| Nucleic acids,as the genetic materials,are the significant components and the basis of species diversity in the lives.Based on the principle of bases sequencing and base-pairing in the molecular biology,nucleic acids can form special secondary structures under the specific conditions.For instance,the guanine-riched DNA or RNA can fold into G-quadruplex secondary structures and then form the multiple stacked Gquartets under the specific environment.Since the sequential and structural diversity in G-quadruplex,studies have shown that the G-quadruplex structures are widely exsisted in organisms and to be involved with the multiple vital processes including the replication,transcription,and translation.Therefore,the researches based on the discovery of G-quadruplex structures and its functions have attracted great attention.Recently,G-quadruplex structures have been proved to be specifically recognized and bind by the G-quadruplex ligands which originated from natural or synthetic small molecule compounds,while many of them have been demonstrated to be capable of inducing and stabilizing the formation of G-quadruplex structures in organisms,thereby these G-quadruplex ligands can regulate the G-quadruplex related biological behaviors.With the advantage of special topological configuration in G-quadruplex,some G-quadruplex ligands have been confirmed to generate efficient enzymatic catalysis or fluorescent signals when binding to the G-quadruplex structure.Hence,these types of G-quadruplex and its ligands are also widely applied in the biosensing.Although the in vitro characterization technology of the G-quadruplex structures has been gradually improved,there are still many difficulties in realizing the systematic exploration of the structures and functions in G-quadruplexes at the cellular level.At present,one popular strategy for discovering the intracellular G-quadruplex structures is to combine the G-quadruplex small molecule fluorogenic probes to realize the visualization and localization of G-quadruplex structures at the cellular level.Despite the G-quadruplex small molecule fluorogenic probes have been currently developed with the advantages of designability,strong specificity,and stable optical properties.However,they still suffered from the imperfection of low transmembrane efficiency,slow response,and poor sensitivities in cell imaging,which may bring great challenges in the real-time tracking and researches on biological function of the target Gquadruplex structures in a single cell.According to these drawbacks of the G-quadruplex small molecule fluorogenic probes,we designed and developed a new type of G-quadruplex small molecule fluorescent probe named ThT-NE by modifying the benzothiazole structure.ThT-NE maintains these advantages from traditional G-quadruplex small molecule fluorogenic probes,while the rational design of ThT-NE also provides a better cell membrane penetration capability in cell imaging.Thus,we have successfully constructed a fluorescent lighting probe named ThT-NE for quickly responding to the HCV RNA Gquadruplex in host cells.Besides,with the advantages of G-quadruplex small molecule probes in biosensing,we also constructed a protease-responsive rolling transcription assay named PRCTA to successfully realize the ultrasensitive visual detection of MMP-2 activities in cell extracts.The research contents of this dissertation are as follows:(1)The construction and characterization of viral RNA G-quadruplex fluorescent lighting probe.As RNA viruses are a huge global threat to human health,it is significant to construct a viral genome visualization system in host cells for virological research and clinical diagnosis.However,it is lack of the chemical tools in response to viral genome that can realize the label-free or gene modification-free in living cells.Therefore,we envisage to construct the viral genome responsive fluorogenic probes to specifically recognize the specific secondary structure in the target viral genome.In this work,the hepatitis C virus(HCV)has been chosen as the research model,and the CG2 a sequence that can form the G-quadruplex structure has been firstly obtained by screening the conserved gene regions in the HCV RNA genome.Secondly,the novel G-quadruplex fluorogenic probe ThT-NE has been rationally designed on the basis of benzothiazole structure and then the fluorescent mechanism of ThT-NE has been systematically demonstrated by Molecular Dynamic Simulations(MDS)and Density Functional Theory(DFT)to inhibit the intramolecular charge transfer and reduce the non-radiative relaxation when binding to the G-quadruplex.The flurorescence results also proved that ThT-NE is capable of specifically binding to the CG2 a by increasing1693 folds fluorescent enhancement,and also shows a capability of sensitive CG2 a detection and the detection limit reaches to 0.008 μM,which can lay the groundwork for HCV RNA genomic visualization in host cells.