2.  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).

4. tenascin-binding aptamer under development by Schering AG for cancer imaging RNA Aptamer Complexed with Vitamin B12 Anti-VEGFR2 aptamer

5. 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

6. 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

7. 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

8. 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

9. Selective Evolution Of Ligands By Exponential Enrichment (SELEX) Tom Strachan,Human molecular genetics

10.  Comparison of the fluorescence of different pools of FITC-labeled DNA binding to NS5A protein or control protein LacZ.

11.  Topmost Sequences Representative The Study

12.  Binding affinity 1:  1. ELONA (Enzyme-Linked Oligonucleotide Assay) Binding affinity of aptamers to recombinant protein NS5A

13. ELONA ( Enzyme-linked Oligonucleotide Assay)

14.  Binding affinity study 2,  The binding affinity of aptamer NS5A-4, NS5A-5 for NS5A protein in lysates of HCV-infected hepatocytes

15. BINDING AFFINITY STUDY 3, APT3-CO-LOCALIZATION OF NS5A-SPECIFIC APTAMERS WITH NS5A PROTEIN IN THE HCV-INFECTED HEPATOCYTES

16. NS5A APTAMER DISRUPTS THE INTERACTION OF NS5A WITH CORE PROTEIN 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

17. Immunopercipitation

18. Co-immunopercipitation

20. 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

21. INHIBITION OF HCV INFECTION BY NS5A- SPECIFIC APTAMERS …….qPCR ANALYSIS

22. Domains I And III Of NS5A Protein Are Involved In The Antiviral Effects Of Ns5a- specific Aptamer NS5A-4

23. Domains I and III of NS5A protein are involved in the antiviral effects of NS5A-specific aptamer NS5A-4