Construction Of Biosensors Of UDG Dectection Based On G-quadruplex Fluorescent Probe Complex

Uracil DNA glycosylase (Uracil DNA Glycosylase, UDG) is a highly reserved repair protein. It removes uracil DNA to prevent the gene mutation in DNA match to maintain the integrity of the gene. Therefore, the detection of enzyme activity is important. The traditional method is time-consuming and polluted. Therefore, the develop new detection methods, such as nucleic acid biosensor for rapid detection of the UDG. Therefore, the development of new and rapid detection methods for UDG is ne cessary, such as nucleic acid biosensor.G-quadruplex DNA refers to the rich guanine (G) in the DNA sequence can be folded to form a four-helix secondary structure. The fluorescence nucleic acid biosensor based on G-quadruplex had wide range of applications in the field of biochemical analysis and diagnosis. In this paper, considering the symmetry of G-quadruplex secondary structure, we designed and synthesized Ci-, C2-, C3-symmetric aromatic-vinyl-substituted pyridine derivatives as G-quadruplex fluorescent probes, and constructed UDG fluorescent biosensors based on G-quadruplex fluorescent probe complex. The main contents and results of the dissertation include:1) Design and synthesis of novel fluorescent probes. For the symmetry structure of G-quadruplex DNA, we designed a series of symmetrical aromatic-vinyl-substituted pyridine derivatives, and obtain these derivatives with simple synthetic procedure. Finally, there are five symmetrical derivatives as G-quadruplex fluorescent probes, which divided into three series, Ci-, C2-, C3-.2) Evaluation and screening of the optimal fluorescent probe. The synthetic derivatives’selectivity, sensitivity and interaction model with G-quadruplex DNA was investigated by fluorescence titration, PAGE experiments, CD spectroscopy, UV titration, FRET melting point experiment. The derivatives investigated in the fluorescence assays were preferentially bound with G-quadruplex DNA compared with other type of nuclei acids, more importantly, the number of substituents, the type of derivative and its impact symmetry and fluorescence response G-quadruplex significantly. Among the derivatives, a C2-symmetric dye (2,6-bis-((E)-2-(1H-indol-3-yl)-vinyl)-1-methylpyridin-l-ium iodide) with indolyl-groups substituted,2a, was screened out from the series giving the best selectivity and sensitivity towards G-quadruplexes DNA (K= 2.17 x 105 M-1, LOD= 33 nM).The compound was also demonstrated as a very selective G-quadruplex fluorescent agent for living cell staining and imaging.3) Construction and evaluation of UDG biosensor. This newly developed analytical method is based on the UDG enzymatic activity to unwind a duplex DNA substrate, and comprises a G-quadruplex-forming sequence (ON1) and uracil-containing DNA strand (ON2) to generate a remarkable fluorescence signal through the specific interaction of 2a with ON1. The application range of the present analytical system is found to be 0.05 to 1.00 U mL-1 UDG with a very low detection limit of 0.005 U/mL. In addition, the recovery study of UDG in real samples gave a very good performance with 75.05%-102.7% recovery and the screening of UDG inhibitors.