Figure 3.
Determination of circRNA immunogenicity in relation to linear mRNA. A) Schematics of RNAs introduced and used in this figure. Linear mRNAs do not contain an IRES or other structured features that may provoke a structure-specific immune response. B) Agarose gel showing progressive modification of circRNA precursor with m1ψ. C) Agarose gel showing purified unmodified and modified RNAs. Modification with m1ψ reduces apparent molecular weight. D) GLuc expression 24 hours after transfection of 293 or A549 cells with unmodified circRNA or m1ψ-circRNA (data presented as mean+SD, n=3). E) Cell viability, GLuc expression stability, and cytokine release from A549 cells transfected with unmodified or m1ψ linear mRNA or circRNA. Cell viability was assessed 3 days after transfection. Cytokine release was assessed 24 hours after transfection (data presented as mean+SD, n=3, ns=not significant p<0.05, ND=not detected). F) N6-methyladenosine modification of circRNA. Left: Agarose gel showing splicing products of unmodified and 10% m6A-modified circRNA with and without the CVB3 IRES. Middle: Agarose gel showing purified circRNAs. Right: Agarose gel showing oligonucleotide-guided RNase H nicking of circ∆IRES. G) GLuc expression 24 hours after transfection of 293 cells with the indicated unmodified or m6A-modified RNAs (data presented as mean+SD, n=3, *p<0.05, see also Figure S3).