Figure 9.

Activity of gapmers designed to SOD1 RNA 4 target in vitro and in HeLa cells. (A) Target SOD1 mRNA sequence containing site of C-to-U SNP (RNA4) and designed to it antisense gapmers. SNP in RNA strand is bolded and underlined, codon in which it occur is marked by red frame. Modified nucleotides within gapmers are marked by colours: blue-LNA, green-2′-O-MeRNA, bolded black- DNA, red-mismatched nucleotides, underlined-SNP site. (B) RNase H in vitro assay results for SOD1 RNA4 target, showing the influence of arrangements of mismatched nucleotides in RNA/ASO duplexes on RNase H cleavage. Red frames mark gapmers which cause undesirable effect of preferential wild type RNA cleavage. Blue frames mark gapmers which cause selective degradation of Mut RNA. (C) Results of ASOs transfection to HeLa cells. The EC₅₀ values for gapmers tested in presence of WT and mutant alleles were estimated based on dose-response curves fitted to experimental data in Origin Lab 8.0 software (for statistics see Supplementary data). The data were gathered from at least three separate experiments involving five concentrations in the range 10–100nM. Comparison of EC₅₀ values for gapmers between WT and Mutant target RNA alleles allowed to evaluate allele-preference for cleavage of tested gapmers. Two last columns of the table present WT/Mut ratio of gapmer selectivity and the selectivity of mismatched gapmers relative to complementary gapmer aKM.