A single mismatch in the LNA/DNA mixmers abrogates the activity of them for exon 7 inclusion. (a) LNA/DNA mixmer sequences used for the experiment. The LNA/DNA mixmers #10–15 contain a single base mismatch at various locations along the sequence. AON #9 consists of 100% LNA. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*. Yellow highlighted: mismatch. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 1 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (c) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). $Significant difference with the non-treated ($p < 0.05, $$p < 0.01, $$$p < 0.005). &Significant difference with AON 4 (&p < 0.05, &&p < 0.01, &&&p < 0.005). %Significant difference with AON 11 (%p < 0.05, %%p < 0.01). (d) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. *p < 0.05, **p < 0.01, ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.