Rational siRNA design for RNA interference
Abstract
Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
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Potent siRNA sequences had been designed and validated against Ppib elsewhere.
“Potent siRNA sequences had also been designed and validated against Ppib elsewhere [28].”
From Elucidating brain transport pathways and cell type-dependent gene silencing of a durable lipid–siRNA conjugate administered into cerebrospinal fluid (2025) · cites this paper as [28]
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“Potent siRNA sequences had also been designed and validated against Ppib elsewhere [ 28 ].”
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- Authors
- Reynolds, A., Leake, D., Boese, Q., Scaringe, S., Marshall, W. S., & Khvorova, A.
- Corresponding author
- Khvorova, Anastasia
- ORCID
- Khvorova, Anastasia
- Venue
- Nature Biotechnology
- Published
- 2004-02-01
- Volume
- 22
- Issue
- 3
- Pages
- 326-330
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- OpenAlex
- W2166802510
- Cited by
- 2,094
- References
- 0
- Citations by year
- 2026: 41 · 2025: 69 · 2024: 67 · 2023: 52 · 2022: 75 · 2021: 58 · 2020: 61 · 2019: 48 · 2018: 67 · 2017: 61 · 2016: 59 · 2015: 88 · 2014: 97 · 2013: 81 · 2012: 135
- FWCI
- 61.02
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (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 › DNA and Nucleic Acid Chemistry
- Keywords
- siRNA design, RNA interference, RNA-induced silencing complex, GC content, inverted repeats, gene knockdown
- MeSH terms
- Animals, Base Sequence, Diptera, Genetic Engineering, Humans, Luciferases, Molecular Sequence Data, Sequence Alignment, Sequence Analysis, RNA, Peptidylprolyl Isomerase, Cyclophilins, RNA Interference, RNA, Small Interfering
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