Abstract
The ss-siRNA activity in vivo requires a metabolically stable 5′-phosphate analog. In this report we used crystal structure of the 5′-phosphate binding pocket of Ago-2 bound with guide strand to design and synthesize ss-siRNAs containing various 5′-phosphate analogs. Our results indicate that the electronic and spatial orientation of the 5′-phosphate analog was critical for ss-siRNA activity. Chemically modified ss-siRNA targeting human apoC III mRNA demonstrated good potency for inhibiting ApoC III mRNA and protein in transgenic mice. Moreover, ApoC III ss-siRNAs were able to reduce the triglyceride and LDL cholesterol in transgenic mice demonstrating pharmacological effect of ss-siRNA. Our study provides guidance to develop surrogate phosphate analog for ss-siRNA and demonstrates that ss-siRNA provides an alternative strategy for therapeutic gene silencing.
Highlights
The demonstration that the reduction of mRNA expression in cells using externally delivered duplex RNA by activating the RISC mechanism enabled the development of siRNA therapeutic [1,2]
It was shown that human Dicer and Ago-2, the enzymes involved in the RNAi pathway, bind short single-stranded RNAs with affinities comparable to siRNAs suggesting that single-stranded RNAs are capable of activating the RNAi pathway [8]
Structural model of cis and trans vinyl phosphonate nucleoside showed that the trans vinylphosphonate can assume a conformation similar to that of the 5 phosphate in the Ago-2 crystal structure (Figure 11)
Summary
The demonstration that the reduction of mRNA expression in cells using externally delivered duplex RNA (siRNA) by activating the RISC mechanism enabled the development of siRNA therapeutic [1,2]. This approach has a potential to treat a wide variety of human diseases through genetic modulation [3]. It was shown that human Dicer and Ago-2, the enzymes involved in the RNAi pathway, bind short single-stranded RNAs with affinities comparable to siRNAs suggesting that single-stranded RNAs are capable of activating the RNAi pathway [8] Consistent with these observations we recently demonstrated single stranded short interfering RNA (ss-siRNA) activity in mice [9,10,11]
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