Biomod/2011/TUM/TNT/Results: Difference between revisions

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Although we cannot present final results for FRET analyses, first single molecule analyses can be provided. For an optimization of the FRET studies, the origami structure needs some slight improvements, like a more rigid base or fluorophores attached nearer to the base. For this optimization, we have laid a thorough fundament not only of experimental results, but also lots of theoretical considerations, which can explain flexibility and correlate observable (via TEM and / or fluorescence measurements: distances, angles) with unobservable (twists) structural changes. <br>
Although we cannot present final results for FRET analyses, first single molecule analyses can be provided. For an optimization of the FRET studies, the origami structure needs some slight improvements, like a more rigid base or fluorophores attached nearer to the base. For this optimization, we have laid a thorough fundament not only of experimental results, but also lots of theoretical considerations, which can explain flexibility and correlate observable (via TEM and / or fluorescence measurements: distances, angles) with unobservable (twists) structural changes. <br>
On the experimental side, one could  try to eliminate some uncertainties regarding the applied concentrations. We did some [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Calculation_of_intercalator_concentrations calculations] to determine the fraction of occupied binding sites even at small concentrations, but as mentioned above, binding could be cooperative and for a proper testing of such a behavior, concentrations of bound DNA binders must be checked experimentally. This is very trying due to the small concentrations and the little fraction of compounds bound compared to those free in solution. We suggest to try some radiolabeled DNA binders, of which the bound fraction can be determined from radioassays. <br>
On the experimental side, one could  try to eliminate some uncertainties regarding the applied concentrations. We did some [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Calculation_of_intercalator_concentrations calculations] to determine the fraction of occupied binding sites even at small concentrations, but as mentioned above, binding could be cooperative and for a proper testing of such a behavior, concentrations of bound DNA binders must be checked experimentally. This is very trying due to the small concentrations and the little fraction of compounds bound compared to those free in solution. We suggest to try some radiolabeled DNA binders, of which the bound fraction can be determined from radioassays.
Using this information, it should be possible to unravel deformation events step by step on even tinier levels. This not only allows for sophisticated understanding of the flexibility of origamis in response to varying triggers, thereby enabling the development of custom-made dynamic structures, but also helps elucidating the mechanic and maybe also mechanistic effects of DNA binders.
 
<h1>Outlook</h1>
<h1>Outlook</h1>
For this last goal, we have the vision of a characteristic plot for DNA binders based on the twist and length changes they cause. In the field of bioscience the Ramachandran plot is a nice way to show typical secondary structures of proteins. In this case we would plot twist against length (fig zzz). <br>
By exploiting the potential of our device to gather new knowledge about DNA-small molecule interactions, it should be possible to unravel structural deformations on even tinier levels. This information does not only allow a more sophisticated understanding of the flexibility of origamis in response to varying triggers, but also helps elucidating the mechanic and maybe also mechanistic effects of DNA binders on DNA. <br>
For this we envisioned a characteristic plot for DNA binders based on the twist and length changes they cause, as depicted in the sketch below (figure 11). Following the example of the famous Ramachandran plot, which enables bioinformaticians to predict secondary structure motifs of proteins with high accuracy, a plot of twist vs. length changes could designate certain regions of increased occurrence. In these regions, the effects of binding molecules within a common binding class would gather. With a modified structure where fluorophore positions have been optimized according to a refined theory of deformation and with an appropriate knowledge base of structural changes due to well-characterized binders, an easy and probably high-throughput procedure for the screening of potential DNA binding molecules could be in closer reach. The folding of already well-designed DNA origamis is, in contrast to the design itself, rather straightforward and requires only basic equipment. By customizing design, folding and purification processes, a wider application would be possible and, as such, attractive e.g. for basic research or pharmaceutical drug development.<br>
Another intriguing feature of our findings is that, with the proper refinements to the underlying model, it should be possible to create a device whose conformational changes can be precisely predetermined. As a result, this would permit to use the principle the other way around. Knowing the outcome of conformational changes of DNA origami using a certain concentration of a well known DNA-binder will provide a valuable tool, advancing the development of custom-made dynamic structures from DNA origami. By altering the concentration of an appropriate binder, movements of susceptible origami parts could be triggered, with the option to reverse to the original state through withdrawal of the binder. <br>


[[Image:Plotslim.png | 600px|center]]<br>
[[Image:Plotslim.png | 600px|center]]<br>

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