Part:BBa_K4829025
Ribotree optimised sequence for the CDS of BBa_K4829018
To make mRNA stable for long periods of time at higher temperatures may be considered one of the ultimate goals of the mRNA vaccine industry today. We are introducing RNA sequences corresponding to all our composite bio-bricks, which are optimised to the best possible stability, by RiboTree, developed by Das labs at Stanford. We have uploaded these sequences separately, as we cannot introduce changes in a sequence to leave out restriction sites if the ultimate goal is to be the 'best possible sequence'! To find the RFC10 compatible DNA sequences, you may check out BBa_K4829018
Usage and Biology
- mRNA optimised by minimising the AUP(Average Unpaired Probability function) and minimising the 'hot spots in the RNA molecule.
- The basis of this is to reduce the chances of hydrolysis and hence breakdown of the RNA molecule.
Summary of RNA Hydrolysis Mechanism:
RNA hydrolysis is a chemical process that results in the cleavage of the RNA backbone. The mechanism involves:
- Initiation by Deprotonation: The hydrolytic cleavage of an RNA backbone phosphodiester bond begins with the deprotonation of the 2′-hydroxyl group on the ribose sugar.
- Transition State Formation: The deprotonated hydroxyl group then attacks the adjacent phosphate group, forming a pentacoordinate transition state. For this state to form, the RNA backbone must adopt a specific conformation where the 2′- hydroxyl group aligns with the departing 5′ oxyanion.
- Cleavage and Strand Break: The 5′ oxyanion leaves, resulting in the formation of a 2′,3′-cyclic phosphate and a break in the RNA strand. This mechanism is universal to RNA and underlies the action of certain ribozymes and protein-based nucleases.
The hydrolysis mechanism is intrinsic to RNA and is influenced by factors like secondary structure. Structured regions within RNA, such as double-stranded areas, can restrict the molecule's conformational flexibility, reducing its propensity for hydrolytic cleavage.
The need for such a tool
Messenger RNA (mRNA) vaccines and therapeutics face challenges related to RNA hydrolysis, impacting their manufacturing, storage, delivery, and in vivo stability. One potential solution to mitigate mRNA hydrolysis is to redesign RNAs to have double-stranded regions, which are resistant to cleavage and degradation, while still encoding the same proteins.
Ribotree introduces calculations for estimating RNA stability against hydrolysis and presents a model connecting the average unpaired probability (AUP) of an mRNA to its hydrolysis rate. The team at Das Labs evaluates the potential stabilization achievable through various mRNA design methods, including crowdsourcing via the OpenVaccine challenge on the Eterna platform. Notably, designs developed on Eterna and certain sophisticated algorithms resulted in 'super folder' mRNAs with low AUP values. These designs have diverse sequence and structure attributes beneficial for translation, biophysical size, and immunogenicity. Importantly, their folding remains stable across different conditions, including temperature variations and changes in the target protein sequence.
The stability of mRNA is crucial, especially in the context of the COVID-19 pandemic where mRNA vaccines have shown promise. However, the inherent chemical instability of RNA poses challenges. RNA degradation is influenced by various factors, including its likelihood to undergo in-line hydrolytic cleavage. The World Health Organization's standards for vaccines highlight the need for formulations that retain efficacy for extended periods under refrigeration. However, traditional mRNA vaccines might not meet these standards, especially under temperature variations or specific storage conditions. One solution is to enhance the RNA's secondary structure, which has been observed to reduce hydrolysis. This research provides a theoretical foundation suggesting that structure-aware design can significantly stabilize COVID-19 mRNA vaccines.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
We will show the comparison of the 2 predicted structures for the standard codon optimised mRNA and the new, revised optimised RNA(via Ribotree):
Figure 1: The structure of the RNA pre-optimisation by Ribotree. 'Loss' function value (AUP) = 482.1750000000001
Figure 2: The structure of the RNA post-optimisation by Ribotree. 'Loss' function value (AUP) = 471.85500000000013
Scale of the AUP
| None |