Selection of hyperfunctional siRNAs with improved potency and specificity

Nucleic Acids Res. 2009 Dec;37(22):e152. doi: 10.1093/nar/gkp864.


One critical step in RNA interference (RNAi) experiments is to design small interfering RNAs (siRNAs) that can greatly reduce the expression of the target transcripts, but not of other unintended targets. Although various statistical and computational approaches have been attempted, this remains a challenge facing RNAi researchers. Here, we present a new experimentally validated method for siRNA design. By analyzing public siRNA data and focusing on hyperfunctional siRNAs, we identified a set of sequence features as potency selection criteria to build an siRNA design algorithm with support vector machines. Additional bioinformatics filters were also included in the algorithm to increase RNAi specificity by reducing potential sequence cross-hybridization or microRNA-like effects. Independent validation experiments were performed, which indicated that the newly designed siRNAs have significantly improved performance, and worked effectively even at low concentrations. Furthermore, our cell-based studies demonstrated that the siRNA off-target effects were significantly reduced when the siRNAs were delivered into cells at the 3 nM concentration compared to 30 nM. Thus, the capability of our new design program to select highly potent siRNAs also renders increased RNAi specificity because these siRNAs can be used at a much lower concentration. The siRNA design web server is available at

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Algorithms*
  • Artificial Intelligence
  • Base Composition
  • Cell Line, Tumor
  • Computational Biology
  • Gene Knockdown Techniques
  • Humans
  • Nucleic Acid Conformation
  • RNA Interference*
  • RNA, Small Interfering / chemistry*
  • Sequence Analysis, RNA


  • RNA, Small Interfering