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Enhancement of Gene Silencing Effect and Membrane Permeability by Peptide-Conjugated 27-Nucleotide Small Interfering RNA.doc

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Enhancement of Gene Silencing Effect and Membrane Permeability by Peptide-Conjugated 27-Nucleotide Small Interfering RNA

Molecules 2012, 17, 11089-11102; doi:10.3390/molecules170911089 OPEN ACCESS molecules ISSN 1420-3049 /journal/molecules Article Enhancement of Gene Silencing Effect and Membrane Permeability by Peptide-Conjugated 27-Nucleotide Small Interfering RNA Takanori Kubo 1,*, Kazuyoshi Yanagihara 2, Yuichiro Sato 1, Yasuhiro Morita 1 and Toshio Seyama 1 1 Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan; E-Mails: sato-y@yasuda-u.ac.jp (Y.S.); morita-y@yasuda-u.ac.jp (Y.M.); seyama@yasuda-u.ac.jp (T.S.) 2 Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; E-Mail: kyanagih@ncc.go.jp * Author to whom correspondence should be addressed; E-Mail: kubo-t@yasuda-u.ac.jp; Tel.: +81-82-878-9447; Fax: +81-82-878-9540. Received: 29 August 2012; in revised form: 10 September 2012 / Accepted: 11 September 2012 / Published: 14 September 2012 Abstract: Two different sizes of siRNAs, of which one type was 21-nucleotide (nt) siRNA containing 2-nt dangling ends and the other type was 27-nt siRNA with blunt ends, were conjugated with a nuclear export signal peptide of HIV-1 Rev at the 5′-sense end. Processing by Dicer enzyme, cell membrane permeability, and RNAi efficiency of the peptide-conjugated siRNAs were examined. Dicer cleaved the peptide-conjugated 27-nt siRNA leading to the release of 21-nt siRNA, whereas the peptide-conjugated 21-nt siRNA was not cleaved. High membrane permeability and cytoplasmic localization was found in the conjugates. Moreover, the peptide-conjugated 27-nt siRNA showed increased potency of RNAi in comparison with the nonmodified 21-nt and 27-nt siRNAs, whereas the peptide-conjugated 21-nt siRNA showed decreased RNAi efficacy. This potent RNAi efficacy is probably owing to acceleration of RISC through recognition by Dicer, as well as to the improvement of cell membrane perme