title: Cyclic up-regulation fluorescence of pyrene excimer for studying polynucleotide kinase activity based on dual amplification.
abstract: 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(-5)UmL(-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.
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: Jing
authors.initials: J
authors.lastname: Xu
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: Yanfang
authors.initials: Y
authors.lastname: Gao
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
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
mesh_terms: D017122:Bacteriophage T4 / Q000737:chemistry / Q000235:genetics; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005453:Fluorescence; D005786:Gene Expression Regulation; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000096:biosynthesis / Q000302:isolation & purification*; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D047408:gamma-Cyclodextrins / Q000737:chemistry
chemical_list: D004277:DNA, Single-Stranded; D011721:Pyrenes; D047408:gamma-Cyclodextrins; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; C023792:gamma-cyclodextrin
keywords: Dual amplification; Fluorescence; Polynucleotide kinase; Pyrene excimer