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

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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.

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.

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References

  1. Foster ER, Downs JA (2010) Histone H2A phosphorylation in DNA double-strand break repair. FEBS J 272(13):3231–3240

    Article  Google Scholar 

  2. Karimi-Busheri F, Daly G, Robins P, Canas B, Pappin DJC, Sgouros J, Miller GG, Fakhrai H, Davis EM, Le Beau MM, Weinfeld M (1999) Molecular characterization of a human DNA kinase. J Biol Chem 274(34):24187–24194

    Article  CAS  PubMed  Google Scholar 

  3. 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 165:653–658

    Article  CAS  PubMed  Google Scholar 

  4. 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 91:631–636

    Article  CAS  Google Scholar 

  5. Lin L, Liu Y, Yan J, Wang X, Li J (2013) Sensitive nanochannel biosensor for T4 polynucleotide kinase activity and inhibition detection. Anal Chem 85(1):334–340

    Article  CAS  PubMed  Google Scholar 

  6. 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(22):8396–8402

    Article  CAS  PubMed  Google Scholar 

  7. Sharma S, Doherty KM, Jr BR (2006) Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability. Biochem J 398(3):319–337

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang LK, Shuman S (2001) Domain structure and mutational analysis of T4 polynucleotide kinase. J Biol Chem 276(29):26868–26874

    Article  CAS  PubMed  Google Scholar 

  9. Bernstein NK, Williams RS, Rakovszky ML, Cui D, Green R, Karimibusheri F, Mani RS, Galicia S, Koch CA, Cass CE (2005) The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase. Mol Cell 17(5):657–670

    Article  CAS  PubMed  Google Scholar 

  10. Hou T, Wang X, Liu X, Lu T, Liu S, Li F, Chem A (2014) Amplified detection of T4 polynucleotide kinase activity by the coupled λ exonuclease cleavage reaction and catalytic assembly of bimolecular beacons. Anal Chem 86(1):884–890

    Article  CAS  PubMed  Google Scholar 

  11. Liu H, Ma C, Wang J, Chen H, Wang K (2017) Label-free colorimetric assay for T4 polynucleotide kinase/phosphatase activity and its inhibitors based on G-quadruplex/hemin DNAzyme. Anal Biochem 517:18–21

    Article  CAS  PubMed  Google Scholar 

  12. Chen W, Tu X, Guo X (2009) Fluorescent gold nanoparticles-based fluorescence sensor for Cu2+ ions. Chem Commun 13(13):1736–1738

    Article  Google Scholar 

  13. Kim TI, Park J, Kim Y (2011) A gold nanoparticle-based fluorescence turn-on probe for highly sensitive detection of polyamines. Chem Eur J 17(43):11978–11982

    Article  CAS  PubMed  Google Scholar 

  14. Tan D, He Y, Xing X, Zhao Y, Tang H, Pang D (2013) Aptamer functionalized gold nanoparticles based fluorescent probe for the detection of mercury (II) ion in aqueous solution. Talanta 113(6):26–30

    Article  CAS  PubMed  Google Scholar 

  15. Liu H, Ma C, Wang J, Wang K, Wu K (2017) A turn-on fluorescent method for determination of the activity of alkaline phosphatase based on dsDNA-templated copper nanoparticles and exonuclease based amplification. Microchim Acta 184(1):1–6

    Article  Google Scholar 

  16. Yeh HC, Sharma J, Iem S, Vu DM, Martinez JS, Werner JH (2012) A fluorescence light-up ag nanocluster probe that discriminates single-nucleotide variants by emission color. J Am Chem Soc 134(28):11550–11558

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Yeh H-C, Sharma J, Han JJ, Martinez JS, Werner JH (2010) A DNA−silver nanocluster probe that fluoresces upon hybridization. Nano Lett 10(8):3106–3110

    Article  CAS  PubMed  Google Scholar 

  18. Latorre A, Somoza Á (2012) DNA-mediated silver nanoclusters: synthesis, properties and applications. Chembiochem 13(7):951–958

    Article  CAS  PubMed  Google Scholar 

  19. Han B, Wang E (2012) DNA-templated fluorescent silver nanoclusters. Anal Bioanal Chem 402(1):129–138

    Article  CAS  PubMed  Google Scholar 

  20. Liu W, Lai H, Huang R, Zhao C, Wang Y, Weng X, Zhou X (2015) DNA methyltransferase activity detection based on fluorescent silver nanocluster hairpin-shaped DNA probe with 5'-C-rich/G-rich-3′ tails. Biosens Bioelectron 68:736–740

    Article  CAS  PubMed  Google Scholar 

  21. Yan X, Sun J, Zhao X-E, Wang R, Wang X, Zuo Y-N, Liu W, Kong R, Zhu S (2018) Molecular beacon-templated silver nanoclusters as a fluorescent probe for determination of bleomycin via DNA scission. Microchim Acta 185(9):403. https://doi.org/10.1007/s00604-018-2939-7

