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{{Biomod/2013/Aarhus/Nano_Creators/Begin|pagetype=Achievements And Future Work|pagename=Achievements And Future Work}}
{{Biomod/2013/Aarhus/Nano_Creators/Begin|pagetype=Achievements And Future Work|pagename=Achievements And Future Work}}


==Achievements and future work==
=='''Achievements and future work'''==


===Origami===
===Origami===
We designed two DNA origamis, a two-layered base plate and a dome that could function as a lid. These were successfully folded and characterized using gel electrophoresis, AFM and TEM. However, connecting the two origamis was not achieved within the time frame of the BIOMOD competition.
Two DNA origamis were designed: a two-layered base plate and a dome that could function as a lid. These were successfully folded and characterized using gel electrophoresis, AFM and TEM. However, connecting the two origamis was not achieved within the time frame of the BIOMOD competition.
ET PÆNT BILLEDE??
Following the BIOMOD competition more experiments in connecting the plate to the dome will be performed, both using staple strands and the peptide lock.


===Cholesterol===
[[image:achievements.png|center|frame|Figure 71. The two components of the origami design. A: AFM image of the origami plate. B: TEM image of the origami dome.]]
In order to attach both the photosensitizer and the cholesterol to DNA strands, the precursor nucleoside for 5-propargylamine ddUTP was successfully synthetized in 6 steps from 2’-deoxyuridine. A cholesterol derivative with an NHS-ester handle was synthetized and subsequently coupled to amine modified DNA strands and successfully incorporated into an origami plate. Cells were treated with the cholesterol-modified plates and visualized using confocal microscopy. As no origamis were visible in these images, due to low concentrations of the sample, this experiment will be repeated.  
Furthermore, to achieve a more direct way of labeling DNA with cholesterols, work will be continued in optimizing the triphosphate synthesis and the following purification. Future work will be done to synthesize a cholesterol modified nucleoside triphosphate which can be used in a cholesterol labeling-kit with TdT.
 
===Photosensitizer===
The photosensitizer was successfully synthesized in five steps from the extracted natural compound and subsequently conjugated to DNA. The photosensitizer’s ability to induce apoptosis in cells was also investigated and the test system showed that photosensitizer-DNA alone could not kill cells, but when in complex with a cholesterol-DNA the system was able to induce apoptosis in cells.
Future experiments will include expanding the cell analyses to anneal the photosensitizer-DNA conjugate into the origami plate and investigate the ability of this system to induce apoptosis.  


Following the BIOMOD competition more experiments in connecting the plate to the dome will be performed, both using staple strands and the peptide lock. Furthermore, the complete structure with its peptide locks will need testing, to see if it can open in response to cleavage by MMP2, possibly by use of Förster resonance energy transfer (FRET).


===Peptide lock===
===Peptide lock===
The synthesis of the peptide lock was attempted using a gradual buildup of DNA, but was unsuccesfull. Coupling of one DNA strand of the lock to the peptide was achieved using click reactions, but the amide coupling of the amine-modified second DNA strand to the peptide was not achieved.  
The synthesis of the peptide lock was attempted using a gradual buildup of DNA, but was unsuccesful. Coupling of one DNA strand of the lock to the peptide was achieved using click reactions, but the amide coupling of the amine-modified second DNA strand to the peptide was not achieved.  
After the BIOMOD competition, amide coupling with standard peptide reagents (HBTU, HOBt, etc.), have been planned to take place in order to obtain the desired molecule. Parallel a design using the succesfull click reaction will be investigated.
After the BIOMOD competition, amide coupling with standard peptide reagents (HBTU, HOBt, etc.), have been planned to take place in order to obtain the desired molecule. Parallel a design using the succesfull click reaction will be investigated.


===sisiRNA and CPPs===
===Chemical modifications===
PEG polymers of two different sizes as well as the cell penetrating peptides GALA and melittin were conjugated to the 5’ ends of both segments of the sisiRNA passenger strand. Cells were transfected with all the different, singly modified duplexes and the activities were compared unmodified sisiRNA or siRNA. In these experiments, it was demonstrated that conjugating these large molecules to the sisiRNA generally did not decrease the activity of the constructs.
The photosensitizer was successfully synthesized in five steps from the extracted natural compound and subsequently conjugated to DNA. A cholesterol derivative with an NHS-ester handle was synthetized and subsequently coupled to amine modified DNA strands. Furthermore, to achieve a more direct way of labeling DNA with cholesterols, work will be continued in optimizing the triphosphate synthesis and the following purification. This can be used to synthesize a cholesterol modified nucleoside triphosphate which can be used in a cholesterol labeling-kit with TdT.
A doubly modified construct with two melittins conjugated to the passenger strand of the sisiRNA was made, and this was tested in high concentrations without a transfection agent. From these experiments it was unclear if the CPPs enabled the sisiRNAs to cross the cell membrane, and it appeared that some component of the assay stimulated an increased expression of the luciferase protein. Experiments to investigate the cause of this effect have been planned after the end of the BIOMOD competition.


