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.
Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, PR China.
Biosens Bioelectron. 2017 May 15;91:631-636. doi: 10.1016/j.bios.2017.01.022. Epub 2017 Jan 12.
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.
T4 多核苷酸激酶（PNK）在修复 DNA 损伤过程中对调节 DNA 磷酸化模式起着关键作用。已经证明 T4 PNK 的异常活性与多种人类病理有关。T4 PNK 活性的敏感检测对临床诊断和治疗都至关重要。在此，通过多功能磁性探针和聚合切口反应介导的超支化滚环扩增（HRCA）开发了一种用于灵敏检测 T4 PNK 活性的背景消除荧光测定法。首先，链霉亲和素-磁性纳米球（MBs）用带有 3'-磷酸基的生物素修饰发夹探针（HP）功能化，形成多功能磁性探针（HP-MBs）。然后，在 T4 PNK 的存在下，HP-MBs 的 3'-磷酸基被水解为 3'-羟基，从而作为引发聚合延伸和切口内切酶切割反应的引物。接下来，从上述“聚合-切口”循环中释放的引物被分离出来，触发随后的 HRCA 过程，产生大量 dsDNA。最后，嵌入染料 SYBR Green I（SG）插入 dsDNA 中，产生增强的荧光信号。在我们的设计中，HP-MBs 在这里共同作为 T4 PNK、DNA 聚合酶和内切酶识别探针，因此避免了使用多个探针设计的要求。此外，它执行初级“聚合-切口”扩增并介导二级 HRCA。除了执行分离功能外，还可以避免 HP-MBs 和 SG 的结合，同时获得低背景。该方法具有出色的灵敏度，检测限为 0.0436 mU/mL，并且能够出色地表征细胞提取物中的 T4 PNK 活性，为生物医学研究和临床诊断提供了有力工具。
