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

• Published in: Nature methods Vol. 3; no. 3; pp. 199 - 204
• Main Authors: Birmingham, Amanda, Anderson, Emily M, Reynolds, Angela, Ilsley-Tyree, Diane, Leake, Devin, Fedorov, Yuriy, Baskerville, Scott, Maksimova, Elena, Robinson, Kathryn, Karpilow, Jon, Marshall, William S, Khvorova, Anastasia
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.03.2006; Nature Publishing Group
• Subjects: 3' Untranslated Regions - drug effects; 3' Untranslated Regions - genetics; Algorithms; Base Pair Mismatch - genetics; Base Pairing - drug effects; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Cell Line; Cell Survival - drug effects; Computational Biology - methods; Databases, Factual; Design; Gene expression; Gene Expression Profiling; Gene Silencing - drug effects; Genetic transcription; HeLa Cells; Humans; Life Sciences; Numerical Analysis, Computer-Assisted; Oligonucleotide Array Sequence Analysis - methods; Physiological aspects; Proteomics; RNA, Messenger - genetics; RNA, Small Interfering - chemical synthesis; RNA, Small Interfering - pharmacology; Sensitivity and Specificity; Sequence Alignment; Silicon - chemistry; Transfection; United States; United States > US
• ISSN: 1548-7091; 1548-7105
• 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.