Exonuclease III-assisted signal amplification strategy for sensitive fluorescence detection of polynucleotide kinase based on poly(thymine)-templated copper nanoparticles. Issue 22 (10th October 2019)

• Record Type: Journal Article
• Authors: Zhao, Han; Yan, Ying; Chen, Mingjian; Hu, Tingting; Wu, Kefeng; Liu, Haisheng; Ma, Changbei
• Is Part Of: Analyst. Volume 144:Issue 22(2019)
• Page Start: 6689
• Page End: 6697
• Publication Date: 2019-10-10
• Subjects: Chemistry, Analytic -- Periodicals; 543
• DOI: 10.1039/c9an01659g
• Languages: English
• ISSNs: 0003-2654

Abstract

Abstract : A facile fluorescent method has been developed for polynucleotide kinase detection based on copper nanoparticles and exonuclease III-assisted signal amplification. 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.