3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets
Abstract
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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siRNA off-target effects are predominantly due to seed (position 2-7 of the guide strand) complementarity in the 3' UTR of other targets.
“siRNA off-target effects are predominantly due to seed (position 2–7 of the guide strand) complementarity in the 3’ UTR of other targets39.”
From A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system (2019) · cites this paper as [39]
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- Authors
- Birmingham, A., Anderson, E. M., Reynolds, A., Ilsley-Tyree, D., Leake, D., Fedorov, Y., Baskerville, S., Maksimova, E., Robinson, K., Karpilow, J., Marshall, W. S., & Khvorova, A.
- Affiliations
- Agilent Technologies (United States) (US)
- Corresponding author
- Khvorova, Anastasia
- Venue
- Nature Methods
- Published
- 2006-02-17
- Volume
- 3
- Issue
- 3
- Pages
- 199-204
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2043828851
- Cited by
- 920
- References
- 1
- Citations by year
- 2026: 21 · 2025: 31 · 2024: 33 · 2023: 24 · 2022: 47 · 2021: 26 · 2020: 20 · 2019: 34 · 2018: 30 · 2017: 31 · 2016: 16 · 2015: 53 · 2014: 47 · 2013: 45 · 2012: 72
- FWCI
- 34.72
- Type
- journal article
- Language
- en
- Update notices
- Open access
- closed
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- siRNA design, 3' untranslated region, seed region, antisense strand, gene silencing
- MeSH terms
- Cell Line, Cell Survival, HeLa Cells, Humans, Numerical Analysis, Computer-Assisted, RNA, Messenger, Sensitivity and Specificity, Silicon, Transfection, Databases, Factual, Sequence Alignment, Computational Biology, Base Pairing, Base Pair Mismatch, Oligonucleotide Array Sequence Analysis, 3' Untranslated Regions, Gene Silencing, Gene Expression Profiling, RNA, Small Interfering
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