title: Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.
abstract: Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research.
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Mangjuan
authors.initials: M
authors.lastname: Tao
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Zhilu
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Xiafei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, 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.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
authors.forename: Baoxin
authors.initials: B
authors.lastname: Li
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods* / Q000706:statistics & numerical data; D002942:Circular Dichroism; D015342:DNA Probes / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D008652:Mesoporphyrins; D021141:Nucleic Acid Amplification Techniques; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis* / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence
chemical_list: D015342:DNA Probes; D008652:Mesoporphyrins; C065420:N-methylmesoporphyrin IX; D011116:Polynucleotide 5'-Hydroxyl-Kinase
keywords: DNA phosphorylation; Fluorescence; Label-free; PNK activity; Signal amplification; Strand displacement reaction