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Microchimica Acta

, 186:3 | Cite as

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

  • Xingxing Zhang
  • Qiang Liu
  • Yan Jin
  • Baoxin Li
Original Paper

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.

Graphical abstract

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.

Keywords

T4 polynucleotide kinase phosphatase activity Fluorescent biosensing Label-free Copper nanoclusters Near-zero background 

Notes

Acknowledgments

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

Compliance with ethical standards

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

Supplementary material

604_2018_3102_MOESM1_ESM.docx (662 kb)
ESM 1 (DOCX 662 kb)

References

  1. 1.
    Allinson SL (2010) DNA end-processing enzyme polynucleotide kinase as a potential target in the treatment of cancer. Future Oncol 6:1031–1042CrossRefPubMedGoogle Scholar
  2. 2.
    Hu L, Lu C-H, Willner I (2015) Switchable catalytic DNA catenanes. Nano Lett 15:2099–2103CrossRefPubMedGoogle Scholar
  3. 3.
    Karimi-Busheri F, Lee J, Tomkinson AE, Weinfeld M (1998) Repair of DNA strand gaps and nicks containing 3′-phosphate and 5′-hydroxyl termini by purified mammalian enzymes. Nucleic Acids Res 26:4395–4400CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Wang LK, Shuman S (2001) Domain structure and mutational analysis of T4 polynucleotide kinase. J Biol Chem 276:26868–26874CrossRefPubMedGoogle Scholar
  5. 5.
    Zhuang J, Lai W, Xu M, Zhou Q, Tang D (2015) Plasmonic AuNP/g-C3N4 nanohybrid-based photoelectro chemical sensing platform for ultrasensitive monitoring of polynucleotide kinase activity accompanying DNAzyme-catalyzed precipitation amplification. ACS Appl Mater Interfaces 7:8330–8338CrossRefPubMedGoogle Scholar
  6. 6.
    Liu S, Liu T, Wang L (2015) Label-free, isothermal and ultrasensitive electrochemical detection of DNA and DNA 3′-phosphatase using a cascade enzymatic cleavage strategy. Chem Commun 51:176–179CrossRefGoogle Scholar
  7. 7.
    Jiang C, Yan C, Jiang J, Yu R (2013) Colorimetric assay for T4 polynucleotide kinase activity based on the horseradish peroxidase-mimicking DNAzyme combined with λ exonuclease cleavage. Anal Chim Acta 766:88–93CrossRefPubMedGoogle Scholar
  8. 8.
    Jiang HX, Kong DM, Shen HX (2014) Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification. Biosens Bioelectron 55:133–138CrossRefPubMedGoogle Scholar
  9. 9.
    Du J, Xu Q, Lu X, Zhang C (2014) A label-free bioluminescent sensor for real-time monitoring polynucleotide kinase activity. Anal Chem 86:8481–8488CrossRefPubMedGoogle Scholar
  10. 10.
    Zhao H, Liu Q, Liu M, Jin Y, Li B (2017) Label-free fluorescent assay of T4 polynucleotide kinase phosphatase activity based on G-quadruplexe-thioflavin T complex. Talanta 165:653–658CrossRefPubMedGoogle Scholar
  11. 11.
    Zhou L, Shen X, Sun N, Wang K, Zhang Y, Pei R (2015) Label-free fluorescence light-up detection of T4 polynucleotide kinase activity using the split-to-intact G-quadruplex strategy by ligation-triggered and toehold-mediated strand displacement release. Analyst 140:5450–5453CrossRefPubMedGoogle Scholar
  12. 12.
    Hou T, Wang X, Liu X, Lu T, Liu S, Li F (2014) Amplified detection of T4 polynucleotide kinase activity by the coupled λ exonuclease cleavage reaction and catalytic assembly of bimolecular beacons. Anal Chem 86:884–890CrossRefPubMedGoogle Scholar
  13. 13.
    Lin L, Liu Y, Zhao X, Li J (2011) Sensitive and rapid screening of T4 polynucleotide kinase activity and inhibition based on coupled exonuclease reaction and graphene oxide platform. Anal Chem 83:8396–8402CrossRefPubMedGoogle Scholar
  14. 14.
    Ma C, Jin S, Wang J, Wang K, Liu H, Wu K (2016) A fluorescence-based assay for T4 polynucleotide kinase/phosphatase activity based on a terminal transferase-aided photoinduced electron transfer strategy. Anal Methods 8:1989–1994CrossRefGoogle Scholar
  15. 15.
    Cen Y, Yang Y, Yu RQ, Chen TT, Chu X (2016) A cobalt oxyhydroxide nanoflake-based nanoprobe for the sensitive fluorescence detection of T4 polynucleotide kinase activity and inhibition. Nanoscale 8:8202–8209CrossRefPubMedGoogle Scholar
  16. 16.
    Wang L, Zhang Q, Tang B, Zhang C (2017) Single-molecule detection of polynucleotide kinase based on phosphorylation-directed recovery of fluorescence quenched by au nanoparticles. Anal Chem 89:7255–7261CrossRefPubMedGoogle Scholar
