CS-SELEX generates high-affinity ssDNA aptamers as molecular probes for hepatitis C virus envelope glycoprotein E2

Abstract

Currently, the development of effective diagnostic reagents as well as treatments against Hepatitis C virus (HCV) remains a high priority. In this study, we have described the development of an alive cell surface--Systematic Evolution of Ligands by Exponential Enrichment (CS-SELEX) technique and screened the functional ssDNA aptamers that specifically bound to HCV envelope surface glycoprotein E2. Through 13 rounds of selection, the CS-SELEX generated high-affinity ssDNA aptamers, and the selected ssDNA aptamer ZE2 demonstrated the highest specificity and affinity to E2-positive cells. HCV particles could be specifically captured and diagnosed using the aptamer ZE2. A good correlation was observed in HCV patients between HCV E2 antigen-aptamer assay and assays for HCV RNA quantities or HCV antibody detection. Moreover, the selected aptamers, especially ZE2, could competitively inhibit E2 protein binding to CD81, an important HCV receptor, and significantly block HCV cell culture (HCVcc) infection of human hepatocytes (Huh7.5.1) in vitro. Our data demonstrate that the newly selected ssDNA aptamers, especially aptamer ZE2, hold great promise for developing new molecular probes, as an early diagnostic reagent for HCV surface antigen, or a therapeutic drug specifically for HCV.

Figure 1. High-affinity aptamers for HCV-E2 glycoprotein were isolated by CS-SELEX.

(A) The E2 stably expressing cell line E2-CT26 was established, and E2-CT26 E2 cell surface expression was identified by a PE-conjugated anti-E2 antibody using flow cytometry. (B) Comparison of binding percentages of different pools of FITC-labeled aptamers with E2-CT26 cells using flow cytometry. Data shown were calculated as mean±SEM, and data are from three independent experiments. (C) Representative results from different pools of FITC-labeled aptamers binding with E2-CT26 cells by flow cytometry.

Figure 2. Selected aptamers specifically bind to HCV E2.

(A) SDS-PAGE (Left) and western blot analysis (Right) of the purified GST-E2 and GST recombinant proteins. (B) Capillary electrophoresis analysis of single aptamer ZE2 binding to different doses of GST-E2 protein. Representative results of CEMSA showed that the binding peak of ZE2 with GST-E2 migrated from left to right, but the binding peak of ZE2 with GST had no change.

Figure 3. The characterization of aptamer ZE2 specificity for E2-expressing cells.

(A) Prediction of ZE2 secondary structure. Arrows represent exchanged bases in ZE2-mut compared to that of the ZE2 aptamer. (B) Fluorescence microscope imaging of E2-expressing cells with FITC-ZE2. FITC-ZE2 bound to E2-HepG2 cells but not to HepG2 cells by confocal immunofluorescence microscopy. (C) Comparison of different doses of FITC-ZE2 or FITC-ZE2-mut binding to the E2-HepG2 or HepG2 cells by confocal immunofluorescence microscopy.

Figure 4. The ssDNA aptamer ZE2 specifically targets HCV particles.

(A) HCVcc could be captured by the aptamer ZE2 in an aptamer dose-dependent manner by sandwich ELISA. Plates were coated with anti-E2 polyclonal antibody and blocked with 1% BSA. After extensive washing, the bound viral particles were added and then revealed using biotin-labeled aptamer ZE2, ZE3 or ZE2-mut as described in Materials and Methods. Data shown were calculated as mean±SEM, and data are from three independent experiments. (B) Comparison of HCV detection methods: HCV E2 antigen-aptamer method and HCV RNA quantification by real time fluorescence quantitative RT-PCR. The aptamer method was performed as above (A), except HCV patients and healthy donor serum samples were added into each 96-well ELISA plate instead of HCVcc. (C) Determination of E2 protein expressions of different genotypes of E1E2 gene stable-expressing HepG2 cells with anti-E2 antibody by western blot analysis. HepG2 was uesd as a control. (D) Different genotypes of E2 detected by aptamer ZE2. Data shown were calculated as mean±SEM and data are from three independent experiments.

Figure 5. DNA aptamer ZE2 and viral receptor CD81 share similar binding sites on HCV E2.

(A) CD81 molecules are expressed on the cellular surface of hepatocytes Huh7.5.1; flow cytometric analysis with PE-labeled anti-CD81 antibody. PE-conjugated rat IgG1 was used as an isotype-matched control antibody (Ab). (B) HCV E2 had a much higher binding affinity for Huh7.5.1 than CT26 cells by flow cytometric analysis. E2-GST or GST proteins were preincubated with 1×10⁶ Huh7.5.1 or CT26 cells, respectively. Anti-GST antibody and FITC-anti-IgG were added and analyzed by flow cytometric analysis. (C) Different pools of aptamers or single DNA aptamer ZE2 can block HCV E2 protein binding to Huh7.5.1 cells. (D) GST and ZE2 do not bind to huh7.5.1 cells by flow cytometric analysis. (E) CD81 competitively blocked FITC-aptamer binding to E2-CT26 cells by flow cytometric analysis. (F) Both ZE2 and CD81 competitively blocked E2 binding to Huh7.5.1 cells, as determined by flow cytometry with FITC-conjugated anti-E2 antibody. All data are mean±SEM from six separate experiments.

Figure 6. Aptamer ZE2 dramatically blocks HCVcc infection of hepatocytes.

(A) Immunofluorescent detection of intracellular HCV mean fluorescence density (MFI) of HCVcc infected Huh-7.5.1 cells with anti-E2 antibody. The data shown were calculated as mean±SEM and data are from three independent experiments. (B) Western blot analysis of intracellular E2 protein expressions of HCVcc infected Huh7.5.1 in the presence of ZE2 or IFN-α with specific anti-E2 antibody; β-actin, a housekeeping gene with constant expression, was used as internal control. β-actin protein was detected with specific anti- β-actin antibody. (C) Quantitation of intracellular HCV RNA was detected using real-time quantitative RT-PCR and normalized to the levels of GAPDH mRNA. GAPDH, a housekeeping gene with constant mRNA expression, was used as internal control. The data shown were calculated as mean±SEM and data are from three independent experiments.