Receptor-mediated delivery of siRNAs by tethered nucleic acid base-paired interactions - PubMed (original) (raw)

Receptor-mediated delivery of siRNAs by tethered nucleic acid base-paired interactions

Kexiong Zhang et al. RNA. 2008 Mar.

Abstract

We report a new strategy for cell-type-specific delivery of functional siRNAs into cells. The method involves the noncovalent attachment of siRNAs to ligand-conjugated oligodeoxynucleotides via nucleic acid base-paired interactions. The resulting complexes can be directly applied to cells, leading to specific cellular uptake and gene silencing. The method is simple, economical, and can be easily adapted for other cell surface receptors. Here we show the application of this method for the delivery of siRNAs to folate receptor-expressing cells.

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Figures

FIGURE 1.

FIGURE 1.

Schematic illustration of the tethering strategy and proposed mechanism of action. The siRNAs specific for the human αV (αV) integrin and survivin (Sur) mRNAs, and a nonsilencing control siRNA used in the studies are shown on the left. The 5′-end of the tethering ODN is covalently conjugated to folate (blue dot). The antisense RNA strands complementary to the target mRNAs are in green. The 3′-ends of the sense RNA strands (red) are extended to form base-paired interactions with the ODN. Following entry into the cell via FR-mediated endocytosis, the siRNA complexes (F-ODN:siRNA) are processed and the released siRNAs incorporated into RNA-induced silencing complex (RISC).

FIGURE 2.

FIGURE 2.

FR expression. (A–C) The representative results of immunofluorescence. The anti-FR staining is in red and the nuclear DAPI staining is in blue. (D–F) The representative results of the folate-delivery assay. The green dots indicate cytoplasmic accumulation of folate-oregon green 488, while the nuclear DAPI staining is in blue.

FIGURE 3.

FIGURE 3.

Cell-type specific gene silencing by tethered siRNAs. HUVECs (A,C,D) and MDA-MB-435S (B) cells were incubated with buffer alone, or with 300 nM of the various siRNAs in the presence of 10% fetal bovine serum. Four hours later, the media were removed and cells were incubated in fresh media containing no siRNAs for an additional 22 h. In C, last column, HUVECs were transfected with poly (I:C) using Lipofectamine 2000. In A–C, total cellular RNAs were extracted and the indicated mRNA levels measured by qRT_–_PCR. Each bar represents mean ± sem (n = 3–6). In D, cell viability and caspase activity of HUVECs were measured following the various treatments above, and the results of an average of two independent experiments were plotted. (Bfr) Buffer alone; (F-ODN:siRNA) siRNA tethered by the folate-conjugated ODN; (F-ODN:siRNA+free folate) F-ODN:siRNA in the presence of 200x molar excess free folate; (ODN:siRNA) siRNA tethered by the same ODN but without folate conjugated; Poly (I:C) cells transfected with poly (I:C).

FIGURE 4.

FIGURE 4.

Specific gene silencing by tethered siRNAs in KB cells. KB cells were incubated with buffer alone, or with 300 nM of the various siRNAs in the presence of 10% fetal bovine serum. Four hours later, the media were removed and cells incubated in fresh media containing no siRNAs for an additional 22 h. In B, last column, KB cells were transfected with poly (I:C) using Lipofectamine 2000. Total cellular RNAs were extracted and the indicated mRNA levels measured by qRT–PCR. Each bar represents mean±sem (n = 3–6). Other labels are the same as in Fig. 3.

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