Theoretical basis for stabilizing messenger RNA through secondary structure design

Abstract

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term ‘superfolder’ mRNAs. These designs exhibit a wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity. Furthermore, their folding is robust to temperature, computer modeling method, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1 and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable.

Cited by papers we hold 7

• Research Advances on the Stability of mRNA Vaccines · 2023
  No notice
• Algorithm for optimized mRNA design improves stability and immunogenicity · 2023
  No notice
• Recent Advances in Lipid Nanoparticles for Delivery of mRNA · 2022
  No notice
• mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability · 2021
  No notice
• An Update on Self-Amplifying mRNA Vaccine Development · 2021
  No notice
• mRNA vaccines for COVID-19: what, why and how · 2021
  No notice
• Challenges of Storage and Stability of mRNA-Based COVID-19 Vaccines · 2021
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.