Characterization and optimization of exon skipping oligonucleotide chemistry for human DMD exon 51. Quantification of exon 51 skipping in human Δ48–50 myoblasts treated gymnotically with the indicated oligonucleotide (10 μM A-C; dose response 0.1, 0.3, 1, 3 or 10 μM D,E). (A) Comparison of activities for oligonucleotides with identical sequence and backbone stereochemistry (stereorandom PS at all positions) with variable numbers of 2′-F modifications in termini. DMD-17 is complementary to mouse Dmd exon 23 and serves as non-targeting control in this experiment. (B) Evaluation of the effect of PO linkages on activity of oligonucleotides with identical sequence and 2′ ribose modification pattern **** P < 0.0001, * P < 0.05 One-way ANOVA with post-hoc comparison to DMD-16 (panel A) or to DMD-100 (panel B), Mean ± s.e.m., n = 2. (C) Evaluation of relationship between PO linkages and 2′-ribose modifications in the center of the oligonucleotide and its activity. **** P < 0.0001, ns, non-significant, One-way ANOVA with post-hoc comparison to DMD-16, Mean ± s.e.m., n = 2. (D) Application of Sp PS backbone stereochemistry and exploration of various 2′-ribose modification patterns in the center of the oligonucleotide. **** P < 0.0001, *** P < 0.001, **P < 0.01,* P < 0.05 Two-way ANOVA with post-hoc comparison to DMD-16, Mean ± s.e.m., n = 2. (E) Impact of PO linkages at various backbone positions on activity. **** P < 0.0001, *** P < 0.001, **P < 0.01,* P < 0.05 Two-way ANOVA with post-hoc comparison to DMD-16, Mean ± s.e.m., n = 2.