Hirano Yu, Komatsu Yasuo
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku Sapporo 062-8517 Japan
RSC Adv. 2022 Aug 30;12(38):24471-24477. doi: 10.1039/d2ra04375k.
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.
我们之前报道过，两侧带有双链体的反义寡核苷酸（ASO）能够长时间高效抑制微小RNA（miRNA）的功能。在本研究中，我们检测了双链结构对ASO亚细胞定位的影响。双链体通过交联以防止解离成单链，并且这种交联双链体（CD）连接在靶向miRNA-21的反义寡核苷酸（AS）的5'或3'末端。将荧光标记的ASO转染到细胞中后，分析其亚细胞分布。虽然单链AS分子迅速转移到细胞核，但5'端带有CD的AS（5'CD-AS）有趣地显示出明显更高的细胞质定位。3'-CD修饰的AS（3'CD-AS）从AS的5'端开始降解，但5'端化学修饰可防止这种降解，从而能够对细胞质定位进行成像。CD修饰显著促进了ASO的细胞质定位，并能够有效敲低存在于细胞质中的miRNA。这些结果表明，双链体结构在控制ASO的亚细胞分布方面具有广阔的应用潜力。
