Moreover, we compared the efficacies between LNA/DNA mixmers and all-LNA oligonucleotides and examined whether LNA-to-DNA substitution could improve the efficacy. When the 13 mer AONs were transfected into the cells at 1 nM, the efficiency of exon inclusion between the mixmers (AONs #4 and #5) and the all-LNA oligonucleotide with the equivalent sequence (AON #9) was not significantly different (Fig. 5b–d). However, when the 18-mer mixmers (AONs #2 and #3) and the all-LNA oligonucleotide with the equivalent sequence (AON #16) were transfected at 0.5 nM, semi-quantitative RT-PCR showed that one of the mixmers (AON #3) had a significantly higher efficacy than the all-LNA oligonucleotide (AON #16) (Fig. 6c). In addition, qPCR revealed that only AON #3 treatment significantly increased the amount of full-length SMN2 compared to the control at 0.5 nM or 1 nM transfection (Fig. 6e). The ratio of full-length to exon 7-deleted SMN2 of AON #3 was significantly larger than that of the all-LNA oligonucleotide (AON #16) at 1 nM transfection (Fig. 6f). These results suggest that the LNA/DNA mixmer could have better efficacies than all-LNA oligonucleotides with an equivalent sequence for SMN2 exon inclusion.Figure 6LNA-to-DNA replacement improves exon 7 inclusion activity. (a) AON sequences used for the experiment. AON #16 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*. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 0.5 or 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. AONs were transfected at 0.5 nM concentration. *p < 0.05. (d) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. AONs were transfected at 1 nM concentration. ***p < 0.005. (e) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data was normalized to non-treated cells. *p < 0.05. (f) Relative expression of full-length SMN2 to ∆7 SMN2 measured by qPCR. The data were normalized to non-treated cells. ***p < 0.005. #$&Significant difference with AON #2, #3, and #16 (1 nM treatment, p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. n.s.: not significant. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.