Fig. 8: Chem-circRNAs containing endocyclic cap engage in the cap-dependent translation.

A, B Show total protein output from circRNA₀₉ and linear (EP₅Cap)RNA₀₉ constructs in A549, HEK293T (HEK), HeLa, and HepG2 (HEP) cells transfected with circular RNAs derived from linear precursors bearing either a 5′ monophosphate (p-) [circ(p)RNA₀₉] or an EP₅Cap structure [circ(EP₅Cap)RNA₀₉]. Protein expression was measured as secreted GLuc activity accumulated over 168 h and normalized to the level obtained with canonical Cap1-RNA₁₁ (=1). C Shows the time-course of GLuc expression in A549 cells transfected with circ(p)RNA₀₉, circ(EP₅Cap)RNA₀₉, respective linear precursors, and linear Cap1-RNA₁₁. D Shows in vitro translation of circ(p)RNA₀₉, circ(EP₅Cap)RNA₀₉, and circ(p)RNA₁₅ in rabbit reticulocyte lysate (RRL) in the presence of the cap-dependent translation inhibitor (m⁷Gp_sppG), demonstrating inhibition of translation and supporting a cap-dependent translation mechanism^40,41. Chem-circ RNAs containing N1-methylpseudouridine can be translationally active (E–H). Cells were transfected with circ(EP₅Cap)RNA₀₉ (Cap), circ(p)RNA₁₅ (EMCV), Cap1-RNA₁₁, and their respective counterparts containing N1-methylpeudouridine (+m¹Ψ). E Shows the time-course of GLuc expression from circRNAs (circ(EP₅Cap)RNA₀₉ and circ(p)RNA₁₅) and linear Cap1-RNA₁₁ over 168 h in A549 cells. F–H Show the total protein output from four cell lines (A549, HeLa, HEK, HepG2) transfected with circ(EP₅Cap)RNA₀₉/circ(EP₅Cap)RNA₀₉ + m¹Ψ (F), circ(p)RNA₁₅/circ(p)RNA₁₅ + m¹Ψ (G), linear Cap1-RNA₁₁/Cap1-RNA₁₁ + m¹Ψ (H), harvested at 168 h post-transfection. In each F–H, values are normalized to the signal from the corresponding uridine-containing mRNA. A, B, F, G, H Data represent mean ± SD of three biological replicates (n = 3), P values were calculated using unpaired Student’s t test. Source data are provided as a Source Data file.