title: Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells.
abstract: RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5' untranslated region (5'UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5'UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2'-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5'UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.
authors: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors: Mariola
authors: M
authors: Dutkiewicz
authors: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors: Agata
authors: A
authors: Ojdowska
authors: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors: Jakub
authors: J
authors: Kuczynski
authors: Institute of Virology, Campus Benjamin Franklin, Charite´-University Medicine, Berlin, Germany.
authors: Vanessa
authors: V
authors: Lindig
authors: Institute of Virology, Campus Benjamin Franklin, Charite´-University Medicine, Berlin, Germany.
authors: Heinz
authors: H
authors: Zeichhardt
authors: Institute of Biotechnology, Department of Applied Biochemistry, Berlin University of Technology, Berlin, Germany.
authors: Jens
authors: J
authors: Kurreck
authors: Department of RNA Biochemistry, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
authors: Jerzy
authors: J
authors: Ciesiołka
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D029822:Enterovirus B, Human / Q000187:drug effects / Q000235:genetics*; D004769:Enterovirus Infections / Q000235:genetics / Q000821:virology; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics*; D016914:Ribonuclease H / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D000998:Antiviral Agents; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D012367:RNA, Viral; D016914:Ribonuclease H