Extended Data Fig. 7: Drug-ID. NLS-SNAP-eGFP-BASU allows better biotinylation in nucleoli.

Determining localization of ASO and biotin ligase via fluorescence microscopy. Analog to Extended Data Fig. 5, 30 nM of ASO containing either MOE or 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 Extended Data Fig. 5, 10 nM BG-ASO were transfected and 20 nM control ASO were spiked in. a) Colocalization of BG-modified ASOs and NLS-SNAP-eGFP-BASU. In case of transfection with LNA-modified ASO, NLS-SNAP-eGFP-BASU is capable of entering nucleoli in contrast to the enzyme without nuclear localization sequence (Extended Data Fig. 5) (N=1). b) Biotin is deposited in nucleoli using NLS-SNAP-eGFP-BASU (N=1). c)-e) SILAC-MS/MS for BG-ASO (20mer containing either MOE or LNA wings) at 30 nM against Lipofectamine 3000. Plotted was the enrichment (log2) of replicate 1 against replicate 2 with swapped SILAC labels. All proteins significantly (p < 0.05) enriched in both replicates are highlighted and colored according to their RNA- and/or DNA-binding function (blue = RNA binding, orange = DNA binding). Furthermore, selected paraspeckle core proteins (brown) and P-body components (gold) are highlighted. c) Enrichment with 10 nM BG-ASO + 20 nM NH₂-ASO (20mer MOE) over Lipofectamine 3000. d) Enrichment with 10 nM BG-ASO + 20 nM NH₂-ASO (20mer LNA) over Lipofectamine 3000. e) Enrichment with 10 nM BG-ASO + 20 nM NH2-ASO (20mer LNA) over 10 nM BG-ASO + 20 nM NH₂-ASO (20mer MOE).