Sequence, Chemical, and Structural Variation of Small Interfering RNAs and Short Hairpin RNAs and the Effect on Mammalian Gene Silencing
Abstract
Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpi…
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Increasing the PS content of individual siRNA duplexes to 80% can limit RISC efficacy and increase toxicity.
“However, increasing the PS content of individual siRNA duplexes to 80% can limit RNA-induced silencing complex (RISC) efficacy 17, 18 and increase toxicity19, 20.”
From A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system (2019) · cites this paper as [20]
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- Authors
- Harborth, J., Elbashir, S. M., Vandenburgh, K., Manninga, H., Scaringe, S. A., Weber, K., & Tuschl, T.
- Affiliations
- Max Planck Institute for Biophysical Chemistry (DE)
- Max Planck Society (DE)
- Venue
- Antisense and Nucleic Acid Drug Development
- Publisher
- SAGE Publications
- Published
- 2003-04
- Volume
- 13
- Issue
- 2
- Pages
- 83-105
- ISSN
- 1087-2906 (ISSN-L)
- OpenAlex
- W2023246459
- Cited by
- 474
- References
- 1
- Citations by year
- 2026: 7 · 2025: 7 · 2024: 8 · 2023: 8 · 2022: 4 · 2021: 9 · 2020: 11 · 2019: 6 · 2018: 7 · 2017: 18 · 2016: 11 · 2015: 17 · 2014: 21 · 2013: 24 · 2012: 32
- FWCI
- 16.09
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Antisense and Nucleic Acid Drug Development (publishedVersion)
- PubMed (publishedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Keywords
- siRNA, shRNA, RNA interference, gene silencing, positional variation, lamin A/C, siRNA duplexes, stem length, HeLa cells
- MeSH terms
- Animals, Base Sequence, Cell Survival, HeLa Cells, Humans, Microscopy, Fluorescence, Models, Chemical, Molecular Sequence Data, Pyrimidines, RNA, Sequence Homology, Nucleic Acid, Thionucleotides, Transfection, Blotting, Western, RNA, Catalytic, Open Reading Frames, Oligonucleotides, Antisense, Protein Isoforms, Reverse Transcriptase Polymerase Chain Reaction, Gene Silencing, RNA, Small Interfering, Lamin Type A, Swiss 3T3 Cells, Cell Line, Tumor, Mice
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