    Article  CAS  Google Scholar 

  22. Fan D, Zhu J, Liu Y, Wang E, Dong S (2016) Label-free and enzyme-free platform for the construction of advanced DNA logic devices based on the assembly of graphene oxide and DNA-templated AgNCs. Nanoscale 8(6):3834–3840

    Article  CAS  PubMed  Google Scholar 

  23. Shah P, Cho SK, Thulstrup PW, Bhang YJ, Ahn JC, Choi SW, Rørvig-Lund A, Yang SW (2014) Effect of salts, solvents and buffer on miRNA detection using DNA silver nanocluster (DNA/AgNCs) probes. Nanotechnology 25(4):111–120

    Article  Google Scholar 

  24. Park KS, Park HG (2015) A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules. Biosens Bioelectron 64:618–624

    Article  CAS  PubMed  Google Scholar 

  25. Zhu Z-M, Yu R-Q, Chu X (2014) Amplified fluorescence detection of T4 polynucleotide kinase activity and inhibition via a coupled lambda exonuclease reaction and exonuclease III-aided trigger DNA recycling. Anal Methods 6(15):6009–6014. https://doi.org/10.1039/c4ay01097c

    Article  CAS  Google Scholar 

  26. Cheng R, Tao M, Shi Z, Zhang X, Jin Y, Li B (2015) Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification. Biosens Bioelectron 73:138–145

    Article  CAS  PubMed  Google Scholar 

  27. Zhang Y, Liu C, Sun S, Tang Y, Li Z (2015) Phosphorylation-induced hybridization chain reaction on beads: an ultrasensitive flow cytometric assay for the detection of T4 polynucleotide kinase activity. Chem Commun 51(27):5832–5835. https://doi.org/10.1039/c5cc00572h

    Article  CAS  Google Scholar 

  28. Hou T, Wang X, Liu X, Pan C, Li F (2014) Sensitive electrochemical assay for T4 polynucleotide kinase activity based on dual-signaling amplification coupled with exonuclease reaction. Sensors Actuators B Chem 202(10):588–593

    Article  CAS  Google Scholar 

  29. Huang Y, Chen J, Shi M, Zhao S, Chen ZF, Liang H (2013) A gold nanoparticle-enhanced fluorescence polarization biosensor for amplified detection of T4 polynucleotide kinase activity and inhibition. J Mater Chem B 1(15):2018–2021

    Article  CAS  Google Scholar 

  30. 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(15):8202–8209

    Article  CAS  PubMed  Google Scholar 

  31. Jiang H, Xu Y, Dai L, Liu X, Kong D (2018) Ultrasensitive, label-free detection of T4 ligase and T4 polynucleotide kinase based on target-triggered hyper-branched rolling circle amplification. Sensors Actuators B Chem 260:70–77

    Article  CAS  Google Scholar 

  32. Zhou L, Cheng H, Wang Jin E, Pei R-J (2016) G-Quadruplex DNAzyme biosensor for quantitative detection of T4 polynucleotide kinase activity by using Split-to-intact G-Quadruplex DNAzyme conversion. Chin J Anal Chem 44(1):13–17. https://doi.org/10.1016/s1872-2040(16)60900-3

    Article  Google Scholar 

  33. Song C, Yang X, Wang K, Wang Q, Liu J, Huang J, He L, Liu P, Qing Z, Liu W (2015) A sensitive detection of T4 polynucleotide kinase activity based on beta-cyclodextrin polymer enhanced fluorescence combined with an exonuclease reaction. Chem Commun 51(10):1815–1818. https://doi.org/10.1039/c4cc08991j

    Article  CAS  Google Scholar 

  34. L-j W, Zhang Q, Tang B, C-y Z (2017) Single-molecule detection of polynucleotide kinase based on phosphorylation-directed recovery of fluorescence quenched by au nanoparticles. Anal Chem 89(13):7255–7261. https://doi.org/10.1021/acs.analchem.7b01783

    Article  CAS  Google Scholar 

  35. Lin L, Shi D, Li Q, Wang G, Zhang X (2016) Detection of T4 polynucleotide kinase based on a MnO2 nanosheet-3,3′,5,5′-tetramethylbenzidine (TMB) colorimetric system. Anal Methods 8(20):4119–4126

    Article  CAS  Google Scholar 

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Acknowledgments

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

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Author notes

  1. Jinlong Li and Jiehua Ma contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, People’s Republic of China

    Jinlong Li, Yongchen Zhang & Zhaoli Zhang

  2. Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing, 210004, People’s Republic of China

    Jiehua Ma

  3. School of Pharmacy, Nanjing Medical University, Nanjing, 211166, People’s Republic of China

    Guangwu He

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  1. Jinlong Li
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  2. Jiehua Ma
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Correspondence to Jinlong Li or Guangwu He.

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Li, J., Ma, J., Zhang, Y. et al. A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe. Microchim Acta 186, 48 (2019). https://doi.org/10.1007/s00604-018-3157-z

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