New Analytical Methods For DNA Damage Identification And Repair Enzymes Activity Research

Cells endure incessant DNA damage, which threatens their viability and genomic stability.1 DNA damage is suggested to contribute to cell aging through its ability to mediate cellular dysfunction. If these DNA lesions remain unrepaired, it will lead to gene instabilities during the encoding of genetic information in DNA and may increase the risk of cancer. So the DNA damage identify and repair have a great significance on maintain the normal cell physiological activities. DNA damage identify is accomplished by Poly(ADP-ribose) polymerases(PARPs) family. In response to DNA-strand damage, PARP catalyze the successive transfer of the ADP-ribose moiety to nuclear protein acceptors and modification of it by the addi tion of chains of poly(ADP-ribose)(PAR), then achieve the DNA damage identify and signal delivery. After recognize there exist DNA damage in intracellular, cell will activate its DNA damage repair process. Among the four types well-known DNA repair mechanisms, the most important path is DNA base excise repair(BER). Uracil DNA glycosylase(UDG) is type of BER enzyme that excise the uracils on DNA strands, and it open the gate of uracil-DNA BER. In view of this, on this paper we make use the fluorescence of super charged green fluorescence protein(sc GFP) can be quenched by negative charged gold nanoclusters(AuNCs), and the PAR can protect the fluorescence of sc GFP from quenched by AuNCs, we established a high performance PARP1 detection platform. Rely on the Guanine-rich DNA strand can form a kind of four-stranded structure known as G-quadruplex, once it bind with hemin(an anionic iron(III) porphyrin), it can exhibit peroxidase catalytic activity, and named G4-DNAzyme. First, a novel and simple strategy based on inhibitory effect of adjacent hybridization(In EAH) to efficiently inhibit the peroxidise-mimic DNAzyme activity was developed here to sensitively assay the BER related enzyme, UDG activity. Making use of DNAzyme can catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfonate disodium salt(ABTS2-) by H2O2 to produce the visual colored ABTS?-, based on the chromatics theory, image analysis of UDG activity using RGB image profiling was invented here.1. In view of activity PARP1 can modify itself with long branched poly(ADP-ribose) polymer(PAR), and the Peptide-templated negative charged gold nanoclusters(AuNCs) can perfectly quench the fluorescence of super charged Green Fluorescence Protein(sc GFP) through nanosurface energy transf er effect(NSET). We were almost the first time take advantage of the negative charged PAR stands can electrostatic coupling with sc GFP with a net charge of +36 and form a negative charged PAR-sc GFP compound, then keep the sc GFP fluorescence away from quenched by the AuNCs, a completely new PARP1 activity analysis method was constructed. By this approach, we creatively directly make use of the most important property of PAR strands and achieve high sensitively approach to assess the PARP's activity. Rely on this platform, accurate PARP1 inhibitors screening and evaluate can also be realized. Since PARP1 inhibitors emerging as a high potential in anticancer therapy research, we firmly believed our research will make its difference on comprehend of the PAR on PARP1 and promote PARP1 inhibitor in anticancer research.2. G4-DNAzyme have the peroxidase activity, and its activity varies when the Guanine-rich DNA sequence different. When the first base(+1) at the antiparallel G4-DNAzyme 3' terminal is adenine, it shows high catalytic activities compare others. But if the adenine on the 3' terminal in hybridized by uracil, the G4-DNAzyme activity decreases about nearly 20 times compared with when the adenine is free. This phenomenon is named inhibitory effect of adja cent hybridization(In EAH).In view of In EAH, a novel and simple strategy based on it to efficiently inhibit the peroxidise-mimic DNAzyme activity was developed here to sensitively assay the UDG activity.3. Guanine-rich DNA probe can form a special G-quadruplex structure with hemin to display peroxidase activity to catalyze the H2O2-mediated oxidation of ABTS2- to the colored ABTS?-, providing a visible signal for UDG detection. In addition, G-quadruplexe can also interact Thioflavin T(Th T) with a high emi ssion enhancement in the visible region. Based on the chromatics theory, a new method of RGB analysis, using the smartphone as the one and all devices, is applied to sense the UDG activity. Through this way, we can directly read out the color value using a smartphone App to reflect the content of UDG with high resolution, providing a new vision for the portable assay strategy. Later a fluorescent UDG detection method based on G-quadruplexes was built here. Furthermore, the utility of this method for screeni ng potential UDG inhibitors was also demonstrated.