Fig. 1: Design and characterization of circRNA-LNP encoding NA.

a Schematic diagram of circRNA vaccine production. Linear RNA precursors encoding N1, N2, and IBV NA were self-spliced by group I catalytic intron to form circRNA. CircRNA was then encapsulated into lipid nanoparticles (LNP). The figure was created by the author using Adobe Illustrator 2020. b Verify the self-splicing junction site of circRNA using Sanger sequencing of the junction site after reverse transcription and PCR amplification. c CircRNA and linear precursor were separated on 1.5% agarose gel. Lane L: Linear precursor. Lane L + R: Linear precursor digested with RNase R. Lane C: circRNA not digested with RNase R. Lane C + R: circRNA digested with RNase R. d MUNANA assay for measuring NA enzyme activity in LNP-transfected cells. The curve shows the fluorescence intensity versus the density of cells in 50 μL. Data was shown as means of four repeated experiments ± SD. e Dynamic light scattering (DLS) to characterize the particle size and dispersion of circRNA-LNP. Representative images of each LNP were shown. f Frequency of NA expression in 293 T cells transfected with NA LNP, Blank LNP as a negative control. IRES: internal ribosome entry site. E1: exon fragment 1 upstream of 5’intron. E2: exon fragment 2 downstream of 3’intron.