Study On The Interaction Between Quinazolinone-coumarin Derivatives And G4s In The Mitochondrial DNA

The G-quadruplex is a special DNA secondary structure through Hoogsteen hydrogen bonds.People have paid much attention to the G-quadruplex because of the close relationship between these G-rich sequences and telomeres、telomerase and many promoter regions.These G-quadruplexes can interact with G-quadruplex binding proteins and participate in the regulation of various biological proces ses.G-quadruplexes are recognized as promising targets for the design of antitumor drugs.So far,there are many small molecules can inhibit telomerase activity,telomere elongation and down-regulate gene expression by stabilizing the nucleic G-quadruplex structure,ultimately inducing cell senes cence and apoptosis.Compared with G4 s in nuclear DNA,little is known about G4 s in mitochondrial DNA(mtDNA),a few G4 s ligands to induce mitochondrial dysfunction via interaction with mtDNA G4 s.Studies have shown that mitochondrial G-quadruplexes are likely to cause genome instability by perturbing replication machinery,which leads to the development of human genetic diseases and cancer.Based on this,we intend to use the quinazolinone-coumarin derivatives CQ and MeCQ as ligands to explore their relationship with the mtDNA G-quadruplex and gene promoter region G-quadruplexes.The results of this work is as follows:1.We studied the interaction of quinazolinone-coumarin derivatives with mtDNA G-quadruplex,proto-oncogene c-myc,c-kit,k-ras three gene promoter region G-quadruplexes and duplex DNA through a series of methods,including CD assay,fluorimetric titration assay and UV-Vis melting assay.The results showed that the quinazolinone-coumarin derivatives CQ can specifically bind to the mitochondrial G-quadruplex and stabilize the mitochondrial G-quadruplex structure.And the CQ hardly bind toward double-stranded DNA.The compound MeCQ showed no specific selectivity for mitochondrial G-quadruplex and double-stranded DNA.CQ also bind to proto-oncogene c-myc,c-kit,k-ras three gene promoter region G-quadruplexes,and increace its fluorescence in the precense of three gene promoter region G-quadruplexes DNA.We propose that the compound CQ can form a conjugated macrocyclic planar structure through intramo lecular hydrogen bonding,resulting in the compound CQ can be embedded in the mitochondrial G-quadruplex,expressing the specificity to the mitochondrial G-quadruplex.MeCQ failed to form intramolecular hydrogen bonds and did not show this specificity.2.Further study showed that compound CQ was no toxicity to HeLa and HepG2 cells by MTT assay.However the final concentration of the compound MeCQ reached 5μM,it showed acute toxicity,and the toxicity mechanism of MeCQ was not clear.The QPCR experiment shows that the expression level of the mitochondrial gene HRCC was down-regulated by compound CQ.In the cell imaging experiment,CQ localize at mitochondria and emit green fluorescence.The results indicated that the compound CQ can be used as a fluorescent probe for detecting mitochondria G-quadruplex in cells.