Nucleic Acids Research Advance Access originally published online on September 18, 2006
Nucleic Acids Research 2006 34(17):4900-4911; doi:10.1093/nar/gkl464
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Nucleic Acids Research, 2006, Vol. 34, No. 17 4900-4911
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification
Department of Chemistry, University of Utah Salt Lake City, UT 84112, USA 1 Ambion, Inc. 2130 Woodward, Austin, TX 78744, USA
*To whom correspondence should be addressed. Fax: +1 801 581 8433; Email: beal{at}chemistry.utah.edu
Received May 25, 2006. Revised June 18, 2006. Accepted June 19, 2006.
The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3'-overhangs, whereas the maximum activation observed for poly(I)•poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N2-benzyl-2'-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.
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