form and purified them.<br><br>

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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>
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