Biomod/2013/StJohns/results: Difference between revisions
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*[[Biomod/2013/StJohns/results#DLS|We have not been able to differentiate bound and unbound complexes via DLS.]] | *[[Biomod/2013/StJohns/results#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. | *[[Biomod/2013/StJohns/results#Selection|We have demonstrated the potential for selecting claws based on their avidity using a chromatography augmented with photocleavable elements.]] | ||
=Data= | =Data= | ||
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Our DLS measurements confirmed that the origami control structure and the capsid were of different sizes. However, when performing DLS on a mix of nonfunctionalized origami and capsid, we found only a single peak rather than peaks showing two differently-sized objects. Based on [[Biomod/2013/StJohns/results#Binding Interaction|data from other experiments]], it is unlikely that this represents a binding interaction. We are currently investigating the source of this anomaly. | Our DLS measurements confirmed that the origami control structure and the capsid were of different sizes. However, when performing DLS on a mix of nonfunctionalized origami and capsid, we found only a single peak rather than peaks showing two differently-sized objects. Based on [[Biomod/2013/StJohns/results#Binding Interaction|data from other experiments]], it is unlikely that this represents a binding interaction. We are currently investigating the source of this anomaly. | ||
[[Image:Lukemanlab-2013-0003.png|thumb|400px|center]] | [[Image:Lukemanlab-2013-0003.png|thumb|400px|center]] | ||
==Selection== | |||
[[Image:Lukemanlab-2013-0004.jpg|thumb|400px|center| The blue boxes highlight where bands would be expected if the photocleaving reaction failed. <br> Note the presence of only functionalized claw in the post-cleavage solution.]] | |||
Revision as of 13:00, 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
Below is a gel demonstrating that versions of the claw tagged with different fluorescent elements can be visually distinguished on a gel.
The six bands on the left are 'blunt' claw and the six on the right are 'sticky' claw.
Odd-numbered lanes contain 0.1 pmol claw, while even-numbered lanes contain 0.2 pmol.
DLS
Our DLS measurements confirmed that the origami control structure and the capsid were of different sizes. However, when performing DLS on a mix of nonfunctionalized origami and capsid, we found only a single peak rather than peaks showing two differently-sized objects. Based on data from other experiments, it is unlikely that this represents a binding interaction. We are currently investigating the source of this anomaly.
Selection
Note the presence of only functionalized claw in the post-cleavage solution.
