文献类型：期刊文献

英文题名：Unveiling the Heterogenous Dephosphorylation of DNA Using an Aerolysin Nanopore

作者：Li, Meng-Yin[1,2];Ying, Yi-Lun[1,2];Li, Shuang[3];Wang, Ya-Qian[3];Wu, Xue-Yuan[1,3];Long, Yi-Tao[1]

机构：[1]Nanjing Univ, Sch Chem & Chem Engn, State Key Lab Analyt Chem Life Sci, Nanjing 210023, Peoples R China;[2]Nanjing Univ, Chem & Biomed Innovat Ctr, Nanjing 210023, Peoples R China;[3]East China Univ Sci & Technol, Sch Chem & Mol Engn, Shanghai 200237, Peoples R China

年份：2020

卷号：14

期号：10

起止页码：12571

外文期刊名：ACS NANO

收录：;EI(收录号：20204809548634);WOS:【SCI-EXPANDED(收录号:WOS:000586793400032)】；

基金：This research was supported by the National Natural Science Foundation of China (21834001), the China Postdoctoral Science Foundation (2020M671429), the Excellent Research Program of Nanjing University (ZYJH004), and the Fundamental Research Funds for the Central Universities (14380239).

语种：英文

外文关键词：phosphorylation; DNA; confined space; nanopore sensors; single-molecule interface

摘要：The simultaneous occurrence of multiple heterogeneous DNA phosphorylation statuses, which include end phosphorylation, 5' end dephosphorylation, 3' end phosphorylation, and 3' lend 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 fusing 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.