Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3.
Li Ka Shing Institute of Virology, Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E1.
Anal Chem. 2020 May 5;92(9):6495-6501. doi: 10.1021/acs.analchem.9b05740. Epub 2020 Apr 16.
The e antigen of hepatitis B (HBeAg) is positively associated with an increased risk of developing liver cancer and cirrhosis in chronic hepatitis B (CHB) patients. Clinical monitoring of HBeAg provides guidance to the treatment of CHB and the assessment of disease progression. We describe here an affinity binding assay for HBeAg, which takes advantage of G-quadruplex aptamers for enhanced binding and stability. We demonstrate a strategy to improve the binding affinity of aptamers by modifying their sequences upon their G-quadruplex and secondary structures. On the basis of predicting a stable G-quadruplex and a secondary structure, we truncated 19 nucleotides (nt) from the primer regions of an 80-nt aptamer, and the resulting 61-nt aptamer enhanced binding affinity by 19 times ( = 1.2 nM). We mutated a second aptamer (40 nt) in one loop region and incorporated pyrrolo-deoxycytidine to replace deoxycytidine in another loop. The modified 40-nt aptamer, with a stable G-quadruplex and two modified loops, exhibited a 100 times higher binding affinity for HBeAg ( = 0.4 nM) than the unmodified original aptamer. Using the two newly modified aptamers, one serving as the capture and the other as the reporter, we have developed an improved sandwich binding assay for HBeAg. Analyses of HBeAg in serum samples (concentration ranging from 0.1 to 60 ng/mL) of 10 hepatitis B patients, showing consistent results with clinical tests, demonstrate a successful application of the aptamer modification strategy and the associated aptamer binding assay.
乙型肝炎病毒 e 抗原 (HBeAg) 与慢性乙型肝炎 (CHB) 患者发生肝癌和肝硬化的风险增加呈正相关。HBeAg 的临床监测为 CHB 的治疗和疾病进展的评估提供了指导。我们在这里描述了一种用于 HBeAg 的亲和结合测定法，该方法利用 G-四链体适体提高了结合和稳定性。我们展示了一种通过修饰其 G-四链体和二级结构来提高适体结合亲和力的策略。基于预测稳定的 G-四链体和二级结构，我们从 80-nt 适体的引物区域截断了 19 个核苷酸 (nt)，所得的 61-nt 适体将结合亲和力提高了 19 倍（= 1.2 nM）。我们在一个环区突变了第二个适体（40 nt），并在另一个环区掺入了吡咯脱氧胞苷以取代脱氧胞苷。修饰后的 40-nt 适体具有稳定的 G-四链体和两个修饰环，与未修饰的原始适体相比，对 HBeAg 的结合亲和力提高了 100 倍（= 0.4 nM）。使用这两个新修饰的适体，一个用作捕获物，另一个用作报告物，我们开发了一种改进的 HBeAg 夹心结合测定法。对 10 名乙型肝炎患者的血清样本（浓度范围为 0.1 至 60 ng/mL）中的 HBeAg 进行分析，与临床测试结果一致，证明了适体修饰策略和相关适体结合测定法的成功应用。
