Nucleic Acids Research, 2002, Vol. 30, No. 8 1757-1766
© 2002 Oxford University Press
Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor
The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, N-0349 Oslo, Norway
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.
* To whom correspondence should be addressed. Tel: +47 22 84 05 32; Fax: +47 22 84 05 01; Email: hans.prydz{at}biotek.uio.no The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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X. G. Xia, H. Zhou, H. Ding, E. B. Affar, Y. Shi, and Z. Xu An enhanced U6 promoter for synthesis of short hairpin RNA Nucleic Acids Res., September 1, 2003; 31(17): e100 - e100. [Abstract] [Full Text] [PDF]
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D. Husken, F. Asselbergs, B. Kinzel, F. Natt, J. Weiler, P. Martin, R. Haner, and J. Hall mRNA fusion constructs serve in a general cell-based assay to profile oligonucleotide activity Nucleic Acids Res., September 1, 2003; 31(17): e102 - e102. [Abstract] [Full Text] [PDF]
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Y. Zeng, R. Yi, and B. R. Cullen MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms PNAS, August 19, 2003; 100(17): 9779 - 9784. [Abstract] [Full Text] [PDF]
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R. Kretschmer-Kazemi Far and G. Sczakiel The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides Nucleic Acids Res., August 1, 2003; 31(15): 4417 - 4424. [Abstract] [Full Text] [PDF]
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F. Czauderna, M. Fechtner, S. Dames, H. Aygun, A. Klippel, G. J. Pronk, K. Giese, and J. Kaufmann Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells Nucleic Acids Res., June 1, 2003; 31(11): 2705 - 2716. [Abstract] [Full Text] [PDF]
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T. Holen, M. Amarzguioui, E. Babaie, and H. Prydz Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway Nucleic Acids Res., May 1, 2003; 31(9): 2401 - 2407. [Abstract] [Full Text] [PDF]
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E. A. Bohula, A. J. Salisbury, M. Sohail, M. P. Playford, J. Riedemann, E. M. Southern, and V. M. Macaulay The Efficacy of Small Interfering RNAs Targeted to the Type 1 Insulin-like Growth Factor Receptor (IGF1R) Is Influenced by Secondary Structure in the IGF1R Transcript J. Biol. Chem., April 25, 2003; 278(18): 15991 - 15997. [Abstract] [Full Text] [PDF]
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M. Hemmings-Mieszczak, G. Dorn, F. J. Natt, J. Hall, and W. L. Wishart Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant Rat P2X3 receptor Nucleic Acids Res., April 15, 2003; 31(8): 2117 - 2126. [Abstract] [Full Text] [PDF]
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O. Heidenreich, J. Krauter, H. Riehle, P. Hadwiger, M. John, G. Heil, H.-P. Vornlocher, and A. Nordheim AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells Blood, April 15, 2003; 101(8): 3157 - 3163. [Abstract] [Full Text] [PDF]
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M. Sohail, G. Doran, J. Riedemann, V. Macaulay, and E. M. Southern A simple and cost-effective method for producing small interfering RNAs with high efficacy Nucleic Acids Res., April 1, 2003; 31(7): e38 - e38. [Abstract] [Full Text] [PDF]
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 K. Strange From Genes to Integrative Physiology: Ion Channel and Transporter Biology in Caenorhabditis elegans Physiol Rev, April 1, 2003; 83(2): 377 - 415. [Abstract] [Full Text] [PDF]
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B. A. Kosciolek, K. Kalantidis, M. Tabler, and P. T. Rowley Inhibition of Telomerase Activity in Human Cancer Cells by RNA Interference Mol. Cancer Ther., March 1, 2003; 2(3): 209 - 216. [Abstract] [Full Text] [PDF]
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T. A. Vickers, S. Koo, C. F. Bennett, S. T. Crooke, N. M. Dean, and B. F. Baker Efficient Reduction of Target RNAs by Small Interfering RNA and RNase H-dependent Antisense Agents. A COMPARATIVE ANALYSIS J. Biol. Chem., February 21, 2003; 278(9): 7108 - 7118. [Abstract] [Full Text] [PDF]
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J. G. Doench, C. P. Petersen, and P. A. Sharp siRNAs can function as miRNAs Genes & Dev., February 15, 2003; 17(4): 438 - 442. [Abstract] [Full Text] [PDF]
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M. Scherr, K. Battmer, T. Winkler, O. Heidenreich, A. Ganser, and M. Eder Specific inhibition of bcr-abl gene expression by small interfering RNA Blood, February 15, 2003; 101(4): 1566 - 1569. [Abstract] [Full Text] [PDF]
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P. L. Splinter, A. I. Masyuk, and N. F. LaRusso Specific Inhibition of AQP1 Water Channels in Isolated Rat Intrahepatic Bile Duct Units by Small Interfering RNAs J. Biol. Chem., February 14, 2003; 278(8): 6268 - 6274. [Abstract] [Full Text] [PDF]
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