Abstract
The discovery of siRNAs as the mediators of RNA interference has led to an increasing interest in their therapeutic applications. Chemical modifications are introduced into siRNAs to optimize the potency, the stability and the pharmacokinetic properties in vivo. Here, we synthesize and test the effects of RNA-3'-PNA chimeras on siRNA functioning and stability. We demonstrate that the chemical modifications are compatible with the siRNA machinery, because all the PNA-modified siRNAs can efficiently mediate specific gene silencing in mammalian cells. Furthermore, we find that the modification on the sense strand of siRNA results in an increased persistence of the activity, whereas modification on both strands results in enhanced nuclease resistance in serum.
Highlights
RNA interference (RNAi) is an endogenous gene-silencing mechanism triggered by doublestranded RNAs that mediates sequence-specific degradation or translational block of the target mRNA [1]
We report the synthesis of siRNAs composed of RNA-Peptide Nucleic Acids (PNAs) chimeras, their chemical-physical characterization and the evaluation of their gene silencing activity in cultured mammalian cells, based on their ability to target the firefly luciferase mRNA
To verify the compatibility of PNA units into siRNA with the RNAi machinery, we have evaluated the different siRNAs for their ability to inhibit firefly luciferase in HeLa cells. 100 nM siRNA duplexes were co-transfected with the reporter plasmid combination pGL2/phRL-TK into HeLa cells using cationic liposomes. pGL2 encodes the target Photinus pyralis luciferase and phRLTK encodes the Renilla reniformis luciferase used as control to normalize data with the efficiency of transfection
Summary
RNA interference (RNAi) is an endogenous gene-silencing mechanism triggered by doublestranded RNAs that mediates sequence-specific degradation or translational block of the target mRNA [1]. Several research groups have attempted to identify chemical modifications that increase the intracellular and extracellular stability of siRNAs by decreasing their susceptibility to nuclease attack, while allowing them to maintain sufficient genesilencing activity for therapeutic use [14] These studies have demonstrated that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications, including terminal and internal modification [15], affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo. We demonstrated that the chemical modifications were compatible with the siRNA machinery and showed that the modification on the sense strand produced an increased persistence of the silencing activity, whereas the modification on both strands yielded an enhanced serum stability These results provide a basis for the development of further PNA-based siRNAs to probe the molecular mechanism of RNAi and to improve their chemical and functional properties for in vivo applications
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