Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection,Analytical Chemistry

当前位置： X-MOL 学术 › Anal. Chem. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-04-26 , DOI: 10.1021/acs.analchem.1c00507
Zhihao Ming _{1,

2} , Wei Zhang ₂ , Meng Lin ₁ , Xiaofeng Tang ₁ , Na Chen ₁ , Na Liu ₁ , Xiaoyan Xin ₂ , Hongbo Wang ₂ , Wenpei Xiang ₁ , Xianjin Xiao _{1,

2}

Affiliation

Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.

中文翻译：

指导链控制的DNA核酸酶具有增强的特异性和可调节的动力学，用于DNA突变检测。

核酸酶是各种生物医学应用中的强大工具，例如基因工程，生物传感和分子诊断。但是，常用的核酸酶（核酸内切酶IV，嘌呤/嘧啶内切核酸酶-1和λ核酸外切酶）易于单链DNA的非特异性裂解，从而使所需的反应产率极低且不可预测。在这里，我们已经开发了指导链控制的核酸酶系统，并构建了理论动力学模型来解释其作用机理。模型显示出与实验结果极好的一致性，从而使动力学具有高度可预测性和可调性。我们的方法抑制了单链探针的非特异性切割，同时保持了双链DNA的高效切割。我们还通过检测从癌症患者血液中提取的基因组DNA样品中的低频突变来证明该方法的临床实用性。PTEN rs121909219的检出限可能为0.01％。我们相信，我们的发现为该领域提供了有力的工具，并且已建立的模型使我们对DNA核酸酶的酶促活性有了更深入的了解。

更新日期：2021-05-11

点击分享查看原文

点击收藏

阅读更多本刊新发论文本刊介绍/投稿指南