Biomod/2013/StJohns/results: Difference between revisions
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*We have synthesized versions of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements. | *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 synthesized versions of the claw with and without fluorescent tags for FRET analysis. | ||
[[Biomod/2013/StJohns/results#Claw Visualization|We have visualized the above versions of the claw on AFM to show that they take the predicted shape.]] | *[[Biomod/2013/StJohns/results#Claw Visualization|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 verified that FRET-tagged origami can be visualized in a gel, providing easy visual discrimination between differently-tagged versions of the claw. | ||
Revision as of 12:09, 24 October 2013
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<html><a href="http://lukemanlab.org"><img src="http://openwetware.org/images/thumb/f/f9/Lukemanlab-toehold-conga-nanny-header.jpg/180px-Lukemanlab-toehold-conga-nanny-header.jpg"></a></html>
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.
‘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)
