Fig. 4: The 42nt apt-VPg reduces nonspecific amplification of VPg saRNA.
A In vitro amplification reactions contained 80 nM His-tagged VPg-pU, 4 μg recombinant huNoV RdRp, 2 μg huNoV 2C-L, and 400 μM NTPs, together with four different huNoV GOI triM-saRNA templates (1 μg each). VPg-tagged mRNA products were enriched via his-tag purification, and representative capillary electrophoresis traces from three independent experiments show the integrity of 5′ VPg-linked progeny strands. B RNAFold-predicted secondary structures of the huNoV 3′UTR and ghrelin mRNA, with the VPg-dependent replication start site indicated (red arrow). BLI analysis evaluated binding affinities of synthesized huNoV 3′UTR and ghrelin mRNA to recombinant VPg (Kd from global fit). The red dashed box highlights truncated VPg-ssRNA species detected by capillary electrophoresis; representative results from three independent experiments are shown. C Schematic model for truncated ssRNA generation during VPg-dependent replication initiation. VPg-pU binds the huNoV 3′UTR to initiate negative-strand synthesis, but AXXC-containing hairpins can nonspecifically recruit VPg-pU, generating short VPg-ssRNA products. Replacing the huNoV 3′UTR with a 42-nt apt-VPg 3′UTR of higher VPg affinity reduces such nonspecific amplification. Alphafold3 predicts the 3D architecture of the 42-nt apt-VPg 3′UTR in complex with VPg. D Representative capillary electrophoresis results from three independent experiments showing VPg-mRNA amplification abundance and strand integrity in 293T cells 24 h after transfection with four huNoV GOI triM-saRNA-LNPs containing the 42-nt apt-VPg 3′UTR; unmodified triM-saRNAs served as controls. E Representative flow-cytometry analyses from three independent experiments showing expression of Ghrelin, EGFP, GSDMDNT, and proc1 in cells transfected with huNoV GOI triM-saRNA-LNPs harboring the 42-nt apt-VPg 3′UTR, compared with unmodified constructs. All cell-based experiments were performed independently three times. Exact p-values are indicated within the figure. Figure 4C was created in BioRender. Dwad, D. (2025) https://BioRender.com/f0yr3ku. Source data are provided as a Source Data file.
