Small interfering RNAs (siRNAs) are considered as potent agents for specific gene silencing; however, nuclease sensitivity of siRNA limits their biomedical applications. Till date, no universal methodology has been developed to improve the nuclease resistance of siRNA, preserving low toxicity and high activity. In this study, we proposed an algorithm for the site-specific modification of siRNAs based on the mapping of their nuclease-sensitive sites in the presence of serum followed by the incorporation of 2'-O-methyl analogs of ribonucleotides at the identified positions of cleavage. We found that the protection of nuclease-sensitive sites considerably enhanced nuclease resistance of siRNA and only slightly reduced the efficiency of silencing. Modification of all nuclease-sensitive sites prolonged the duration of the silencing effect of the siRNA compared to nonmodified, partially modified, or randomly modified siRNA of the same sequence. This study showed that the targeted chemical modification of nuclease-sensitive sites could provide highly efficient siRNA-based therapeutics for the control of disease-related genes.
The ability of chemically modified siRNAs targeted to MDR1 mRNA to inhibit P glyco protein expression and to restore sensitivity of cancer cells to antibiotic vinblastine was investi gated. The effects of chemical modifications on RNA stability in cell culture medium and inhibition of MDR1 gene expression were tested. We found that siRNAs containing 2´ O methyl ribonucleotides within either sense or/and antisense strands display high stability in serum but exhibit a significant reduction in the biological activity. The protection of 3´ ends of siRNA by introduction of 3´-3´ inverted phosphodiester bonds and two 2´ O methyl ribonucleotides in protruding 3´ ends considerably increase their biological activity, which allows a 30 fold decrease in the cytostatic agent concentration required for cancer cell death. The data obtained demonstrate that the chemically modified siRNAs can be considered as potential therapeutics, which enhances the efficiency of cancer chemotherapy.
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