Fig. 6: Chemically circularized RNAs (chem-circRNAs) are compatible with translation.

A–E Translation activity and protein production profiles of linear and circular mRNAs based on two sequence designs: RNA₁₂ (A, B) and RNA₁₃ (C–E), both containing EMCV IRES elements upstream of the GLuc coding region. Circular RNAs were generated from linear precursors bearing either a 5′-monophosphate (p-) or ethylenediamine motif (EP₅). A, C Show total protein output measured in A549, HEK293T (HEK), HeLa, and HepG2 (HEP) cells over 168 h post-transfection, normalized to canonical Cap1-RNA₁₁ (=1). B, D Show time-course of luciferase activity in A549 cells transfected with circ(p), circ(EP₅), lin(p), lin(EP₅), and control RNAs. A–D Data represent mean ± SD from three biological replicates (n = 3). A, C P values were calculated using unpaired Student’s t test. E Shows normalized protein levels obtained from RNA₁₃ calculated as a protein level at a specific timepoint divided by the total protein collected over 168 h. The bars (left graph) represent the relative levels of the reporter protein in the cell culture medium, determined at the indicated time points and normalized to the level obtained for the reference canonical mRNA (Cap1-RNA₁₁), means ± SEM, n = 3, * P < 0.05, *** P < 0.001, **** P < 0.0001, ordinary one-way ANOVA with Tukey’s multiple comparisons test (4 h, 96 h, 120 h, 144 h, 168 h) or Kruskal–Wallis test with Dunn’s multiple comparisons test (24 h, 48 h, 72 h). The right graph shows time-dependent protein accumulation (percentage of protein produced at all timepoints from 0 h up to the specific timepoint) calculated for each timepoint and fitted to a one-phase decay curve to determine the protein production half-life (the time after which half of the total protein level was reached). Source data are provided as a Source Data file.