3' UTR seed matches, but not overall identity, are associated with RNAi off-targets

Nat Methods. 2006 Mar;3(3):199-204. doi: 10.1038/nmeth854.


Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3' untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2-7 or 2-8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

MeSH terms

  • 3' Untranslated Regions / drug effects
  • 3' Untranslated Regions / genetics*
  • Base Pair Mismatch / genetics*
  • Base Pairing / drug effects*
  • Cell Line
  • Cell Survival / drug effects
  • Computational Biology / methods
  • Databases, Factual*
  • Gene Expression Profiling
  • Gene Silencing / drug effects
  • HeLa Cells
  • Humans
  • Numerical Analysis, Computer-Assisted
  • Oligonucleotide Array Sequence Analysis / methods*
  • RNA, Messenger / genetics
  • RNA, Small Interfering / chemical synthesis
  • RNA, Small Interfering / pharmacology*
  • Sensitivity and Specificity
  • Sequence Alignment
  • Silicon / chemistry
  • Transfection


  • 3' Untranslated Regions
  • RNA, Messenger
  • RNA, Small Interfering
  • Silicon