Published online 3 February 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 691-699
© 2004 Oxford University Press
Effects on RNAi of the tight structure, sequence and position of the targeted region
1 Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan and 2 Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
*To whom correspondence should be addressed at Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 3 5841 8828 or +81 29 861 3015; Fax: +81 3 5841 8828 or +81 29 861 3019; Email: taira{at}chembio.t.u-tokyo.ac.jp
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
RNA interference (RNAi) is a gene-silencing phenomenon that involves the double-stranded RNA-mediated cleavage of mRNA, and small interfering RNAs (siRNAs) can cause RNAi in mammalian cells. There have been many attempts to clarify the mechanism of RNAi, but information about the relationship between the sequence and structure, in particular, a tight structure, of the target RNA and the activities of siRNAs are limited. In the present study, we examined this relationship by introducing the TAR element, which adopts a very stable secondary structure, at different positions within target RNAs. Our results suggested that the activities of siRNAs were affected by the tight stem–loop structure of TAR. In contrast, the position of the target within the mRNA, the binding of the Tat protein to the TAR, and the location of the target within a translated or a noncoding region had only marginal effects on RNAi. When the target sequence was placed in two different orientations, only one orientation had a significant effect on the activities of siRNA, demonstrating that the presence of certain nucleotides at some specific positions was favorable for RNAi. Systematic analysis of 47 different sites within 47 plasmids under identical conditions indicated that it is the target sequence itself, rather than its location, that is the major determinant of siRNA activity.
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