(2)Viral RNA G-quadruplex fluorescent lighting probe in cellular visualization.RNA shows an important function in the living system.As the original nonfluorescence property of RNA,it will provide a powerful tool in cellular RNA visualization and the research on RNA function with developing the strategy of RNA lighting up that combines specific RNA structures with small molecule fluorogenic probes.Based on the capability of ThT-NE in specifically recognizing the HCV RNA G-quadruplex,the imaging effect of ThT-NE in host cells and a systematic optimization of the imaging conditions of ThT-NE will be proceeded in this chapter.The optimal results have proved that ThT-NE shows the best fluorescence imaging contrast and the capability of quick identification of the HCV RNA genome when compared to the other G-quadruplex small molecule fluorogenic probes.In addition,ThT-NE also shows a good biocompatibility and the capability of photobleaching resistance in host cells,which can provide a better condition for real-time imaging of the HCV RNA genome in host cells.Thereby,the excellent performances of ThT-NE are expected to realize the fluorescent lighting on HCV RNA genome and the systematic research and realtime diagnosis of HCV.(3)The applications of viral RNA G-quadruplex fluorescent lighting probe.Based on the high specificity,high contrast and quick response imaging properties of ThTNE,the life cycle of natural HCV in host cells has been systematically studied in this chapter.Firstly,the cellular distribution of the HCV RNA genomes has been analyzed by comparing the fluorescent colocalization foci of ThT-NE with different subcellular organelles.Secondly,through further calculating the number of fluorescent foci of ThTNE and the lit-up cells,the replication cycles and infectious distributions can be well identified during the visual monitoring process of natural HCV infection.Finally,as ThT-NE keeps the capability of inducing and stabilizing the potential G-quadruplex sequences into G-quadruplex structure formation,the results prove that ThT-NE also provide an anti-viral efficiency for inhibiting the replication and translation process of HCV.Therefore,on account of these application researches of HCV life cycle,it shows that ThT-NE is a potential chemical tool for the HCV diagnosis and therapy in living cells,and fluorescent lighting on viral genome with ThT-NE also provides a novel strategy for virus analysis,medical diagnosis,and drug development in virology research.(4)Proteolysis-responsive rolling circle transcription assay for MMP-2 activities detection.As proteases perform vital roles in the malignant progression of tumors,they have been regarded as the biomarkers for many cancers.Currently,although protease assays such as immunoassays and fluorogenic substrate-based probes have been developed,it still remains challenges for them to give consideration on both sensitivity and accuracy in protease detection.To solve this problem,a proteolysis-responsive rolling circle transcription assay named PRCTA has been developed in this chapter for target protease response by rational integration of a protease-responsive RNA polymerase(PR)and rolling circle transcription(RCT),which enables to specifically identify the target protease by the substrate peptide of PR and generate the ultrasensitive signal outputs by RCT reaction at the same time.Taking cancer biomarker matrix metalloproteinase-2(MMP-2)as the research model,the PRCTA that can transduce and amplify each proteolytic catalyzed signal by MMP-2 into the multiple tandem RNA G-quadruplex by in vitro transcription and then generating the fluorescence signal outputs through binding to the ThT.Such a rational integration greatly enhances the signal gain in PRCTA,and it enables an ultrasensitive MMP-2activities detection with the limit of detection of MMP-2 as low as 3 f M.The feasibility of PRCTA has been validated by the sensitive analysis of cellular MMP-2 activities of different cell lines with good accuracy,and the readout can be further visualized to distinguish the normal cells and cancer cells as well as the sensitive visual detection of MMP-2 in cancer cells by a fluorescence imaging system.PRCTA exhibits a promising potential in biomedicine research and cancer diagnosis.Meanwhile the novel design concept and the highly efficient,sensitive,and intuitive performances of PRCTA also provide an ideal research plateform for protease analysis,drug screening,and prognosis assessment. |
