Unveiling the Heterogenous Dephosphorylation of DNA Using an Aerolysin Nanopore.,ACS Nano

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Unveiling the Heterogenous Dephosphorylation of DNA Using an Aerolysin Nanopore.
ACS Nano ( IF 15.8 ) Pub Date : 2020-08-03 , DOI: 10.1021/acsnano.0c03215
Meng-Yin Li _{1,

2} , Yi-Lun Ying _{1,

2} , Shuang Li ₃ , Ya-Qian Wang ₃ , Xue-Yuan Wu _{1,

3} , Yi-Tao Long ₁

Affiliation

The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include 5′ end phosphorylation, 5′ end dephosphorylation, 3′ end phosphorylation, and 3′ end dephosphorylation, is crucial for regulating numerous cellular processes. Although there are many methods for detecting a single type of DNA phosphorylation, the direct and simultaneous identification of DNA phosphorylation/dephosphorylation on the 5′ and/or 3′ ends remains a challenge, let alone the unveiling of the heterogeneous catalysis processes of related phosphatases and kinases. Taking advantage of the charge-sensitive aerolysin nanopore interface, herein, an orientation-dependent sensing strategy is developed to enhance phosphorylation-site-dependent interaction with the nanopore sensing interface, enabling the direct and simultaneous electric identification of four heterogeneous phosphorylation statuses of a single DNA. By using this strategy, we can directly evaluate the heterogeneous dephosphorylation process of alkaline phosphatase (ALP) at the single-molecule level. Our results demonstrate that the ALP in fetal bovine serum preferentially catalyzes the 3′ phosphate rather than both ends. The quantification of endogenous ALP activity in fetal bovine serum could reach the submilli-IU/L level. Our aerolysin measurements provide a direct look at the heterogeneous phosphorylation status of DNA, allowing the unveiling of the dynamic single-molecule functions of kinase and phosphatase.

中文翻译：

使用Aerolysin纳米孔揭示DNA的异质去磷酸化。

同时发生多种异质DNA磷酸化状态，包括5'末端磷酸化，5'末端脱磷酸化，3'末端磷酸化和3'末端脱磷酸，对于调节众多细胞过程至关重要。尽管有很多方法可以检测单一类型的DNA磷酸化，但直接和同时鉴定5'和/或3'末端的DNA磷酸化/去磷酸化仍然是一个挑战，更不用说相关磷酸酶的多相催化过程的揭开了。和激酶。在本文中，利用电荷敏感的溶血素溶血素纳米孔界面，开发了一种方向依赖性的传感策略，以增强与纳米孔传感界面的磷酸化位点依赖性相互作用，可以直接和同时电识别单个DNA的四个异质磷酸化状态。通过使用此策略，我们可以在单分子水平上直接评估碱性磷酸酶（ALP）的异质去磷酸化过程。我们的结果表明，胎牛血清中的ALP优先催化3'磷酸而不是两端。胎牛血清中内源性ALP活性的定量可以达到submilli-IU / L水平。我们的气溶素测量结果可以直接观察DNA的异质磷酸化状态，从而揭示激酶和磷酸酶的动态单分子功能。我们可以直接评估单分子水平上碱性磷酸酶（ALP）的异质去磷酸化过程。我们的结果表明，胎牛血清中的ALP优先催化3'磷酸而不是两端。胎牛血清中内源性ALP活性的定量可以达到submilli-IU / L水平。我们的气溶素测量结果可以直接观察DNA的异质磷酸化状态，从而揭示激酶和磷酸酶的动态单分子功能。我们可以直接评估单分子水平上碱性磷酸酶（ALP）的异质去磷酸化过程。我们的结果表明，胎牛血清中的ALP优先催化3'磷酸而不是两端。胎牛血清中内源性ALP活性的定量可以达到submilli-IU / L水平。我们的气溶素测量结果可直接观察DNA的异质磷酸化状态，从而揭示激酶和磷酸酶的动态单分子功能。

更新日期：2020-08-03

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