Biomod/2011/TUM/TNT/Results

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Spermine causes a positive twist (46°) of double stranded DNA, and additionally decreases the length of DNA (base step rise reduced from 0.34nm to 0.29nm; [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras/References#DNA_binders Tari et.al.]). According to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras/References#DNA_binders Salerno et.al.], each bound molecule of ethidium bromide increases the length of a DNA double helix by 3.4nm, which is exactly the length of one base pair. Additionally, it induces a twist of -27°, in contrast to the +36° twist of one base pair. <br>
Spermine causes a positive twist (46°) of double stranded DNA, and additionally decreases the length of DNA (base step rise reduced from 0.34nm to 0.29nm; [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras/References#DNA_binders Tari et.al.]). According to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras/References#DNA_binders Salerno et.al.], each bound molecule of ethidium bromide increases the length of a DNA double helix by 3.4nm, which is exactly the length of one base pair. Additionally, it induces a twist of -27°, in contrast to the +36° twist of one base pair. <br>
Although both DNA binders induce length changes in opposite directions on DNA helices, both shorten the whole origami structure. The crosslinking between the helices in theU alters the type of deformation compared to an isolated double helix. One could assume that local changes in twist and length combine in an origami, causing a length change effect with all local deformations integrated. <br>
Although both DNA binders induce length changes in opposite directions on DNA helices, both shorten the whole origami structure. The crosslinking between the helices in theU alters the type of deformation compared to an isolated double helix. One could assume that local changes in twist and length combine in an origami, causing a length change effect with all local deformations integrated. <br>
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Regarding the measured twist angles, for small concentrations no effects can be seen with spermine. Without spermine, as well with ca. 5% and 14% occupied binding sides, the angle remains ca. 9°. For higher occupations (50% and 67%), the angle increases to 12°. Additional data points will be needed to fit these findings, but we suggest that a cooperative behavior would be an appropriate explanation. Within DNA origamis, not only a single helices needs to be twisted, but large bundles of helices with many crosslinks. This makes the single helices more rigid, consequently hindering an induced fit of spermine molecules. Only higher concentrations could excert enough force to overcome the local restraints and induce a global twist. <br>
+
Regarding the measured twist angles, for small concentrations no effects can be seen with spermine. Without spermine, as well with ca. 5% and 14% occupied binding sides, the angle remains ca. 9°. For higher occupations (50% and 67%), the angle increases to 12°. Additional data points will be needed to fit these findings, but we suggest that a cooperative behavior would be an appropriate explanation. Within DNA origamis, not only a single helix needs to be twisted, but large bundles of helices with many crosslinks. This makes the single helices more rigid, consequently hindering an induced fit of spermine molecules. Only higher concentrations could excert enough force to overcome the local restraints and induce a global twist. <br>
To put these considerations in a nutshell, new theoretical approaches are needed to correlate effects on a single helix with effects on a huge system of interconnected helices.  
To put these considerations in a nutshell, new theoretical approaches are needed to correlate effects on a single helix with effects on a huge system of interconnected helices.  
<h2>Twisted Positive Control is good Comparison for Deformation by Ethidium Bromide</h2>
<h2>Twisted Positive Control is good Comparison for Deformation by Ethidium Bromide</h2>
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One approach to gain further insights and a solid experimental fundament for this goal was the investigation of an intrinsically twisted structure as positive control. In average every 21bp an additional base was inserted, resulting in global deformations that were easily observable in the TEM. Effects on length cannot be examined in this way, since the positive control needed a longer scaffold than the normal theU structure, but it is a good examination object for the angles between the arms. Ethidium bromide lends itself for a comparison, since both every bound ethidium bromide and every additional base cause comparable elongation and they differ only in the twist they cause on a double stranded DNA. Thus this effect can be examined isolated. Regarding our angle distributions from the TEM data, the mean global twist for one additional base every 21bp is 21°, compared to 11° induced by one molecule ethidium bromide every 21bp. One could argue that our method is error-prone due to the angle measurement by hand, but the width of the distributions is in good agreement with the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Thermal_Fluctuation_of_the_Arms calculated thermal fluctuations], so these data can be regarded as reliable. It will be necessary to check further DNA binders, but the direction of twist should be of high importance for the angle deformation. Positive twists add to the existing pitch, while the negative twist by ethidium bromide needs to work against the intrinsic direction of helical rotation. One needs to consider also that the direction of the total twist of the structure cannot be determined from the 2D projections analyzed in this study.  
+
One approach to gain further insights and a solid experimental fundament for this goal was the investigation of an intrinsically twisted structure as positive control. In average every 21bp an additional base was inserted, resulting in global deformations that were easily observable in the TEM. Effects on length cannot be examined in this way, since the positive control needed a longer scaffold than the normal theU structure, but it is a good examination object for the angles between the arms. We compared the data with those from ethidium bromide, since every bound ethidium bromide as well as every additional base cause comparable elongation and they differ only in the twist they cause on a double stranded DNA. Thus this effect can be examined isolated. Regarding our angle distributions from the TEM data, the mean global twist for one additional base every 21bp is 21°, compared to 11° induced by one molecule ethidium bromide every 21bp. One could argue that our method is error-prone due to the angle measurement by hand, but the width of the distributions is in good agreement with the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Thermal_Fluctuation_of_the_Arms calculated thermal fluctuations], so these data can be regarded as reliable. It will be necessary to check further DNA binders, but the direction of twist should be of high importance for the angle deformation. Positive twists add to the existing pitch, while the negative twist by ethidium bromide needs to work against the intrinsic direction of helical rotation. One needs to consider also that the direction of the total twist of the structure cannot be determined from the 2D projections analyzed in this study. Therefore, FRET measurements would be an appropriate method.  
<h2>New Practical Methods and Theories will be needed</h2>
<h2>New Practical Methods and Theories will be needed</h2>
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Therefore, FRET measurements would be an appropriate method. 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. For this, 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. <br>
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.  
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.  

Revision as of 23:09, 2 November 2011

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