title: Sensitive detection of T4 polynucleotide kinase activity based on multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification.
abstract: T4 polynucleotide kinase (PNK) plays critical roles in regulating DNA phosphorylation modes during the repair of DNA lesions. The aberrant activity of T4 PNK has been proven to be associated with a variety of human pathologies. Sensitive detection of T4 PNK activity is critical to both clinical diagnosis and therapeutics. Herein, a background-eliminated fluorescence assay for sensitive detection of T4 PNK activity has been developed by multifunctional magnetic probes and polymerization nicking reactions mediated hyperbranched rolling circle amplification (HRCA). First, the streptavidin-magnetic nanobeads (MBs) were functionalized with the biotin modified hairpin probe (HP) with 3'-phosphoryl, forming multifunctional magnetic probes (HP-MBs). Then, in the presence of T4 PNK, the 3'-phosphoryl of HP-MBs was hydrolyzed to 3'-hydroxyl, thus serving as primers to initiate the polymerization extension and nicking endonuclease cleavage reaction. Next, the primers released from above "polymerization-nicking" cycles were separated out to trigger the subsequently HRCA process, producing plenty of dsDNA. Finally, the intercalating dye SYBR Green I (SG) was inserted into the dsDNA, generating enhanced fluorescence signals. In our design, the HP-MBs here serve together as the T4 PNK, DNA polymerase, and endonuclease recognition probe, and thus avoid the demands of utilizing multiple probes design. Moreover, it performed primary "polymerization-nicking" amplification and mediate secondary HRCA. In addition to, performing the separation function, the binding of HP-MBs and SG could be avoided while a low background was acquired. This method showed excellent sensitivity with a detection limit of 0.0436 mU/mL, and accomplished exceptional characterization T4 PNK activity in cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China; Department of Chemistry, Liaocheng University, Liao cheng 252059, PR China.
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Chemistry, Liaocheng University, Liao cheng 252059, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Song
authors.affiliation: Department of Chemistry, Liaocheng University, Liao cheng 252059, PR China.
authors.forename: Qingwang
authors.initials: Q
authors.lastname: Xue
authors.affiliation: Department of Chemistry, Liaocheng University, Liao cheng 252059, PR China; Department of Biomedical Engineering, Florida International University, Miami, FL 33174, USA. Electronic address: licz@fiu.edu.
authors.forename: Chenzhong
authors.initials: C
authors.lastname: Li
authors.affiliation: Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China. Electronic address: wjiang@sdu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D015374:Biosensing Techniques / Q000379:methods*; D015342:DNA Probes / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D059346:Magnets / Q000737:chemistry; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D015342:DNA Probes; D019809:Streptavidin; D011116:Polynucleotide 5'-Hydroxyl-Kinase
keywords: Fluorescence detection; Hyperbranched rolling circle amplification; Multifunctional magnetic probes; Polymerization-nicking; T4 polynucleotide kinase