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{{TUMNanUHeaderProject}}
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<font size="6"> Project </font size="6">
<font size="6">Project</font size="6">
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<h1>The Idea</h1>
<h1>The Idea</h1>
<h2>Relevance of DNA - Small Molecule Interactions</h2>


<h2>Relevance of DNA - Small Molecule Interactions</h2>
<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">Containing the blueprint for every function and structure of life, DNA plays an essential role in most fields of the biosciences. Because of this reason, detailed knowledge of compounds that interact with DNA is of great importance. For instance, small DNA binding molecules including intercalators and minor/ major groove binders can cause changes in the geometry of double-helical DNA domains. Such changes can affect the dynamics of transcriptional regulation and the activity of nucleases. To name an example, the minor binder Netropsin is a potent inhibitor of bacterial DNA gyrases with a broad range of activity, including the notorious nosocomial pathogen Pseudomonas aeruginosa.<br>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">Containing the blueprint for every function and structure of life, DNA plays an essential role in most fields of the biosciences. Because of this reason, detailed knowledge of compounds that interact with DNA is of great importance. For instance, small DNA binding molecules including intercalators and minor / major groove binders can cause changes in the geometry of double-helical DNA domains. Such changes can affect the dynamics of transcriptional regulation and the activity of nucleases. To name an example, the minor groove binder Netropsin is a potent inhibitor of bacterial DNA gyrases with a broad range of activity, including the notorious nosocomial pathogen Pseudomonas aeruginosa.<br>
Hence, small DNA binding molecules can represent candidate compounds with antibiotic or tumor-repressing activity.  Elucidating the microscopic structural changes caused by small molecules can thus help to select candidate molecules as well as shed light on functional mechanisms.</td><td>[[Image:DNA Netropsin.png|thumb|200x200px|right|Fig. 1: Crystal structure of the minor groove binder Netropsin bound to DNA.]]</td></tr>
Hence, small DNA binding molecules can represent candidate compounds with antibiotic or tumor-repressing activity.  Elucidating the microscopic structural changes caused by small molecules can thus help to select candidate molecules as well as shed light on functional mechanisms.</font color="#555555"></td><td>[[Image:DNA Netropsin.png|thumb|200x200px|right|Fig. 1: Crystal structure of the minor groove binder Netropsin bound to DNA.]]</td></tr>
</table>
</table>


<h2>Why DNA Origami?</h2>
<h2>Why DNA Origami?</h2>
The working principle of DNA origami is based on the highly specific interaction behavior of DNA. Because of this specificity, the binding behavior of two strands can be encoded in the base sequence of said strands. By including sequences complementary to two strands on another, a trimolecular system can be created where one strand binds two different ones together. By including more and more complementary sequences on a single strand, increasingly complex configurations can be created. DNA Origami uses this modular behavior to encode the shape of the planned structure in the sequences of the used DNA strands. Designing a new structure is generally done according to the following steps. During the computer-aided design of the structure, a long single stranded DNA called the 'scaffold' is first laid out such that it fills out the shape of the planned structure. Next, short DNA strands called 'staples' are chosen such that they are complementary to partial sequences of the scaffold. By binding to those complementary sequences, the staples force the scaffold into a set conformation, removing degrees of freedom. With each bound staple, more and more helices with fixed relative positions are created. Once all bases of the scaffold are hybridized, the scaffold is forced into one specific shape.<br>
The working principle of DNA origami is based on the highly specific interaction behavior of DNA. Because of this specificity, the binding behavior of two strands can be encoded in the base sequence of said strands. By including sequences complementary to two strands on another, a trimolecular system can be created where one strand binds two different ones together. By including more and more complementary sequences on a single strand, increasingly complex configurations can be created. DNA Origami uses this modular behavior to encode the shape of the planned structure in the sequences of the used DNA strands. Designing a new structure is generally done according to the following steps. During the computer-aided design of the structure, a long single stranded DNA called the 'scaffold' is first laid out such that it fills out the shape of the planned structure. Next, short DNA strands called 'staples' are chosen such that they are complementary to partial sequences of the scaffold. By binding to those complementary sequences, the staples force the scaffold into a set conformation, removing degrees of freedom. With each bound staple, more and more helices with fixed relative positions are created. Once all bases of the scaffold are hybridized, the scaffold is forced into one specific shape.<br>
If the design of an origami contains certain targeted insertions and deletions of base pairs, it is possible to create a built-in twist or curvature [[http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras#References Dietz et. al., 2009]]. This work was inspiring because it implies the general idea of an origami's overall shape to be liable to distortions of it's microstructure. According to this concept, the above mentioned constraints within the DNA Origami allow the effects of multiple DNA binders to be transported along the structure so that they sum up, amplifying otherwise immeasurable influences. Furthermore, the unparalleled positional control of DNA Origami allows us to place probe molecules on the structure with high accuracy, permitting the observation of minute changes.
If the design of an origami contains certain targeted insertions and deletions of base pairs, it is possible to create a built-in twist or curvature [[http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras#References Dietz et. al., 2009]]. This work was inspiring because it implies the general idea of an origami's overall shape to be liable to distortions of its microstructure. According to this concept, the above mentioned constraints within the DNA Origami allow the effects of multiple DNA binders to be transported along the structure so that they sum up, amplifying otherwise immeasurable influences. Furthermore, the unparalleled positional control of DNA Origami allows us to place probe molecules on the structure with high accuracy, permitting the observation of minute changes.


