Development of Fluorescent Probes Based on Spiropyran in Situ Switching for G-quadruplex DNA and Their Biological Applications

Author:Li Jin

Supervisor:guo zuo

Database:Doctor

Degree Year:2019

Download:79

Pages:181

Size:12298K

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G-quadruplex(G4)DNA structure,a non-classical secondary DNA structure,formed from DNA guanine-rich sequences,which has been confirmed to be stably present in human live cells.The four-stranded structured DNA is viewed as an emerging anticancer target.c-MYC G4 DNA and c-KIT1 G4 DNA are present in oncogene promoters.c-MYC oncogene is associated with cell growth,proliferation and a range of malignant tumors such as osteosarcoma,breast cancer and so on.And overexpression of c-KIT oncogene plays a role in oncogenic transformation of certain cell types and are found in several highly malignant human cancers,notably gastrointestinal stromal tumors(GIST).Stabilization of these two G-quadruplex structures can suppress their corresponding oncogenes transcription and expression in cancer cells.Therefore,nondestructive fast detection of these endogenous DNA G4s can provide specific real-time information,which is of particular interest for clinic accurate diagnosis.In this regard,cell-permeable small-molecule fluorescent probes have been great attention in view of their advantages of high selectivity,high resolution and real-time live-cell imaging.Recently,a number of excellent small-molecule fluorescent probes targeting DNA G4s have been developed.Most of them were designed to be fluorescence light-up probes with positive charges due to the negatively charged phosphate backbone and loops of DNA G4s.However,these highly charged probes are not taken up rapidly by live cells because of the strongly lipophilic and hydrophobic nature of cell membranes’phospholipid bilayers.So far,these probes often spend a long time imaging G4 DNA in live cells.Lysosomes,acidic organelles present in essentially all eukaryotic cells,employ more than70 hydrolytic enzymes to degrade biological macromolecules taken up by endocytosis,phagocytosis and autophagy.Abnormal lysosomal pH values have been associated with some common diseases including neurodegenerative,cancer and Alzheimer’s diseases.Therefore,monitoring pH changes in lysosomes is critical for understanding cellular status and pathological processes.At present,many fluorescent probes that can target lysosomes have become a powerful tool for observing the behavior of lysosomes in cells.As an energy plant of cells,mitochondria are another important subcellular organelle.They participate in normal physiological processes such as regulation of cell signaling and cell death.Meanwhile,they also affect the growth and survival of cancer cells.Therefore,the dynamic monitoring of mitochondria in live cells can provide some guidance for visualizing physiological and pathological activities,as well as early screening and treatment of cancer.In recent years,mitochondria-targetable fluorescent probes have become a focus of research.Spiropyran derivatives belong to a class of organic photochromes that have been studied extensively for application in the construction of novel dynamic materials.Upon environmental stimuli such lipophilic spiropyran can be converted to its charged open form which has aπ-extended conjugation structure.In view of this,a spiropyran in situ switching(SIS)strategy for real-time fluorescent sensing of certain important biomarkers in live cells is proposed in this paper.That is,through reasonable design,the spiropyran can pass through the cell membrane and nuclear membrane readily,and can also be in situ activated by G4 DNA,H~+under the acidic lysosomal environment or mitochondrial membrane potential.Upon switching forms from closed to open,the resulting system causes a red shift in the fluorescent emission wavelength,thereby achieving real-time imaging of G4 DNA or subcellular organelles.With the aid of the SIS strategy,three spiropyran-linked fluorescent probes were designed and synthesized:QIN for the real-time imaging of endogenous G4 DNA and lysosomes,HAN for tracking dynamic changes of live-cell lysosomal pH and TANG for targeting endogenous G4 DNA and mitochondria.The specific research work is as follows:1.A classical nitrospiropyran derivative(1)was modified through structure-based screening and optimization via 2,3 and 4 to obtain a lead probe QIN which exhibited superior detection performance for G4 DNA,especially c-MYC G4 DNA.Therefore,the binding of QIN to c-MYC G4 DNA was studied by fluorescence spectra,UV-Vis spectra,circular dichroism spectra and pH titration systematically.The pKa of QIN is 5.9,which means that it exists predominantly in spirocyclic form at physiological pH and emits blue fluorescence.When QIN selectively binds to c-MYC G4 DNA,the pKa value increases to 7.4,which forces the probe to switch to its open form at physiological pH and emit red fluorescence.The G4 DNA induced p Ka shift mechanism was further verified by molecular docking and mass spectrometry.The targeting of G4 DNA in cancer cells by QIN was proved by digestion experiments of DNase I and RNase A and the positive control experiment of G4stabilizer PDS(pyridostain).Live-cell super-resolution fluorescent imaging suggests that the SIS-based probe has high photostability.The lipophilic QIN can enter live cells readily within 15 s,thereby achieving the second-level real-time imaging of endogenous DNA G4s.Furthermore,QIN with the appropriate pKa can also specifically label lysosomes.2.Encouraged by QIN,a fluorescent probe HAN for selectively sensing c-MYC G4 DNA was designed by molecular docking and synthesized.Unsurprisingly,from the docking results and in vitro studies,HAN exhibited high binding affinity towards c-MYC G4 DNA.As a spiropyran derivative,HAN’s reversible isomerization can be also achieved by pH.The pKa values of HAN were calculated to be 5.0 based on the Henderson-Hasselbalch equation,suggested that the sensitive response of HAN to pH matched well with the physiological pH range of lysosomes in live cells.However,confocal images of all the cells treated with HAN showed no response to endogenous DNA G4s.For lysosomes colocalization,the fluorescence of HAN at red channel almost completely overlaid with that of LysoTracker Green DND-26 and the Pearson’s correlation coefficient was higher than 0.94,confirming that HAN had a high lysosome-targeting capability.Using the lysosomal probe,clear super-resolution images of lysosomes in A549 cells was also obtained.Moreover,HAN could be used to successfully monitor lysosomal pH changes in stressed cells undergoing chloroquine stimulation.3.After analyzing the structural characteristics of c-KIT1 G4 DNA and its small molecular ligands,a novel fluorescent probe TANG was designed by molecular docking.The probe was synthesized via six steps from commercial chemicals.Consistent with the simulation results,TANG showed better recognition of the parallel structure G-quadruplex,especially c-KIT1 G4 DNA.At pH 7.4,TANG was induced partially by c-KIT1 G4 DNA to adopt its open form,causing a red shift in the fluorescent emission wavelength.The CD signal showed that the conformation of parallel c-KIT1 G4 DNA changed at pH 4.0.Upon the addition of TANG,the hybrid G-quadruplex structure was converted into the original parallel structure,further illustrating the capability of TANG for recognization of the parallel G-quadruplex structure.The SIS-based probe can achieve detection of endogenous G4 DNA in GIST cells with overexpression of c-KIT.In colocalization studies,TANG failed to label lysosomes,which can be attributed to the low p Ka of the probe(4.3)mismatching pH windows of lysosomes.However,TANG is effective in specifically labeling mitochondria in live cells,probably because the strong negative membrane potential of cellular mitochondria induced the formation of positively charged open form.