3' UTR seed matches, but not overall identity, are associated with RNAi off-targets.
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.
Birmingham
Amanda
A
Dharmacon Research, 2650 Crescent Drive, #100, Lafayette, Colorado 80026, USA.
Anderson
Emily M
EM
Reynolds
Angela
A
Ilsley-Tyree
Diane
D
Leake
Devin
D
Fedorov
Yuriy
Y
Baskerville
Scott
S
Maksimova
Elena
E
Robinson
Kathryn
K
Karpilow
Jon
J
Marshall
William S
WS
Khvorova
Anastasia
A
D020413:3' Untranslated Regions / Q000187:drug effects / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics*; D020029:Base Pairing / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D019295:Computational Biology / Q000379:methods; D016208:Databases, Factual; D020869:Gene Expression Profiling; D020868:Gene Silencing / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D009716:Numerical Analysis, Computer-Assisted; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D012825:Silicon / Q000737:chemistry; D014162:Transfection
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012825:Silicon