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

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;">We have addressed <span 

style="font-weight: bold;">two

problems</span> that became apparent when we tried to implement this idea, namely <span style="font-weight: bold;">ZFP solubility</span> and <span style="font-weight: bold;">specific tight binding</span>. First, in our hands but also in other reports, several ZFPs are difficult to handle <span style="font-style: italic;">in vitro</span> since they are prone to aggregation and are difficult to isolate in the soluble form. We solved this problem by the addition of two solubility enhancement domains (MBP and GST), which provide an additional tag for the purification of those recombinant proteins and can serve as a spacer for 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 six 6 zinc fingers, which therefore recognize 18 bp DNA target sequence. This increases affinity of ZFPs into the picomolar range 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. tiol 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 <span 

style="font-weight: bold;">combine addressing specific

DNA sequence and selected functions targeting them to selected positions on DNA nanostructures</span>. 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 <span 

style="font-weight: bold;">vertical stacking of

DNA origami layers</span>. 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 bifunctional ZFP protein tethers and designed heterodimerizing protein tethers we didn't have time to test them experimentally. We think that 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 formation of protein-tethered DNA origami stacks since the cooperativity of several ZFP-DNA interactions between neighboring DNA layers shifts the dissociation constant towards an extremely low value.</span><br 

style="font-family: Arial;">

<span style="font-family: Arial;"></span><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 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 presence of the excess of staple strands. Further potential refinement of our fabrication procedures may involve stabilization of DNA origami layers with psoralen, which covalently photo-crosslinks nucleotides of separated chains (Rajendran, 2011).</span><br 

style="font-family: Arial;">

<br style="font-family: Arial;"> <span style="font-family: Arial;">The most direct 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, which might find 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;">In conclusion we have demonstrated the proof of principle of the functionalization of DNA origami that open new avenues for real world applications.</span><br 

style="font-family: Arial;">

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

style="font-family: Arial;"><br>

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.<br> 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:

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