Fluorescence Analysis Of T4 Polymeric Nucleotide Kinase Activity And Inhibition

T4polynucleotide kinase (T4PNK) that is a protein encoded by pseT gene of T4phage can phosphorylated the5’-hydroxyl end of nucleic acid, which is closely related to the recombination, replication and repair of the nucleic acid. It plays an important role in the process of molecular biology, drug discovery, cancer diagnosis and so on. Therefore, research on T4PNK has attracted considerable attention. And the development of simple and reliable methods for detection of T4PNK activity has become an important research area. This thesis aimed to develop novel methods for homogeneous and rapid detection of T4PNK activity. Based on different ways of signal conversion caused by DNA phosphorylation, three methods have been established for fast and sensitive monitoring T4PNK activity and inhibition. The main content is summarized as the following three parts.1. Research on fluorescence assay of T4polynucleotide kinase activity and inhibition via enzyme-assisted signal amplificationAn enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4PNK. A hairpin oligonucleotide was designed as a probe whose stem can be degraded from5’to3’direction by lambda exonuclease (λ exo) only when its5’ end was phosphorylated by PNK. So, initiator strand was released to open a molecular beacon that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by endonuclease Nt.BbvCI to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of1.0×10-4U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK.2. Research on T4PNK activity and inhibition by photo-induced electron transferA novel fluorescence method has been proposed to study the activity and inhibition of T4PNK based on photo-induced electron transfer (PIET) beween deoxyguanosine and fluorescence dye by using carboxyfluorescein-labeled DNA (FDNA) as a probe. In the absence of PNK, fluorescence of FDNA was quenched by deoxyguanosine of its complementary DNA because stable DNA can not digest by lambda exonuclease without phosphorylation, which led to an effective PIET. In the presence of T4PNK, lambda exonuclease digested DNA duplex from5’to3’, resulting in a fluorescence recovery. Therefore, the activity of PNK can be monitored by the change in fluorescence intensity. Research on inhibition efficiency of different inhibitors indicated the potential of screening inhibitors. Therefore, it is a simple, cost-effective and fast method for studying T4PNK activity and screening inhibitors.3. Research on label-free assay of T4polynucleotide kinase activity and inhibitionA label-free fluorescence strategy has been developed to study PNK activity and inhibition based on lambda exonuclease (X exo) aided signal change by using SYBR Green I (SGI) as a molecular probe. Fluorescence intensity of SGI significantly increased when it mixed with double-stranded (dsDNA) substrate due to its high affinity to dsDNA in the absence of PNK. However, in the presence of PNK, an obvious decrease in fluorescence intensity was observed due to the release of SGI from dsDNA substrate by λ exo which can degrade5’-phosphorylated dsDNA from5’to3’direction. Therefore, T4PNK activity can be sensitively, rapidly and accurately detected by monitoring the change in fluorescence. Morevoer, the influence of three inhibitors on PNK activity have been investigated, indicating that it also can be utilized to study PNK inhibition and screen inhibitors. Gel electrophoresis and circular dichroism analysis further verified the feasibility and reliability of sensing mechanism. Therefore, it provides a simple, selective and sensitive method for assay of kinase activity, as well as has a potential to screen the inhibitor of kinase.