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Team:Warwick/Parts
From 2014.igem.org
5' Promoter
This is derived from the 5i UTR of the HCV virus strain 1b isolate Con1. The secondary structure of this sequence acts as a binding site and initiates replication of the system by RdRp. It also includes the first 16 amino acids of the first gene of HCV as this has been shown to increase the efficacy of binding of the RdRp to the 5' promoter.
IRES
This acts as an initiation for eukaryotic ribosomes and begins translation of the following protein sequence. We compared two different IRESs: the classical EMCV IRES used in many papers,which has been shown to be compatible with replicons and the NKRF IRES derived from the 3’UTR of the mammalian NF-kappaB repressing factor, which we also chose to test because during investigation regarding the efficacy and strength of the EMCV IRES, the NKRF derived IRES was shown to be 30-fold more efficient however had never been previously used in Huh7.
MS2 Box
The MS2 box acts as a binding site for the MS2 coat protein, formed downstream in the replicon, which represses translation hence preventing exponential growth of the replicon.
Neomycin Resistance
This was included in order to select for the cells which had been successfully transfected with the replicon. This is commonly used in human cell studies and allows survival of eukaryotic cells in the prescence of geneticin.
Aptazyme
This is an RNA enzyme which self- cleaves in the presence of theophylline. Theophylline is not endogenous to mammalian cells hence acts as a selective “off-switch” in an instance such as, Hepatitis C infection or tumour formation which is exacerbated by lack of DPP-IV.
siRNA
This is the functional element of our replicon and shows a potential area for RNA experimentation. This sequence was specifically designed using RNA fold and investigating the minimum free energy bond formation, in order to form a secondary structure in the positive sense that would not include a length of double stranded RNA longer than 16 bases and hence would not be recognised by Dicer and our replicon would remain intact for further replication. However, in the negative sense the secondary structure will form a hairpin recognised by Dicer which will result in cleavage and release of the siRNA from the RNA strand allowing it to bind in a complimentary fashion to the 3’UTR of DPP-IV mRNA and cause RNA silencing hence reducing the amount of DPP-IV protein present in the cell. We used siRNA sequences from various papers to design this hairpin:
MS2 Coat Protein
This coding sequence forms a protein which binds to the MS2 box, hairpin type structure, and represses translation of the replicon.
P2A
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RdRp
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3' Promoter
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