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

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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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[[Image:DAPI 7 histo gaussian.png|390px]]
[[Image:DAPI 7 histo gaussian.png|390px]]


The distribution of angles in the control has two populations, one where the two arms are exactly above each other which leads to very small angles and one where the two arms are considerably spread. This leads to a gaussian distribution around an finite angle. The width of this distribution is in good agreement with the calculated [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project/Theory#Fluctuation_of_the_measured_angles thermal fluctuations], which yield deviation of ca. 4.4°. <br>
The distribution of angles in the control has two populations, one where the two arms are exactly above each other which leads to very small angles and one where the two arms are considerably spread. This leads to a gaussian distribution around an finite angle. The width of this distribution is in good agreement with the calculated [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project/Theory#Fluctuation_of_the_measured_angles thermal fluctuations], which yield deviations of ca. 4.4°. <br>
The positive control with an internally induced twist by additional base pairs in each helix (these additional base pairs lead to a net torque in each helix and therefore a macroscopic deformation of the structure) displays much higher angles. The population around zero is maybe due to deformed structures which had no second arm and could not be excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. Here the angle of the positive control is shifted to higher values by approximately a factor of 2 because of the induced twist. So in principle this way of measuring the deformation of our structure in dependence of induced stress works. <br>
The positive control with an internally induced twist by additional base pairs in each helix (these additional base pairs lead to a net torque in each helix and therefore a macroscopic deformation of the structure) displays much higher angles. The population around zero is maybe due to deformed structures which had no second arm and could not be excluded. This results in many angles around zero. The other population around the finite angle is now the more spread structure. Here the angle of the positive control is shifted to higher values by approximately a factor of 2 because of the induced twist. So in principle this way of measuring the deformation of our structure in dependence of induced stress works. <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.
The measured angles φ for negative and positive control, <math>\phi_{neg} \approx 9</math>° and <math>\phi_{pos} \approx 21</math>°, can be related to a torsion α of the base according to the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project/Theory#Theoretical_considerations_2 theoretical considerations for the base twist]:
 
<h2>Including Base-Twist Theory</h2>
 
The measured angles φ for negative and positive control, <math>\phi_{neg} \approx 9</math>° and <math>\phi_{pos} \approx 21</math>°, can be related to a torsion α of the base according to the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project/Theory#Theoretical_considerations_2 Theoretical considerations of the base twist]:


<math>
<math>
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</math>
</math>


The theory determines the torsion for these particular φ-values to <math>\alpha_{neg} \approx 33</math>° and <math>\alpha_{pos} \approx 93</math>°. This corresponds to a torsion of 5° per base-pair in the base.
The theory determines the torsion for these particular φ-values to <math>\alpha_{neg} \approx 33</math>° and <math>\alpha_{pos} \approx 93</math>°. This corresponds to a torsion of 5° per basepair in the base of theU. <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>

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