===System in action===
The ability of the photosensitizers to induce apoptosis in cells was investigated and it was shown that photosensitizer-DNA alone could not kill cells, but was functional in complex with a cholesterol-DNA.
Future experiments will include expanding the cell analyses to anneal the photosensitizer-DNA conjugate into the origami plate and investigate the ability of this system to induce apoptosis.
Cholesterol- and photosensitizer modified DNA strands were successfully incorporated into an origami plate. Cells were treated with the cholesterol-modified plates and visualized using confocal microscopy. As no origamis were visible in these images due to low concentrations of the sample, this experiment will be repeated.


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Latest revision as of 04:34, 2 November 2015

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Achievements and future work

Origami

Two DNA origamis were designed: a two-layered base plate and a dome that could function as a lid. These were successfully folded and characterized using gel electrophoresis, AFM and TEM. However, connecting the two origamis was not achieved within the time frame of the BIOMOD competition.

Figure 71. The two components of the origami design. A: AFM image of the origami plate. B: TEM image of the origami dome.

Following the BIOMOD competition more experiments in connecting the plate to the dome will be performed, both using staple strands and the peptide lock. Furthermore, the complete structure with its peptide locks will need testing, to see if it can open in response to cleavage by MMP2, possibly by use of Förster resonance energy transfer (FRET).

Peptide lock

The synthesis of the peptide lock was attempted using a gradual buildup of DNA, but was unsuccesful. Coupling of one DNA strand of the lock to the peptide was achieved using click reactions, but the amide coupling of the amine-modified second DNA strand to the peptide was not achieved. After the BIOMOD competition, amide coupling with standard peptide reagents (HBTU, HOBt, etc.), have been planned to take place in order to obtain the desired molecule. Parallel a design using the succesfull click reaction will be investigated.

Chemical modifications

The photosensitizer was successfully synthesized in five steps from the extracted natural compound and subsequently conjugated to DNA. A cholesterol derivative with an NHS-ester handle was synthetized and subsequently coupled to amine modified DNA strands. Furthermore, to achieve a more direct way of labeling DNA with cholesterols, work will be continued in optimizing the triphosphate synthesis and the following purification. This can be used to synthesize a cholesterol modified nucleoside triphosphate which can be used in a cholesterol labeling-kit with TdT.


System in action

The ability of the photosensitizers to induce apoptosis in cells was investigated and it was shown that photosensitizer-DNA alone could not kill cells, but was functional in complex with a cholesterol-DNA. Future experiments will include expanding the cell analyses to anneal the photosensitizer-DNA conjugate into the origami plate and investigate the ability of this system to induce apoptosis. Cholesterol- and photosensitizer modified DNA strands were successfully incorporated into an origami plate. Cells were treated with the cholesterol-modified plates and visualized using confocal microscopy. As no origamis were visible in these images due to low concentrations of the sample, this experiment will be repeated.

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</style> </head> <body> <div id="indexing"> <div id="sitemap"> <p id="sitemapTitle">SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University</p> <div id="footer-contents"> <div class="footer-section"> <p class="footer-section-title">INTRODUCTION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus">Home, abstract, animation and video</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Introduction">Introduction</a></li </ul> </div> <div class="footer-section"> <p class="footer-section-title">RESULTS AND DISCUSSION</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Results_And_Discussion/System_In_Action">System in action</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">MATERIALS AND METHODS</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Origami">Origami</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Peptide_lock">Peptide lock</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Chemical_Modification">Chemical modification</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/sisiRNA">sisiRNA</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/System_In_Action">System in action</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Materials_And_Methods/Methods">Methods</a></li> </ul> </div> <div class="footer-section"> <p class="footer-section-title">SUPPLEMENTARY</p> <ul> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Team_And_Acknowledgments">Team and acknowledgments</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Optimizations">Optimizations</a></li> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Data">Supplementary data</a></li> 

                                               <li><a

href="/wiki/Biomod/2013/Aarhus/Supplementary/Supplementary_Informations">Supplementary informations</a> <li><a href="/wiki/Biomod/2013/Aarhus/Supplementary/References">References</a></li> </ul> </div> </div> <div> <p id="copyright">Copyright (C) 2013 | BIOMOD Team Nano Creators @ Aarhus University | Programming by: <a href="mailto:pvskaarup@gmail.com?Subject=BIOMOD 2013:">Peter Vium Skaarup</a>.</p> </div> </div>

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