“…As model sequences, we selected SSOs previously developed to treat EPP. , To quantify the oligonucleotides in cells and subcellular compartments following free uptake, we employed CL-qPCR, ,, a technique we established as superior to Splint ligation-qPCR at detecting an ASO with MOE PS chemistry (Figure C,D). Using CL-qPCR, we detected approximately 50,000 to 200,000 ASO molecules per nucleus following free uptake at low micromolar concentrations for 24 h (Table S2), which is consistent with previous reports. , Then, we covalently conjugated different NLS peptides to a lead MOE PS oligonucleotide using thiol–maleimide chemistry. , Using CL-qPCR, we identified one conjugate with better nuclear accumulation relative to the parent SSO (Figures E, E). However, in contrast to the parent, unconjugated SSO, the conjugates were inactive at splice correction under free uptake conditions in vitro .…”