Nature Methods
- 3, 199 - 204 (2006)
Published online: 17 February 2006; | doi:10.1038/nmeth854
There is an Addendum (June 2006) associated with this Article.
There is a Corrigendum (June 2007) associated with this Article.
3' UTR seed matches, but not overall identity, are associated with RNAi off-targetsAmanda Birmingham1, Emily M Anderson1, Angela Reynolds1, Diane Ilsley-Tyree2, Devin Leake1, Yuriy Fedorov1, Scott Baskerville1, Elena Maksimova1, Kathryn Robinson1, Jon Karpilow1, William S Marshall1 & Anastasia Khvorova11
Dharmacon Research, 2650 Crescent Drive, #100, Lafayette, Colorado 80026, USA. 2
Agilent Technologies, 3500 Deer Creek Rd., MS 25U-7, Palo Alto, California 94034, USA.
Correspondence should be addressed to Anastasia Khvorova khvorova.a@dharmacon.com 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.
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