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Nucleic Acids Research Advance Access published online on January 21, 2008
Nucleic Acids Research, doi:10.1093/nar/gkm1043
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© 2007 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.

Methods Online

Short-hairpin RNAs synthesized by T7 phage polymerase do not induce interferon

Takuma Gondai1, Kazuya Yamaguchi1, Naoko Miyano-Kurosaki1,2, Yuichirou Habu1,3 and Hiroshi Takaku1,2,*

1Department of Life and Environmental Science, 2High Technology Research Center, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-0016 Japan, and 3Japanese Foundation for AIDS Prevention, 1-3-12 Misakicho, Chiyoda, Tokyo 101-0061, Japan

*To whom correspondence should be addressed: Tel: +81 47 478 0407; Fax: +81 47 471 8764; Email: hiroshi.takaku{at}it-chiba.ac.jp

Received April 5, 2007. Revised October 31, 2007. Accepted November 1, 2007.

RNA interference (RNAi) mediated by small-interfering RNAs (siRNAs) is a highly effective gene-silencing mechanism with great potential for gene-therapeutic applications. siRNA agents also exert non-target-related biological effects and toxicities, including immune-system stimulation. Specifically, siRNA synthesized from the T7 RNA polymerase system triggers a potent induction of type-I interferon (IFN) in a variety of cells. Single-stranded RNA also stimulates innate cytokine responses in mammals. We found that pppGn (n = 2,3) associated with the 5'-end of the short-hairpin RNA (shRNA) from the T7 RNA polymerase system did not induce detectable amounts of IFN. The residual amount of guanine associated with the 5'-end and hairpin structures of the transcript was proportional to the reduction of the IFN response. Here we describe a T7 pppGn (n = 2,3) shRNA synthesis that does not induce the IFN response, and maintains the full efficacy of siRNA.

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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