Biomembrane-permeable and ribonuclease-resistantsiRNA with enhanced activity
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Type I insulin-like growth factor receptor (IGF-IR) plays a pivotal role in the IGF signaling pathway. In addition to its role in development and cell cycle progression, the IGF-IR may also regulate early phases of tumorigenicity. Down-regulation of IGF-IR causes massive apoptosis of cells and regression of wild-type tumor. siRNAs are short 21-25 nt dsRNAs which can incorporate into RNA-induced silencing complex (RISC) and exert silencing in a variety of forms. Because of its ability to knock down the expression of a target gene in an effective, specific, and reproducible manner, siRNA has tremendous value in both genetic studies and therapeutics. Modifying the 2 ' hydroxyl group of the ribose with dinitrophenyl (DNP) group is a convenient technique developed in our lab. The DNP-modification is found to enhance the membrane-permeability and RNase resistance of native RNA. In order to test whether the DNP-modification can also improve the membrane-permeability and stability of siRNA without loss of its gene-silencing effect, a series of new experiments were carried out. Native and DNP-modified siRNAs were incubated with T98G cells and in situ hybridization (ISH) was performed to detect the presence of the siRNAs inside the cell, using a biotin-labeled DNA probe. In the absence of oligofectamine, it was found that DNP-ssRNA and DNP-dsRNA crossed the plasma membrane while the native ssRNA and dsRNA did not. With the help of oligofectamine, both DNP-modified and native siRNA can get into the cell within 8 hours. The DNP-siRNAs that entered the cells were localized in the cytoplasm where they can easily reach their target mRNA. The RNase digestion assays showed that the stability of different siRNAs decreases in the following order: DNP-dsRNA [approximate] DNP-ssRNA > native dsRNA > native ssRNA. To test their intracellular stability, native and DNP-modified siRNAs of the S2 sequence were delivered respectively into the T98G cells with the help of oligofectamine and incubated for different periods of time. The DNP-siRNAs can be detected by ISH even after incubation for 4 days whereas native siRNAs were completely degraded after incubation for 2 days. Two different IGF-IR targeting siRNAs, S1 and S2 were tested for their efficacy in inhibition of cell growth. Experimental data from T98G, A549 and MCF-7 cell lines show that DNP-modified siRNAs are more effective than the corresponding native dsRNAs in inhibition of tumor cell growth in the presence of oligofectamine. Without the help of oligofectamine, DNP-siRNA still can inhibit the growth of tumor cells, though a much higher dosage is required. In comparison, native siRNA shows no inhibitory effect in the absence of oligofectamine. The S2 sequence is more effective than the S1 sequence, and dsRNA is more potent than the corresponding ssRNA of the same sequence. The sequence-independent toxicity of the DNP-RNA is low. RT-PCR and Western blotting experiments were also conducted and the results are similar to those of the cell growth inhibition experiments. These data also show that DNP-modified dsRNAs are more potent than the corresponding native dsRNAs in promoting the clearance of IGF-IR mRNAs, and in reducting in synthesis of the IGF-IR protein. It was found that the S2 sequence is more effective than the S1 sequence and dsRNA is more effective than the corresponding ssRNA of the same sequence.