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Microchimica Acta

, 186:48 | Cite as

A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe

  • Jinlong Li
  • Jiehua Ma
  • Yongchen Zhang
  • Zhaoli Zhang
  • Guangwu He
Original Paper
  • 103 Downloads

Abstract

The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5′ hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL−1), a low detection limit (0.01 U·mL−1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.

Graphical abstract

A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.

Keywords

Phosphorylation T4 PNK Proximity effect Cell extracts Clinical diagnostics Inhibitor Lambda exonuclease DNA-AgNCs Na2HPO4 ATP 

Notes

Acknowledgments

This work is supported by the National Natural Science Foundation of China (Grant No. 81703088).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2018_3157_MOESM1_ESM.docx (156 kb)
ESM 1 (DOCX 156 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Laboratory Medicine, The Second Hospital of NanjingNanjing University of Chinese MedicineNanjingPeople’s Republic of China
  2. 2.Women’s Hospital of Nanjing Medical UniversityNanjing Maternity and Child Health Care HospitalNanjingPeople’s Republic of China
  3. 3.School of PharmacyNanjing Medical UniversityNanjingPeople’s Republic of China

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