摘要
G-三链体(G3)由三段连续G碱基的富G序列组成的,因其潜在的生物学功能和特殊的结构受到越来越多的关注。G3序列能结合硫磺素T(ThT)发出强荧光,以此作信号报告分子,本文提出了一种基于G3分子信标(MB)的T4多聚核苷酸激酶(PNK)荧光检测新方法。有T4 PNK时,P1的5'末端被磷酸化,P1/G3MB双链结构中的P1被外切酶酶切,封闭在G3MB中的G3序列不能结合ThT,得到弱荧光信号。无T4 PNK时,P1/G3MB双链结构能阻止外切酶酶切,G3MB被打开,释放G3序列结合ThT荧光增强。该方法对T4 PNK检测的线性范围为0.0002~0.01 U/μL,检测限为0.0002 U/μL。该方法具有操作简便、成本低和选择性高等优点,并可用于T4 PNK酶活性抑制剂的评估。
G-triplex(G3)is formed by G-rich sequences with only three G-tracts,has attracted great attention due to its potential biological function and special structure.G3 can bind to thioflavin T(ThT)and emit strong fluorescence,which can act as a signal reporter.In this paper,a new method for fluorescence detection of T4 polynucleotide kinase(PNK)based on G3-based molecular beacon(MB)is proposed.In the presence of T4 PNK,the 5'end of P1 is phosphorylated,P1 in the P1/G3MB double-stranded structure is cleaved by exonuclease,and the G3 sequence enclosed in G3MB cannot bind ThT,resulting in a weak fluorescent signal.In the absence of T4 PNK,the double-stranded structure of P1/G3MB can prevent exonuclease cleavage,G3MB is opened,and the G3 sequence is released to enhance the fluorescence of ThT.The linear range of this method is 0.0002~0.01 U/μL,and the detection limit is 0.0002 U/μL.This method has the advantages of simple operation,low cost and high selectivity,and is suitable for the evaluation of T4 PNK enzyme activity inhibitors.
作者
朱文平
刘硕硕
李志怡
ZHU Wenping;LIU Shuoshuo;LI Zhiyi(College of Chemistry and Chemical Engineering,Zhoukou Normal University,Zhoukou 466001,China)
出处
《周口师范学院学报》
CAS
2022年第5期52-56,共5页
Journal of Zhoukou Normal University
基金
周口师范学院校本项目“DNA碱基切除修复蛋白超灵敏检测的电化学传感新方法研究”(ZKNUB1201701)。