Team:Warwick/Parts

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         <!--CONTENT START-->
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<a href="https://2014.igem.org/Main_Page"> <img class = "headerImage" style = "width: 12%;"
 +
src="https://static.igem.org/mediawiki/2014/2/22/Logo2014v2.png"> </a>
          
          
         <a href="/Team:Warwick"> <img class = "headerImage" style = "width: 30%;" src="https://static.igem.org/mediawiki/2014/f/ff/RepliconLogoON.png"> </a>
         <a href="/Team:Warwick"> <img class = "headerImage" style = "width: 30%;" src="https://static.igem.org/mediawiki/2014/f/ff/RepliconLogoON.png"> </a>
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         <a href="/Team:Warwick/DMB"> <img class = "headerImage" style = "width: 12%;" src="https://static.igem.org/mediawiki/2014/f/f6/Warwick_logo.png"> </a>
+
         <a href="/Team:Warwick"> <img class = "headerImage" style = "width: 12%;"  
 +
src="https://static.igem.org/mediawiki/2014/f/f6/Warwick_logo.png"> </a>
         <div id = "mainMenuBar">
         <div id = "mainMenuBar">
 +
            <li> <a href = "/Team:Warwick"> HOME </a> </li>
             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Project"> PROJECT </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
             <li> <a href = "/Team:Warwick/Team"> TEAM </a> </li>
-
             <li> <a href = "/Team:Warwick/Parts"> PARTS </a> </li>
+
             <li> <a href = "/Team:Warwick/Parts"> <span> PARTS </span> </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Modelling"> MODELLING </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
             <li> <a href = "/Team:Warwick/Notebook"> NOTEBOOK </a> </li>
-
             <li> <a href = "/Team:Warwick/Human"> HUMAN PRACTICES </a> </li>
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             <li> <a href = "/Team:Warwick/Human"> POLICY & PRACTICES </a> </li>
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             <li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>
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             <!--<li> <a href = "/Team:Warwick/Measurements"> MEASUREMENTS </a> </li>-->
             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
             <li> <a href = "/Team:Warwick/Interlab"> INTERLAB </a> </li>
-
             <li> <a href = "/Team:Warwick/Attributions"> SPONSORS </a> </li>
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             <li> <a href = "/Team:Warwick/Attributions"> ATTRIBUTIONS </a> </li>
 +
         
