title: Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.
abstract: Double-stranded short interfering RNAs (siRNA) induce post-transcriptional silencing in a variety of biological systems. In the present study we have investigated the structural requirements of chemically synthesised siRNAs to mediate efficient gene silencing in mammalian cells. In contrast to studies with Drosophila extracts, we found that synthetic, double-stranded siRNAs without specific nucleotide overhangs are highly efficient in gene silencing. Blocking of the 5'-hydroxyl terminus of the antisense strand leads to a dramatic loss of RNA interference activity, whereas blocking of the 3' terminus or blocking of the termini of the sense strand had no negative effect. We further demonstrate that synthetic siRNA molecules with internal 2'-O-methyl modification, but not molecules with terminal modifications, are protected against serum-derived nucleases. Finally, we analysed different sets of siRNA molecules with various 2'-O-methyl modifications for stability and activity. We demonstrate that 2'-O-methyl modifications at specific positions in the molecule improve stability of siRNAs in serum and are tolerated without significant loss of RNA interference activity. These second generation siRNAs will be better suited for potential therapeutic application of synthetic siRNAs in vivo.
authors.affiliation: Atugen AG, Otto Warburg Haus (No. 80), Robert-Roessle-Strasse 10, 13125 Berlin, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Czauderna
authors.forename: Melanie
authors.initials: M
authors.lastname: Fechtner
authors.forename: Sibylle
authors.initials: S
authors.lastname: Dames
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Aygün
authors.forename: Anke
authors.initials: A
authors.lastname: Klippel
authors.forename: Gijsbertus J
authors.initials: GJ
authors.lastname: Pronk
authors.forename: Klaus
authors.initials: K
authors.lastname: Giese
authors.forename: Jörg
authors.initials: J
authors.lastname: Kaufmann
mesh_terms: D004722:Endoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D016376:Oligonucleotides, Antisense / Q000235:genetics; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases / Q000096:biosynthesis / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D034622:RNA Interference; D020871:RNA Stability; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004722:Endoribonucleases; D016376:Oligonucleotides, Antisense; D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D010744:Phosphoric Monoester Hydrolases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C494929:PTEN protein, human