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 competition assay with excess small molecule, (+)-JQ1. 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 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 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. Implementing the current state-of-the-art in ASO design, this MALAT1-ASO had a gapmer design, containing a fully PS backbone with terminal wings of five 2′methoxy-ethyl (MOE) sugar modifications (Figure ). In the gapmer design, the central region of DNA oligonucleotides are recognized by RNase H, while the flanks of 2′-modified sugars are RNase H-inactive, but enhance nuclease stability and target binding. As with the SSO, we prepared the (+)-JQ1 conjugate utilising copper-catalysed click chemistry, using the same (+)-JQ1-alkyne and a 5′-azide-modified MALAT1 gapmer. The 5′-azide MALAT1 gapmer was also prepared using azidoacetic acid-NHS ester functionalisation of a 5′-terminally amine-modified gapmer ASO. Following copper-catalysed click chemistry, the (+)-JQ1-MALAT1 gapmer was produced in >90% reaction yields and >95% purity, after HPLC 20 purification (SI Figure ).