The pandemic has created a unique situation for science. We only had lab access starting the middle of July and acquiring funding became a new challenge with the pandemic’s effect on the general financial situation. Regardless, the students of UC Davis took this challenge and continued to pursue discovery. Below is a summary of our findings/advancements from our Dry Lab, Integrated Human Practices, and Wet Lab.
Project Cargo created a functioning computational pipeline based on the ViennaRNA package that is able to take an RNA secondary structure in a fasta format and iterate through an RNA sequence to find the best insertion location. This RNA sequence should be broken up into the RNA secondary structure of interest, 5’ UTR, and coding sequence. From here it takes the RNA secondary structure, iterates the secondary structure at every position of the 5’ UTR to generate a Unique sequence. All these sequences are stored in a single fasta file. That comprehensive fasta file is then fed into fold-fast-alike, a piece of code provided by Dr. Radecki, which parses the fasta file and creates base pair probabilities for each sequence that gets stored as individual folders.
To determine the best candidates, we parse their base pair probabilities to see how likely an RNA structure is likely to fold and not conflict with native RNA structures. We also look at the dot-plot graph generated also by fold-fast-alike and allows us to observe general folding trends to select out candidates with the possibility of global folding resulting in a piece of RNA that will “kiss” onto itself and compromise biological function.
From our folding simulations, we determined that position 17 and 35 were stable locations in the 5’ UTR to place our IRE for our constructs. We also had access to the Moderna and Pfizer-BioNTech mRNA vaccine sequence. We determined
To validate our folding pipeline we compared it to nature and took wildtype ferritin heavy chain genes from humans on the NCBI database. We removed the highly conserved wildtype IRE sequences from and fed those sequences through our pipeline seeing if we would re-insert the IRE back into their native location. We successfully did this with the human ferritin gene where it is naturally found at position 32 and our pipeline predicted position 32 to be most ideal as well. This is under the assumption that natural selection has placed the IRE into a location that is conformationally the most stable.
We took great lengths to document the mechanics of our code and create a comprehensive guide on GitHub for future RNA folding endeavors. We hope that our package can be a useful tool for future iGEM and research purposes.
Integrated Human Practice
Vaccines with reduced side effects would help people with aging immune systems, chronic conditions, and disabilities achieve immunity while minimizing the side effects. Knowing this we felt simply doing a survey that asked people an almost rhetorical question “if a vaccine had reduced side effects, would you prefer to take that one?” was not productive. We felt instead that it would be better to explore the sources of vaccine hesitancy in the USA in Black and/or disabled people. Historically these communities have had to be more cautious of US medical initiatives due to a history of exploitation or inequity.
We worked very closely with the wonderful Mr. Glenn Ellis to connect us to seven, also wonderful, people who come from the Black 50+ years old community with varying degrees of chronic illness and disability. They came from the states of Alabama and Pennsylvania. The concerns from people of these communities were not fully addressed in the pandemic response and this led to slower vaccine distribution thus a less effective herd immunity.
We conducted phone interviews with these seven individuals. We found stories ranging from delayed vaccine access to impoverished communities to one individual who received one of the rarest side effects from the mRNA vaccine; paralysis. Simply summarizing their stories and experiences here does not do them justice at all. We highly encourage readers to go to our “Human Outreach” Tab to learn and read their stories.
In a brief summary, what we found is that four of the seven people experienced side effects from a mRNA vaccine with three cases that were more severe than previous vaccinations and three people who did not have side effects from either mRNA or previous vaccines. Aside from side effects we had strong leads into what other socioeconomic and historical factors could contribute to low vaccination rates in Black Americans.
Project Cargo believes science is only as valuable as the good it can do for people. So like our RNA folding platform, hopefully we laid down the groundwork for future research efforts.
We aimed to verify our mRNA folding pipeline with wet lab data. However, as of currently writing this, we are still working to assemble our plasmid for transfection and we hope to be able to report on our cumulative translation and translation rate for the judging session. We hope to bring exciting results then!
Our goal was to take an IRE that would bind to IRBP1, IRBP2, a mutant IRE that could bind to both, or a mutant that could bind to neither to assess the effect of initial translation rates of our designed mRNA constructs. We aimed to replicate vaccine conditions as closely as possible by utilizing a lipid particle based delivery of our DNA plasmids via lipofectamine into mammalian cells.
Rather than encoding for a spike protein like the real vaccine, our coding region (“cargo”) would have encoded for a membrane integrating protein (VSV-G) fused with an HA tag for HA antibody staining to assess total translation amount. Separated by a P2A cleavage sequence that allows for a single piece of RNA to encode for another protein. Downstream of the P2A site, the translation is stopped and the next sequence begins translation. Our second protein was a destabilized YFP protein that can be induced to fluorescence with the addition of Trimethoprim (TMP).
We would transfect CHO DG44 cells with our various plasmids with the different mutated IRE’s at the locations of 17 and 35 in the 5’ UTR. Transfected cells would be monitored in Nikon Eclipse Ti inverted microscope installed in Dr. Albeck’s lab. This microscope is able to act as both an incubator for mammalian cell growth and measure the change in fluorescence over time with a mobile camera that can capture image data through several different light channels over the course of days. The Cy5 channel were used to monitor mCardinal fluorescence and YFP for the venus fluorescence.
Just because the wiki freese has passed, that does not mean our work is done! We have a new plan on how to assemble our plasmid for transfection and imaging. We have taken every step we can at the moment to validate all the computational, wet lab protocols, data collection, and plotting methods we need to. Thus we will be ready to collect data as soon as we build our plasmid fully.