Hepatitis C virus nonstructural protein 5B (NS5B) is a RNA-dependent RNA polymerase (RdRp) which initiates de novo RNA synthesis with the help of specific RNA promoters. In our project, the RNA interference is conditional because the pri-miRNA analogue which partially pairs with an inhibitory strand is unable to be processed by DROSHA. Once NS5B is introduced to catalyze a substitutional hybridation to release the pri-miRNA in the nucleus, the analogue gets to evolve into miRNA and interferes with the target gene mRNA.
Since the existent NS5B from Standard Parts collection which can not translocate into the nucleus does not suit our needs, we tend to design our own part that is able to do its job in the nucleus. After deep consideration, we decided to improve the function of the part submitted by iGEM14Warwick (BBa_K1442028). We load a nuclear location sequence (NLS) to the N terminal of NS5B (NS5BNLS) to transport the RdRP into the nucleus (BBa_K2624002). We demonstrate this improvement by indirect immunofluorescence. As the following figure shows, NS5B with NLS (NLS+) have a higher ability of nuclear translocation than NS5B without NLS (NLS-).