Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification,Analytical Chemistry

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Binding Activity of Prokaryotic Argonaute for Background-Suppressed Exponential Isothermal Amplification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-01-03 , DOI: 10.1021/acs.analchem.3c05263
Qiuyuan Lin ₁ , Xin Ye ₂ , Hongyuan Chen ₃ , Xueen Fang ₄ , Hui Chen ₄ , Jilie Kong ₄

Affiliation

Prokaryotic Argonautes (pAgos) have been recently used in many nucleic acid biosensing applications but have rarely been used for regulating the isothermal amplification system. Herein, we reported Thermus thermophilus Argonaute (TtAgo)-mediated background-suppressed exponential isothermal amplification (EXPAR) as the first example to explore the binding activity of pAgos toward regulation of the amplification template. It was demonstrated that thermophilic pAgos efficiently eliminated nonspecific hybridization between templates by their binding affinity with the template, resulting in greatly enhancing the specificity of EXPAR. TtAgo-mediated, background-suppressed EXPAR was employed to detect miRNA with a detection limit of 10^–15 M, which was 1000 times and 100 times more sensitive than that of traditional RT-PCR and EXPAR, respectively. This method further showed good performance in discriminating cancer patients from healthy individuals, indicating its potential for practical clinical applications.

中文翻译：

原核Argonaute对背景抑制指数等温扩增的结合活性

原核Argonautes (pAgos)最近已被用于许多核酸生物传感应用，但很少用于调节等温扩增系统。在此，我们报道了嗜热栖热菌Argonaute (TtAgo) 介导的背景抑制指数等温扩增 (EXPAR)，作为探索 pAgos 对扩增模板调节的结合活性的第一个例子。结果表明，嗜热pAgos通过与模板的结合亲和力有效地消除了模板之间的非特异性杂交，从而大大增强了EXPAR的特异性。采用TtAgo介导、背景抑制的EXPAR检测miRNA，检测限为^10-15M ，分别比传统RT-PCR和EXPAR灵敏1000倍和100倍。该方法在区分癌症患者与健康个体方面进一步表现出良好的性能，表明其具有实际临床应用的潜力。

更新日期：2024-01-03

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