基于线性荧光探针对T4 DNA连接酶的活性分析

高等学校化学学报 ›› 2010, Vol. 31 ›› Issue (3): 463.

基于线性荧光探针对T4 DNA连接酶的活性分析

刘斌, 羊小海, 王柯敏, 谭蔚泓

湖南大学化学生物传感与计量学国家重点实验室, 化学化工学院, 生命科学与技术研究院, 生物纳米与分子工程湖南省重点实验室, 长沙 410082

收稿日期:2009-11-09 出版日期:2010-03-10 发布日期:2010-03-10
通讯作者: 王柯敏, 男, 博士, 教授, 博士生导师, 主要从事纳米及分子水平上的生物分析化学及纳米生物技术研究. E-mail: kmwang@hnu.cn
基金资助:
国家自然科学基金(批准号: 90606003, 20805012), 国家“八六三”计划项目(批准号: 2007AA022007)和湖南省杰出青年科学基金(批准号: 08JJ1002)资助.

Activity Analysis of T4 DNA Ligase Based on Linear Fluorescence Probes

LIU Bin, YANG Xiao-Hai, WANG Ke-Min*, TAN Wei-Hong

State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China

Received:2009-11-09 Online:2010-03-10 Published:2010-03-10
Contact: WANG Ke-Min. E-mail: kmwang@hnu.cn
Supported by:
国家自然科学基金(批准号: 90606003, 20805012), 国家“八六三”计划项目(批准号: 2007AA022007)和湖南省杰出青年科学基金(批准号: 08JJ1002)资助.

摘要/Abstract

摘要：

利用线性荧光探针作为核酸连接反应的模板和信号分子, 通过实时监测荧光信号的降低来表征连接产物的生成过程, 从而建立了一种连续、简单且特异性高的T4 DNA连接酶活性分析的新方法, 检出限可达1.2 U/mL; 同时, 该方法还可用于快速考察金属离子和化学药物对酶促反应的影响. 实验结果表明, 该法不仅为灵敏、实时监测核酸连接反应提供了一种简便快捷的非同位素分析方法, 也为开展核酸连接酶活性分析、反应动力学机制探讨和药物快速筛选提供了一种新技术.

关键词: 线性荧光探针; T4 DNA连接酶; 实时监测

Abstract:

A continuous, simple method with high specificity for analyzing T4 DNA ligase activity is introduced by monitoring the fluorescence change in real time. Linear DNA probes that can occur in fluorescent resonance energy transfer were used as templates and detecting probes in a homogeneous solution. The limit of detection of T4 DNA ligase was 1.2 U/mL, the K_M and K_cat were (1.18±0.06) μmol/L and 40 min^-1, respectively. In addition, the method was applied to investigate the influence of chemical drugs and metal ions on T4 DNA ligase activity. This method not only overcame the drawbacks of traditional radioactive assays, such as discontinuity and being time-consuming and complicated, but also can be applied to the activity assay, kinetics investigation and drug screening of other ligases.

Key words: Linear fluorescence probe; T4 DNA ligase; Real-time monitoring

TrendMD:

刘斌, 羊小海, 王柯敏, 谭蔚泓. 基于线性荧光探针对T4 DNA连接酶的活性分析. 高等学校化学学报, 2010, 31(3): 463.

LIU Bin, YANG Xiao-Hai, WANG Ke-Min*, TAN Wei-Hong. Activity Analysis of T4 DNA Ligase Based on Linear Fluorescence Probes. Chem. J. Chinese Universities, 2010, 31(3): 463.

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