         </div>
         </div>
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<div id="secondaryMenu">
 +
<li> <a href = "/Team:Warwick/Parts/Aptazyme"> APTAZYME </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/IRES"> IRES </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/sirna"> siRNA </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/Neomycin"> NEOMYCIN </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/T7"> T7 </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/RdRp"> RdRp </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/P2a"> P2A </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/MS2"> MS2 </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/3promoter"> RNA PROMOTERS </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/Testing"> TESTING MODULES </a> </li>
 +
<li> <a href = "/Team:Warwick/Parts/bb"> EXISTING BIOBRICK </a> </li>
 +
</div>
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             <div id = "pageContent">
             <div id = "pageContent">
             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
             <!-- THIS IS WHERE YOUR MAIN BODY GOES -->
 +
<p> Below is an interactive schematic of our system in RNA (for simplicity, we have omitted in the drawing a T7 promoter and its terminator to transcribe the whole operon), hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results. You can find the full list of parts we've submitted <a href="http://parts.igem.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2014&group=Warwick">here</a>. The parts labelled 'favourite' are our best parts. </p>
 +
<div id="pop1" class="popbox">
 +
    <h2>5' RNA Promoter</h2>
 +
    <p>This is derived from the 5' UTR of the HCV virus strain 1b isolate Con1. It contains the reverse complement of the RdRp initiation sequence. The secondary structure of this sequence acts as a binding site and initiates replication of the minus strand 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. We define an RNA promoter as a sequence located at the 3' end of the transcript, consisting of a RdRp initiation sequence and a ribozyme that cleaves after the initiation sequence without leaving any scar. We foresee that such regulatory elements will allow constructing novel post-transcriptional circuits by adding operator sites in the promoters. Such operator sites could be for instance the MS2 hairpin or any other RNA sequence known to bind to a regulator. This is the RNA equivalent to a transcription factor.</p>
 +
</div>
 +
<div id="pop2" class="popbox">
 +
    <h2>IRES</h2>
 +
    <p>This
-
<h1> PARTS </h1> <br> <br>
+
acts as an initiation for eukaryotic
-
<h2>IRES comparison</h2>
+
-
<b>NF-kappaB repressing factor (Homo sapiens)</b>
+
-
<p>This IRES (internal ribosome entry site) is derived from the long 5’ untranslated region of the NF-kB repressing factor, a multifunctional cytokine interferon-β. This forms a secondary structure, shown below with sequence, which directs ribosomes to the downstream start codon by a cap-dependent mechanism. Following experimentation this was shown to have a 30-fold higher than picornaviral IRESs (Oumard, 2000). This was compared to the EMCV IRES and the poliovirus IRES in HeLa cells, murine embryonic stem cells and embryonic fibroblasts, using the firefly luciferase. The level of fluorescence seen was 92-fold higher than EMCV and 130-fold more active than the poliovirus IRES. NKRF acts in a distance independent manner and has a very high efficiency of translation initiation.</p>
+
-
<div style="text-align: center"><img src="https://static.igem.org/mediawiki/2014/7/74/Warwick_Sequence_image_1.jpg"></div>
+
ribosomes and begins translation of the
-
<p><font color ="blue" size="1pt"><div style="text-align: center">Figure 1: Sequence and seconary structure of the NRF 5'UTR (EMBL accession number AJO11812) Oumard, 2000</div></font></p>
+
following protein sequence. We  
-
<p>The sequence seen in Figure 1 is the one we used. This was cloned with a green fluorescent protein (iGEM part BBa_E0040) which we codon optimised for use in human cells and compared with a similar construct using EMCV IRES in place of the NKRF. We transfected the RNA of these constructs into Huh 7.5 cells in which, to our knowledge, the NKRF IRES has not been tested.</p>
+
-
<h3> RNA Dependent RNA polymerase </h3>
+
compared two different IRESs: the  
-
<b> Background </b>
+
-
<P> RdRp is an enzyme which catalyses the replication of RNA from an RNA template, an essential protein for all viruses with an RNA genome. RdRp in Hepatitis C virus is also referred to as the NS5B protein, with a total molecular weight of 65 kDa. Heterelogous expression of NS5B has been achieved in insect and bacterial hosts, with RNA-dependent RNA synthesis initiated  de novo (Behrens et al., 1996; Lohmann et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause insertion into the membrane, essential for HCV RNA replication (Moradpour et al., 2004). Amino acid substitution experiments of the 21 amino acid insertion sequence indicate functions beyond a membrane anchor role, with intracellular protein-protein interactions implicated (Moradpour 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 viral RNA synthesis  (Hong et al., 2001).
+
-
RdRp initiates viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several amino acid residues implicated in nucleotide  triphosphate contact (Bressanelli et al., 2002). NS5B activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al., 1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003).
+
classical EMCV IRES used in many papers in our target cells,which has been shown to be compatible
-
We have designed an RdRp linked to an MS2 domain through a P2A linker, the MS2 domain is able to bind to the MS2 box and act in a negative-feedback loop to regulate RdRp expression in both ''E. coli'' and Huh 7.5 cells  </P>
+
with replicons and the NKRF IRES derived from the 3’UTR of
 +
 
 +
the mammalian NF-kappaB repressing
 +
 
 +
factor. We chose to test it also 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, it had never been previously used in Huh7.</p>
 +
</div>
 +
<div id="pop3" class="popbox">
 +
    <h2>MS2 Box</h2>
 +
    <p>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. This "copy number control" also allows a switching off of the RdRp once a steady state is reached, which minimizes the possible interferences between RdRp and the ribosome.</p>
 +
</div>
 +
<div id="pop4" class="popbox">
 +
    <h2>Neomycin Resistance</h2>
 +
    <p>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.</p>
 +
</div>
 +
<div id="pop5" class="popbox">
 +
    <h2>Aptazyme</h2>
 +
    <p>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.</p>
 +
</div>
 +
<div id="pop6" class="popbox">
 +
    <h2>siRNA</h2>
 +
    <p>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.</p>
 +
</div>
 +
<div id="pop7" class="popbox">
 +
    <h2>MS2 Coat Protein</h2>
 +
    <p>This coding sequence forms
 +
 
