Fig 2: Evaluating the ability of clamp-G PNA to bind and inhibit TTR mRNA(A) Graphic representation of target site on TTR mRNA. The table contains PNA oligomer sequences to the TTR target site. PNA4-LBA, cGPNA5-L, and ScR-PNA6-L contain succinic acid (SA) at the 5′ end (N terminus) for conjugation with LBA ligand. Lysine (K) was added at the 3′ end (C terminus) of the PNA, and OOO represents the trioxo-miniPEG linker.(B) Dose-dependent gel shift assay of TTR target with PNA4, cGPNA5, and ScR-PNA6 at indicated concentration. Samples were incubated in physiological buffer (2 mmol/L MgCl2, 150 mmol/L KCl, and 10 mmol/L NaPi) for 16 h at 37°C, followed by PAGE separation.(C) Schematic representation of PNA inhibiting translation initiation of TTR mRNA. Plasmid containing TTR coding region, nucleotides, and T7 polymerase were used to make TTR mRNA via in vitro transcription. TTR mRNA was incubated with different concentrations of PNA, and in vitro translation was performed using reticulocyte lysate.(D) Representative western blot analysis (top) of TTR protein after PNA and TTR mRNA incubation in reticulocyte lysate. The graph (bottom) represents the quantification of TTR protein fold change relative to the control. ImageJ was used for quantification, the graph shows mean ± SEM (n = 3), and the p value is from two-way ANOVA.(E) Fold change in TTR mRNA levels in HepG2 cells treated with indicated doses of PNA4 and PNA4-L in comparison to ScR-PNA6.(F) Relative change in TTR mRNA levels in HepG2 cells treated with indicated doses of cGPNA5 and cGPNA5-L doses in comparison to ScR-PNA6. Graph shows mean ± SEM (n = 3) and p value is from two-way ANOVA.