LNA, or 2′-O,4′-C-methylene-bridged nucleic acid (2′,4′-BNA), is an artificial nucleic acid developed independently by Wengel’s group and Obika’s group in the late 1990s (Fig. 1a)20, 21, 30. Although LNAs have been used for various gene silencing techniques, e.g. antisense gapmer, short interfering RNA, blocking of microRNA, and triplex-forming oligonucleotides22, 24, 31, 32, it has not been used as an SSO until recently. Singh et al. showed a 14-mer all-LNA oligonucleotide targeting ISS-N1 induced exon inclusion of SMN2 33. In addition, it has been reported that alternating LNA/deoxyribose oligonucleotides, or LNA/DNA mixmers, efficiently induce exon skipping in vitro 23, 34. In contrast to Singh et al.33, SSOs only containing LNAs showed very low ability to induce exon skipping in the DMD exon 5823. Shimo et al. and Yamamoto et al. hypothesized that it is probably because LNA-only-SSOs possess extremely high binding affinity to mRNAs and that the high binding affinities of LNAs reduce the dissociation rate from targeted mRNAs and prevent the efficient turnover of SSOs23, 35. Meanwhile, our data indicated that the proportion and the positions of LNA and DNA in mixmers affected the exon inclusion efficiency. Interestingly, AON #3, which is composed of LNAs with DNA being substituted for LNA at every third nucleotide position, was more effective than AON #2 (DNA substitution at every other nucleotide) and AON #16 (all-LNA oligonucleotide) (Fig. 6). It suggests that the ratio and the position of LNAs and DNAs are critical factors to determine the efficacies of LNA/DNA mixmers.