A Fluorimetric Nanoplatform Based On Metal–organic Frameworks And Coupled Exonuclease Reaction For Determination Of T4 Polynucleotide Kinase Activity And Its Inhibition Screening

As a vital enzyme in DNA phosphorylation,T4 polynucleotide kinase（T4 PNK）catalyzes the transfer ofγ-phosphate from adenosine triphosphate（ATP）to the5’-hydroxyl terminal of DNA,which is an important process in DNA replication,recombination and restoration.The hindrance of DNA phosphorylation will affect the cellular responses to DNA damage and is directly related to some human diseases,such as Bloom’s syndromes,Werner syndromes,and Rothmund-Thomson syndromes.According to previous reports,the T4 PNK inhibition is likely to improve the efficiency ofγ-radiation treatment on human tumors.Therefore,it is of great significance to construct a sensitive and facile strategy to determine the T4 PNK activity in biological matrix and screen its inhibitors.A nanoplatform based on metal–organic frameworks（MOFs）and Lambda exonuclease（λexo）for the fluorimetric determination of T4 polynucleotide kinase（T4 PNK）activity and inhibition was developed.Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA（ss DNA）over double-stranded DNA（ds DNA）and its quenching property.Briefly,FAM-labeled ds DNA（FAM-ds DNA）was made up of FAM-labeled ss DNA（FAM-ss DNA）and its complementary DNA.In the presence of T4 PNK,FAM-ds DNA was phosphorylated on its 5’-terminal.λexo then recognized and cleaved the phosphorylated strand to yield FAM-ss DNA.The fluorescence of the produced FAM-ss DNA was quenched due to Fe-MIL-88’s adsorption to FAM-ss DNA.On the contrary,in the absence of T4 PNK,the phosphorylation and cleavage processes cannot take place.Therefore,the fluorescence of FAM-ds DNA still remained.The fluorescence intensity was detected at maximum emission wavelength of 524 nm under the maximum excitation wavelength of 488 nm.The related experimental conditions were optimized,such as concentration of Fe-MIL-88,concentration of ATP,phosphorylation time,the p H,Mg²⁺concentration andλexo units.The synthesized Fe-MIL-88 was characterized by transmission electron microscope,X-ray photoelectron spectroscopy,Fourier infrared spectroscopy and so on.In addition,the mechanism and feasibility of the assay were investigated by fluorescence lifetime data,Stern-Volmer curves,agarose gel electrophoresis,etc.The interference study demonstrated that the nanoplatform possessed excellent selectivity.The assay of T4 PNK based on the fluorescence quenching of FAM-ss DNA achieves a linear relationship in the range of 0.010-5.0 U?m L^-1with a detection limit of 0.0089 U?m L^-1in buffer.The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix.It shows a good linear relationship ranging from 0.050 to 10.0 U?m L^-1in biological samples.The results also reveal the great ability of the assay in T4 PNK inhibitor screening.