Biomod/2011/Slovenia/BioNanoWizards/discussion

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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;">Discussion</span></big></big></big></big><br>

<br><br> <span style="font-family: Arial;">Results of this project represent proof of the concept for the use of protein domains as versatile DNA origami "add-ons" for the position-specific binding to DNA origami and their enhancement with different functions of protein domains. The main advantage of ZFPs is that there are hundreds of already characterized ZFPs available (<a href="http://bindr.gdcb.iastate.edu:8080/ZiFDB/">ZiFDB database</a>) and potentially hundreds of thousands of variants which can be used to simultaneously address many different positions on DNA origami.</span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;">We have experimentally addressed <strong>two problems</strong> that became apparent when we tried to implement this idea, namely <strong>ZFP solubility</strong> and <strong>specific tight binding</strong>. <br><br> First, in our hands but also in other reports, several ZFPs are difficult to handle <em>in vitro</em> since they are prone to aggregation and are difficult to isolate in the soluble form. We solved this problem by the addition of one of the two different solubility enhancement domains (MBP and GST), which provide an additional tag for the purification of ZFP-chimeric recombinant proteins and can serve as a spacer for the precise separation of vertical DNA origami stacks. </span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">The second problem, binding of ZFPs to their target DNA at low micromolar affinities may present a problem, since the bound ZFP may slowly dissociate from the DNA, in contrast to very tight interactions used previously, e.g. biotin and streptavidin with sub-picomolar affinity. We solved this problem by extension of zinc finger domains from 3 to 6 zinc fingers, which therefore recognize 18 bp DNA target sequence. This increases the affinity of ZFPs into the picomolar range(Papworth, 2003; Sera, 2002), making it comparable to the quasi-irreversible affinity of the biotin-streptavidin pair. Additionally, once established, the connection between ZFP and target DNA could be made covalent by introduction of appropriate reactive functional groups, e.g. thiol groups, while retaining the ability to simultaneously address different positions on DNA origami.</span> <br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">Proteins are able to perform an impressive set of different functions in living organisms and have important technological potential. They can recognize specific structures, catalyze different chemical reactions, absorb and emit light, perform redox reactions, exhibit fluorescence, nucleate growth of magnets or other inorganic materials etc. Recombinant DNA techniques allow us to prepare protein chimeras that <strong>combine specific DNA-binding sequence targeting them to selected positions on DNA nanostructures with desired protein functions</strong>. We have envisioned several potential applications of DNA origami-protein hybrids that could harvest the combined power of DNA origami and protein functions, such as <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/applabonchip">lab-on-a-nanochip</a>,

<a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosynthteticcompartments">biosynthetic

compartments</a>, <a 

href="http://openwetware.org/wiki/Biomod/2011/Slovenia/BioNanoWizards/appbiosensors">biomolecular

sensors</a> and many others.</span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">The second extension of DNA origami towards applications was the idea of <strong>vertical stacking of DNA origami layers</strong>. We have experimentally demonstrated formation of two perfectly aligned DNA rectangles that were vertically tethered by DNA. Protein tethers have the advantage that they are more rigid than DNA duplex, separating the derivatized DNA layers by a defined distance. While we have produced twin ZFP protein tethers and designed heterodimerizing protein tethers we didn't have time to test them experimentally. We think that the successful perfect alignment of DNA-tethered stacks was ensured by the use of 10 different DNA tethers, which may be achievable but technically demanding to achieve with proteins. On the other hand we could probably use shorter ZFPs for the formation of protein-tethered DNA origami stacks since the cooperativity of several ZFP-DNA interactions between neighboring DNA layers will shift the dissociation constant to subpicomolar value and therefore support the binding specificity.</span> <br style="font-family: Arial;"> <br> <span style="font-family: Arial;">Formation of multilayered DNA arrays from several molecules has been reported recently, however the positioning of those layers relied on interactions with magnesium ions. Such multilayers do not allow any spacers and no provisions were taken for the precise positioning of the overlay of discrete self-assembled DNA objects (Koyfman, 2009).</span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial;">Self-assembly of vertical stacks demonstrated that reannealing at high temperatures may disrupt the already formed DNA origami, which we solved by the excess of staple strands. Further potential refinement of our fabrication procedures may involve stabilization of DNA origami layers with psoralen or other chemical crosslinking reagents, which covalently photo-crosslinks nucleotides of separate chains (Rajendran, 2011).</span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;">The applications of vertical stacking approach that we discussed in our brainstorming sessions are in nanoelectronics, where the closely separated conductive plates may be used as nanocapacitors, while stacking of two DNA origamis modified with different metals or metallic oxides could be used as nanobatteries. Extension of vertical stacks from two plates to the selected number and order is straightforward, and we also thought of additional applications such as manufacturing of ID tags or others.</span><br style="font-family: Arial;"> <br style="font-family: Arial;"> <span style="font-family: Arial; font-weight: bold">In conclusion, we have demonstrated the proof of principle of the functionalization of DNA origami by protein domains that opens new avenues for real world applications.</span><br 

style="font-family: Arial;">

<br><br> <hr style="width: 100%; height: 2px;"><span 

style="font-family: Arial;">

<ul style="font-size: small;"> <li>Koyfman AY, Magonov SN, Reich NO (2009) Self-assembly of DNA arrays into multilayer stacks. <span style="font-style: italic;">Langmuir</span> 25: 1091-6.</li> <li>Papworth M, Moore M, Isalan M, Minczuk M, Choo Y, Klug A (2003) Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors. <em>Proc. Natl. Acad. Sci.</em> 100:1621-6.</li> <li>Rajendran A, Endo M, Katsuda Y, Hidaka K, Sugiyama H (2011) Photo-cross-linking-assisted thermal stability of DNA origami structures and its application for higher-temperature self-assembly. <span 

style="font-style: italic;">J Am Chem Soc</span> 133:

14488-14491.</li> <li>Sera T, Uranga C (2002)Rational design of artificial zinc-finger proteins using a nondegenerate recognition code table. <em>Biochemistry</em> 41:7074-81.</li>

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