<h1>Project Specification</h1>
<h1>Project Specification</h1>
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*Provide means for quantifying how the target molecule changes helical pitch and length of double-helical DNA domains.<br>
*Provide means for quantifying how the target molecule changes helical pitch and length of double-helical DNA domains.<br>
We set out to develop a self-assembled DNA origami device that amplifies microscopic structure changes imposed on its constituent double-helical DNA domains by a large global conformational change. The global structure alteration becomes detectable either by direct imaging with transmission electron microscopy (TEM) but ideally also in a simple fluorometric assay with equipment that is available to many laboratories in the world.<br>
We set out to develop a self-assembled DNA origami device that amplifies microscopic structure changes imposed on its constituent double-helical DNA domains by a large global conformational change. The global structure alteration becomes detectable either by direct imaging with transmission electron microscopy (TEM) but ideally also in a simple fluorometric assay with equipment that is available to many laboratories in the world.<br>
We have focussed on changes in helical pitch and tested our device with a set of different DNA binders from the three major classes, including <b>spermine (major groove binding), ethidium bromide (intercalating),</b> and <b>DAPI (minor groove binding)</b> in different concentrations by direct imaging with TEM. In accordance to computational models that we made for our device, the local deformations led to observable global structural changes. In the light of the time constraints of this project we therefore consider the goals accomplished by our proof-of-principle experiments with TEM alone.<br>
We have focussed on changes in pitch and length and tested our device with a set of different DNA binders from the three major classes, including <b>spermine (major groove binding), ethidium bromide (intercalating),</b> and <b>DAPI (minor groove binding)</b> in different concentrations by direct imaging with TEM. In accordance to computational models that we made for our device, the local deformations led to observable global structural changes. In the light of the time constraints of this project we therefore consider the goals accomplished by our proof-of-principle experiments with TEM alone.<br>
However, since access to TEM microscopes is limited, for practical application of the device it is desirable that it can function in simple fluorometric assays. We have therefore also taken first steps toward using fluorescent signals to report on structural changes that our device undergoes upon binding of small molecules. We present some promising preliminary results obtained with single-molecule FRET microscopy and bulk photometry. 
However, since access to TEM microscopes is limited, for practical application of the device it is desirable that it can function in simple fluorometric assays. We have therefore also taken first steps toward using fluorescent signals to report on structural changes that our device undergoes upon binding of small molecules. We present some promising preliminary results obtained with single-molecule FRET microscopy and bulk photometry. 


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<h2>caDNAno</h2>
<h2>caDNAno</h2>
[[Image:Cross section the U structure.tif|right|x170px | thumb | Fig. 2 Cross section. Orange circles: helices of the two arms. Grey circles: helices of the base.]]


