To confirm that the improved splice switching was induced by BET protein-mediated nuclear import rather than nonspecific binding to cellular proteins, we carried out a blockade assay with excess small molecule, (+)-JQ1. We hypothesized that excess (+)-JQ1 would saturate BET bromodomain binding sites, blocking (+)-JQ1-SSO conjugate binding and resulting in a loss of activity. At low concentrations of (+)-JQ1 (5 nM) the enhanced activity of the (+)-JQ1-SSO conjugate at 25 and 50 nM was effectively inhibited (Figure ). Consistent with the idea that the (+)-JQ1-ASO conjugate acts via an interaction 35 with BET proteins, even higher concentrations of 200 nM (+)-JQ1 were required to inhibit the enhanced activity of the (+)-JQ1-SSO at 100 nM and 200 nM (Figure , SI Figure ). Thus, we demonstrated that splice-switching ASO activity can be doubled via a specific interaction with the BET proteins for enhanced nuclear import. Once we achieved an improvement in splice-switching activity, we wanted to test whether this approach could be extended to RNase H-mediated gene knockdown (Figure ). Our test system was a 20mer ASO that targets the gold-standard knockdown target metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a nuclear-enriched long non-coding RNA. MALAT1 plays key roles in gene regulation and metastasis in cancer, and is primarily retained within the nucleus. 5