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

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<h1>Survey of results</h1>
<h1>Survey of Results</h1>
We successfully designed a structure that folds properly with high yields [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification (link)] and is suitable for observing the structural deformations. Comprehensive TEM analysis yielded insights into global structural deformations and allowed for statistical evaluation of angle and length distributions dependent on DNA binder concentrations [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis (link)]. Our structure could be labeled with fluorescent dyes and a huge variety of different approaches to fluorescence measurements was tested. In single molecule measurements FRET events could be observed. [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements (link)]. Based on these experimental data and also our structure simulations and calculations, we gained new insights into the structural properties of DNA origamis especially with regards to binding of small molecules [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Discussion (link)].  
We successfully designed a structure that folds properly with high yields [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification (link)] and is suitable for observing the structural deformations. Comprehensive TEM analysis yielded insights into global structural deformations and allowed for statistical evaluation of angle and length distributions dependent on DNA binder concentrations [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#TEM_Image_Analysis (link)]. Our structure could be labeled with fluorescent dyes and a huge variety of different approaches to fluorescence measurements was tested. In single molecule measurements FRET events could be observed. [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Fluorescence_Measurements (link)]. Based on these experimental data and also our structure simulations and calculations, we gained new insights into the structural properties of DNA origamis especially with regards to binding of small molecules [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Discussion (link)].  


<h1> Folding & purification </h1>
<h1> Folding & Purification </h1>


The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar results as the 15_65 ramp.  
The U structure was folded using the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Setting_up_folding_reactions 15_65] ramp. This ramp was the fastest of the tested ones and also led to proper folded origamis as shown in figure 1. There is only one major band visible in the agarose gel, indicating that no significant amounts of byproducts (like dimers) have been formed. The results of the slower ramps 2D_H3_ML and 5D_H3_ML yielded similar results as the 15_65 ramp.  
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<h1>TEM image analysis</h1>
<h1>TEM Image Analysis</h1>
<h2>Distribution of Angles</h2>
<h2>Distribution of Angles</h2>
When we inspected the structure in the TEM, we saw a spread of the arms in the uprightly orientated structures (figure 3). The magnitude of this spread seemed to be correlated to the amount of DNA binding molecules.  
When we inspected the structure in the TEM, we saw a spread of the arms in the uprightly orientated structures (figure 3). The magnitude of this spread seemed to be correlated to the amount of DNA binding molecules.  
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For small spermine concentrations, no significant peak shift can be observed. Only at higher concentration, the maximum of the distribution is shifted noticeably towards higher angles. For the series negative control - ethidium bromide every 21 bp - ethidium bromide every 7 bp, the data display no systematic shift, while for DAPI, the mean angle decreases continuously. <br>
For small spermine concentrations, no significant peak shift can be observed. Only at higher concentration, the maximum of the distribution is shifted noticeably towards higher angles. For the series negative control - ethidium bromide every 21 bp - ethidium bromide every 7 bp, the data display no systematic shift, while for DAPI, the mean angle decreases continuously. <br>


<h2>Length measurements</h2>
<h2>Length Measurements</h2>
We also measured the lengths of the origami structures on the TEM images. Histograms of the length distributions display a gaussian shape (figure 5). For increasing concentrations of spermine, the length decreases steadily (figure 6a, for raw data see this file: [[Image:TEM length measurements raw data.xlsx]]). Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is known to increase the length of a simple double stranded DNA (figure 6b). It seems, that origami structures react here in another manner then single helices. <br>
We also measured the lengths of the origami structures on the TEM images. Histograms of the length distributions display a gaussian shape (figure 5). For increasing concentrations of spermine, the length decreases steadily (figure 6a, for raw data see this file: [[Image:TEM length measurements raw data.xlsx]]). Surprisingly, for rising concentrations of ethidium bromide, the length decreases as well, although addition of ethidium bromide is known to increase the length of a simple double stranded DNA (figure 6b). It seems, that origami structures react here in another manner then single helices. <br>


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<h1>Fluorescence measurements</h1>
<h1>Fluorescence Measurements</h1>


<h2>FRET bulk measurements</h2>
<h2>FRET Bulk Measurements</h2>
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Helix_MH_255/256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Helix_MH_255/256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful.  
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful.  
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To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.
To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.


<h2>Single molecule measurements at the fluorescence microscope</h2>
<h2>Single Molecule Measurements at the Fluorescence Microscope</h2>


<h3>FRET measurement</h3>
<h3>FRET Measurement</h3>


We designed the structure in such a way that a small change of angle in the base, which is a 30 helix bundle in a honey comb lattice, is amplified by the two arms, which are both 10 helix bundles and therefore should twist as well. To measure the change in twist and angle two fluorophores were attached to the two arms so that a deformation should cause a change in distance between them. We chose a donor and an acceptor fluorophore, namely Atto 550 and Atto 647N, so a change in distance between them leads to a change in  FRET-efficiency.  
We designed the structure in such a way that a small change of angle in the base, which is a 30 helix bundle in a honey comb lattice, is amplified by the two arms, which are both 10 helix bundles and therefore should twist as well. To measure the change in twist and angle two fluorophores were attached to the two arms so that a deformation should cause a change in distance between them. We chose a donor and an acceptor fluorophore, namely Atto 550 and Atto 647N, so a change in distance between them leads to a change in  FRET-efficiency.  
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<h3>Fluorescence tracking</h3>
<h3>Fluorescence Tracking</h3>


Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength.
Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength.
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<h1>Discussion</h1>
<h1>Discussion</h1>
<h2>Origamis respond in another way than single DNA helices on local deformations</h2>
<h2>Origamis Respond in Another Way than Single DNA Helices on Local Deformations</h2>


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>
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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>


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. 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.  


<h2>New practical methods and theories will be needed</h2>
<h2>New Practical Methods and Theories will be needed</h2>


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>
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>

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