Publications

Seed Sequence-matched Controls Reveal Limitations of Small Interfering RNA Knockdown in Functional and Structural Studies of Hepatitis C Virus NS5A-MOBKL1B Interaction

Chung H, et al., 88(19):11022-33, J Virol, 2014

The authors describe a very important concept pertaining to siRNA-mediated gene silencing experiments. It shows an alternate dimension called "siRNA off-target effect" that could lead to false interpretations of the biological data. In a previous investigation, authors identified a cellular protein called MOBKL1B as a binding partner for NS5A protein of hepatitis C virus. They used MOBKL1B siRNA (in the absence of sufficient control experiments) to demonstrate its ability to reduce viral replication. Although they believed MOBKL1B-NS5A interaction is crucial for the siRNA effects observed, a variant lacking this interaction was not been able to replicate after treatment with MOBKL1B siRNA. By repeating the experiments with appropriate controls the authors were able to demonstrate that the MOBKL1B siRNAs previously reported had off-target inhibitory effects on viral replication. The BLI assays helped in the identification of a highly conserved binding site in NS5A comprising residues implicated in CypA-dependent HCV RNA replication. The BIACORE 2000 and ForteBio Octet RED 384 instruments were helpful in determining the binding interactions.

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