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

作者：Takata Ryohei; Makado Gouki; Kitamura Ayaka; Watanabe Hajime; Wada Tadashi^*

来源：RNA Biology, 2016, 13(3): 279-289.

DOI：10.1080/15476286.2015.1119364

• untranslated region
• LNA
• dual lock
• poly(A)
• Antisense

Nuclear factor kappa B (NF kappa B), which is composed of the RelA and p50 subunits, binds to NF kappa B 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) ofthe RelAmRNA were generated; these molecules were named 3 '-LNA and 5 '-LNA, respectively. To evaluate their effects on NF kappa B 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 NF kappa B activity by 30-40%, without affectingRelAmRNA accumulation. Concomitant transfection of HeLa cells with 5 '-LNA and 3 '-LNA resulted in a 70% reduction in NF kappa B 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 NF kappa B activity similarly to a combination of 5 '-LNA and 3 '-LNA. In addition, an in vitro translation 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.

• 出版日期2016-3-3