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We have successfully synthesized and characterized this structure with (‘sticky’) and without (‘blunt’) single-stranded binding sites. Below are AFM images of the 100nm armed claws.
=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 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.
 
Below are AFM images of the 100nm armed claws.
<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>


Revision as of 11:56, 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 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.
Below are AFM images of the 100nm armed claws.

<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 Sticky/WT capsids.

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


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.

<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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