siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.

Abstract:

:A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three 'top hits' from our screen have been demonstrated to result from off-target gene silencing. Two of the three 'siRNA hits' were found to directly trigger down-regulation of hif-1alpha mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1alpha mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1alpha was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3'-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving 'hits-follow-up' strategies for future large-scale knockdown experiments.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Lin X,Ruan X,Anderson MG,McDowell JA,Kroeger PE,Fesik SW,Shen Y

doi

10.1093/nar/gki762

keywords:

subject

Has Abstract

pub_date

2005-08-09 00:00:00

pages

4527-35

issue

14

eissn

0305-1048

issn

1362-4962

pii

33/14/4527

journal_volume

33

pub_type

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