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.
We came up with this idea by synthesizing research ideas from two different sources. We heard about a friend of ours, Sean Sullivan who was a graduate researcher at Tufts. He had written a research proposal about using a modified cas9 protein (referred to as cas13a from hereafter) in order to produce an amplified signal output from a toehold switch in order to detect mRNA in antibiotic resistant bacteria.
We also read about Boston University’s igem project from 2017, and realized we might be able to adapt their toeholds for use with the cas13a as signal output in order to detect small amounts of miRNA in the bloodstream with high sequence specificity.
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.
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.
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.