Fluorescence Sensitive Detection Of DNA Glycosylase And MicroRNA

The early detection of cancer can significantly reduce cancer mortality and saves lives.Thus,a great deal of effort has been devoted to the exploration of new technologies to detect early signs of the disease.Tumour biomarkers are present in cancer tissues or serum and encompass a wide variety of molecules,including DNA,m RNA,enzymes,metabolites,transcription factors,and cell surface receptors.The goal of the cancer biomarker field is to develop reliable,cost-effective,powerful detection and monitoring strategies for cancer risk indication,early cancer detection,and tumour classification;so that the patient can receive the most appropriate therapy and that physicians can monitor the disease progression,regression,and recurrence.Due to their low content in the complex biological samples,traditional detection methods are insufcient to satisfy the requirements for monitoring those biomolecules,therefore,it is urgent to develop new technology and new methods for the detection of cancer biomarkers that are sensitive,accurate,simple and rapid.In recent years,the fluorescence methods have received more and more attention due to the advantages of simple operation,fast response,high sensitivity and good selectivity,and the signal amplifcation can signifcantly enhance the fuorescence signal and sensitivity of the fluorescence methods.In this thesis,a series of tumour biomarkers detection methods with high sensitivity and specificity were developed based on DNA glycosylases and micro RNA combined with signal amplification-based fluorescence detection.The main contents of this thesis are as follows:1.Nucleobase oxidation and alkylation can destroy Watson-Crick base-pairing to challenge the genomic integrity.Human 8-oxoguanine glycosylase 1（h OGG1）and alkyladenine glycosylase（h AAG）are evolved to counter these two cytotoxic lesions through base-excision repair,and their deregulations are implicated with multifactorial diseases and cancers.Herein,we demonstrate activatable self-dissociation of Watson-Crick structures with fluorescent nucleotides for sensing multiple human glycosylases at single-cell level.The presence of h OGG1 and h AAG catalyzes 8-oxo G and deoxyinosine removal in functional probe1 to release two trigger probes.Then,trigger probes hybridize with functional probe 2 to activate the autocatalytic degradation of functional probes 2（Cycle I）and 3（Cycle II）,replicating abundant trigger probes and releasing two fluorophores（2-aminopurine（2-AP）and pyrrolo-d C（P-d C））.New trigger probes,in turn,hybridize with free functional probes 2 and 3,repeating Cycles I and II turnovers.Through multicycle self-dissociation of Watson-Crick structures,2-AP and P-d C are exponentially accumulated for the simultaneous quantification of h OGG1 and h AAG.This nanodevice exhibits high sensitivity with a detection limit of 2.9×10^-3U/m L for h OGG1 and 1.5×10^-3U/m L for h AAG,and it can measure enzymatic kinetics,identify potential inhibitors,discriminate glycosylases between cancer and normal cell lines,and even quantify glycosylase activities in a single He La cell.Moreover,this assay may be rapidly and isothermally performed in one tube with only one tool enzyme in a quencher-free manner,promising a simple and powerful platform for multiple human glycosylase detection.2.Micro RNAs（mi RNAs）are non-protein-coding single-stranded RNAs with the capability of inhibiting the translation of corresponding proteins by completely/incompletely matching with target messenger RNAs（m RNAs）,and they regulate various cellular processes including differentiation and apoptosis.Conventional DNA catabolic strategies are restrained with the fragile and unclear enzymatic reactions that might lead to inefficient and uncontrollable digestion of DNA scaffolds and thus might bring undesirable side effects to the sophisticated biosystems.Here,we demonstrate activatable self-dissociation of multifunctional DNAzyme nanodevice based on rolling circle amplification for micro RNA quantification and imaging in living cells.In the presence of primer and circular template,RCA amplification is initiated,which will produce a long DNA strand containing PTK7 protein aptamer and DNAzyme.With the addition of capture probe,its 5′-end will bind to the rolling circle product,accompanied by the generation of Cy5 fluorescence.The verified multifunctional DNAzyme nanodevice preparation incubates with cells and enters the cell by binding to the PTK7 protein on the cell membrane.With the addition of Zn²⁺,the DNAzyme sequence on the DNAzyme nanostructure will be cleavaged and the capture probe will be released.Similarly,in the presence of the target mi R-21,it binds to the 3′-end of capture probe,producing Cy3fluorescence.The biosensor provides sensitive detection of mi RNAs and it can be used for imaging low abundance micro RNA in living cells,with a detection limit of 21.92 a M.Moreover,it can absolutely quantify the mi R-21 expression in different types of lung cancer cells and even discriminate healthy individuals from NSCLC patients,which may greatly contribute to fundamental biomedical research and clinical disease diagnosis.