Project Cargo set out to create a folding package that is able to assist optimize mRNA vaccine development for stability, regulation, and conformity. However, there are secondary structures than just IREs and more applications than just mRNA vaccines. Thus we designed the Foldase package to be an easy to use tool for even non-coding background researchers to insert RNA secondary structures.
Foldase is open source and we encourage further developments of it so far. As of now, Project Cargo has more ideas on how to further improve our code. Such as including a color coded heat map of base pairs to better visualize pairings and further automation steps to make everything more intuitive. Our Foldase bundle can be found on GitHub and is ready for download. It comes with a how to use guide written by our primary software engineer, Ricardo Valdarago, guiding users through installation, execution, intepreration, and troubleshooting. Our hope is that after a few more iterations and stress testing sessions, we can share our coding package to not just other iGEM teams, but also other researchers. Many biotech companies are looking into the potential of mRNA biology research and development with the success of mRNA vaccines in light of the pandemic. As such we feel that Foldase can be a valuable tool for projects looking into manipulating mRNA structure.
Validating mRNA Structure
The team sure wishes we had more than just one summer. There are just more avenues we want to explore when it comes to RNA structure prediction and real time translation monitoring. One thing we did not get a chance to explore more in depth was how we would validate successful mRNA folding outside of expected translation rate behavior from mRNA constructs. One possibility of assessing mRNA structure is through the use of hyperchromicity plots in a spectrophotometer. The theory behind this would be to heat solutions of purified RNA molecules in a spectrophotometer to measure the change in absorbance of wavelengths of 260 nm light. The absorbance is measured as a function of temperature and should show a curve where the chance center point would show the melting temperature (Tm) of the specific RNA molecule. The idea is that more structured mRNA absorbs less 260 nm light as the internal bonds are less available for excitation. As the RNA melts, the secondary structures become more undone, resulting in a rise of absorbance.
With specialized equipment, we could run a differential scanning calorimetry which follows a similar principle, but instead utilizes two separate chambers of solution. One containing a reference solution of typically water and a sample solution containing purified RNA. an electrical current is applied to measure the change in heat of the samples. The advantage of this method is that it gives an instant read output of heat capacity which can be used to calculate the enthalpy of unfolding of the RNA molecule as its secondary structures unwind. This can then be further derived to produce a ΔS value and a ΔCp for a given RNA sequence and its secondary structures.
Minimum Inoculum Dose and Medical Safety
Here we discuss how we would assess the effect of vaccine efficacy when an IRE is added. Despite our constructs and the real mRNA vaccines utilizing beta-globin sequences in their 3’ UTR mRNA sequences to prolong the mRNA half life, all mRNA must be digested at some point. With a set life span of mRNA sequences, we would have to address the issue of total protein translation lost in the first 10-hour half life cycle lost as mRNA becomes available to translate as IRBP unbinds.
We initially spoke with a professor at UC Davis about meeting a minimum innoclulam dose. However, there was no availability in the schedule to explore this topic further. The first step to ensuring that our vaccine can enlist sufficient immunity, we would look into comparing our results of our HA-tagged VSV-G protein in our constructs with no IRE. Here we would demonstrate how much initial translation would be lost from the IRE system compared to a translation curve of a construct with no IRE. This no IRE system would be the closest we would be able to replicate real vaccine conditions in our lab.
When we have that data, we can address the total loss of translation by adjusting the amount of mRNA construct included in a vaccine dose. This way we can maintain the initial gradual translation and translate enough protein to meet the minimum inoculum dose. Eventually, we hope to see our IRE system utilized in mRNA vaccines in clinical trials.