Theoretical basis for stabilizing messenger RNA through secondary structure design.

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinformatics

Background:

• Messenger RNA (mRNA) hydrolysis poses significant challenges for vaccine and therapeutic manufacturing, storage, delivery, and in vivo stability.
• Reducing mRNA degradation is crucial for the efficacy and practicality of mRNA-based technologies.
• Current strategies for enhancing mRNA stability are limited, necessitating novel approaches.

Purpose of the Study:

• To explore RNA redesign as a strategy to mitigate mRNA hydrolysis by forming protective double-stranded regions.
• To develop methods for estimating RNA stability and linking it to structural properties like average unpaired probability (AUP).
• To compare the effectiveness of different algorithmic approaches and crowdsourcing in optimizing mRNA structure for stability.

Main Methods:

• Developed calculations to estimate RNA stability against hydrolysis.
• Introduced a model correlating average unpaired probability (AUP) with mRNA hydrolysis rate.
• Compared AUP optimization using conventional methods, sophisticated algorithms, and crowdsourcing (OpenVaccine on Eterna).

Main Results:

• Identified 'superfolder' mRNAs, generated through rational design and advanced algorithms, exhibiting significantly low AUP.
• Demonstrated that superfolder mRNAs possess diverse sequence and structure features potentially beneficial for translation, size, and immunogenicity.
• Showcased the robustness of superfolder mRNA folding across various conditions and its adaptability to new target protein sequences, including SARS-CoV-2 variants.

Conclusions:

• RNA redesign into stable structures ('superfolder' mRNAs) is an effective strategy to combat hydrolysis.
• Advanced computational algorithms and rational design approaches, including crowdsourcing, can yield highly stabilized mRNA constructs.
• Superfolder mRNAs offer a promising avenue for improving the stability and applicability of mRNA-based therapeutics and vaccines, with potential for at least a two-fold increase in in vitro half-life.