Systematic evaluation of 2′- <i>O</i> -methyl RNA antisense oligonucleotides with limited phosphorothioate linkages for efficient splice switching

Herein, we report a systematic exploration of 2'-<i>O</i>-methyl (2'-OMe) antisense oligonucleotides (ASOs) with a stereo-random phosphodiester-phosphorothioate (PO-PS) backbone regarding their design, RNA binding affinity, nuclease resistance, and exon skipping efficacy. The results showed that ASOs having PO linkages arranged at the 3'-end exhibited higher exon skipping efficacy and slightly higher target binding affinity than their counterparts with PO linkages arranged at the 5'-end; and the residual length of ASOs can be still well protected from 3'-exonuclease after initial hydrolysis of as many as four successive PO linkages from the 3'-end. These findings provide insights and guidance for rational design of splice-switching ASOs with limited PS linkages.