Application of RNAi Gene Silencing Platform for The Development of Novel Antiviral Compounds Targeting Hepatitis C Virus
M. Mutso University of Tartu, Estonia, Nooruse 1, Tartu 50411, Estonia
Abstract:
Background: Hepatitis C virus (HCV, a positive-strand RNA virus) high mutation rate result in rapid development of drug-resistant virus strains. RNAi antisense technology directly targets the viral RNA. We used RNAi approach for identification of target sites in the coding region of HCV RNA. We used best sites to design DNA oligonucelotides incorporating modified nucleobases and assayed their effects on HCV RNA replication. Our approach considerably boosts the flexibility of potential target sites selection for antisense nucleic acid inhibitors.
Methods: 40 different siRNAs targeting HCV RNA were designed by using specialized algorithm-driven software. The siRNAs or modified oligonucleotides were reverse-transfected into Huh-luc/neo-ET cell line (ReBlikon GmbH) using LipofectamineT RNAiMAX (Invitrogen) reagent and luciferase activity was measured after 48 hours.
Results: Two siRNAs were found to be the most potent HCV replication inhibitors in a cell culture experiment and hence corresponding target sites were chosen as the primary sites for different antisense oligonucleotides. From variety of tested antisense compounds the oligonucleotides with modified nucleobases caused much higher decrease of HCV RNA replication compared with the normal and LNA- oligonucleotides.
Conclusion: The results clearly demonstrate that the oligonucleotides with nucleobase modifications are much more potent antiviral compounds than oligonucleotides without such modifications.
