Team:Warwick/Project

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<h4> Aptazyme </h4>
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An aptazyme is an RNA based switch  that operates by ribozyme-mediated cleavage of RNA. This original sequence for this part derives from Hartig et al., 2002 and requires the addition of theophylline to induce hammerhead ribozyme activation and cleavage, as depicted in Figure 1a. The part sequence is modified to contain a stop codon at the end, as an RBS is present. 
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Revision as of 10:34, 8 September 2014

PROJECT



Disrupting the flow of biological information at the level of mRNA is a safer alternative to conventional gene therapy, wherein insertional mutagenesis can occur through integrating vectors. In addition, the ability to regulate the level of expression of a gene using such vectors proves difficult. Therefore, we aim to create a modular, self-replicating RNA system using Hepatitis C Virus (HCV) derived RNA dependent RNA polymerase (RdRp). This drives production of siRNA directed against the enzyme dipeptidyl peptidase-IV (DPP-IV) which is elevated in type 2 diabetes and is the target of major drug studies. The replicon contains control modules, exhibiting a negative feedback mechanism provided by: an MS2 domain linked to RdRp, thereby controlling RdRp translation and therefore controlling replication, and an aptazyme switch to regulate expression levels of our siRNA. Validation of our system and the testing of modules will be performed in human (Huh 7.5) and E. coli cells.

RNA Dependent RNA Polymerase

RNA dependent RNA polymerase (RdRp) is an enzyme which catalyses the replication of RNA from an RNA template. An essential protein encoded within viruses that lack a DNA phase and replicate using negative sense RNA. The submitted RdRp part derives from the Hepatitis C virus con1 strain, RdRp is also referred to as non-structural protein 5B (NS5B). Part sequence was derived from Lohmann et al., 1997, with the authors achieving full expression and activity of RdRp in a baculovirus expression system. Membrane association of RdRp is essential for replication of HCV subgenomic regions, with the C-terminal tail containing 21 amino acids which confer high hydrophobicity and mediate insertion into the membrane (Moradpour et., 2004). The 21 amino acid residues have been removed in the part sequence, ensuring cytoplasmic RdRp activity, in line with previous analysis showing no significant loss of nucleotide polymerization activity (Vo et al., 2004). The C-terminal tail preceding the C-terminal hydrophobic insertion sequence interacts with structural elements including the β-hairpin loop of NS5b (Leveque et al., 2003). The β-hairpin loop inserts into the active site, believed to position the 3’ terminius of HCV viral RNA to initiate RNA synthesis (Hong et al., 2001). RdRp initiates RNA synthesis with nucleotide transfer activity found within the palm motif (Figure 1a & b), with several amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). RdRp requires 5’ and 3’ untranslated regions (UTRs) found within the HCV genome to direct RNA synthesis. The UTRs form ordered RNA structures and are evolutionary conserved.

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Aptazyme

An aptazyme is an RNA based switch that operates by ribozyme-mediated cleavage of RNA. This original sequence for this part derives from Hartig et al., 2002 and requires the addition of theophylline to induce hammerhead ribozyme activation and cleavage, as depicted in Figure 1a. The part sequence is modified to contain a stop codon at the end, as an RBS is present. HTML5 Icon HTML5 Icon