Screening Of G-quadruplex Ligand Electrochemical Method

It is found that the single-stranded DNA at 3'end of telomere can form G-quadruplex DNA structures under optimum condition. This secondary structure can not be recongnisized by telomerase, and can halt tumor cell proliferation. Thus, telomere can be used as the target of anticancer drugs. Molecules that induce human telomere DNA to form G-quadruplex are potential anticancer drugs for cancer treatment. In this thesis, flavonoids, alkaloids, and anthraquinones were chosen as model anticancer drugs. Based on the structure tansformation of telomere DNA caused by specific interaction between drugs and telomere DNA, Two electrochemical methods for screening the G-quadruplex ligands have been developed. The contents and results of research in this thesis contain the following two parts:1. Electrochemical selection of G-quadruplex-binding ligand based on structure-switching of GDNAAn oligonucleotide with four repeats of the human telomeric sequence was used as capture probe and labeled with ferrocene and thiol at its 5'and 3' ends, respectively. An electrochemical method for selection of G-quadruplex-binding ligand has been established based on the signal change resulted from the structures change of GDNA from dsDNA to G-quadruplexes. The capture probe firstly self-assembled on the electrode via Au-S affinity, and then hybridized with a short complementary DNA (cDNA). The rigidity of part duplex DNA prevented the Fc tag from approaching the electrode surface and peak current was low. In the prsence of ligand, configuration conversion of GDNA from DNA duplex to G-quadruplex shortened the distance between terminal Fc tag and electrode, resulting in an increase in current. Seven natural drugs were investigated as potential ligands using electrochemical method, and the CD was used to illustrate the structure conversion before and after DNA interaction with drug. The reasults indicated that daidzein, berberine, emodin, aloe-emodin, rhein and chrysophanol selected by this electrochemical method could induce the GDNA to fold into G-quadruplex. At the same time, DNA modified electrode could be regenerated. The reasults demonstrated that the proposed method could effectively identify ligands with potential anticancer activity.2. Label-free electrochemical selection of G-quadruplex-binding ligands based on structure-switching of GDNAA label-free electrochemical method has been established based on the difference of electraostatic interaction between RuHex and DNA using RuHex as electrochemical indicator. The GDNA/cDNA hybids were immobilized on the gold electrode via Au-S self-asembly. The voltammetric signals of RuHex were investigated at GDNA/cDNA hybrids modified electrodes because of electrostatic interaction between anionic phosphate backbones of DNA and RuHex. In the presence of quadruplex-binding ligand, the GDNA folded into G-quadruplex and released from the electrode, which reduced the adsorption amount of RuHex and decreased the peak current of RuHex. Ten natural drugs were investigated as potential ligands using this electrochemical method. Structure conversion of GDNA before and after DNA interacting with drugs has been verified by CD measurements. Results of research indicated that natural drugs, including emodin, aloe-emodin, rhein chrysophanol, daidzein, kaempferol, Chrysin and berberine, selected by this electrochemical method could induce the GDNA to fold into G-quadruplex. It offers a simple, lable-free, and effective electrochemical method for selection anti-tumor drugs.