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PMID: 11937629 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

Nucleic acids research ·Vol. 30 ·No. 8 ·2002-04-15 ·Pages 1757-66

Holen T, Amarzguioui M, Wiiger MT, Babaie E, Prydz H

Abstract

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

MeSH Terms
Animals Base Pair Mismatch Base Sequence COS Cells Cell Line Gene Silencing Gene Targeting HeLa Cells Humans Kinetics RNA Processing, Post-Transcriptional RNA, Messenger/analysis RNA, Small Interfering RNA, Untranslated/chemistry,metabolism,pharmacology Thromboplastin/antagonists & inhibitors,biosynthesis,genetics Transfection
Chemicals
RNA, Messenger RNA, Small Interfering RNA, Untranslated Thromboplastin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Holen Torgeir
The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway.
Amarzguioui Mohammed
Wiiger Merete T
Babaie Eshrat
Prydz Hans
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/30.8.1757
Published
2002-04-15
Pages
1757-66
Language
English
Region
England
NLM ID
0411011
PMCID
PMC113209
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/30.8.1757 PMC Full Text View journal details
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