Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway.

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.

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

[2]  Phillip D Zamore,et al.  Ancient Pathways Programmed by Small RNAs , 2002, Science.

[3]  Michael T. McManus,et al.  Small Interfering RNA-Mediated Gene Silencing in T Lymphocytes1 , 2002, The Journal of Immunology.

[4]  A. Fire,et al.  Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[5]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[6]  Phillip D Zamore,et al.  Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. , 2002, Molecular cell.

[7]  Paul Ahlquist,et al.  RNA-Dependent RNA Polymerases, Viruses, and RNA Silencing , 2002, Science.

[8]  Chunja Lee,et al.  The human cytochrome P450 1A1 mRNA is rapidly degraded in HepG2 cells. , 2000, Archives of biochemistry and biophysics.

[9]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

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

[11]  F. Buchholz,et al.  Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Rahaus,et al.  Varicella-zoster Virus (VZV) Mediates a Delayed Host ShutoffIndependent of Open Reading Frame (ORF) 17 Expression , 2004, Virus genes.

[13]  A. Caudy,et al.  Fragile X-related protein and VIG associate with the RNA interference machinery. , 2002, Genes & development.

[14]  Ronald H. A. Plasterk,et al.  RNA Silencing: The Genome's Immune System , 2002, Science.

[15]  H. Prydz,et al.  Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes. , 2000, Nucleic acids research.

[16]  M. Amarzguioui,et al.  Tolerance for mutations and chemical modifications in a siRNA. , 2003, Nucleic acids research.

[17]  T. Miyata,et al.  Regulation of human tissue factor expression by mRNA turnover. , 1993, The Journal of biological chemistry.

[18]  K. Kemphues,et al.  par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed , 1995, Cell.

[19]  P. Zamore,et al.  ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway , 2001, Cell.

[20]  Henning Urlaub,et al.  Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi , 2002, Cell.

[21]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[22]  M. Siomi,et al.  A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. , 2002, Genes & development.

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

[24]  R. Plasterk,et al.  mut-7 of C. elegans, Required for Transposon Silencing and RNA Interference, Is a Homolog of Werner Syndrome Helicase and RNaseD , 1999, Cell.

[25]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[26]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.