We synthesised the (+)-JQ1-G3139 conjugate using the same copper-catalysed click methodology as the previous ASOs. The 5′-azide-G3139 was synthesised from the commercially-obtained 5′-amine PS ASO using azideoacetic acid-NHS ester functionalisation, and the (+)-JQ1 conjugate was synthesised through a copper-catalysed click reaction with the (+)-JQ1-20 alkyne (SI Figure ). We transfected ASOs into HEK293T cells and measured the BCL-2 transcript and protein levels, using RT-qPCR and western blotting, respectively, after 24 hours. Our (+)-JQ1-G3139 conjugate dramatically outperformed the unconjugated G3139 ASO at all concentrations tested. We measured 43.9%, 51.3%, 50.9%, and 64.5% less transcript using the conjugated (+)-JQ1-G3139 compared to the unconjugated G3139, at 50, 100, 200, and 500 nM respectively (Figure ). We also observed a concomitant marked reduction in comparative protein levels, especially at lower ASO concentrations (Figure ). We then conducted a competition assay using excess small molecule (+)-JQ1, similar to the approach used with the other conjugates. At 5 nM (+)-JQ1, the enhanced activity of (+)-JQ1-G3139 at 100 nM showed a significant reduction, and at 200 nM (+)-JQ1, the enhanced activity of (+)-JQ1-G3139 was fully inhibited. As observed with the previous (+)-JQ1 conjugates, no increase in cellular toxicity was observed for (+)-JQ1-G3139, compared to the unconjugated ASO (SI Figure ). two most prominent mechanisms of action: splice switching and RNase H-mediated gene knockdown. These findings build upon the previous literature surrounding the development of (+)-JQ1 as a nuclear importer. Our data underscores the importance of precisely targeting therapeutic agents to their site of action. By inducing ASO enrichment in the nucleus, we have improved their functional efficacy and therapeutic potential. In this way, we have shown that nuclear localization is crucial for maximizing the therapeutic potential of ASOs for splice switching and RNase H-mediated knockdown.