Suzanne de Bruijn

154 Chapter 3.2 cleave 2’-O-methyl RNA bases was exploited to improve allele-specificity. In view of the preferred cleavage preference of RNase H1 22 , an asymmetric wing design was preferred for AON 6 (7-10-3), whereas symmetric wings were chosen for AON 7 (5-10-5) ( Figure 1 ). Additionally, the AON 7 gapmer sequence was shifted by one nucleotide compared to AON 2. Treatment with AON 6 and 7 both led to a significant reduction of mutant RIPOR2 transcript levels (33%and 51%, respectively) as compared to treatment with transfection reagent only. Only AON 6 showed allele-specific knockdown as no significant reduction of wildtype transcripts was observed ( Figure 2B ). Figure 1. Design of RIPOR2 -targeting AONs. (A) In silico prediction of the most-probable structure of the mutant RIPOR2 (pre-)mRNA. The 12-nucleotide target deletion breakpoint-nucleotides are marked in red. The mRNA conformation was analyzed using the mfoldWeb Server, which revealed a mixture of open (non-base paired) and closed nucleotides. (B) Design of 20-mer antisense oligonucleotides (AONs) spanning the 12-nucleotide target deletion (dotted lines). AONs 1-3 consist of a complete phosphorothioate (PS)-linked DNA backbone, whereas AONs 6 and 7 are PS-linked gapmer molecules that contain a DNA gap flanked by 2’-O-methyl RNA wings (2’-OMe, depicted in blue).