Poster Presentation Australasian RNA Biology and Biotechnology Association 2025 Conference

Engineering mRNA for Improved Longevity and Protein Expression (#62)

Tessa Swain 1 2 3 , Sania Zahan 1 3 , John Blinco 1 2 , Hayley Ingram 1 2 , Jessica Kretzmann 1 , Max Ward 1 3 , Archa Fox 1 2 3
  1. The University of Western Australia, Crawley, WA, Australia
  2. RNA Innovation Foundry (RIF), Crawley, Western Australia
  3. Australian Centre for RNA Therapeutics in Cancer (ACRTC), Crawley

mRNA has emerged as a promising therapeutic modality, exemplified by the success of COVID-19 vaccines. However, the development of mRNA-based therapies is still limited by challenges in achieving sufficient stability and optimal protein expression. To address these limitations, we aim to investigate how specific mRNA structural features influence transcript longevity and translational efficiency. It has previously been established that longer mRNA half-lives are associated with highly structured coding sequences (1) and overly structured regions surrounding the start codon are linked to poorer translation (2). What is less well understood is whether interfering with the highly conserved structure of the UTRs (3) by altering the structure of synthetic mRNA coding sequences might affect the overall stability and translation of the molecule. To test this hypothesis, we will design a series of mRNA constructs encoding a truncated GFP reporter protein (4) where the coding region is structurally optimised but this optimisation will either take the UTR structure into account, or it will be ignored. Thus, providing different templates where UTR folding will be disrupted or maintained. Protein expression levels will be evaluated through high-throughput flow cytometry screening in vitro, while mRNA stability will be assessed using RT-qPCR time-course experiments. Insights from this study will contribute to the rational design of mRNA transcripts with enhanced translation efficiency and stability, facilitating the development of more effective mRNA therapeutics for clinical applications.

  1. Leppek K, Byeon GW, Kladwang W, Wayment-Steele HK, Kerr CH, Xu AF, et al. Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. Nat Commun. 2022 Mar 22;13(1):1536.
  2. Ringnér M, Krogh M. Folding Free Energies of 5′-UTRs Impact Post-Transcriptional Regulation on a Genomic Scale in Yeast. PLOS Computational Biology. 2005 Dec 9;1(7):e72.
  3. Leppek K, Das R, Barna M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat Rev Mol Cell Biol. 2018 Mar;19(3):158–74.
  4. Liang GT, Lai C, Yue Z, Zhang H, Li D, Chen Z, et al. Enhanced small green fluorescent proteins as a multisensing platform for biosensor development. Front Bioeng Biotechnol. 2022 Oct 17;10:1039317.