The design of the three dimensional shape was done with caDNAno (a free program for easily designing 3D DNA origamis; see http://cadnano.org/ for further information). So we created a suited cross section, routed the scaffold's path through the cross section, broke the staples and added final modifications like adapter staples for the immobilization on glass slides.
The design of the three dimensional shape was done with caDNAno (a free program for easily designing 3D DNA origamis; see http://cadnano.org/ for further information). So we created a suited cross section, routed the scaffold's path through the cross section, broke the staples and added final modifications like adapter staples for the immobilization on glass slides.
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<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">theU consists in principle of two 10 helix bundles which are connected on the broad side with a third 10 helix bundle. So we have two long deformable arms connected by a base. The cross section can be seen in fig. 2. The orange circles are the helices of the two arms and the 10 grey ones the additional helices of the base. The long arms are now able to amplify the deformation of the single helices in our structure.</td><td></td></tr>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">theU consists in principle of two 10 helix bundles which are connected on the broad side with a third 10 helix bundle. So we have two long deformable arms connected by a base. The cross section can be seen in fig. 2. The orange circles are the helices of the two arms and the 10 grey ones the additional helices of the base. The long arms are now able to amplify the deformation of the single helices in our structure.</font color="#555555"></td><td>[[Image:Cross section the U structure.tif|right|x170px | thumb | Fig. 2 Cross section. Orange circles: helices of the two arms. Grey circles: helices of the base.]]</td></tr>
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<h3>Scaffold Routing</h3>


[[Image:Cross section path.tif|x170px|left | thumb | Fig. 3 Scaffold path]]
[[Image:Cross section path.tif|x170px|left | thumb | Fig. 3 Scaffold path]]


<h3>Scaffold Routing</h3>
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<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">Based on the cross section, we could begin routing the circular scaffold through the pattern of helices. The scaffold first goes through one 10 helix bundle and then changes over to the next 10 helix bundle as one can see in the fig. 3 (the red arrows indicate the direction of the scaffold path). It is commonly known that this enables easy folding of the structure. (refer to our results from [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results#Folding_.26_Purification folding and purification]).
</font color="#555555"></table>


<table cellspacing="0" cellpadding="2">
{{-}}
<tr bgcolor="#f5f5f5"><td valign="top">Based on the cross section, we could begin routing the circular scaffold through the pattern of helices. The scaffold first goes through one 10 helix bundle and then changes over to the next 10 helix bundle as one can see in fig. 3 (the red arrows indicate the direction of the scaffold path). It is commonly known that this enables easy folding of the structure.
(refer to our results from [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results/Folding_Purification folding and purification])
</table>


<h3>Breaking the Staples</h3>
<h3>Breaking the Staples</h3>


With the completion of the routed scaffold we just have to add complimentary pieces of DNA which fit into the honey comb pattern of theU. But there are several problems which had to be considered: <br>
With the completion of the routed scaffold we just have to add complementary pieces of DNA which fit into the honey comb pattern of theU. But there are several problems which had to be considered: <br>
* Staples must not be longer than 50 bases, because of rising inaccuracy of the sequence with rising staples length.
* Staples must not be longer than 50 bases, because of rising inaccuracy of the sequence with rising staples length.
* Staples must not be shorter than 14 bases, because a certain length is needed so that the staple is most of the time bound at room. temperature
* Staples must not be shorter than 14 bases, because a certain length is needed so that the staple is most of the time bound at room. temperature
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<html><a href="http://openwetware.org/wiki/Image:The_U_v02_18.json">The U v02 18.json</a></html>
<html><a href="http://openwetware.org/wiki/Image:The_U_v02_18.json">The U v02 18.json</a></html>


In the json file one can find the complete design of our structure which is based on the p7560 scaffold. With this file we also performed the finite element simulation on [http://cando.dna-origami.org/ canDo] (see [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#Cando canDo simulations] for more details)


<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">The grey staples belong to the core of our structure and are needed to build the basic shape. The staples marked in red can be added to the folding reaction if uncontrolled aggregation arises (if included the single stranded poly-T domains build random coils which suppress the base stacking interaction with another structure). In our case basically no aggregation occurred and we did not need to add the extra poly-T staples.</td><td>[[Image:cadnano_design.tif|x330px | thumb | Fig. 4 caDNAno design. Grey staples: build the basic shape. Red staples: if uncontrolled aggregation arises.]]</td></tr>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">The grey staples belong to the core of our structure and are needed to build the basic shape. The staples marked in red can be added to the folding reaction if uncontrolled aggregation arises (if included the single stranded poly-T domains build random coils which suppress the base stacking interaction with another structure). In our case basically no aggregation occurred and we did not need to add the extra poly-T staples.</font color="#555555"></td><td>[[Image:cadnano_design.tif|x330px | thumb | Fig. 4 caDNAno design. Grey staples: build the basic shape. Red staples: if uncontrolled aggregation arises.]]</td></tr>
</table>
</table>


