Fig. 5: Stability and cellular expression of selected highly structured RNA designs in solution and formulated with polyplex.

a Schematic for testing the synergy between RNA modifications and mRNA design rules on downstream stability and protein output. mRNAs were in vitro synthesized with or without ψ and subjected to degradation conditions. Samples were collected overtime and the RNA was purified before being transfected into HEK293T cells. Luciferase activity was measured 24 h after transfection. b Luciferase activity of the reference Nluc sequence and DegScore-optimized CDS with or without ψ after being subjected to in-solution degradation. mRNA half-lives (t_1/2) per construct are given in hours (hrs). Plotted on y-axis are the geometric mean of Nluc/Fluc reporter activity ratios normalized to time zero. Error bars indicate geometric standard deviation. n = 4 biologically independent samples. c Schematic for testing the effect of RNA formulation on downstream stability and protein output from selected RNA designs. mRNAs were in vitro synthesized, formulated with polyplex (PLX), and subjected to degradation conditions and/or expression analysis. Samples were collected over time and the formulated RNA was added to HEK293T cells. d In vitro stability of RNAs formulated with polyplex over 14 days at 5 °C. RNA half-lives were calculated based on the degradation slopes: Nluc start (reference) (14 days), Genewiz_1 (30 days), BugacMan’s_Lost_LD + finetuning_mod_Deg-2-ed (58 days), RLT-10 (69 days) and Ribotree_LinearDesign_degscoreall_1 (46 days). Results correspond to technical duplicates. e Expression of Nluc from HEK293T cells transfected with selected RNA designs formulated with polyplex. Expression was measured by fluorescence after the RNAs were formulated with polyplex, incubated at 5 °C in degradation conditions for 0 and 14 days, and then added to the medium of the cultured cells. Results correspond to technical replicates; normalized Nluc/Fluc activity ± SD. n = 3; ns not significant. *p ≤ 0.05 was considered significant (two-tailed unpaired Student’s t-test; ns: p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001).