A naked antisense oligonucleotide with phosphorothioate linkages is taken up intracellularly more efficiently but functions less effectively
A naked antisense oligonucleotide with phosphorothioate linkages is taken up intracellularly more efficiently but functions less effectively
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DOI：
10.1016/j.bbrc.2021.08.035
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发表时间：
2021-08-17
影响因子：
3.1
通讯作者：
Nashimoto，Masayuki
中科院分区：
文献类型：
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作者：
Takahashi，Masayuki;Seki，Mineaki;Nashimoto，Masayuki
我们一直在开发一种基因沉默技术，利用tRNA 3′加工核糖核酸内切酶，tRNase ZL，与反义寡核苷酸。在这里，为了进一步改进这种技术，我们通过流式细胞术和荧光显微镜研究了裸寡核苷酸的长度和修饰如何影响它们被HeLa、HEK 293和HL 60细胞摄取的效率。检查了7-30-nt Alexa-Fluor-568标记的具有硫代磷酸酯键的DNA和7-30-nt Alexa-Fluor-568标记的不具有硫代磷酸酯键的2′-O-甲基化RNA，总体上，较长的寡核苷酸显示出更有效地被细胞内摄取。此外，还检测了不含硫代磷酸酯键的2′-O-甲氧基乙基化RNA、不含硫代磷酸酯键的2′-氟化RNA、含硫代磷酸酯键的2′-O-甲基化RNA和含硫代磷酸酯键和5′/3′-末端核苷酸LNA修饰的2′-O-甲基化RNA。具有硫代磷酸酯键的寡核苷酸比没有键的寡核苷酸更有效地被细胞吸收。此外，我们使用具有和不具有硫代磷酸酯键的22-nt抗miR 16寡核苷酸来检查寡核苷酸的硫代磷酸酯键如何影响其反义效应。后一种寡核苷酸比前一种寡核苷酸更强烈地降低miR 16水平，尽管后者在细胞内的吸收效率低得多。这些观察结果可能不是普遍的，并且根据寡核苷酸和细胞类型的特征而不同。总之，这些结果表明，需要考虑反义寡核苷酸的生产性摄取效率以选择其长度和修饰。
We have been developing a gene silencing technology by harnessing a tRNA 3′ processing endoribonuclease, tRNase ZL, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7–30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7–30-nt Alexa-Fluor-568-labeled, 2′-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2′-O-methoxyethylated RNA without phosphorothioate linkages, a 2′-fluoriated RNA without phosphorothioate linkages, a 2′-O-methylated RNA with phosphorothioate linkages, and a 2′-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5′-/3′-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-ntanti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.