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 (MOFs) and lambda exonuclease (λ 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. λ 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. Graphical abstract 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; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Sensitive fluorescence sensing of T4 polynucleotide kinase activity and inhibition based on DNA/polydopamine nanospheres platform.

5'-Polynucleotide kinase (PNK) is a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-OH termini and this phosphorylation reaction has been involved in many important cellular activities. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, the polydopamine nanospheres (PDANS) could adsorb single-stranded DNA (ssDNA) through π-π stacking or hydrogen bonding between nucleobases and aromatic groups of PDANS, while the interaction between double-stranded DNA (dsDNA) with PDANS was weakened due to the changed conformation. Hence, a novel DNA/PDANS platform was constructed for the sensitive and selective determination of T4 PNK activity based on the preferential binding properties of PDANS for ssDNA over dsDNA and the excellent fluorescence quenching property of PDANS. The dye-labeled dsDNA was phosphorylated by T4 PNK and then digested by λ exonuclease, yielding dye-labeled ssDNA, which would be adsorbed on the surface of the PDANS and the fluorescence was greatly quenched by PDANS. Because of the preferential binding properties of PDANS for ssDNA over dsDNA and the high quenching property of PDANS, the developed DNA/PDANS platform exhibited good analytical performance for T4 PNK sensing in complex biological matrix and applied to screening inhibitors. The proposed DNA/PDANS based platform is promising in developing high-throughput assays for drug screening and clinical diagnostics.