 +
a protein which binds to the MS2 box - a hairpin
 +
 
 +
type structure, and represses translation of the
 +
 
 +
replicon.</p>
 +
</div>
 +
<div id="pop8" class="popbox">
 +
    <h2>P2A</h2>
 +
    <p>This is derived from the Porcine
 +
 
 +
Teschovirus-1 genome and cleaves with high
 +
 
 +
efficiency and leaves no scar following the
 +
 
 +
formation of a polyprotein MS2 coat protein,
 +
 
 +
P2A and RdRp. This is required as the HCV
 +
 
 +
genome has one open reading frame
 +
 
 +
from which one long polyprotein is produced.
 +
 
 +
RdRp is the final protein in this polygenic
 +
 
 +
mRNA and hence does not have a start codon,
 +
 
 +
adding a start codon could be disastrous for
 +
 
 +
the function of RdRp.</p>
 +
</div>
 +
<div id="pop9" class="popbox">
 +
    <h2>RdRp</h2>
 +
    <p>This is a polymerase derived from Hepatitis C Virus Strain
 +
 
 +
1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in
 +
 
 +
the contact of the HCV genome. Heterelogous expression of NS5B has been achieved in insect and
 +
 
 +
bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann
 +
 
 +
et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause
 +
 
 +
insertion into the membrane with other intracellular protein-protein interactions implicated (Moradpour
 +
 
 +
et al., 2004) making the final 63 bases essential for HCV RNA replication in eukaryotic cells
 +
 
 +
(Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. RdRp initiates
 +
 
 +
viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several
 +
 
 +
amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). NS5B
 +
 
 +
activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al.,
 +
 
 +
1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains
 +
 
 +
ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for
 +
 
 +
viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for
 +
 
 +
RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003).</p>
 +
</div>
 +
<div id="pop10" class="popbox">
 +
    <h2>3' RNA Promoter</h2>
 +
    <p>Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The promoter is "insulated" by using a ribozyme on its 3' end that self-cleaves without leaving a scar (and, therefore, producing the right 3' end for RdRp initiation).  The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such initiation sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative RNA promoter sequences previously identified by Heinz et al. as templates for replication by the HCV RdRP. </p>
 +
</div>
 +
 
 +
<table border="0" cellpadding="0" cellspacing="0">
 +
<tr>
 +
 
 +
<td>
 +
<a href="/Team:Warwick/Parts/3promoter" class="popper" data-popbox="pop1"><img src="https://static.igem.org/mediawiki/2014/7/7f/5%27_promoter-01-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/IRES" class="popper" data-popbox="pop2"><img src="https://static.igem.org/mediawiki/2014/0/01/IRES-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/MS2" class="popper" data-popbox="pop3"><img src="https://static.igem.org/mediawiki/2014/5/50/MS2_box-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/Neomycin" class="popper" data-popbox="pop4"><img src="https://static.igem.org/mediawiki/2014/6/6c/Neomycin_Resistance-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/Aptazyme" class="popper" data-popbox="pop5"><img src="https://static.igem.org/mediawiki/2014/d/d7/Aptazyme-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/sirna" class="popper" data-popbox="pop6"><img src="https://static.igem.org/mediawiki/2014/0/0c/SiRNA-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/IRES" class="popper" data-popbox="pop2"><img src="https://static.igem.org/mediawiki/2014/f/fa/IRES_2-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/MS2" class="popper" data-popbox="pop7"><img src="https://static.igem.org/mediawiki/2014/a/a5/MS2_coat_protein-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/P2a" class="popper" data-popbox="pop8"><img src="https://static.igem.org/mediawiki/2014/c/c5/P2A-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/RdRp" class="popper" data-popbox="pop9"><img src="https://static.igem.org/mediawiki/2014/3/38/RdRp-01.png" width="100%"></a>
 +
</td>
 +
<td>
 +
<a href="/Team:Warwick/Parts/3promoter" class="popper" data-popbox="pop10"><img src="https://static.igem.org/mediawiki/2014/c/c7/3%27_promoter-01.png" width="100%"></a>
 +
</td>
 +
</tr>
 +
</table
             </div>
             </div>
 +
 +
 +
 +
<!--This is a popbox test.  <a href="#" class="popper" data-popbox="pop1">Hover here</a> to see how it works.  You can also hover <a href="#" class="popper" data-popbox="pop2">here</a> to see a different example.-->
 +
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<footer>
 +
  <p>Created by: iGEM Warwick</p>
 +
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 +
  igem.warwick@gmail.com</a></p>
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Latest revision as of 03:48, 18 October 2014