  17. 17.
    Xu M, Li B (2015) Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR green I as probe. Spectrochim Acta A 151:22–26CrossRefGoogle Scholar
  18. 18.
    Hu H, Zhang J, Ding Y, Zhang X, Xu K, Hou X, Wu P (2017) Modulation of the singlet oxygen generation from the double strand DNA-SYBR green I complex mediated by T-melamine-T mismatch for visual detection of melamine. Anal Chem 89:5101–5106CrossRefPubMedGoogle Scholar
  19. 19.
    Li X, Xu X, Song J, Xue Q, Li C, Jiang W (2017) Sensitive detection of T4 polynucleotide kinase activity based on multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification. Biosens Bioelectron 91:631–636CrossRefPubMedGoogle Scholar
  20. 20.
    Zhu W, Zhao Z, Li Z, Jiang J, Shen G, Yu R (2013) A graphene oxide platform for the assay of DNA 3′-phosphatases and their inhibitors based on hairpin primer and polymerase elongation. J Mater Chem B 1:361–367CrossRefGoogle Scholar
  21. 21.
    Liao H, Liu G, Liu Y, Li R, Fu W, Hu L (2017) Aggregation-induced accelerating peroxidase-like activity of gold nanoclusters and their applications for colorimetric Pb2+ detection. Chem Commun 53:10160–10163CrossRefGoogle Scholar
  22. 22.
    Huang L, Zhang W, Chen K, Zhu W, Liu X, Wang R, Zhang X, Hu N, Suo Y, Wang J (2017) Facet-selective response of trigger molecule to CeO2{110} for up-regulating oxidase-like activity. Chem Eng J 330:746–752CrossRefGoogle Scholar
  23. 23.
    Huang L, Chen K, Zhang W, Zhu W, Zhu X, Liu X, Wang J, Wang R, Hu N, Suo Y, Wang J (2018) ssDNA-tailorable oxidase-mimicking activity of spinel MnCo2O4 for sensitive biomolecular detection in food sample. Sensors Actuators B Chem 269:79–87CrossRefGoogle Scholar
  24. 24.
    Yuan Z, Chen YC, Li HW, Chang HT (2014) Fluorescent silver nanoclusters stabilized by DNA scaffolds. Chem Commun 50:9800–9815CrossRefGoogle Scholar
  25. 25.
    Huang J, Lin L, Sun D, Chen H, Yang D, Li Q (2015) Bio-inspired synthesis of metal nanomaterials and applications. Chem Soc Rev 44:6330–6374CrossRefPubMedGoogle Scholar
  26. 26.
    Hu X, Liu T, Zhuang Y, Wang W, Li Y, Fan W, Huang Y (2016) Recent advances in the analytical applications of copper nanoclusters. Trends Anal Chem 77:66–75CrossRefGoogle Scholar
  27. 27.
    Xu F, Shi H, He X, Wang K, He D, Guo Q, Qing Z, Yan L, Ye X, Li D, Tang J (2014) Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection. Anal Chem 86:6976–6982CrossRefPubMedGoogle Scholar
  28. 28.
    Ge J, Zhang L, Dong ZZ, Cai QY, Li ZH (2016) Sensitive and label-free T4 polynucleotide kinase/phosphatase detection based on poly(thymine)-templated copper nanoparticles coupled with nicking enzyme-assisted signal amplification. Anal Methods 8:2831–2836CrossRefGoogle Scholar
  29. 29.
    Zhang L, Zhao J, Zhang H, Jiang J, Yu R (2013) Double strand DNA-templated copper nanoparticle as a novel fluorescence indicator for label-free detection of polynucleotide kinase activity. Biosens Bioelectron 44:6–9CrossRefPubMedGoogle Scholar
  30. 30.
    Qing Z, He X, He D, Wang K, Xu F (2013) Poly(thymine)-templated selective formation of fluorescent copper nanoparticles. Angew Chem Int Ed 52:9719–9722CrossRefGoogle Scholar
  31. 31.
    Song Q, Shi Y, He D, Xu S, Ouyang J (2015) Sequence−dependent dsDNA−templated formation of fluorescent copper nanoparticles. Chem Eur J 21:2417–2422CrossRefPubMedGoogle Scholar
  32. 32.
    Wei W, Lu Y, Chen W, Chen S (2011) One-pot synthesis, photoluminescence, and electrocatalytic properties of subnanometer-sized copper clusters. J Am Chem Soc 133:2060–2063CrossRefPubMedGoogle Scholar
  33. 33.
    Liu G, Shao Y, Peng J, Dai W, Liu L, Xu S, Wu F, Wu X (2013) Highly thymine-dependent formation of fluorescent copper nanoparticles template by ssDNA. Nanotechnology 24:345502–345508CrossRefPubMedGoogle Scholar
  34. 34.
    Qing T, Qing Z, Mao Z, He X, Xu F, Wen L, He D, Shi H, Wang K (2014) dsDNA-templated fluorescent copper nanoparticles: poly(AT-TA)-dependent formation. RSC Adv 4:61092–61095CrossRefGoogle Scholar
  35. 35.
    Zhou F, Meng R, Liu Q, Jin Y, Li B (2016) Photoinduced electron transfer-based fluorescence quenching combined with rolling circle amplification for sensitive detection of microRNA. ChemistrySelect 1:6422–6428CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical EngineeringShaanxi Normal UniversityXi’anChina

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