Design,Synthesis And Tumor Diagnosis And Treatment Of Mitochondrial DNA G-Quadruplex Fluorescent Ligands

In recent years,the formation of intracellular G-quadruplexes has been considered a new target for antitumor drug development due to its close connection to tumor development and apoptosis.For studying the effect of mitochondrial DNA G-quadruplex formation on tumor therapy and the intracellular regulatory mechanism,one of the most effective methods is to develop fluorescent ligands combined with fluorescence microscopy imaging technology to achieve real-time fluorescent tracing.However,the development of fluorescent ligands based on mitochondrial DNA G-quadruplex is currently in the preliminary stage of development,and relevant ligands are still underdeveloped.Based on these,we designed and synthesized the benzothiazole-triphenylamine derivative,named SPN,and investigated the interaction and biological activity between SPN with mitochondrial DNA G-quadruplex using spectroscopic and biochemical methods.The main research contents of this paper are as follows.（1）The benzothiazole-triphenylamine derivatives,SPN and SIN,were designed and synthesized through theoretical calculation and structural characterization.（2）The binding selectivity,sensitivity,fluorescence performance,fluorescence stability,and the binding properties between the compound SPN and mitochondrial DNA G-quadruplex are investigated using fluorescence spectroscopy and visual observation.（3）The binding mode and interaction mechanism of SPN with mitochondrial DNA G-quadruplexes were analyzed by CD spectroscopy,CD melting point,UV titration,fluorescence titration,and Job plot method.（4）The targeting ability and targeting mechanism of compounds on mitochondria were investigated by cytotoxicity,cell imaging,fluorescence analysis of mitochondrial isolation,and subcellular co-localization imaging.（5）The fluorescence-responsive binding sites of SPN in cells were determined by enzymatic digestion and intracellular competition assays.（6）The potential application of SPN as a diagnostic probe for tumors was investigated by cell imaging.These results show that SPN has good selectivity and affinity for the binding of mitochondrial DNA G-quadruplex,and can stabilize the conformation of mitochondrial DNA G-quadruplex.The probe SPN can bind to mitochondrial DNA G-quadruplex in solution and cells and emit red fluorescence,mainly distributed in cytoplasmic mitochondria.This project has successfully developed a highly selective,sensitive,and stable mitochondrial DNA Gquadruplex fluorescent ligand SPN,which provides a good tool for mitochondrial DNA Gquadruplex and mechanism exploration.Its imaging effect on tumors further validates mitochondrial DNA G-quadruplex as an ideal target for tumor diagnosis.Numerous studies have been performed for tumor phototherapy using different mitochondrial targeting strategies,but certain limitations still exist.Meanwhile,interfering with genomic stability by relying solely on the stability of DNA G-quadruplexes（G4）usually has a limited therapeutic effect on tumors.Based on this background,this paper proposes mitochondria-based photodynamic therapy to synergize stabilize mitochondrial G4 therapy to achieve efficient tumor ablation.The oxidative damage of naked mitochondrial DNA by PDT and the synergistic enhancement of the stability of mitochondrial DNA G-quadruplexes leads to genomic disorders,which may improve the poor antitumor activity of PDT alone and stabilized G-quadruplex alone.The specific experiments of the project are as follows.（1）The ROS generation efficiency of the compound was analyzed by UV spectroscopy.（2）The tumor proliferation,genomic stability,and tumor migration ability after stabilizing mitochondrial DNA G-quadruplex by compound SPN were initially investigated by CCK-8 assay,population doubling assay,and cell scratching assay.（3）The intracellular ROS generation capacity and cytotoxic photolysis products of compound SPN were further explored.（4）Finally,the synergistic antitumor activity of a single compound SPN with mitochondrial DNA Gquadruplex stabilization and mitochondrial PDT was investigated by CCK-8 assay and cell live-death staining.The derivative SPN showed efficient ROS generation efficiency in tumor therapy in both solution and cellular systems.The compound SPN exhibited the ability to stabilize mitochondrial DNA G-quadruplexes in CD melting point assays and selective antitumor activity against tumor cells intracellularly.These may be attributed to the fact that the compound stabilizes mitochondrial DNA G-quadruplexes in tumor cells after stabilization by affecting mitochondrial DNA replication,transcription,and expression,thus inducing apoptosis.Finally,the cytotoxicity assay and Calcein-AM/PI staining assays demonstrated that the compound SPN was more efficient and complete in ablating tumors when combined with PDT.This multi-effective synergistic single-molecule compound targets mitochondrial DNA and synergistically enhances the antitumor activity of mitochondrial G-quadruplex DNA after stabilization by PDT.