Figure 1. An ASO‐based strategy to identify a short regulatory motif located within intron 7 of human SMN2. (a) Most common chemical modifications of RNA ASOs. All ASOs used in these studies carried these modifications; (b) Initial screening with 15‐mer ASOs (overlapping bars) targeting intron 7 of human SMN2. The ASO (shown in green) restores SMN2 exon 7 inclusion due to blocking of ISS‐N1, a negative element [ 7]; (c) Strategy of ultra‐refined antisense microwalk with small ASOs targeting ISS‐N1. The hnRNP A1 motifs are described in Ref. [ 8], and sequences of ASOs are given in Ref. [ 10]. The green bars represent ASOs that promote SMN2 exon 7 inclusion; the intensity of the green color reflects the strength of the stimulatory effect, while tan bars represent ASOs that have no effect on SMN2 exon 7 inclusion. The area highlighted in pink represent a GC‐rich sequence in the first half of human intron 7; area highlighted in light blue represent the core sequence of the antisense target. Detailed results of concentration‐dependent effect of ASOs on splicing of endogenous SMN2 were reported [ 10]; (d) Splicing pattern of transcripts derived from endogenous SMN2 in SMA type I patient fibroblasts (GM03813) treated with 40 nM of representative ASOs. 3UP8 was the shortest ASO to show a stimulatory response (highlighted by the green box). Sequences of ASOs are given in Table 2; (e) Relative positioning of ISS‐N1, GC‐rich sequence in the context of predicted RNA structure. The green bar represents 3UP8; (f) Validation of the specificity of the antisense effect. The left‐hand panel depicts the diagrammatic representation of intron 7 of SMN2 minigene and its mutant, SMN2/64A. Sequences of ASOs are given in Table 1. The right‐hand panel shows the results of RT‐PCR. HeLa cells were transfected with 50 nM of a given ASO and 0.1 µg of minigene. Splicing products were determined 24 h after transfection. The results were analyzed as described in Ref. [ 10].