Biomod/2011/Slovenia/BioNanoWizards/applabonchip

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style="color: black; font-weight: bold;">Lab-on-a-nanochip</span></big></big></big></big><br>

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     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 37: Representation of an idea of using protein-DNA origami hybrid for the excision of a DNA segment on a single DNA origami nanochip.</span></td>
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     <td style="text-align: justify;"><span
style="font-weight: bold;">Figure 38: Representation of an idea of using protein-DNA origami hybrids for the site specific insertion of DNA fragments.</span></td>
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 </tbody>

</table> <span style="font-family: Arial;"> Current trends in molecular biology encourage integration of several different biochemical reactions as well as their parallelization into single chip setups, which have dimensions of only millimeters or a few square centimeters. Those devices have several advantages:</span> <ul>

 <li>short distances account for shorter reaction times,</li>
 <li>fast heating due to high surface to volume ratio allows for better and faster reaction control, </li>
 <li>systems are adequate for massive parallelization due to compactness,</li>
 <li>low fluid volumes are needed, thus minimizing reagent consumption and waste production, which allows for low costs of analysis and production.</li>

</ul><br> <span style="font-family: Arial;"> <strong>We can envision that the proposed protein DNA origami add-ons approach can scale down the size of a lab-on-a-chip into nanometric dimensions.</strong><br><br> Molecular cloning is still by and large a very labor intensive and lengthy procedure, which can be a nightmare of students in molecular biology labs. Molecular cloning protocols, for example, might be simplified using ZFP chimeras fused with nuclease, ligase, phosphatase, recombinase and other polynucleotide modifying domains, to name just a few.<br> The main advantage of zinc finger proteins is an almost unlimited variability of sequences available for targeting to different positions.<br><br> The simplest implementation of this idea is to use a combination of ZFP and a nonspecific nuclease FokI immobilized at the surface of DNA origami at defined positions. This, in fact, creates a new meganuclease that cleaves the DNA at a position that is separated by a defined distance from the recognition site, which is defined by an immobilized zinc finger or any other DNA binding protein.<br><br> An additional illustration of the potential application of DNA-modifying DNA orgami-based lab is a specific one-step excision system, as depicted in <strong>Figure 38</strong>. The idea is universal in terms, that we could target almost any sequence in the target DNA, and specific, because binding sites used for the excision can be as long as necessary to achieve the specific targeting of a selected segment.<br><br> In the first step, we assemble the restriction-ligation device on top of the DNA origami. Specific DNA-binding zinc fingers, nonspecific nuclease (e.g. FokI nuclease) and DNA ligase are positioned at the defined positions on the surface of DNA origami using ZFP domains. The target DNA binds to the two binding sites (ZFPs), depicted as hands. FokI nuclease cleaves the DNA at the position that depends on the distance between the FokI nuclease and each of the two ZFPs. On the other side the DNA ligase positioned at the same distance or closer than FokI nuclease ligates the cleaved segment. Release of the modified DNA can be accomplished by the addition of EDTA that extracts zinc from the ZFP and weakens the interaction. <br><br></span>

<span style="font-family: Arial;">A different setup could allow for the site-specific insertion of the selected DNA fragments into the target DNA. In this case two pairs of ZFPs are used to arrange the two DNA segments for a restriction and ligation step.</span><br>

<span style="font-family: Arial;"> We note that similar principles could be used for many other reactions involving multiple reactions, such as for example proteolytic degradation into defined size of polypeptides using positioning on DNA origami as a ruler.</span>

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