Biomod/2013/StJohns/introduction: Difference between revisions
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In our proof-of-principle approach, the origami structure is a three-pronged DO ‘claw’<sup>[[Biomod/2013/StJohns/References|[2]]]</sup> with sticky-ended DNA strands complementary to the surface of a modified<sup>[[Biomod/2013/StJohns/References|[3]]]</sup> bacteriophage MS2 capsid substrate. | In our proof-of-principle approach, the origami structure is a three-pronged DO ‘claw’<sup>[[Biomod/2013/StJohns/References|[2]]]</sup> with sticky-ended DNA strands complementary to the surface of a modified<sup>[[Biomod/2013/StJohns/References|[3]]]</sup> bacteriophage MS2 capsid substrate. | ||
====Near-Term Goals==== | |||
* [[Biomod/2013/StJohns/approaches#Claw|The basic claw design itself:]] | * [[Biomod/2013/StJohns/approaches#Claw|The basic claw design itself:]] | ||
**Synthesis of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements. | **Synthesis of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements. | ||
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**Visualize these claws on AFM to show that they take the desired shape. | **Visualize these claws on AFM to show that they take the desired shape. | ||
**Verify that different FRET-tagged claw versions can be visually distinguished on a gel. | **Verify that different FRET-tagged claw versions can be visually distinguished on a gel. | ||
**Demonstrate that FRET occurs when the claw is bound to the capsid substrate. | |||
**Show that the binding interaction between the claw and the virus capsid produces a visible change in gel mobility. | **Show that the binding interaction between the claw and the virus capsid produces a visible change in gel mobility. | ||
**Show that the functional claw preferentially binds to the functionalized substrate, not other objects. | **Show that the functional claw preferentially binds to the functionalized substrate, not other objects. | ||
Revision as of 16:07, 26 October 2013
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Motivation
There are many ways to detect viruses, but they rely on detecting small pieces of the virus rather than the whole structure. If we could reliably detect the entire virus rather than just a part, this could be applied to the rapid diagnosis of viral diseases as well as rapid detection of infectious agents in the environment.
Project Overview
The goal of this project is to design and characterize a DNA origami[1] (DO) structure that undergoes significant conformational changes when bound to objects ranging in size from 10-100 nm.
Since binding-specific conformational change can be transduced into a signal, this should enable the design of nanometre-scale sensors for viruses.
In our proof-of-principle approach, the origami structure is a three-pronged DO ‘claw’[2] with sticky-ended DNA strands complementary to the surface of a modified[3] bacteriophage MS2 capsid substrate.
Near-Term Goals
- The basic claw design itself:
- Synthesis of the claw with (‘sticky’) and without (‘blunt’) single-stranded binding elements.
- Synthesis of the claw with and without fluorescent tags for FRET analysis.
- Show that these claws fold into single structures (by testing for single band formation on electrophoresis gel).
- Visualize these claws on AFM to show that they take the desired shape.
- Verify that different FRET-tagged claw versions can be visually distinguished on a gel.
- Demonstrate that FRET occurs when the claw is bound to the capsid substrate.
- Show that the binding interaction between the claw and the virus capsid produces a visible change in gel mobility.
- Show that the functional claw preferentially binds to the functionalized substrate, not other objects.
- Differentiate bound and unbound claw/capsid mixtures via DLS.
- Methods of selecting the most avid claws from a ‘library’ of designs with different binding-site geometries:
- Provide proof of principle for the use of a selection model to optimize claw avidity in the next phase of the project.
- The potential use of immunoglobulins as binding elements in future claw designs:
- Demonstrate that we can isolate anti-Flag FAB fragments reliably and effectively.
- Attach small model ketones to DNA strands as a preliminary to attaching FAB fragments.
- Methods of controlling the vertex angles of DO structures for precise hinge angles:
- Synthesize small DO triangle elements with precise geometries, and verify these geometries on AFM.
To characterize the conformational changes and binding interactions of this system, we will use four primary methods:
- Gel Electrophoresis
- Atomic Force Microscopy
- Förster Resonance Energy Transfer (FRET) analysis
- Dynamic Light Scattering
Youtube Video
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