Difference between revisions of "Team:Tufts/Description"
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<li> First, a mRNA linked to a particular disease would bind to the switch region of the toehold</li> | <li> First, a mRNA linked to a particular disease would bind to the switch region of the toehold</li> | ||
<li>This would cause the collapse of the toehold, freeing the start codon and allowing for translation.</li> | <li>This would cause the collapse of the toehold, freeing the start codon and allowing for translation.</li> | ||
| − | <li>The protein being translated would be Cas13a, which when activated by a guide RNA, cleaves RNA | + | <li>The protein being translated would be Cas13a, which when activated by a guide RNA, cleaves RNA nondiscriminatorily.</li> |
| − | <li> | + | <li>The RNA cut would be bound to a fluorescent probe and quencher. When cleaved, the quencher is split from the fluorescent probe, allowing the original activation of the toehold switch to be activated by fluorescence.</li> |
</ul> | </ul> | ||
</div> | </div> | ||
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<div class="column third_size" > | <div class="column third_size" > | ||
<div class="highlight decoration_A_full"> | <div class="highlight decoration_A_full"> | ||
| − | <h3> | + | <h3>Why is this project important?</h3> |
| − | <p> | + | <p>There are many disease processes in the body that cannot be detected early on in the human body with current methods. These include hairline fractures, which can be too small for X-rays to detect, cancers, such as ovarian cancer, that currently have no early-stage imagine available, and autoimmune illnesses, such as multiple sclerosis, that must have active brain lesions present before the disease can be diagnosed. However, while miRNAs are frequently cited as being important in RNA silencing and post transcriptional regulation, they are also released in almost every disease process as a result. While they are already in use in diagnostics through microarrays and next generation sequencing, these processes are expensive and time consuming. Developing a stable toehold switch that can be used in quick diagnostics will increase the viability of miRNAs as a widespread diagnostic tool. </p> |
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<div class="column two_thirds_size" > | <div class="column two_thirds_size" > | ||
| − | <h3> | + | <h3>Challenges</h3> |
<p> | <p> | ||
| − | + | While we still wholeheartedly believe in the ability of miRNAs to be used rapidly and inexpensively in sensitive biological tests, there is much more research needed before our model can be implemented. While we achieved partial success in the assembly of the cas13a DNA sequence, we were not able to successfully assemble the toehold switch or use a kit to turn in into the more usable RNA form. Our inability to use the project to its full capability does not mean that the project is impossible, rather that more time and more trial and error are needed before it can be made a reality. | |
</p> | </p> | ||
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<div class="column third_size"> | <div class="column third_size"> | ||
<h3>References</h3> | <h3>References</h3> | ||
| − | <p> | + | <p>https://onlinelibrary.wiley.com/doi/full/10.1002/jcp.25056 insert more later.</p> |
</div> | </div> | ||
Revision as of 23:10, 15 October 2018
Description
Our project was based on the concept of a toehold switch. A toehold switch is a hairpin of RNA that is able to be used as a mechanism to activate the translation of certain proteins. The toehold consists of a ribosome binding site, a start codon, and a switch region. In our project, the RNAs complementary to the switch region were theoretically those miRNAs that are emitted when disease processes occur in the human body. Thus, activating the switch with these miRNAs would allow for detection of disease.
How exactly would this work?
- First, a mRNA linked to a particular disease would bind to the switch region of the toehold
- This would cause the collapse of the toehold, freeing the start codon and allowing for translation.
- The protein being translated would be Cas13a, which when activated by a guide RNA, cleaves RNA nondiscriminatorily.
- The RNA cut would be bound to a fluorescent probe and quencher. When cleaved, the quencher is split from the fluorescent probe, allowing the original activation of the toehold switch to be activated by fluorescence.
Why is this project important?
There are many disease processes in the body that cannot be detected early on in the human body with current methods. These include hairline fractures, which can be too small for X-rays to detect, cancers, such as ovarian cancer, that currently have no early-stage imagine available, and autoimmune illnesses, such as multiple sclerosis, that must have active brain lesions present before the disease can be diagnosed. However, while miRNAs are frequently cited as being important in RNA silencing and post transcriptional regulation, they are also released in almost every disease process as a result. While they are already in use in diagnostics through microarrays and next generation sequencing, these processes are expensive and time consuming. Developing a stable toehold switch that can be used in quick diagnostics will increase the viability of miRNAs as a widespread diagnostic tool.
Challenges
While we still wholeheartedly believe in the ability of miRNAs to be used rapidly and inexpensively in sensitive biological tests, there is much more research needed before our model can be implemented. While we achieved partial success in the assembly of the cas13a DNA sequence, we were not able to successfully assemble the toehold switch or use a kit to turn in into the more usable RNA form. Our inability to use the project to its full capability does not mean that the project is impossible, rather that more time and more trial and error are needed before it can be made a reality.
References
https://onlinelibrary.wiley.com/doi/full/10.1002/jcp.25056 insert more later.