Poster Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

Optimising mRNA Design by Rethinking UTR Structural Constraints (#151)

Tessa Swain 1 2 3 , Danielle Rudler 1 2 , Kirti Giridhar 1 2 3 , Faiza Chowdhury 1 3 , John Blinco 1 3 , Nikolay Shirokikh 1 2 , Max Ward 2 , Archa Fox 1 2 3
  1. The University of Western Australia, Crawley, WA, Australia
  2. Australian Centre for RNA Therapeutics in Cancer (ACRTC), Crawley
  3. RNA Innovation Foundry (RIF), Crawley, Western Australia

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 have investigated 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 (CDS)1 and overly structured regions surrounding the start codon are linked to poorer translation2. However, it remains unclear whether preserving the highly conserved secondary structure of untranslated regions (UTRs)3, by altering the structure of the CDS, influences transcript stability and protein expression. To test this hypothesis, we designed three groups of multiple miniGFP-encoding mRNA ('short' CDS), or d2eGFP mRNA ('long' CDS) with structurally optimised CDSs, but each group varied in how the UTR sequence information was considered. In the ‘Ignore_UTR’ group, the design algorithm was not given the UTR sequences when optimising the CDS. In contrast, in the ‘Protect_UTR’ group, the algorithm was provided with the 5’ and 3’UTR sequence information and any generated CDSs were penalised if they interfered with UTR structure. Finally, in the ‘Unprotected_UTR’ group, the UTR sequence context was again provided but no penalty was applied, so potential structural interference may have occurred between CDS and UTR. Thus a series of different mRNAs were synthesised in which UTR secondary structure was either maintained or disrupted without altering the underlying UTR sequence. These parameters are a new capability that no other mRNA design algorithm incorporates, as they typically focus on the CDS alone, and optimise this regardless of the UTRs that flank it.

Initial testing of several short miniGFP mRNAs found only modest differences in protein expression and mRNA stability (direct RNA sequencing and INDEGRA/TagGen analysis4,5) between design groups, with Ignore_UTR slightly outperforming Protect_UTR. In contrast, optimisation of the longer d2eGFP sequence, which has greater potential for disrupting UTR structure, revealed substantially improved translation efficiency and mRNA integrity in the Ignore_UTR compared to the Protect_UTR group. Contrary to our hypothesis, preserving UTR structure during coding sequence optimisation did not improve mRNA performance. These findings suggest that incorporating UTR structural constraints into mRNA design algorithms may be unnecessary, enabling simpler and less computationally intensive optimisation while maintaining or improving mRNA stability and translation. This work provides new insights for the rational design of more effective therapeutic mRNAs.

 

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  5. Chowdhury, F. et al. TagGen: High-Performance Barcode Generator and Demultiplexer for High-Throughput and Long-Read Sequencing Applications. F1000Research, 15 (2026).