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

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<h2>Twist of the Arms</h2>
<h2>Twist of the Arms</h2>


<h3>Theoretical considerations</h3>
<h3>Theoretical Considerations</h3>


[[Image:TUM_arm_twist_mod.png | left | 300px |thumb| Fig. 9 Cylindric model for the twist of the arms. ]]
[[Image:TUM_arm_twist_mod.png | left | 300px |thumb| Fig. 9 Cylindric model for the twist of the arms. ]]
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<h3>Derivation</h3>
<h3>Derivation</h3>


<h4>Fluctuation of one Arm</h4>
<h4>Fluctuation of One Arm</h4>


The probability of finding a rod with a 10 helix bundle cross section at a certain angle <math>\phi</math>  
The probability of finding a rod with a 10 helix bundle cross section at a certain angle <math>\phi</math>  
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As FRET labels we use the fluorophores [https://www.atto-tec.com/attotecshop/product_info.php?info=p103_ATTO-550.html&XTCsid=ucvoaehx Atto 550] and [https://www.atto-tec.com/attotecshop/product_info.php?info=p114_ATTO-647N.html&XTCsid=ucvoaehx Atto 647N]. The Förster distance for this pair is 6.5 nm according to AttoTec. Both dyes are commercially available linked to ddNTPs, so they can be attached to oligonucleotides using terminal transferase. The fluorophores not only exhibit a high stability against photobleaching, but also have excitation and fluorescence spectra that fit to the setup of the self-made fluorescence microscope in our lab. Thus we have not only the possibility to measure FRET at the photospectrometer and the more sensitive real time PCR, but can also perform single molecule experiments at our TIRF microscope. <br>
As FRET labels we use the fluorophores [https://www.atto-tec.com/attotecshop/product_info.php?info=p103_ATTO-550.html&XTCsid=ucvoaehx Atto 550] and [https://www.atto-tec.com/attotecshop/product_info.php?info=p114_ATTO-647N.html&XTCsid=ucvoaehx Atto 647N]. The Förster distance for this pair is 6.5 nm according to AttoTec. Both dyes are commercially available linked to ddNTPs, so they can be attached to oligonucleotides using terminal transferase. The fluorophores not only exhibit a high stability against photobleaching, but also have excitation and fluorescence spectra that fit to the setup of the self-made fluorescence microscope in our lab. Thus we have not only the possibility to measure FRET at the photospectrometer and the more sensitive real time PCR, but can also perform single molecule experiments at our TIRF microscope. <br>


<h2>FRET-pair Positions</h2>
<h2>FRET-Pair Positions</h2>
[[Image:Cross section the U.tif|right|x180px | thumb | Fig. 21: FRET-pair positions marked on the cross section. ]]
[[Image:Cross section the U.tif|right|x180px | thumb | Fig. 21: FRET-pair positions marked on the cross section. ]]
Since theU is a 3D object, there are many different options for positioning the fluorophores. <br>
Since theU is a 3D object, there are many different options for positioning the fluorophores. <br>
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Second, different positions along the Z-axis are possible. The relevance of different Z-positions lies in the fact that the fixed basis of theU causes the arms' twist to increase with increasing distance from the base. This way we can adjust the mean displacement due to small molecule binding so it always lies within the linear FRET range. In general, the honeycomb lattice used for theU's construction allows for FRET pairs positioned every 21 basepairs, which is visualized in figure 22 below. Symmetric solutions align without X-axis shift, whereas asymmetric solutions would expose a 7 basepair shift.
Second, different positions along the Z-axis are possible. The relevance of different Z-positions lies in the fact that the fixed basis of theU causes the arms' twist to increase with increasing distance from the base. This way we can adjust the mean displacement due to small molecule binding so it always lies within the linear FRET range. In general, the honeycomb lattice used for theU's construction allows for FRET pairs positioned every 21 basepairs, which is visualized in figure 22 below. Symmetric solutions align without X-axis shift, whereas asymmetric solutions would expose a 7 basepair shift.


[[Image:Fluorphore positions cadnano.png|left|x150px | thumb | Fig. 22 Flourophore positions shown in the caDNAno file.]]
[[Image:Fluorphore positions cadnano.png|left|x150px | thumb | Fig. 22: Flourophore positions shown in the caDNAno file.]]


At last, some strategies for attaching the fluorophores to the structure deserved some consideration. Shortening the respective staple to accomodate the labeled nucleotide has the advantage of a well-defined length of the staple. But it is also the less flexible solution because changing the fluorophore's position is not straightforward. On the other hand, extending the existing staple with the labelled nucleotide has the opposite merit profile. Flexibility was more important to us, so we chose to extend the existing staples for labeling.
At last, some strategies for attaching the fluorophores to the structure deserved some consideration. Shortening the respective staple to accomodate the labeled nucleotide has the advantage of a well-defined length of the staple. But it is also the less flexible solution because changing the fluorophore's position is not straightforward. On the other hand, extending the existing staple with the labelled nucleotide has the opposite merit profile. Flexibility was more important to us, so we chose to extend the existing staples for labeling.
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