Journal
ANALYTICA CHIMICA ACTA
Volume 766, Issue -, Pages 88-93Publisher
ELSEVIER
DOI: 10.1016/j.aca.2012.12.034
Keywords
Polynucleotide kinase; lambda exonuclease; The horseradish peroxidase-mimicking; DNAzyme; Colorimetric assay
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Funding
- Natural Science Fundation of China [21025521, 20875027]
- National Key Basic Research Program [2011CB911000]
- CSIRT Program
- Natural Science Fundation of Hunan Province [10JJ7002]
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T4 polynucleotide kinase (PNK) plays a critical role in various cellular events. Here, we describe a novel colorimetric strategy for estimating the activity of PNK and screening its inhibitors taking advantage of the efficient cleavage of lambda exonuclease and the horseradish peroxidase-mimicking DNAzyme (HRPzyme) signal amplification. A label-free hairpin DNA with the sequence of HRPzyme was utilized in the assay. The 5'-hydroxyl terminal of the hairpin DNA was firstly phosphorylated in the presence of PNK and then digested by lambda exonuclease. As a result, the blocked 'HRPzyme' sequence of the hairpin DNA was released due to the removal of its completely complementary sequence. Using this strategy, the assay for PNK activity was successfully translated into the detection of HRPzyme. Because of the completely blocking and efficiently releasing of HRPzyme, the colorimetric method exhibited an excellent performance in PNK analysis with a low detection limit of 0.06 U mL(-1) and a wide detection range from 0.06 to 100 U mL-1. Additionally, the effects of different inhibitors on PNK activity were also evaluated. The proposed strategy holds great potential in the development of high-throughput phosphorylation investigation as well as in the screening of the related drugs. (C) 2012 Elsevier B.V. All rights reserved.
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