title: Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA.
abstract: Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA-DNA-LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2'-O-methyl RNA-DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA-DNA-LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1-green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 approximately 70 nM). In contrast, the efficiency of a 2'-O-methyl-modified oligonucleotide (IC50 approximately 220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA-DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.
authors.affiliation: Freie Universität Berlin, Institut für Chemie-Biochemie, Thielallee 63, D-14195 Berlin, Germany.
authors.forename: Arnold
authors.initials: A
authors.lastname: Grünweller
authors.forename: Eliza
authors.initials: E
authors.lastname: Wyszko
authors.forename: Birgit
authors.initials: B
authors.lastname: Bieber
authors.forename: Ricarda
authors.initials: R
authors.lastname: Jahnel
authors.forename: Volker A
authors.initials: VA
authors.lastname: Erdmann
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
mesh_terms: D000818:Animals; D019556:COS Cells; D015870:Gene Expression; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D020128:Inhibitory Concentration 50; D008164:Luminescent Proteins / Q000235:genetics; D008745:Methylation; D009696:Nucleic Acids / Q000737:chemistry / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics*; D020320:Oligoribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D011955:Receptors, Drug / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry / Q000235:genetics; D014162:Transfection
chemical_list: D008164:Luminescent Proteins; D009696:Nucleic Acids; D016376:Oligonucleotides, Antisense; D020320:Oligoribonucleotides, Antisense; D034741:RNA, Small Interfering; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D049452:Green Fluorescent Proteins