Zhang Meiling, Song Jiahang, Zhang Huawen, Li Yang, Hun Xu
Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, Ministry of Education, College of Chemistry and Molecular Engineering, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Shandong Key Laboratory of Biochemical Analysis, Qingdao University of Science and Technology, Qingdao, 266042, PR China.
Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, Ministry of Education, College of Chemistry and Molecular Engineering, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Shandong Key Laboratory of Biochemical Analysis, Qingdao University of Science and Technology, Qingdao, 266042, PR China.
Anal Chim Acta. 2025 Oct 1;1369:344357. doi: 10.1016/j.aca.2025.344357. Epub 2025 Jun 22.
Rolling circle amplification (RCA) is a powerful nucleic acid detection method widely used in molecular diagnosis. The circular template, which is an essential component of RCA, is typically generated via enzymatic ligation. However, enzymatic ligation has limitations such as high cost and strict reaction conditions, and nucleic acid detection usually relies on large instruments, which is difficult to promote in places with limited resources. Therefore, it is of great significance to construct a rapid, simple and efficient non-enzymatic ligation detection method.
Herein, a click chemistry-based method was developed for joining the sticky ends of DNA molecules in RCA. This approach leverages a 2-cyano-6-aminobenzothiazole (CBT) and cystine (Cys-Cys) modification at the nucleic acid termini (CBT-nucleic acid-Cys-Cys). When the HAV is present, a tightly packed circular DNA is formed. After the addition of glutathione, the disulfide bond is reduced, facilitating chemical linkage between CBT and Cys. Subsequently, RCA was initiated using phi29 polymerase. The RCA product and dsDNA magnetic conjugation sequence replacement enables the release of hCG-ssDNA2 for hepatitis A virus (HAV) quantification via pregnancy test strip (PTS) detection. The process was validated using mass spectrometry, gel electrophoresis and pregnancy test strip (T-line) signals. The method demonstrated a detection limit of 3.2 fM for HAV, with a linear range from 10.0 nM to 10.0 fM, showcasing its high sensitivity and specificity.
CBT-Cys click ligation has a high rate constant (13.82 M S), offering better kinetic characteristics than the traditional enzyme-catalyzed ligation reaction, providing a milder and more flexible choice for DNA ligation methods. This efficient non-enzymatic ligation system exhibits promising potential for nucleic acid detection and clinical diagnostics, offering a promising approach for rapid, portable and cost-effective testing in future biomedical settings.
滚环扩增（RCA）是一种强大的核酸检测方法，广泛应用于分子诊断。环状模板是RCA的重要组成部分，通常通过酶促连接产生。然而，酶促连接存在成本高和反应条件苛刻等局限性，并且核酸检测通常依赖大型仪器，在资源有限的地方难以推广。因此，构建一种快速、简单且高效的非酶促连接检测方法具有重要意义。
在此，开发了一种基于点击化学的方法用于在RCA中连接DNA分子的粘性末端。该方法利用核酸末端的2-氰基-6-氨基苯并噻唑（CBT）和胱氨酸（Cys-Cys）修饰（CBT-核酸-Cys-Cys）。当甲型肝炎病毒（HAV）存在时，形成紧密堆积的环状DNA。加入谷胱甘肽后，二硫键被还原，促进CBT与Cys之间的化学连接。随后，使用phi29聚合酶启动RCA。RCA产物与双链DNA磁共轭序列置换使得能够通过验孕棒（PTS）检测释放出人绒毛膜促性腺激素单链DNA2（hCG-ssDNA2）以定量甲型肝炎病毒（HAV）。该过程通过质谱、凝胶电泳和验孕棒（T线）信号进行了验证。该方法对HAV的检测限为3.2 fM，线性范围为10.0 nM至10.0 fM，显示出其高灵敏度和特异性。
CBT-Cys点击连接具有高反应速率常数（13.82 M⁻¹S⁻¹），比传统的酶催化连接反应具有更好的动力学特性，为DNA连接方法提供了更温和、更灵活的选择。这种高效的非酶促连接系统在核酸检测和临床诊断中展现出有前景的潜力，为未来生物医学环境中的快速、便携且经济高效的检测提供了一种有前景的方法。
