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Department Of Medical Biotechnology School Of Allied Medicine
IUMS
INHIBITION OF HEPATITIS C VIRUS INFECTION BY NS5A-SPECIFIC APTAMER
Soheila Shokrollahzade
MSc student
Hepatitis C virus (HCV) infects 170 million people worldwide
up to 80% of those infected become chronic infection.
HCV infection can cause chronic hepatitis, liver cirrhosis and even
hepatocellular carcinoma (Laucer and Walker, 2001).
no vaccine available
peginterferon alpha (Peg-IFN-a)-based therapy is the current
treatment for the patients (Hoofnagle and Seeff, 2006).
tenascin-binding aptamer under development by Schering AG for cancer imaging
RNA Aptamer Complexed with Vitamin B12
Anti-VEGFR2 aptamer
Hepatitis C Virus
single positive-strand RNA virus genome: about 9.6 kb in length encodes a single long polyprotein which
is processed into
Structural proteins : core, E1 , E2
Non structural proteins: NS2, NS3, NS4A, NS4B,NS5A and NS5B
Master regulatory of HCV replication and assembly; NS5A protein
Domain 1:
i. an anchorage for connecting the protein to ER membrane.
ii. Contains a zinc binding domain (ZBD) to eliminate the ability of replication
Domain2:
i. interaction with RNA dependant- RNA polymerase
ii. ISRD region (Interferon sensitivity determining region)
Domain 3 :
Viral particle production PDB code:4DMB
Why Aptamers?Aptamers Vs Antibodies
(1) High stability of aptamers
(2) Production of aptamers (synthesis/modification)
(3) Low immunogenicity of aptamers
(4) Variety of target
Expression And Purification Of Hcv NS5A Protein
Western blot analysis of purified NS5A protein. His-tagged NS5A was expressed by IPTG induction in E. coli BL21 (DE3) and then confirmed using mouse anti-His monoclonal antibody (left) or anti-NS5Amonoclonal antibody (right) via western blot
Selective Evolution Of Ligands By Exponential Enrichment (SELEX)
Tom Strachan ,Human molecular genetics
Comparison of the fluorescence of different pools of FITC-labeled DNA binding to NS5A protein or control protein LacZ.
Topmost Sequences Representative The Study
Binding affinity 1:
1. ELONA (Enzyme-Linked Oligonucleotide Assay)Binding affinity of aptamers to recombinant protein NS5A
ELONA (Enzyme-linked Oligonucleotide Assay)
Binding affinity study 2,
The binding affinity of aptamer NS5A-4, NS5A-5 for NS5A protein in lysates of HCV-infected hepatocytes
BINDING AFFINITY STUDY 3,APT3-CO-LOCALIZATION OF NS5A-SPECIFIC APTAMERS WITH NS5A PROTEIN IN THE HCV-INFECTED HEPATOCYTES
NS5A APTAMER DISRUPTS THE INTERACTION OF NS5A WITH CORE
PROTEIN
Interaction of NS5A with core protein is essential for
infectious virus production (Masaki et al., 2008).
Disruption the interaction of NS5A with core protein impairs
infectious virus production (Jiang and Luo, 2012).
Co-immunopercipitation
Immunopercipitation
Co-immunopercipitation
CORE protein in immuno- precipitates with equal amount of NS5A protein in aptamer NS5A-4 or NS5A-5-treated hcv infected Huh7.5 cells was lower than that in library-treated group
INHIBITION OF HCV INFECTION BY NS5A-SPECIFIC APTAMERS …….qPCR ANALYSIS
Domains I And III Of NS5A Protein Are Involved In The Antiviral Effects Of Ns5a-specific Aptamer NS5A-4
Domains I and III of NS5A protein are involved in the antiviral effects of NS5A-specific aptamer NS5A-4