Ligase-mediated Isothermal Amplification Approach For Polynucleotide Kinases Detection

Polynucleotide kinase?PNK?is a kind of repair enzyme and mainly participate in the base excision repair pathway?BER?and non-homologous end-joining repair pathway?NHEJ?.The PNK can catalyze the transfer of phosphate group from ATP to the 5'-hydroxyl terminus of DNA/RNA to generate a 5'-phosphate terminus,which plays crucial roles in many fundamental cellular processes such as DNA recombination,DNA replication and DNA damage repair.The deregulation of PNK activity may disturb the stability of cellular repair system and cause various human diseases including Thomson syndrome,Werner syndrome,neurodegenerative disorder and cancers.Therefore,PNK is regarded as a potential biomarker for the diagnosis and treatment of diseases,and there is an urgent need for efficient and sensitive PNK assay.However,the conventional PNK detection methods are usually laborious and time-consuming with relatively poor sensitivity.In order to overcome these shortcomings,we develop a simple,selective and sensitive method for PNK detection.In this paper,we introduce a ligase-mediated isothermal amplification approach for PNK detection.We used duplex-specific nuclease as a tool enzyme for signal amplification under isothermal conditions,avoiding any precise and expensive thermal cycling procedures and greatly reducing the analysis time.We designed a bifunctional quencher/fluorophore dual-labeled Taqman probe,which functions as both a template for the ligation reaction and a signal probe for signal output,greatly reducing the design complexity.In the presence of target PNK,it catalyzes the phosphorylation of the 5'-hydroxylated donor RNA to generate a 5'-phosphoryl terminus.Subsequently,the ligation of two short RNA probes?donor RNA and acceptor RNA?to form a longer RNA trigger strand,which efficiently triggers duplex-specific nuclease-mediated cleavage of Taqman probes to generate obvious fluorescence signal.The proposed method can be used for the sensitive detection of PNK with a low detection limit of 5?10^-6U/mL,and it can be further used for the screening of PNK inhibitors and the accurate detection of PNK in complex biological samples.Importantly,this method can be extended to the accurate quantification of other biomolecules such as DNAs,proteins and ATP,providing a new approach for biomedical research and clinical diagnosis.