To confirm that the enhanced ASO activity was a result of increased nuclear accumulation, we quantified the levels of G3139 and (+)-JQ1-G3139 conjugate within the nucleus and cytoplasm using sub-cellular fractionation. To enable simple fluorescent-based detection of ASO localisation, we synthesised a (+)-JQ1-G3139-Cy3 conjugate. The (+)-JQ1-G3139-Cy3 conjugate was synthesised as above, from the commercially available 5'-terminal amine-and 3'-terminal Cy3-modified G3139 PS ASO. A 5'-azide was installed using azideoacetic acid-NHS ester functionalisation, and the final (+)-JQ1-G3139-Cy3 30 conjugate was synthesised through a copper-catalysed click reaction of the 5'-azide-G3139-3'-Cy3 with the (+)-JQ1-alkyne (SI Figure ). We first verified, using RT-qPCR, that the addition of the Cy3 moiety at the 3'-end of G3139 or (+)-JQ1-G3139 did not alter the knockdown activity observed above (SI Figure ). We then transfected the cells with either G3139-Cy3 or (+)-JQ1-Cy3 for 24 hours, followed by sub-cellular fractionation (SI Figure ), and measured the fluorescence intensity of the cytoplasmic and nuclear fractions via a plate reader. We observed a significant increase in nuclear 35 accumulation of the (+)-JQ1-G3139-Cy3 conjugate compared to its unmodified counterpart at both 200 nM and 500 nM (Figure , SI Figure ). Furthermore, the nuclear and cytoplasmic distribution of G3139 and (+)-JQ1-G3139 at 200 nM and 500 nM was consistent with the activity trends measured at the protein and RNA level. Discussion/Future perspectives Our work demonstrates that covalent conjugation of the small molecule (+)-JQ1 to ASOs can enhance their activity in their 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 20 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.