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. Author manuscript; available in PMC: 2020 Oct 3.
Published in final edited form as: Mol Cell. 2019 Aug 29;76(1):96–109.e9. doi: 10.1016/j.molcel.2019.07.016

Figure 3. m6A-modified circFOREIGN attenuates immune response in vitro and in vivo.

Figure 3.

A. (Left) Model of unmodified and m6A-modified circFOREIGN effects of immunogenicity. (Right) Transfection of unmodified circFOREIGN into wild-type HeLa cells stimulates immune response, but m6A-modified circFOREIGN does not. Graph showing gene expression of innate immune genes 24 hours following RNA transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3), *p < 0.05, Student’s t-test, comparing gene stimulation of linear RNA to indicated RNA.

B. Transfection of circFOREIGN plasmid lacking RRACH m6A consensus motifs stimulates immune response at a greater level than circFOREIGN. RRACH motifs (n = 12 sites) were mutated to RRUCH throughout the exon sequence. Mutating every adenosine to uracil within the first 200 bases (n = 37 sites) after the splice junction further increased immunogenicity. Graph showing gene expression of innate immune genes following DNA plasmid transfection. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3), **p < 0.01, ***p < 0.001, Student’s t-test, comparing circFOREIGN to indicated RNA transfection.

C. Transfection of circFOREIGN plasmid with all adenosines replaced by uracil results in elevated immunogenicity. Relative expression of the indicated mRNA and transfected RNA are measured by qRT-PCR, results normalized to expression following mock transfection. Means ± SEM are shown (n = 3), *p<0.01, Student’s t-test, comparing circFOREIGN to indicated RNA transfection.

D. m6A-modified circFOREIGN attenuates anti-OVA T cell responses following primary vaccination. Means are shown (n = 10), *p < 0.05, Anova-Tukey’s test.

E. m6A-modified circRNA attenuates anti-OVA antibody titers following secondary vaccination. Means are shown (n = 10), *p < 0.05, ANOVA-Tukey’s test.