Design And Synthesis Of Novel Flurescent Probes Targeting On G-quadruplex DNA

The development of highly sensitive and selective probes to qualitatively and quantitatively detect nucleic acids is extremely important for a wide range of investigations, such as the active research areas of chemical biology, biochemistry, and clinical diagnosis. The prototypical nucleic acid DNA, apart from forming primarily double helical structures, is able to adopt higher-ordered and functionally-useful structures. G-quadruplex is one such distinctive structure, which forms an unique four-stranded structure containing guanine-rich nucleic acids sequences. G-quadruplexes can be divided into three primary topologies: parallel, antiparallel, and hybrid-type structures. In recent years, G-quadruplex structures have received attention because their unique structural features are believed to be able to provide biological significance in telomere maintenance, transcription regulation, and anti-tumor chemotherapy. G-quadruplex binders have great potential for development as anticancer drugs with the expected outcome for research in this area being eventual translation to the clinic. Since G-rich telomeric DNA sequences synthesized by telomerase have been shown to form G-quadruplexes in vivo, telomeric G-quadruplexes therefore attract tremendous interest and research effort. Moreover, G-quadruplexes were demonstrated as versatile building blocks that offer substantial advantages on fluorescence sensors. Fluorescence-based technology development of instruments and techniques, such as fluorescence microscopy cell imaging technology, has been widely used in various fields of biochemistry analysis and biomedical research. Therefore, there is the use of small molecule fluorescent probes specific for binding to cells, through fluorescence microscopy, real-time tracer G-quadruplex structures in cell behavior is the study of the molecular mechanism of its biological function good choice.Thiazole orange is also an example of a dye that has the favorable physical characteristics of a reticulocyte dye:it has a large fluorescence enhancement and high quantum yield upon binding to nucleic acids, it apparently binds tightly and is nonprecipitating, and it permeates live cell membranes. Derivatives having good membrane permeability, high quantum yields, but with a variety of DNA (including double-stranded and G-quadruplexes) can produce strong fluorescence signal upon binding, poor selectivity. Styrene substituted quinoline derivative is another fluorescent DNA probes. We hope that by introducing a structural fragment based on styrene thiazole orange on looking to improve their selectivity.The main work of the paper is:(1) The rational design and synthesis of a series of thiazole orange derivatives. (2) The fluorescence properties of derivatives with different types of nucleic acid (3) The effects of the compounds to G-quadruplex structure were investigated by CD spectroscopy and FRET experiments. (4) The binding mode of TO or TO derivatives with G-quadruplex/duplex DNA were investigated by molecular docking, (5) gel electrophoresis and cell imaging method confirmed as G-quadruplex DNA probes; (6) The application of derivatives as RNA fluorescent probes were investigated by UV spectra, cell imaging and fluorescence spectra.The results indicate that the introduced styryl substituent is able to induce remarkable selectivity for G-quadruplex DNA and show significant G-quadruplex structure stabilization in terms of large △Tm values obtained. In addition, the effects of the styryl moiety on the G-quadruplexes DNA binding behavior were further investigated with modelling study. The docking results show distinctive influence of the ancillary substituents which introduced at the 2-position of thiazole orange ligand on the binding discrimination of dsDNA and G-quadruplex DNA. Their photo-physical properties, equilibrium binding constants, selectivity and sensitivity, and photo-stability with G-quadruplex DNA were comprehensively investigated. Native PAGE experiments, FRET, CD analysis, and live cell staining were also studied to demonstrate the performance and potential applications of this class of thiazole orange based fluorescent probes in biomolecular sensing and cell imaging. A new and robust RNA-specific switch-on fluorescent dye 4j was developed from ligand integration of a classical nucleic acid fluorescent probe(TO) and a DNA dye(C61)moieties. At the same time the report has synthesized a fluorescent RNA probes E36,4j, E36 and commercialization SYTO RNASelect contrast experiment. The study demonstrated the concept of ligand integrationand proved that the dye is able to offer excellent RNA specificity, photo-stability, and cell tolerance. The dye shows very good counterstain compatibility with DAPI for specific RNA-DNA colocalization investigations in live cells. Also,the dye was successfully utilized in staining of nucleolus RNA for cell imaging andits performance is found much better than the commercial SYTO RNA Select dye.