Below is an interactive schematic of our system in RNA (for simplicity, we have omitted in the drawing a T7 promoter and its terminator to transcribe the whole operon), hover your mouse over any part to see a brief description, and click to follow through to the Registry and see a more in depth description, and information regarding sequences and results. You can find the full list of parts we've submitted here. The parts labelled 'favourite' are our best parts.

5' RNA Promoter

This is derived from the 5' UTR of the HCV virus strain 1b isolate Con1. It contains the reverse complement of the RdRp initiation sequence. The secondary structure of this sequence acts as a binding site and initiates replication of the minus strand 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. We define an RNA promoter as a sequence located at the 3' end of the transcript, consisting of a RdRp initiation sequence and a ribozyme that cleaves after the initiation sequence without leaving any scar. We foresee that such regulatory elements will allow constructing novel post-transcriptional circuits by adding operator sites in the promoters. Such operator sites could be for instance the MS2 hairpin or any other RNA sequence known to bind to a regulator. This is the RNA equivalent to a transcription factor.

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 in our target cells,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. We chose to test it also 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, it 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. This "copy number control" also allows a switching off of the RdRp once a steady state is reached, which minimizes the possible interferences between RdRp and the ribosome.

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 - a hairpin type structure, and represses translation of the replicon.

P2A

This is derived from the Porcine Teschovirus-1 genome and cleaves with high efficiency and leaves no scar following the formation of a polyprotein MS2 coat protein, P2A and RdRp. This is required as the HCV genome has one open reading frame from which one long polyprotein is produced. RdRp is the final protein in this polygenic mRNA and hence does not have a start codon, adding a start codon could be disastrous for the function of RdRp.

RdRp

This is a polymerase derived from Hepatitis C Virus Strain 1b isolate Con1 which catalyses the replication of RNA from an RNA template, reffered to as NS5B in the contact of the HCV genome. Heterelogous expression of NS5B has been achieved in insect and bacterial hosts, with RNA-dependent RNA synthesis initiated de novo (Behrens et al., 1996; Lohmann et al., 1997). Structural studies indicate the hydrophobic C-terminal 21 amino acid residues cause insertion into the membrane with other intracellular protein-protein interactions implicated (Moradpour et al., 2004) making the final 63 bases essential for HCV RNA replication in eukaryotic cells (Moradpour et al., 2004). In prokaryotic cells the final 21 amino acids are expendable. RdRp initiates viral RNA synthesis with nucleotidyl transfer activity found within palm motifs A and C, with several amino acid residues implicated in nucleotide triphosphate contact (Bressanelli et al., 2002). NS5B activity has been demonstrated ''in vitro'', with synthesis of full length HCV RNA (Lohmann et al., 1997; Ferrari et al., 1999). 5’ and 3’ untranslated regions (UTRs) of the HCV genome contains ordered RNA structures, which are evolutionary conserved and contain crucial cis-acting elements for viral RNA replication. 150 nt in the 3’ termini of HCV RNA contains elements which are essential for RdRp binding and replication of viral RNA (Cheng et al., 1999; Yi and Lemon, 2003).

3' RNA Promoter

Each unique RNA dependent RNA polymerase (RdRP) initiates de novo replication of a RNA strand by interacting with RdRP-specific RNA sequences, henceforth called RdRP/RNA promoters. The promoter is "insulated" by using a ribozyme on its 3' end that self-cleaves without leaving a scar (and, therefore, producing the right 3' end for RdRp initiation). The RdRP chosen for our project is taken from the Hepatitis C virus (HCV) and it recognizes a limited set of such initiation sequences. All of them possess a few common characteristics: an initiation cytidylate at the 3’ end, where the replication starts; and a stable secondary structure – single stranded tail and a stem of various length. For our project in addition to the indigenous to HCV RdRP promoters, we designed alternative RNA promoter sequences previously identified by Heinz et al. as templates for replication by the HCV RdRP.

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