title: Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions.
abstract: Contradictory reports in the literature have emphasised either the sequence of small interfering RNAs (siRNA) or the structure of their target molecules to be the major determinant of the efficiency of RNA interference (RNAi) approaches. In the present study, we analyse systematically the contributions of these parameters to siRNA activity by using deliberately designed mRNA constructs. The siRNA target sites were included in well-defined structural elements rendering them either highly accessible or completely involved in stable base-pairing. Furthermore, complementary sequence elements and various hairpins with different stem lengths and designs were used as target sites. Only one of the strands of the siRNA duplex was found to be capable of silencing via its respective target site, indicating that thermodynamic characteristics intrinsic to the siRNA strands are a basic determinant of siRNA activity. A significant obstruction of gene silencing by the same siRNA, however, was observed to be caused by structural features of the substrate RNA. Bioinformatic analysis of the mRNA structures suggests a direct correlation between the extent of gene-knockdown and the local free energy in the target region. Our findings indicate that, although a favourable siRNA sequence is a necessary prerequisite for efficient RNAi, complex target structures may limit the applicability even of carefully chosen siRNAs.
authors.affiliation: Free University Berlin, Institute for Chemistry (Biochemistry), Thielallee 63, 14195 Berlin, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schubert
authors.forename: Arnold
authors.initials: A
authors.lastname: Grünweller
authors.forename: Volker A
authors.initials: VA
authors.lastname: Erdmann
authors.forename: Jens
authors.initials: J
authors.lastname: Kurreck
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D015195:Drug Design; D004770:Enterovirus / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005818:Genetic Engineering; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D051573:Proto-Oncogene Proteins c-pim-1; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C496637:Pim1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1