Aptamer Binding Assay for the E Antigen of Hepatitis B Using Modified Aptamers with G-Quadruplex Structures

Area of Science:

• Biochemistry
• Molecular Biology
• Medical Diagnostics

Background:

• Hepatitis B e antigen (HBeAg) is a key biomarker in chronic hepatitis B (CHB) infection, linked to liver cancer and cirrhosis risk.
• Accurate HBeAg monitoring is crucial for guiding CHB treatment and assessing disease progression.

Purpose of the Study:

• To develop an enhanced affinity binding assay for HBeAg detection.
• To improve G-quadruplex aptamer binding affinity and stability through sequence modification.

Main Methods:

• G-quadruplex aptamer sequence optimization via truncation and loop modification with pyrrolo-deoxycytidine.
• Development of a sandwich binding assay utilizing two modified aptamers as capture and reporter molecules.
• Validation of the assay using HBeAg-positive human serum samples.

Main Results:

• Truncating an 80-nt aptamer to 61-nt increased HBeAg binding affinity 19-fold (Kd = 1.2 nM).
• Modifying a 40-nt aptamer's loops and incorporating pyrrolo-deoxycytidine enhanced binding affinity 100-fold (Kd = 0.4 nM).
• The developed sandwich assay accurately detected HBeAg in patient serum samples (0.1–60 ng/mL).

Conclusions:

• Aptamer modification strategies significantly enhance binding affinity and stability for HBeAg detection.
• The improved aptamer binding assay offers a sensitive and reliable method for HBeAg clinical monitoring.
• This work demonstrates the potential of modified aptamers in advancing diagnostic tools for hepatitis B.