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<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">Since we also wanted to perform single molecule FRET measurements with our structure, we finally had to add adapter staples to the base of theU. We used biotin labeled staples to bind theU to the surface of a microscope slide.</td><td>[[Image:adaptors.png|right|x200px | thumb | Fig. 5 Adaptors]]</td></tr>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">Since we also wanted to perform single molecule FRET measurements with our structure, we finally had to add adapter staples to the base of theU. We used biotin labeled staples to bind theU to the surface of a microscope slide.</font color="#555555"></td><td>[[Image:adaptors.png|right|x200px | thumb | Fig. 5 Adaptors]]</td></tr>
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</table>


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<table cellspacing="0" cellpadding="2">
<table cellspacing="0" cellpadding="2">
<tr bgcolor="#f5f5f5"><td valign="top">In order to have a positive control, we designed a structure which has an internally induced twist. This was done by adding an extra base pair every 21 base pairs in every single helix of theU. Since there is a cross over to another helix every 7 base pairs, this additional base pairs induces a local torque which leads to a macroscopic twist in the structure. This conformational shift could later clearly be seen in finite elements computer simulations and TEM images. In fig. 6 the 21 base pair tokens are indicated by the 2 black dashed lines and the additional base pairs by the little loops with '1' inside the scaffold.</td><td>[[Image:loop.png|right|x200px | thumb | Fig. 6 Reference structure. 21 base pair tokens: 2 black dashed lines. Additional base pairs: loops with '1' inside the scaffold.]]</td></tr>
<tr bgcolor="#f5f5f5"><td valign="top"><font color="#555555">In order to have a positive control, we designed a structure which has an internally induced twist. This was done by adding an extra base pair every 21 base pairs in every single helix of theU. Since there is a crossover to another helix every 7 base pairs, this additional base pairs induces a local torque which leads to a macroscopic twist in the structure. This conformational shift could later clearly be seen in finite elements computer simulations and TEM images. In fig. 6 the 21 base pair tokens are indicated by the 2 black dashed lines and the additional base pairs by the little loops with '1' inside the scaffold.</font color="#555555"></td><td>[[Image:loop.png|right|x200px | thumb | Fig. 6 Reference structure. 21 base pair tokens: 2 black dashed lines. Additional base pairs: loops with '1' inside the scaffold.]]</td></tr>
</table>
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<h2>Cando</h2>
<h2>Cando</h2>


In addition to our structural considerations we also did computer simulations on our structures. They were performed with [http://cando.dna-origami.org/ Cando] (a finite elements simulation based on our *.json files and mechanical properties of dsDNA). Figures 7a and b show the simulation of the nontwisted structure from two views. In comparison figures 8 a and b show the pre-twisted structure from the same projections. One can clearly see the twist and spread of the arms in fig. 8 a,b. These simulations were used for further theoretical and experimental considerations.
In addition to our structural considerations we also did computer simulations on our structures. They were performed with [http://cando.dna-origami.org/ canDo] (a finite elements simulation based on our *.json files and mechanical properties of dsDNA). Figures 7a and b show the simulation of the nontwisted structure from two views. In comparison figures 8 a and b show the pre-twisted structure from the same projections. One can clearly see the twist and spread of the arms in fig. 8 a,b. These simulations were used for further theoretical and experimental considerations.


<table cellspacing="0">
<table cellspacing="0">
<tr bgcolor="#f5f5f5"><td>[[Image:BM2_1.GIF‎|370px|thumb| Fig. 7a cando simulation of the structure BM2 view 1.]]</td><td>[[Image:BM2_3.GIF‎|370px | thumb | Fig. 7b  cando simulation of the structure BM2 view 2.]]</td></tr>
<tr bgcolor="#f5f5f5"><td>[[Image:BM2_1.GIF‎|385px|thumb| Fig. 7a cando simulation of the structure BM2 view 1.]]</td><td>[[Image:BM2_3.GIF‎|385px | thumb | Fig. 7b  cando simulation of the structure BM2 view 2.]]</td></tr>


