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PreprintCited by 199RNA and protein synthesis mechanismsRNA Research and SplicingRNA Interference and Gene Delivery

Theoretical basis for stabilizing messenger RNA through secondary structure design

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Wayment-Steele, Hannah K., Kim, Do Soon, Choe, Christian A., Nicol, John J., Wellington-Oguri, Roger, Watkins, Andrew M., Sperberg, R. Andres Parra, Huang, Po-Ssu +2 more·2020·Nucleic Acids Research
DOI 10.1101/2020.08.22.262931PMC PMC7457604

Preprint — published as 10.1093/nar/gkab764 · Paper page

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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. These computational tests were carried out on both model mRNAs and COVID-19 mRNA vaccine candidates. 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 wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity, and their folding is robust to temperature, 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. Significance statement: Messenger RNA (mRNA) medicines that encode and promote translation of a target protein have shown promising use as vaccines in the current SARS-CoV-2 pandemic as well as infectious diseases due to their speed of design and manufacturing. However, these molecules are intrinsically prone to hydrolysis, leading to poor stability in aqueous buffer and major challenges in distribution. Here, we present a principled biophysical model for predicting RNA degradation, and demonstrate that the stability of any mRNA can be increased at least two-fold over conventional design techniques. Furthermore, the predicted stabilization is robust to post-design modifications. This conceptual framework and accompanying algorithm can be immediately deployed to guide re-design of mRNA vaccines and therapeutics to increase in vitro stability.

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Authors
Wayment-Steele, H. K., Kim, D. S., Choe, C. A., Nicol, J. J., Wellington-Oguri, R., Watkins, A. M., Sperberg, R. A. P., Huang, P.-S., Participants, E., & Das, R.
Affiliations
  • Eterna Massive Open Laboratory (US)
  • Northwestern University (US)
  • Stanford University (US)
Corresponding author
Das, Rhiju
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2020-08-24
Volume
49
Issue
18
Pages
10604-10617
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2020.08.22.262931
PMC
PMC7457604
OpenAlex
W3200792349
Cited by
199
References
0
Citations by year
2026: 37 · 2025: 52 · 2024: 34 · 2023: 31 · 2022: 27 · 2021: 16 · 2020: 2
FWCI
8.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • National Science Foundation
  • Northwestern University
  • Oregon State University
  • National Institute of Advanced Industrial Science and Technology
  • Baidu
  • National Institutes of Health
  • National Institute of General Medical Sciences (R35GM122579)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
mRNA stabilization, RNA hydrolysis, mRNA vaccines, mRNA therapeutics, SARS-CoV-2 spike protein, mRNA half-life

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