Promotion of cytoplasmic localization of oligonucleotides by connecting cross-linked duplexes.

We previously reported that antisense oligonucleotides (ASOs) flanked by duplexes can suppress microRNA (miRNA) function with high efficiency for a long duration. In this study, we examined the effect of the double-stranded structure on the subcellular localization of ASOs. Double strands were cross-linked to prevent dissociation into single strands, and this cross-linked duplex (CD) was connected at the 5' or 3' termini of an antisense-targeting miRNA-21 (AS). The subcellular distribution of fluorescently labelled ASOs was analyzed following transfection into cells. While single-stranded AS molecules promptly moved to the nucleus, AS with the CD at the 5'-end (5'CD-AS) interestingly showed significantly higher cytoplasmic localization. The 3'-CD-modified AS (3'CD-AS) was degraded from the 5'-end of the AS, but the degradation was prevented by 5'-end chemical modifications, thereby allowing the imaging of the cytoplasmic localization. The CD modification significantly promoted the cytoplasmic localization of ASOs and enabled the effective knockdown of miRNA existing in cytoplasm. These results reveal that the duplex structure has promising potential to control the subcellular distribution of ASOs.