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Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters

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Abstract

A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3′-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570 nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07 U mL−1 to 15 U mL−1 and a detection limit of 0.06 U mL−1.

The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant no. 21775099 and 21475083).

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Authors and Affiliations

  1. Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, China

    Xingxing Zhang, Qiang Liu, Yan Jin & Baoxin Li

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  1. Xingxing Zhang
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  2. Qiang Liu
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  3. Yan Jin
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  4. Baoxin Li
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Correspondence to Baoxin Li.

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Zhang, X., Liu, Q., Jin, Y. et al. Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters. Microchim Acta 186, 3 (2019). https://doi.org/10.1007/s00604-018-3102-1

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  • DOI: https://doi.org/10.1007/s00604-018-3102-1

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