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Functional polarity is introduced by Dicer processing of short substrate RNAs

Rose, Scott D. and Kim, Dong-Ho and Amarzguioui, Mohammed and Heidel, Jeremy D. and Collingwood, Michael A. and Davis, Mark E. and Rossi, John J. and Behlke, Mark A. (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Research, 33 (13). pp. 4140-4156. ISSN 0305-1048. http://resolver.caltech.edu/CaltechAUTHORS:ROSnar05

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Abstract

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes (1). Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the ‘right’ (‘R’) and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the ‘left’ (‘L’). Interestingly, the ‘R’ version of the asymmetric 27mer is generally more potent in reducing target gene levels than the ‘L’ version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the ‘R’ form favoring S strand and the ‘L’ form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.


Item Type:Article
Additional Information:© The Author 2005. Published by Oxford University Press. All rights reserved The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oupjournals.org Received May 13, 2005; Revised and Accepted July 6, 2005; Published online 26 July 2005. We thank Brian Elliott for assistance with ESI-MS analysis and Stephanie McConahay for assistance with preparation of the figures. D. Kim is a Beckman Fellow. M. Amarzguioui is a postdoctoral fellow of the Norwegian Research Council. This work was supported in part by a grant from the Arnold and Mabel Beckman Foundation and the National Institutes of Health (AI29329, AI42552 and HL074704 to J.J.R.). Funding to pay the Open Access publication charges for this article was provided by Integrated DNA Technologies, Inc. Conflict of interest statement. Scott D. Rose, Michael A. Collingwood and Mark A. Behlke are employed by Integrated technologies, Inc. (IDT), which has filed at least one patent application on the inventions described in this manuscript, and which offers oligonucleotides for sale similar to the oligonucleotides described in this manuscript. IDT is, however, not a publicly traded company, and Scott D. Rose, Michael A. Collingwood and Mark A. Behlke personally do not own any shares or equity in IDT. None of the authors have any conflicts to declare.
Record Number:CaltechAUTHORS:ROSnar05
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:ROSnar05
Alternative URL:http://dx.doi.org/10.1093/nar/gki732
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:742
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:23 Sep 2005
Last Modified:26 Dec 2012 08:41

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