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. 2012 Sep 14;17(9):11089-102.
doi: 10.3390/molecules170911089.

Enhancement of gene silencing effect and membrane permeability by Peptide-conjugated 27-nucleotide small interfering RNA

Takanori Kubo  1 , Kazuyoshi Yanagihara, Yuichiro Sato, Yasuhiro Morita, Toshio Seyama
Affiliations

Enhancement of gene silencing effect and membrane permeability by Peptide-conjugated 27-nucleotide small interfering RNA

Takanori Kubo et al. Molecules. .

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 permeability and intracellular accumulation.

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Figures

Scheme 1
Scheme 1
Synthesis of peptide-conjugated ssRNAs.
Figure 1
Figure 1
(A) Sequences of siRNAs. Two different sizes of siRNAs (21-nt and 27-nt) were designed and targeted to exogenous Renilla luciferase; (B) Structures of Peptide-siRNAs. HIV-1 Rev NES peptide was covalently attached to the 21-nt and 27-nt siRNAs at the 5′-end of the sense strand; (C) PAGE analysis of 21-nt Peptide-siRNA and 27-nt Peptide-siRNA.
Figure 2
Figure 2
Dicer-substrate siRNAs including peptide conjugates. 21-nt siRNA (A), 27-nt siRNA (B), 21-nt Peptide-siRNA (C), and 27-nt Peptide-siRNA (D) were reacted with the recombinant Dicer enzyme for 12 h at 37°C. The reaction products were electrophoresed on 20% PAGE and visualized by silver staining.
Figure 3
Figure 3
Comparative study of RNAi efficacy of Peptide-siRNAs and nonmodified siRNAs. The Peptide-siRNAs and nonmodified siRNAs (0.5, 1, 2, and 5 nM each) were transfected by LF2000 to HeLa cells, and controls were given only PBS(−). RNAi efficacies of the Peptide-siRNAs and nonmodified siRNAs were evaluated to detect the luminescence of Renilla luciferase activity, which was normalized by the luminescence of Firefly luciferase activity, after 48 h incubation. The mean and SD values are from three independent experiments.
Figure 4
Figure 4
Confocal microscopic images and FACS analysis of HeLa cells incubated for 6 h with 21-nt and 27-nt Peptide-siRNAs (200 nM), including nonmodified siRNAs, labeled with FAM in the presence of LF2000. FL, fluorescence image; Merge, merged image of FL and Transmission; FACS, FACS analysis. In the FACS analysis, the logarithm of the fluorescence intensity is shown on the horizontal axis, and the number of cells is shown on the longitudinal axis. The percentage of cell population separated into four parts (a–d) along with the fluorescence intensity.
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