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Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles

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Abstract

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5′-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL−1 with a low detection limit of 2 × 10−4 U mL−1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.

Graphical abstract: Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles

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Publication details

The article was received on 27 Aug 2019, accepted on 03 Oct 2019 and first published on 07 Oct 2019


Article type: Paper
DOI: 10.1039/C9AN01659G
Analyst, 2019, Advance Article

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    Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles

    H. Zhao, Y. Yan, M. Chen, T. Hu, K. Wu, H. Liu and C. Ma, Analyst, 2019, Advance Article , DOI: 10.1039/C9AN01659G

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