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Anal Biochem. 2014 Nov 1;464:63-9. doi: 10.1016/j.ab.2014.07.008. Epub 2014 Jul 21.

Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification.

Author information

1
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, and Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
2
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, and Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China. Electronic address: jinyan@snnu.edu.cn.

Abstract

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 5' to 3' direction by lambda exonuclease (λ exo) when its 5' end is phosphorylated by PNK. So, the 3' stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0×10(-4)U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases.

KEYWORDS:

DNA phosphorylation; Polynucleotide kinase activity; Signal amplification

PMID:
25058928
DOI:
10.1016/j.ab.2014.07.008
[Indexed for MEDLINE]
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