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Quantification and Optimization of Circular RNA Stability for Improved RNA Therapeutics

CSEF · 2023 Biochemistry/ Molecular Biology First Award

Kosaraju Reshma · 11

Overview

As mRNA-based therapeutics like COVID-19 vaccines rapidly evolve, degradation remains a key limitation. Circular RNAs (circRNAs) possess a closed-loop structure, conferring enhanced stability. This study aims to 1. Quantify the stability of circRNA in vitro to identify ideal storage conditions and 2. Improve circRNA stability through optimizations of molecular structure. CircRNAs of length ~1,700 and ~3,350 nucleotides were found to remain stable at room temperature (RT) for 2 weeks and at -20°C and -80°C for 7.5 months. Stability did not improve with storage buffers commonly used for mRNA. CircRNA withstood 10 freeze-thaw cycles without degradation or reduced protein expression. In comparison, mRNA is stable for several hours to a few days at RT and up to 6 months at -80°C, degrading after 5 freeze-thaw cycles. For structural optimization, 14 modified nucleoside triphosphates found to enhance mRNA stability were tested on an mNeonGreen circRNA at a 5% substitution rate. Stability and expression were evaluated relative to unmodified circRNA. Stability improved over 16-fold for all optimizations. Expression levels of certain optimizations outperformed the unmodified circRNA 24 hours after digestion, but all performed worse after 96 hours. CircRNA is far more stable than mRNA in a laboratory environment, allowing greater flexibility for scientists and enabling easier storage/transportation, thus improving accessibility of therapeutics. CircRNA-based therapeutics like vaccines and CRISPR gene editing may benefit from increased stability and decreased expression to ensure longevity while reducing common side effects such as toxicity and immune responses that result from overexpression of target proteins.

Source coverage

This record comes from a published award list, not a complete project archive. Its abstract comes from CSEF's public project showcase as archived by the Internet Archive before judging (https://web.archive.org/web/20230401224130/https://ca-csef.zfairs.com/showcase/ShowcaseInfo?f=838e60b7-ea75-46e8-865c-fde4864244b3); the version presented may differ.

Awards (1)

Competition history

  • CSEF 2023 Biochemistry/ Molecular Biology · Entry S0529

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