Identification of the best chimeric ASO structure. AO2+1 comprised 12 ENA and 6 2′-OMe RNA components. To find the most effective structure of AO2+1, AO2+1 was modified by replacing ENA with 2′-OMe RNA. Ultimately, three derivatives of AO2+1 were synthesized (AO2+1a, AO2+1b, and AO2+1c). The sequences of these ASOs are described in the top left panel. Red uppercase and black lowercase letters represent ENA and 2′-OMe RNA, respectively. Green marking of 2′-OMe RNA indicates the replaced nucleotide. These ASOs were examined for their ability to inhibit the production of mature MSTN mRNA in CRL-2061 cells by RT–PCR amplification (bottom). The amount of product was calculated and is expressed as the ratio of MSTN to GAPDH. The MSTN/GAPDH ratio was lowest in AO2+1-treated cells. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 vs. AO2+1 treatment.