Citation: ZHOU Lu, CHENG Hui, WANG Jin-E, PEI Ren-Jun. G-Quadruplex DNAzyme Biosensor for Quantitative Detection of T4 Polynucleotide Kinase Activity by Using Split-to-Intact G-Quadruplex DNAzyme Conversion[J]. Chinese Journal of Analytical Chemistry, ;2016, 44(1): 13-18. doi: 10.11895/j.issn.0253-3820.150480 shu

G-Quadruplex DNAzyme Biosensor for Quantitative Detection of T4 Polynucleotide Kinase Activity by Using Split-to-Intact G-Quadruplex DNAzyme Conversion

1.
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China

Corresponding author: PEI Ren-Jun,
Received Date: 11 June 2015
Available Online: 24 August 2015
Fund Project: 本文系国家自然科学基金(Nos.31301495,21275156,21305154)资助 (Nos.31301495,21275156,21305154)

A biosensor for detection of T4 polynucleotide kinase (PNK) activity was fabricated by using a split-to-intact DNAzyme conversion strategy. G-quadrplex-forming G-rich sequence PS5.M was chosen as a model DNAzyme and splitted into two short parts of S₁OH and S₂OH, which both had hydroxyl groups at 3' and 5'terminals, and could hybridize to another sequence, helper DNA (SH), to form a duplex structure. In the presence of T4 PNK, 5'-end of S₂OH was phophorylated to obtain S₂P. Then S₁OH and S₂P could be linked by T4 DNA ligase to intact PS5.M. In the presence of exonuclease Ⅲ (Exo Ⅲ), SH chain in the duplex of PS5.M-SH could be cleaved into nucleotides, releasing free PS5.M. The released PS5.M could form catalytically active G-quadruplex DNAzyme upon binding hemin in the presence of K⁺, which could catalyze the oxidation of 2,2'-azino bis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) by H₂O₂. Quantitative detection of T4 PNK activity could be achieved by measuring the maximal absorbance of product at 418 nm. The absorbance of the sensing system at 418 nm exhibited a linear relationship with T4 PNK activity in the range of 0.02-0.3 U/mL with a detection limit of 0.014 U/mL (S/N=3). The biosensor was also applied to the determination of PNK activity in Hela cell and HEK 293 cell, and good recoveries of 95.6%-105.7% were obtained at three spiked levels.
- T4 polynucleotide kinase,
- DNAzyme,
- Split-to-intact G-quadruplex,
- Biosensor
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沈阳化工大学材料科学与工程学院沈阳 110142