Fig. 4: Integration of 5′/3′ UTRs, structure-optimized CDSs, and pseudouridine (ψ) together enhance mRNA stability and translational output.
From: Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics
a CDS and 5′/3′UTR combinations differentially impact protein synthesis. Six mRNA constructs were in vitro synthesized and luciferase activity was measured 6 or 24 h post-transfection. Inclusion of ψ was tested on two selected constructs. Bars indicate the geometric mean of Nluc/Fluc reporter activity ratios normalized relative to Nluc start/hHBB UTRs. Error bars indicate geometric standard deviation. n = 4 biologically independent samples. b Workflow for different approaches to design the CDS variants tested in (c). c Variations in CDS design facilitate high in-solution stability and differential protein expression. In vitro transcribed mRNAs (24 in total) were subjected to in-solution degradation or transfected into HEK293T cells for 6 and 24 h. In-solution half-lives and luciferase activity are normalized to the Nluc start reference construct. Predicted secondary structures are shown for select constructs with colors indicating DegScore at each nucleotide. Designs derived from LinearDesign solutions are marked with a purple triangle. Asterisks correspond to two-sided significance tests with ****p < 0.0001, ***p < 0.001, **p < 0.01. Exact p-values are provided in Supplementary Data 5. Bars indicate the mean of Nluc/Fluc reporter activity ratios normalized relative to Nluc start. Error bars indicate standard deviation across n ≥ 3 biologically independent samples. d Predicted secondary structure overview of Ribotree_LinearDesign_degscoreall_1. Zoomed boxes indicate sequence optimizations and subsequent structural changes made by DegScore to the reference LinearDesign construct. e Increased in-solution half-life correlates with DegScore. Significance test for Spearman correlation value: two-sided p-value for a hypothesis test whose null hypothesis is that two sets of data are uncorrelated, n = 24. Error bars indicate standard deviation across n ≥ 3 biologically independent samples.