</table>
</table>


<table cellspacing="0">
<table cellspacing="0">
<tr bgcolor="#f5f5f5"><td>[[Image:BM24_3.GIF‎|370px|thumb| Fig. 8a cando simulation of the twisted control BM21 view 1.]]</td><td>[[Image:BM24_1.GIF‎|370px | thumb | Fig. 8b  cando simulation of the twisted control BM21 view 2.]]</td></tr>
<tr bgcolor="#f5f5f5"><td>[[Image:BM24_3.GIF‎|385px|thumb| Fig. 8a cando simulation of the twisted control BM21 view 1.]]</td><td>[[Image:BM24_1.GIF‎|385px | thumb | Fig. 8b  cando simulation of the twisted control BM21 view 2.]]</td></tr>


</table>
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For a detailed list of all structures, please consult the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page labbook entry]. <br>
For a detailed list of all structures, please consult the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page labbook entry]. <br>
One of the most important structures for this project was <b>BM2</b>, which is a simple U shaped origami as described above. Most of the TEM analyses concentrated on this structure. <br>
One of the most important structures for this project was <b>BM2</b>, which is a simple U shaped origami as described above. Most of the TEM analyses concentrated on this structure. <br>
Other important structures were <b>BM12, BM13</b> and <b>BM14</b>, which contain FRET labels for twist measurements at the three positions mentioned earlier. Each of these structures additionally contained several adapter staples at the bottom, which could be used for immobilization via biotin / neutravidin. These structures are suited for single molecule fluorescence microscopic examination. <br>
Other important structures were <b>BM12, BM13</b> and <b>BM14</b>, which contain FRET labels for twist measurements at positions that will later be explained in detail. Each of these structures additionally contained several adapter staples at the bottom, which could be used for immobilization via biotin / neutravidin. These structures are suited for single molecule fluorescence microscopic examination. <br>
<b>BM21</b> is designed very similar, but with FRET labels positioned for length measurement instead of twist. Finally, <b>BM24</b> (unlabeled) and <b>BM25</b> (labels for twist analysis) are intrinsically twisted reference structures.
<b>BM21</b> is designed very similar, but with FRET labels positioned for length measurement instead of twist. Finally, <b>BM24</b> (unlabeled) and <b>BM25</b> (labels for twist analysis) are intrinsically twisted reference structures.


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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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</math>
</math>


[[Image:Distance_torsion_ab_twist+a.png | 380px | left | thumb | Fig. 16a: Distance of FRET-pair C1→C2 for a counterclockwise (in the view from the arms in direction to the base) torsion.]]
<table cellspacing="0" cellpadding="2">
[[Image:Distance_torsion_ab_twist-a.png | 380px | right | thumb | Fig. 16b: Distance of FRET-pair C1→C2 for a clockwise torsion.]]
<tr bgcolor="#f5f5f5"><td valign="top">[[Image:Distance_torsion_ab_twist+a.png | 375px | left | thumb | Fig. 16a: Distance of FRET-pair C1→C2 for a counterclockwise (in the view from the arms in direction to the base) torsion.]]</td><td>[[Image:Distance_torsion_ab_twist-a.png | 375px | right | thumb | Fig. 16b: Distance of FRET-pair C1→C2 for a clockwise torsion.]]</td></tr>
</table>
 


{{-}}
{{-}}


Fig. 16a & 16b correspond to FRET-pair positions C1→C2 as described in the article about [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project/Structure#FRET_labels FRET-pair positions] and to the structure [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM12_to_BM14:_Fluorophore-labeled_structures_for_FRET_measurements.2C_including_adapters_for_immobilisation BM14_5/20].
Fig. 16a & 16b correspond to FRET-pair positions C1→C2 as described in the article about [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project#FRET-Pair_Positions FRET-pair positions] and to the structure [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM12_to_BM14:_Fluorophore-labeled_structures_for_FRET_measurements.2C_including_adapters_for_immobilisation BM14_5/20].


The parameters describing the FRET-pair position are:
The parameters describing the FRET-pair position are:
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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. ]]
<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>
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|right|x150px | thumb | Fig. 22 Flourophore positions shown in the caDNAno file.]]<br>
</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.
 
<table cellspacing="0" cellpadding="2">
<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.]]
 


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>

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