"Construction of a simple, localized and homogeneous fluorescence detection platform for T4 PNK activity based on tetrahedral DNA nanostructure-mediated primer exchange reaction"

作者全名："Xie, Zuowei; Wang, Xingyu; Chen, Siyi; Zhao, Zixin; Zhao, Shuhui; Zhang, Wenxiu; Luo, Lijuan; Yi, Gang"

作者地址："[Xie, Zuowei; Wang, Xingyu; Chen, Siyi; Zhao, Zixin; Zhao, Shuhui; Zhang, Wenxiu; Luo, Lijuan; Yi, Gang] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China"

通信作者："Yi, G (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China."

来源：MICROCHEMICAL JOURNAL

ESI学科分类：CHEMISTRY

WOS号：WOS:000868296300006

JCR分区：Q1

影响因子：4.8

年份：2022

卷号：183

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：Localization; Sensitive; T4 polynucleotide kinase; Tetrahedral DNA nanostructure; Primer exchange reaction

摘要："T4 polynucleotide kinase (T4 PNK), which acts as a bifunctional enzyme, is widely involved in important phosphorylation and dephosphorylation processes. Sensitive and specific detection of T4 PNK activity will help to elucidate its critical importance in disease diagnosis and treatment. The conventional detection method uses hairpin or single-strand primers modified with phosphorylation group as the substrates for T4 PNK hydrolysis. However, considering to the large spatial distances and disordered reaction orientation among the substrates and enzymes, the efficiency and sensitivity of the method are somewhat limited. Here, we propose a simple and sensitive signal amplification strategy, integrating the tetrahedral DNA nanostructure with primer exchange reaction (TDN-PER), which ensures different reaction direction and improves the local reaction concentrations. Compared to dispersed single PER, localized TDN-PER provides more primers for initiating signal amplification. Benefiting from the unique structural transformation from dispersion to localization, this strategy provided ultrahigh sensitivity, achieving a detection limit of 1.8 x 10-5 U/mL, while maintaining excellent accuracy and specificity in complex human serum and cell lysates environments. The method successfully evaluated the inhibitory effects of (NH4)2SO4 and Na2HPO4 on T4 PNK activity. Finally, the practical application capability of this strategy was verified in the detection of cell-free extracts, providing a promising analytical platform for T4 PNK-related biological and medical research."

基金机构：Key Project of Science and Technology Research Program of Chongqing Education Commission [KJZD-K202000404]; Special Fund Project Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education)

基金资助正文：This work was supported by the Key Project of Science and Technology Research Program of Chongqing Education Commission (Grant No. KJZD-K202000404) and the Special Fund Project Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education) .