The Fluorescence Of The G-tetramer Small Molecule Ligand Screening Method

Anticancer drugs play a significant role in the treatment of cancer. The study of anticancer drug screening method is an important section for the research of anticancer drugs. The research shows that small molecular ligands of G-quadruplex are potential anticancer drugs. Currently, the screening of potential small molecular anticancer drugs as the G-quadruplex for target is the research focus. Herein, we established two types of fluorescence method for screening of the G-quadruplex small molecular ligands.1. Fluorescence screening method for G-quadruplex small molecular ligands based on fluorescence quenching effect of single-walled canbon nanotubesSingle-walled canbon nanotubes (SWNTs) have unique physical and chemical properties, which have the good quenching for fluorophore. Single-stranded DNA can be adsorbed on the surface of SWNTs, when adding SWNTs to the fluorescein (FAM) labeled single-stranded GDNA (F-GDNA) solution, dye-tagged probe DNA sticks to the SWNTs so that the fluorescence of single-stranded probes is quenched. After adding quadruplex-binding ligands, the formation of G-quadruplex leads to releasing the probe DNA from SWNTs and the fluorescence enhancement is observed, as for the non quadruplex ligands, the formation of F-GDNA does not change and the fluorescence intensity still weak. So we can screen the quadruplex-binding small molecular ligands based on the change of fluorescence intensity when adding the small molecular ligands before and after. Several small molecular ligands were studied by the proposed method. Fluorescence experiments, control experiments and CD measuements indicated that we provided a simple and effective method for the sceening of potential anticancer drugs.2. Label-free fluorescence method for screening G-quadruplex small molecular ligandsHere, we development a label-free fluorescence method for screening G-quadruplex ligands. It is based on the G-rich single-stranded DNA can bind with hemin to produce G-quadruplex-hemin complex (also called G-quadruplex-based DNAzyme), possessing high peroxidase activity as well as HRP. The G-quadruplex-based DNAzyme is found to effecyively catalyze the H2O2-mediation oxidation of p-hydroxyphenylancetic acid (HPA). HPA is an excellent non-fluorescent substrate. But bi-p', p-hydroxyphenylacetic acid (the oxidation product of HPA with H2O2) is a strong fluorescent compound (exicitation/emnssion wavelength maxima, ca.313 nm/405 nm). So, the G-quadruplex-based DNAzyme can induce the strong fluorescence emission of HPA-H2O2 system, when a strong G-quadruplex ligand is added into G-quadruplex-hemin complex system, the ligand can displace hemin from the G-quadruplex-based DNAzyme. The peroxidase activity of the G-quadruplex-based DNAzyme is two orders of magnitude higher than that of hemin alone. Consequently, the binding ability of the given ligand to G-quadruplex can be easily monitored according to the decrease of the fluorescencent intensity of HPA-H2O2 system. Based on this, we established a novel approach to screening G-quadruplex ligands in a label-free fluorescent manner. In addition, the method was validated by positive experiment, negative experiment, and circular dichroism.