Fig. 4: isASO-ID overcomes intracellular localization limitations of Drug-ID.

a, Microscopy as in Fig. 3e. LNA-modified and MOE-modified ASO (red channel) were transfected at a final concentration of 30 nM (10 nM BG–ASO and 20 nM control ASO) into transgenic HeLa cells expressing SNAP-eGFP-BASU (green channel). White arrows indicate co-localization of Atto594–ASO and the biotin ligase (n = 1). b, SILAC–MS/MS for 10 nM BG–ASO and 20 nM control ASO for the LNA-modified ASO against Lipofectamine 3000. Plotted was the enrichment (log₂) of replicate 1 against replicate 2 with swapped SILAC labeling (n = 2). c, SILAC–MS/MS for 10 nM BG–ASO and 20 nM control ASO for the LNA-modified ASO over the MOE-modified ASO. Plotted was the enrichment (log₂) of replicate 1 against replicate 2 with swapped SILAC labeling. Statistical significance of hits in b and c was calculated with respect to the distance of the median of the distribution of all protein ratios as well as protein intensities using Perseus. Significance was set to P < 0.05 in b and c. d, Schematic overview of isASO-ID experiments. e, Fluorescence microscopy using Halo-BASU-His for biotinylation. Either 25 nM MOE-modified or LNA-modified Halo-ASO (20-mer) was transfected, followed by conjugation of Halo-BASU-His. To visualize ASO localization, 2 nM Atto488-labeled ASO (green channel) was spiked into transfections of BG–ASO. Biotin deposition was stained with Atto594–streptavidin (red channel). Cell nuclei were stained with DAPI (blue channel). White arrows indicate the appearance of structures with co-localization of Atto488–ASO and the biotin signal (n = 2). f, SILAC–MS/MS with 25 nM CA-ASO (20-mer LNA) over Lipofectamine 3000. Plotted was the enrichment (log₂) of replicate 1 against replicate 2 with swapped SILAC labeling. g, Fluorescence microscopy using SNAP-BASU-His for biotinylation. Microscopy was carried out as described in e, transfecting either 25 nM MOE-modified or LNA-modified BG–ASO (20-mer) (n = 2). h, SILAC–MS/MS for 25 nM BG–ASO (20-mer LNA) over Lipofectamine 3000. i, SILAC–MS/MS experiment for comparison of enrichment after transfection with either 30 nM MOE-modified ASO or LNA-modified ASO. In h and i, the normalized enrichment (log₂) of replicate 1 against replicate 2 with swapped SILAC labeling was plotted. rep, replicate.