Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

Sujiet Puthenveetil, Landon Whitby, Jin Ren, Kevin Kelnar, Joseph F. Krebs, Peter A. Beal

Research output: Contribution to journalArticle

58 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4900-4911
Number of pages12
JournalNucleic Acids Research
Volume34
Issue number17
DOIs
StatePublished - Oct 2006
Externally publishedYes

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ASJC Scopus subject areas

  • Genetics

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