A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition
A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition
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基于金属有机框架和偶联核酸外切酶反应的纳米平台，用于荧光测定 T4 多核苷酸激酶活性和抑制
DOI：
10.1007/s00604-020-4194-y
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发表时间：
2020-03-23
影响因子：
5.7
通讯作者：
Cen, Yao
中科院分区：
文献类型：
--
作者：
Chai, Yuying;Cheng, Xia;Cen, Yao
介绍了一种基于金属有机骨架(MOF)和波长外切酶(Lambda Exo)的纳米平台，用于荧光测定T4多核苷酸激酶(T4 PNK)的活性和抑制。Fe-MIL-88与单链DNA(SsDNA)的结合能力明显优于双链DNA(DsDNA)，且具有猝灭特性，因此被选为纳米材料。用透射电子显微镜、扫描电子显微镜和X射线光电子能谱对合成的Fe-MIL-88进行了表征。在T4 PNK存在下，FAM标记的dsDNA(FAM-dsDNA)在其5‘端被磷酸化。然后，Lambda exo识别并切割磷酸化的链，产生FAM标记的单链DNA(FAM-ssDNA)。由于Fe-mIL-88‘S吸附在FAM-SSDNA上，产生的FAM-SSDNA的荧光被猝灭。相反，在没有T4 PNK的情况下，磷酸化和切割过程不能发生。因此，FAM-dsDNA的荧光仍然存在。在最大激发波长为488 nm的情况下，在最大发射波长524 nm处检测到荧光强度。基于荧光猝灭的T4PNK在缓冲液中的线性范围为0.0089.0 1~5.0 U m L(-1)，检出限为5.0 0 U m L(-1)。该方法可用于复杂生物基质中T4 PNK活性的测定。建立了一种基于Fe-MIL-88和偶联核酸外切酶反应的纳米平台，用于T4多核苷酸激酶活性的荧光检测。FAM-ssDNA，FAM标记的单链DNA；cDNA，互补DNA；lambda exo，lambda外切酶；T4 PNK，T4多核苷酸激酶
A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (lambda exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5 '-terminal. lambda exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL(-1) with a detection limit of 0.0089 U mL(-1) in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening.Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; lambda exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase