Nanopore Single-molecule Detection Of DNA Phosphorylation And T4 Polynucleotide Kinase

Exposure to environmental toxicants and stressors,radiation,drugs,inflammation,cellular respiration,and conventional DNA metabolism can cause cytotoxic DNA strand breaks.And the DNA strands often lack 5'-phosphate and 3'-hydroxyl for DNA synthesis and connection after breaks.If the damage to the terminal of the DNA cannot be solved,it will lead to abnormal DNA replication and repair,resulting in genomic instability,mutagenesis,neurological diseases,aging and cancer.T4 polynucleotide kinase?T4 PNK?is one of the common kinases in the organism,with 5'-kinase and 3'-phosphatase activity.The 5'-kinase activity of T4 PNK can transfer the ATP's?-phosphate to the 5'-hydroxyl end of the polynucleotide,and it is an important biological enzyme for repairing biological gene damage.Traditionally,the determination the phosphorylation of DNA and T4 PNK activity was based on radioisotope ³²P-labeling,polyacrylamide gel electrophoresis and autoradiography.However,these methods are cumbersome,time-consuming,and radioactive labels may cause health problems.In recent years,nanopore single-molecule technology has become a very promising detection method in the field of analytical chemistry because of its advantages such as label-free,high throughput,and low cost.In this paper,by using?-hemolysin??-HL?protein nanopore as the sensing element,the DNA hairpins are designed to enable the analysis of DNA phosphorylation and T4polynucleotide kinase through single molecule detection.The main research contents of this paper are as follows:1. Research reviewThis chapter briefly reviews the development history of nanopore analytical technology,detection principle,classification of nanopores,research progress of?-hemolysin nanopores in epigenetic detection and single-molecule enzyme reaction analysis,and the introduction of T4polynucleotide kinase.2. Using?-HL as the sensing element and designing DNA with hairpin structure to realize the detection of DNA phosphorylation.In this chapter,we designed DNA hairpins for phosphorylation detection.We have explored the characteristics of four types of unphosphorylated and phosphorylated DNA by?-HL?WT?₇nanopores.It is found that the stability of 3'-phosphorylated and 5'-phosphorylated DNA hairpins is much lower than that of corresponding unphosphorylated DNA hairpins,and the duration of phosphorylated DNA hairpins is about one-tenth of that of unphosphorylated DNA hairpins.Through the experiments with varying voltage and salt concentrations,we think that the electrostatic repulsion between the phosphoric acid at the 5'-or 3'-end and the phosphate ester on the main chain reduces the stability of the double chain and reduces the duration.As the number of base pairs increases,the log?Duration?ms??values of 5'-hp DNA and 3'-hp DNA gradually approach and the stability gradually approaches.By analyzing the signal of DNA-doxorubicin complex,it is found that the addition of doxorubicin enhanced the stability of the DNA hairpins.Due to the interaction between the 5'-phosphorylated phosphate group at the end of the DNA hairpins and doxorubicin,the effect weakens the electrostatic repulsion of the phosphate group and the main chain phosphate group,making the stability of DNA-doxorubicin complex increase more obviously.Therefore,the system can be used to quickly detect and analyze the structural stability of unphosphorylated and phosphorylated DNA hairpins,and to study the combination of DNA hairpins and drug molecules.Compared with other detection methods,this strategy is simple,fast,and does not require radiolabeling and nanopore modification.This detection scheme provides a new way to detect DNA phosphorylation and analyze the effects of phosphorylation and drug molecules on DNA structure.3. Using?-HL as the sensing element and hairpin DNA as the substrate of T4 polynucleotide kinase to realize the detection and analysis of T4 polynucleotide kinase and its inhibitors.Based on the results of analysis of unphosphorylated and 5'-phosphorylated DNA hairpins,we use DNA hairpins as the substrate for T4 PNK,and make single-molecule detection of the reaction products by?-HL?WT?₇ in this chapter.Through further analysis of the DNA hairpins'signal before and after phosphorylation,we found that the concentration of T4 PNK?0.01?0.10U/?L?was linearly negatively correlated with the proportion of the signal in the long duration area.The detection limit of T4 PNK is 8.6×10^-4 U/?L.It is found that the strategy has no response to many interfering proteins,with high selectivity,and is suitable for the detection of serum samples.The standard recovery rate is 95%?107%.The T4 PNK activity decreases with the increasing concentration of inhibitor?NH₄?₂SO₄ for the same T4 PNK concentration,and the half-inhibitory concentration of?NH₄?₂SO₄ is 10.5 m M.This solution has the advantages of simple design,rapid detection,no radioactive ³²P labeling,low cost,and has high sensitivity and accuracy.The result provides an important reference for the relevant research of T4 PNK in vivo,with great potential for application to enzyme detection as a new strategy.