Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions.

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.

[1]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[2]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[3]  Michaela Scherr,et al.  Gene silencing mediated by small interfering RNAs in mammalian cells. , 2003, Current medicinal chemistry.

[4]  T. Tuschl,et al.  Analysis of gene function in somatic mammalian cells using small interfering RNAs. , 2002, Methods.

[5]  V. Erdmann,et al.  Comparative Study of DNA Enzymes and Ribozymes against the Same Full-length Messenger RNA of the Vanilloid Receptor Subtype I* , 2002, The Journal of Biological Chemistry.

[6]  Mouldy Sioud,et al.  Gene silencing by systemic delivery of synthetic siRNAs in adult mice. , 2003, Journal of molecular biology.

[7]  B. Berkhout,et al.  Inhibition of virus replication by RNA interference. , 2003, Journal of biomedical science.

[8]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[9]  Thomas Tuschl,et al.  siRNAs: applications in functional genomics and potential as therapeutics , 2004, Nature Reviews Drug Discovery.

[10]  Sangdun Choi,et al.  Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy , 2005, Nature Biotechnology.

[11]  M. Amarzguioui,et al.  Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. , 2002, Nucleic acids research.

[12]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[13]  Ola Snøve,et al.  Many commonly used siRNAs risk off-target activity. , 2004, Biochemical and biophysical research communications.

[14]  Kazunari Taira,et al.  Effects on RNAi of the tight structure, sequence and position of the targeted region. , 2004, Nucleic acids research.

[15]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[16]  A. Reynolds,et al.  Rational siRNA design for RNA interference , 2004, Nature Biotechnology.

[17]  K Taira,et al.  siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells , 2003, Nucleic acids research.

[18]  S. Fesik,et al.  Specificity of short interfering RNA determined through gene expression signatures , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Sioud.  Therapeutic siRNAs. , 2004, Trends in pharmacological sciences.

[20]  Tyra G. Wolfsberg,et al.  Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Wei Ge,et al.  Synthetic shRNAs as potent RNAi triggers , 2005, Nature Biotechnology.

[22]  Anastasia Khvorova,et al.  RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  K. Ui-Tei,et al.  Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. , 2004, Nucleic acids research.

[24]  V. Erdmann,et al.  Cellular uptake and localization of a Cy3-labeled siRNA specific for the serine/threonine kinase Pim-1. , 2003, Oligonucleotides.

[25]  Robert H. Silverman,et al.  Activation of the interferon system by short-interfering RNAs , 2003, Nature Cell Biology.

[26]  R. Iggo,et al.  Induction of an interferon response by RNAi vectors in mammalian cells , 2003, Nature Genetics.

[27]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[28]  Mamoru Watanabe,et al.  Inhibition of intracellular hepatitis C virus replication by synthetic and vector‐derived small interfering RNAs , 2003, EMBO reports.

[29]  Howard Y. Chang,et al.  Genomewide view of gene silencing by small interfering RNAs , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Heinz,et al.  Small interfering RNA molecules as potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism. , 2004, Antiviral research.

[31]  A. Szallasi,et al.  Biochemical pharmacology of the vanilloid receptor TRPV1. An update. , 2004, European journal of biochemistry.

[32]  V. Erdmann,et al.  Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs. , 2005, Journal of molecular biology.

[33]  M. Amarzguioui,et al.  An algorithm for selection of functional siRNA sequences. , 2004, Biochemical and biophysical research communications.

[34]  C. F. Bennett,et al.  Efficient Reduction of Target RNAs by Small Interfering RNA and RNase H-dependent Antisense Agents , 2003, The Journal of Biological Chemistry.

[35]  G. Sczakiel,et al.  Local RNA target structure influences siRNA efficacy: a systematic global analysis. , 2005, Journal of molecular biology.

[36]  E. Southern,et al.  The Efficacy of Small Interfering RNAs Targeted to the Type 1 Insulin-like Growth Factor Receptor (IGF1R) Is Influenced by Secondary Structure in the IGF1R Transcript* , 2003, The Journal of Biological Chemistry.

[37]  Vivek Mittal,et al.  Improving the efficiency of RNA interference in mammals , 2004, Nature Reviews Genetics.

[38]  A. Szallasi,et al.  Biochemical pharmacology of the vanilloid receptor TRPV1 , 2004 .

[39]  Georg Sczakiel,et al.  The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. , 2003, Nucleic acids research.

[40]  J. Kurreck.  Antisense and RNA interference approaches to target validation in pain research. , 2004, Current opinion in drug discovery & development.

[41]  B. Li,et al.  Expression profiling reveals off-target gene regulation by RNAi , 2003, Nature Biotechnology.

[42]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[43]  Huifang M. Zhang,et al.  Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand , 2005, Journal of Virology.

[44]  V. Erdmann,et al.  Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA, phosphorothioates and small interfering RNA. , 2003, Nucleic acids research.