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Cyclic up-regulation fluorescence of pyrene excimer for studying polynucleotide kinase activity based on dual amplification

Xu, Jing, Gao, Yanfang, Li, Baoxin, Jin, Yan
Biosensors & bioelectronics 2016 v.80 pp. 91-97
biosensors, detection limit, enzymatic reactions, fluorescence, gamma-cyclodextrin, monitoring, phosphorylation, recycling, single-stranded DNA, transfer DNA
Due to its important biological and clinical roles of polynucleotide kinase (PNK), accurate monitoring of PNK activity and inhibition is highly desirable. Herein, a homogeneous and sensitive fluorescence assay has been proposed for the detection of PNK activity by integrating target recycling signal amplification of DNA toehold strand displacement reaction (TSDR) with gamma-cyclodextrin (γ-CD) enhancement of pyrene excimer. A label-free hairpin DNA1 (H1) and two singly pyrene-labelled DNA, H2 and H3, are designed. Accompanying the occurrence of the efficient enzyme reactions, namely phosphorylation-actuated λ exonuclease reaction, a single-stranded DNA as a trigger DNA (tDNA) of TSDR can be released from H1. Then, tDNA drives circulatory interactions between H2 and H3 to continuously form H2/H3 duplex, resulting in formation of pyrene excimer and a “turn on” fluorescence signal of pyrene excimer. Furthermore, the fluorescence of pyrene excimer is further amplified by introducing gamma-cyclodextrin (γ-CD), which can regulate the space proximity of two pyrene molecules. Thus, TSDR-induced cyclic formation of pyrene excimer and γ-CD enhancement can specifically up-regulate the fluorescence of pyrene excimer for detection of PNK activity, the detection limit is 9.3×10⁻⁵UmL⁻¹, which is superior to those of most existing approaches. Moreover, the proposed strategy can also be successfully utilized to study inhibition efficiency of different PNK inhibitors as well. Therefore, a dual amplification approach is provided for nucleic acid phosphorylation related researches.