Extended Data Fig. 5: Drug-ID. Localization of the ASO-SNAP-eGFP-BASU conjugate depends on ASO dose and ASO chemistry.

Determining localization of ASO and biotin ligase via fluorescence microscopy. Analog to Supplementary Fig. 5, increasing amounts of BG-ASO (Gapmers containing MOE and LNA wings) were transfected with Lipofectamine 3000 into transgenic HeLa cells expressing SNAP-eGFP-BASU (green channel). To visualize ASO localization, 2 nM Atto594-labeled ASO (red channel) were spiked into transfections of BG-ASO. Nuclei were stained with DAPI (blue channel). As in Supplementary Fig. 5 the concentration of BG-ASO was kept at 10 nM and control ASO (lacking BG) was co-transfected to increase total ASO amount. ASOs not having accessed the nuclei (arrow a), cytoplasmic foci (arrow b), nuclear foci (arrow c), and nucleolar aggregates (arrow d) are indicated with white arrows in all panels. a) In contrast to the 20mer MOE-ASO, the 20mer containing LNA wings accumulated already at 10 nM in nuclear foci and shifts to the nucleoli at the highest concentration (100 nM). b) The 20mer MOE ASO is shown for comparison at a concentration of 30 nM, further data see Supplementary Fig. 5. Transfection with Lipofectamine 3000 served as negative control.