Off-target Effects by SiRNA Can Induce Toxic Phenotype

Overview

Journal RNA

Specialty Molecular Biology

Date 2006 May 10

PMID 16682561

Citations 196

Authors

Yuriy Fedorov

Emily M Anderson

Amanda Birmingham

Angela Reynolds

Jon Karpilow

Kathryn Robinson

Devin Leake

William S Marshall

Anastasia Khvorova

Affiliations

Soon will be listed here.

Abstract

Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.

Citing Articles

Landscape of small nucleic acid therapeutics: moving from the bench to the clinic as next-generation medicines.

Liu M, Wang Y, Zhang Y, Hu D, Tang L, Zhou B Signal Transduct Target Ther. 2025; 10(1):73.

PMID: 40059188 PMC: 11891339. DOI: 10.1038/s41392-024-02112-8.

From Origin to the Present: Establishment, Mechanism, Evolutions and Biomedical Applications of the CRISPR/Cas-Based Macromolecular System in Brief.

Yuan Z Molecules. 2025; 30(4).

PMID: 40005257 PMC: 11858448. DOI: 10.3390/molecules30040947.

RNAi-based biocontrol for crops: a revised expectation for a non-recent technology.

Septiani P, Pramesti Y, Ghildan M, Aprilia K, Awaludin R, Medina S Planta. 2025; 261(2):44.

PMID: 39862243 DOI: 10.1007/s00425-025-04625-0.

Targeting NUCKS1 with a fragment of tRNA of Chinese yew for the treatment of colorectal cancer.

Cao K, Zhang D, Bai L, Yan T, Chen Y, Jiang Y Noncoding RNA Res. 2024; 11:38-47.

PMID: 39736854 PMC: 11683283. DOI: 10.1016/j.ncrna.2024.11.002.

RNA Interference Applied to Crustacean Aquaculture.

Fajardo C, De Donato M, Macedo M, Charoonnart P, Saksmerprome V, Yang L Biomolecules. 2024; 14(11).

PMID: 39595535 PMC: 11592254. DOI: 10.3390/biom14111358.

References

Gao F, Carson C, Levine T, Keene J . Selection of a subset of mRNAs from combinatorial 3' untranslated region libraries using neuronal RNA-binding protein Hel-N1. Proc Natl Acad Sci U S A. 1994; 91(23):11207-11. PMC: 45196. DOI: 10.1073/pnas.91.23.11207. View

Meister G, Landthaler M, Patkaniowska A, Dorsett Y, Teng G, Tuschl T . Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell. 2004; 15(2):185-97. DOI: 10.1016/j.molcel.2004.07.007. View

Hartsel S, Kitchen D, Scaringe S, Marshall W . RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry. Methods Mol Biol. 2004; 288:33-50. DOI: 10.1385/1-59259-823-4:033. View

Shimizu N, Ohta M, Fujiwara C, Sagara J, Mochizuki N, Oda T . Expression of a novel immediate early gene during 12-O-tetradecanoylphorbol-13-acetate-induced macrophagic differentiation of HL-60 cells. J Biol Chem. 1991; 266(19):12157-61. View

Haley B, Zamore P . Kinetic analysis of the RNAi enzyme complex. Nat Struct Mol Biol. 2004; 11(7):599-606. DOI: 10.1038/nsmb780. View

Elbashir S, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T . Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 2001; 20(23):6877-88. PMC: 125328. DOI: 10.1093/emboj/20.23.6877. View

Jing Q, Huang S, Guth S, Zarubin T, Motoyama A, Chen J . Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell. 2005; 120(5):623-34. DOI: 10.1016/j.cell.2004.12.038. View

Levine T, Gao F, King P, Andrews L, Keene J . Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs. Mol Cell Biol. 1993; 13(6):3494-504. PMC: 359819. DOI: 10.1128/mcb.13.6.3494-3504.1993. View

Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S . Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 2005; 11(3):263-70. DOI: 10.1038/nm1191. View

10.

King P . RNA-binding analyses of HuC and HuD with the VEGF and c-myc 3'-untranslated regions using a novel ELISA-based assay. Nucleic Acids Res. 2000; 28(7):E20. PMC: 102803. DOI: 10.1093/nar/28.7.e20. View

11.

Jackson A, Burchard J, Leake D, Reynolds A, Schelter J, Guo J . Position-specific chemical modification of siRNAs reduces "off-target" transcript silencing. RNA. 2006; 12(7):1197-205. PMC: 1484422. DOI: 10.1261/rna.30706. View

12.

Lin X, Ruan X, Anderson M, McDowell J, Kroeger P, Fesik S . siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 2005; 33(14):4527-35. PMC: 1184219. DOI: 10.1093/nar/gki762. View

13.

Lim L, Lau N, Garrett-Engele P, Grimson A, Schelter J, Castle J . Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005; 433(7027):769-73. DOI: 10.1038/nature03315. View

14.

Collins M, Irvine B, Tyner D, Fine E, Zayati C, Chang C . A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml. Nucleic Acids Res. 1997; 25(15):2979-84. PMC: 146852. DOI: 10.1093/nar/25.15.2979. View

15.

Reynolds A, Leake D, Boese Q, Scaringe S, Marshall W, Khvorova A . Rational siRNA design for RNA interference. Nat Biotechnol. 2004; 22(3):326-30. DOI: 10.1038/nbt936. View

16.

Judge A, Sood V, Shaw J, Fang D, McClintock K, MacLachlan I . Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 2005; 23(4):457-62. DOI: 10.1038/nbt1081. View

17.

Jackson A, Bartz S, Schelter J, Kobayashi S, Burchard J, Mao M . Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol. 2003; 21(6):635-7. DOI: 10.1038/nbt831. View

18.

Birmingham A, Anderson E, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y . 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods. 2006; 3(3):199-204. DOI: 10.1038/nmeth854. View