Analysis of siRNA specificity on targets with double-nucleotide mismatches

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ∼35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.

[1]  Tomoyuki Yamada,et al.  dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference , 2005, Nucleic Acids Res..

[2]  Claes Wahlestedt,et al.  A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites , 2005, Nucleic acids research.

[3]  L. Lim,et al.  Widespread siRNA "off-target" transcript silencing mediated by seed region sequence complementarity. , 2006, RNA.

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

[5]  Anastasia Khvorova,et al.  Corrigendum: 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets , 2007, Nature Methods.

[6]  John J Rossi,et al.  Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase , 2004, Nature Biotechnology.

[7]  N. Caplen,et al.  Mismatched siRNAs downregulate mRNAs as a function of target site location , 2006, FEBS letters.

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

[9]  A. Aigner.  Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo , 2007, Applied Microbiology and Biotechnology.

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

[11]  J. Schelter,et al.  Designing siRNA That Distinguish between Genes That Differ by a Single Nucleotide , 2006, PLoS genetics.

[12]  C. Wahlestedt,et al.  No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA. , 2006, Biochemical and biophysical research communications.

[13]  J. Rossi,et al.  Progress and prospects: RNA-based therapies for treatment of HIV infection , 2007, Gene Therapy.

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

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

[16]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

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

[18]  Anastasia Khvorova,et al.  3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets , 2006, Nature Methods.

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

[20]  M. Woodle,et al.  Modulation of angiogenesis with siRNA inhibitors for novel therapeutics , 2005, Trends in Molecular Medicine.

[21]  Terran Lane,et al.  A computational study of off-target effects of RNA interference , 2005, Nucleic acids research.

[22]  Michael R. Green,et al.  Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs). , 2004, RNA.

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

[24]  C. Wahlestedt,et al.  Validating siRNA using a reporter made from synthetic DNA oligonucleotides. , 2004, Biochemical and biophysical research communications.