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. 2023 Jan 16;51(4):e24. doi: 10.1093/nar/gkac1252

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Comparison of reporter expression and immunogenicity. (A) Structure illustration of linear mRNAs and circRNAs used in these evaluations. All mRNAs code EGFP as a reporter gene. (B) Comparison of reporter expression between circRNAs and linear mRNAs with the same sequence component. 0.3 pmol of each reporter mRNA and transfection control, iRFP670-coding mRNA were transfected. (C) Comparison of circRNAs and linear mRNAs (Cap-EGFPs) without any other structural features corresponding to CVB3 IRES and self-splicing ribozyme. 0.3 pmol of each reporter EGFP-coding mRNA and transfection control, iRFP670-coding mRNA were transfected. (D) Evaluation of immune response-related genes by RT-qPCR after 24 h of transfection in A549 cells. 0.6 pmol of each mRNA was transfected. 200 ng poly I:C was transfected as a positive control. Levels of significance are denoted as **P < 0.01, ***P < 0.001 (Dunnett's test). N.S. (non-significant, P > 0.05) pairs were not denoted on the graph. (E) Evaluation of cytotoxicity by WST-1 assay was performed with a 96-well format. 0.15 pmol of each mRNA was transfected. For toxic control, the cells were cultured in 1 μg/ml (HEK293FT, HeLa) or 2 μg/ml (A549) puromycin for 1 day before the measurement. All data in this figure are presented as mean ± SD, n = 3.