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

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<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. ]]
<table cellspacing="0" cellpadding="2">
Since theU is a 3D object, there are many different options for positioning the fluorophores. <br>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">Since theU is a 3D object, there are many different options for positioning the fluorophores. <br>
First, they can have different positions in the X-Y-plane, each referring to a particular helix the fluorophores are attached to. We considered a total of 4 symmetric and 3 asymmetric solutions as seen in the figure to the right. The following positions are at the arms' interface: A1 (helix 8), B2 (helix 4), C2 (helix 5), D1 (helix 7) on one arm and A2 (helix 23), B1 (helix 29), C1 (helix 20), D2 (helix 22) on the other. The four symmetric solutions are: A1→A2 and B1→B2 with a distance of 12 nm as well as C1→C2 and D1→D2 with a distance of 5 nm. For our experiments we chose the symmetric solutions A1→A2, B1→B2 and C1→C2, because they complement each other and are more straightforward to analyze due to their symmetry. The expected twist of the arms as seen in the simulation of the naturally (-) twisted positive control is counterclockwise when seen from above. So the pairs B1→B2 and C1→C2 move towards each other with increasing twist until they eclipse, while A1→A2 move apart. For a substance which causes a (+) twist thus deforming the structure clockwise, the opposite pattern could be observed. <br>
First, they can have different positions in the X-Y-plane, each referring to a particular helix the fluorophores are attached to. We considered a total of 4 symmetric and 3 asymmetric solutions as seen in the figure to the right. The following positions are at the arms' interface: A1 (helix 8), B2 (helix 4), C2 (helix 5), D1 (helix 7) on one arm and A2 (helix 23), B1 (helix 29), C1 (helix 20), D2 (helix 22) on the other. The four symmetric solutions are: A1→A2 and B1→B2 with a distance of 12 nm as well as C1→C2 and D1→D2 with a distance of 5 nm. For our experiments we chose the symmetric solutions A1→A2, B1→B2 and C1→C2, because they complement each other and are more straightforward to analyze due to their symmetry. The expected twist of the arms as seen in the simulation of the naturally (-) twisted positive control is counterclockwise when seen from above. So the pairs B1→B2 and C1→C2 move towards each other with increasing twist until they eclipse, while A1→A2 move apart. For a substance which causes a (+) twist thus deforming the structure clockwise, the opposite pattern could be observed. </font color="#555555"></td><td>[[Image:Cross section the U.tif|right|x180px | thumb | Fig. 21: FRET-pair positions marked on the cross section. ]]</td></tr>
</table>
 
<br>
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.


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<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">
[[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.
 
 
</font color="#555555"></td><td>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.</td></tr>
</table>
 
 
[[Image:Fluorphore positions cadnano.png|left|x150px | thumb | Fig. 22: Flourophore positions shown in the caDNAno file.]]

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