Biomod/2013/StJohns/results: Difference between revisions

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*We have visualized the above versions of the claw on AFM to show that they take the predicted shape.
*We have visualized the above versions of the claw on AFM to show that they take the predicted shape.
*We have shown that the above versions of the claw form tight bands on a gel, indicating a single primary product of the anneal.
*We have shown that the above versions of the claw form tight bands on a gel, indicating a single primary product of the anneal.
*We have verified that FRET-tagged origami can be visualized in a gel, providing easy visual discrimination between differently-tagged versions of the claw.
*We have demonstrated a binding interaction between the functionalized claw and functionalized capsid as well as a lack of interaction between the nonfunctionalized claw and capsid.
*We have demonstrated a binding interaction between the functionalized claw and functionalized capsid as well as a lack of interaction between the nonfunctionalized claw and capsid.
*We have not been able to differentiate bound and unbound complexes via DLS.
*We have not been able to differentiate bound and unbound complexes via DLS.
*We have generated and isolated FAB fragments for use as binding elements in future claw designs.
*We have generated and isolated FAB fragments for use as binding elements in future claw designs.
*We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements.
*We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements.




=Data=
=Data=
Below are AFM images of the 100nm armed claws.
==Claw Visualization==
Below are AFM images of the claws alone, with and without binding elements.
<html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html>
<html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html>


Additionally, we have used gel electrophoresis to characterize the binding of Sticky/Blunt DO to
==Binding Interaction==
Sticky/WT capsids.
We used gel electrophoresis to characterize the binding interaction between origami structures and capsids with and without binding elements.


[[Image:Lukemanlab-2013-0001.png|thumb|400px|center|No binding between WT capsids and any DO,<br>
[[Image:Lukemanlab-2013-0001.png|thumb|400px|center|No binding between WT capsids and any DO,<br>
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however, blunt DO binds sticky capsids.]]
however, blunt DO binds sticky capsids.]]


 
==FRET==
We use FRET as a molecular ruler to measure the distance between the claw and capsid during binding.
The following gel images demonstrate FRET fluoresnce.


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Revision as of 12:06, 24 October 2013

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For optimal viewing experience, please enlarge this window to at least 1024 pixels.

Summary

  • We have synthesized versions of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements.
  • We have synthesized versions of the claw with and without fluorescent tags for FRET analysis.
  • We have visualized the above versions of the claw on AFM to show that they take the predicted shape.
  • We have shown that the above versions of the claw form tight bands on a gel, indicating a single primary product of the anneal.
  • We have verified that FRET-tagged origami can be visualized in a gel, providing easy visual discrimination between differently-tagged versions of the claw.
  • We have demonstrated a binding interaction between the functionalized claw and functionalized capsid as well as a lack of interaction between the nonfunctionalized claw and capsid.
  • We have not been able to differentiate bound and unbound complexes via DLS.
  • We have generated and isolated FAB fragments for use as binding elements in future claw designs.
  • We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements.


Data

Claw Visualization

Below are AFM images of the claws alone, with and without binding elements. <html><center><table><tbody align="center"><tr><td><img src="http://openwetware.org/images/thumb/6/66/Lukemanlab-Bluntclaw_afm.png/200px-Lukemanlab-Bluntclaw_afm.png"></td><td><img src="http://openwetware.org/images/thumb/9/9f/Lukemanlab-Stickyclaw_afm.png/200px-Lukemanlab-Stickyclaw_afm.png"></td></tr><tr><td>"Blunt" claw</td><td>"Sticky" claw</td></tr></tbody></table></center></html>

Binding Interaction

We used gel electrophoresis to characterize the binding interaction between origami structures and capsids with and without binding elements.

No binding between WT capsids and any DO,
‘sticky’ DO bind sticky capsids strongly;
however, blunt DO binds sticky capsids.

FRET

<html> <center> <table> <tbody align="center"> <tr> <td> <img src="http://openwetware.org/images/thumb/d/d5/1a_ch1_FRET.tif/345px-1a_ch1_FRET.tif.png"> </td> <td> <img src="http://openwetware.org/images/thumb/6/69/1a_ch2_DONOR.tif/345px-1a_ch2_DONOR.tif.png"> </td> <td> <img src="http://openwetware.org/images/thumb/c/ce/1a_ch3_ACCEPTOR.tif/345px-1a_ch3_ACCEPTOR.tif.png"> </td> </tr> <tr> <td>FRET</td><td>DONOR</td><td>ACCEPTOR</td> </tr> </tbody> </table> </center> </html>


To determine FRET, FRET intensity - DONOR intensity - ACCEPTOR intensity DA(i,j) = DA(i,j) - beta_leak * DD(i,j) - beta_dir * AA (i,j)

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