title: 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.
abstract: 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 the RelA mRNA 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 affecting RelA mRNA 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, 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.
authors.affiliation: a Nucleic Acid Regulation (Yoshindo) Joint Research Laboratory and.|b Bioenvironmental Science , Department of Biotechnology, Osaka University , Suita, Osaka , Japan.|c Research and Development, Yoshindo , Haginoshima, Fuchu-machi, Toyama , Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Takata
authors.affiliation: a Nucleic Acid Regulation (Yoshindo) Joint Research Laboratory and.|c Research and Development, Yoshindo , Haginoshima, Fuchu-machi, Toyama , Japan.
authors.forename: Gouki
authors.initials: G
authors.lastname: Makado
authors.affiliation: a Nucleic Acid Regulation (Yoshindo) Joint Research Laboratory and.|b Bioenvironmental Science , Department of Biotechnology, Osaka University , Suita, Osaka , Japan.|c Research and Development, Yoshindo , Haginoshima, Fuchu-machi, Toyama , Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Kitamura
authors.affiliation: a Nucleic Acid Regulation (Yoshindo) Joint Research Laboratory and.|b Bioenvironmental Science , Department of Biotechnology, Osaka University , Suita, Osaka , Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: a Nucleic Acid Regulation (Yoshindo) Joint Research Laboratory and.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Wada
mesh_terms: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D015536:Down-Regulation; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D009841:Oligonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics*; D051996:Transcription Factor RelA / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D016328:NF-kappa B; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C498143:RELA protein, human; D012333:RNA, Messenger; D051996:Transcription Factor RelA; C477371:locked nucleic acid
keywords: Antisense; LNA; dual lock; poly(A); untranslated region