Supplementary Figure 11: Analysis of off-target editing sites with increased editing yield upon ASO treatment.

a) Besides the targeted site in GAPDH, 9 off-target editing sites were identified in ASO + IFN-α-treated cells compared to the control (no ASO + IFN-α). Six of them (CHARC1, SOD2 #1-#5) were known editing sites and found to be already edited in the control, N=2 independent experiments. b) and c) The regions around the off-target sites were aligned to the ASO-interacting region (40 nt) of the GAPDH transcript using MUSCLE (ebi.ac.uk/tools/msa/muscle/). The red A indicates the edited site and nucleotides matching to the target sequence of the ASO in GAPDH are highlighted in turquois. The sequence alignment suggests that the editing at the three novel editing sites (PRR11, GPR64, EFHD2) is clearly induced by misguiding through the ASO. Notably, the strongest off-target (PRR11) might be controllable by further chemical modification of the specificity domain of the ASO. Four of the nine off-target sites (SOD2 #2-5) lack any strong homology to target region, also the edited codon is different from 5´-UAG. This makes it very unlikely that the off-target editing at such sites was induced by the ASO via direct binding to the off-target site, also because those sites all reside in secondary RNA structure (Alu elements). However, we found a potential ASO binding site in the 3´-UTR of SOD2 (panel d) around nt 2100ff. (refering to NM_000636.3) that resides around 300 nt 5´ to the first Alu element (nt 2380-2670) and around 1300 nt 5´ to the second Alu element (nt 3400-3525). Since all SOD2 off-target sites reside in the two Alu elements one could imagine an ASO-dependent induction of the editing by either increase of the local ADAR concentration or by assisting the formation of an editable RNA secondary structure in the Alu element.