A novel dual lock method for down-regulation of genes, in which a target mRNA is captured at 2 independent positions by linked locked nucleic acid antisense oligonucleotides.
A novel dual lock method for down-regulation of genes, in which a target mRNA is captured at 2 independent positions by linked locked nucleic acid antisense oligonucleotides.
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一种用于下调基因的新型双锁方法，其中通过连接的锁核酸反义寡核苷酸在2个独立位置捕获靶mRNA。
DOI：
10.1080/15476286.2015.1119364
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发表时间：
2016
期刊：
影响因子：
4.1
通讯作者：
and Wada T.
中科院分区：
文献类型：
--
作者：
Takata R.;Makado G.;Kitamura A.;Watanabe H.;and Wada T.
核因子κB（NFκB）由RelA和p50亚基组成，与NFκB反应元件（NRE）结合，刺激炎症相关基因的转录。在此，产生了与RelAmRNA的3′-和5′-非翻译区（UTR）末端互补的锁核酸（LNA）反义寡核苷酸（ASO）;这些分子分别被命名为3′-LNA和5′-LNA。为了评估它们对NFκB活性的影响，将LNA ASO和携带NRE的荧光素酶报告基因共转染HeLa细胞。用3′-LNA转染细胞可使NFκB活性降低30- 40%，而不影响RelAmRNA的积累。同时转染5′-LNA和3′-LNA可使HeLa细胞NFκB活性降低70%。此外，在LNA ASO转染的细胞中发生部分聚（A）尾缩短。我们还使用三甘醇作为间隔基连接5′-LNA和3′-LNA。报告基因分析显示间隔子连接的LNA阿索与5′-LNA和3′-LNA的组合类似地降低NFκB活性。此外，体外翻译试验显示间隔子连接的LNA ASO以特异性方式抑制靶mRNA的翻译。总之，这项研究描述了一种新的反义方法捕获靶mRNA在独立的位置。
Nuclear factor κB (NFκB), which is composed of the RelA and p50 subunits, binds to NFκB response elements (NREs) and stimulates the transcription of inflammation-related genes. Here, locked nucleic acid (LNA) antisense oligonucleotides (ASOs) complementary to the termini of the 3′- and 5′-untranslated regions (UTRs) of theRelAmRNA were generated; these molecules were named 3′-LNA and 5′-LNA, respectively. To evaluate their effects on NFκB activity, HeLa cells were co-transfected with the LNA ASOs and a luciferase reporter gene carrying an NRE. Transfection of the cells with 3′-LNA reduced NFκB activity by 30–40%, without affectingRelAmRNA accumulation. Concomitant transfection of HeLa cells with 5′-LNA and 3′-LNA resulted in a 70% reduction in NFκB activity. Furthermore, partial poly(A) tail shortening occurred in LNA ASO-transfected cells. We also employed triethylene glycol as a spacer to link 5′-LNA and 3′-LNA. Reporter gene assays showed that the spacer-linked LNA ASO reduced NFκB activity similarly to a combination of 5′-LNA and 3′-LNA. In addition, anin vitrotranslation assay revealed that spacer-linked LNA ASOs inhibited the translation of a target mRNA in a specific manner. In summary, this study describes a novel antisense method capturing the target mRNA at independent positions.