Study On Catalytic Performance Of DNA/Hemin Mimic Enzyme And Its Biologicalanalysis Applications

DNAzyme is an important artificial mimic enzyme with the advantages of small molecular weight,easy synthesis,low cost,strong tolerance and programmable.However,there is still a certain gap between the catalytic performance of DNA mimic enzymes and traditional proteases,which greatly limits its wide application.Hence,improving the catalytic performance of mimic enzymes,constructing efficient and stable DNA mimic enzyme structures,and applying them to the fields of precise disease diagnosis,molecular imaging,tumor diagnosis and treatment,are key issues in the research of DNA mimic enzymes.This dissertation integrates the latest research results of clinical laboratory diagnostics,bioanalytical chemistry,and nanotechnology,in-depth discussion,and elaboration of the catalytic activity and reaction mechanism of DNA/hemin mimic enzymes;developing a series of a new type of DNA mimic enzyme with high-efficiency catalytic ability,stable properties,and strong environmental tolerance;establishing a new method for highly sensitive detection of multiple disease markers such as nucleic acid,small molecules,and cell membrane proteins.This research provides new theoretical and technical support for the enzyme-free detection system and new ideas for the development of artificial mimic enzymes.This research mainly includes the following three parts:1.DNA-grafted hemin with preferable catalytic properties than G-quadruplex/hemin for fluorescent miRNA biosensingThe mimetic enzyme based on the covalent binding of hemin to the oligonucleotide chain(DNA-Hemin DNAzyme)has attracted widespread attention as a catalyst in biosensing applications.However,its structural allosteric mode and catalytic performance still need to be further explored and studied.This study proposes a conceptual analogy analysis strategy to compare the catalytic and analytical performance of the DNA covalently bound hemin DNAzyme(DGH)and the classic G4/Hemin DNAzyme.First,two similar target-triggered fluorescent catalytic systems were designed to compare their performance as molecular recognition probes and catalysts.The results showed that although G4/Hemin showed better catalytic ability,DGH has the advantages of low background,fast catalytic rate,good tolerance and high efficiency of target-triggered conformation conversion.Besides,when miRNA-21 is used as a model analyte,the detection limit of the DGH system is 0.17 nM,which is about 20 times that of G4/Hemin,indicating that the DGH system has better sensitivity and dose-response capabilities in fluorescent biosensing strategies.Therefore,the DGH enzyme is an excellent non-enzyme signal transduction tool with broad application prospects.2.Dynamic DNA self-assembly activated hemin-mimetic enzyme system for versatile fluorescent biosensingIn this work,a hemin mimic enzyme system activated by dynamic DNA self-assembly was established to realize multifunctional fluorescent biosensing detection.DNA covalently bound hemin(DGH)is not only used as a module of dynamic DNA self-assembly but also as a tunable tool for simulating enzymes.When the target is present,the entropy-driven dynamic DNA assembly initiates the circuit,dissociating the inactivated hemin dimer labeled at the end of the DNA strand into activated monomers.The activated DNA-hemin mimic enzyme can catalyze the non-fluorescent tyramine substrate to generate a fluorescent signal,realizing simple,fast,and enzyme-free detection.The detection limit and linear range of this strategy for detecting the target GBS gene are 78 pM and 0.1-50 nM,respectively,and the detection time is 40 minutes.Besides,this method is also used to detect small molecule cocaine with a detection limit of 4.85 ?M and a detection time of 70 minutes.The advantages of the detection system are tunable analog enzyme activation mechanism,simple design of oligonucleotide modules for DNA assembly,and no need to add additional hemin for rapid assembly.The simple and universal target-triggered DNA-hemin enzyme system proposed in this work can achieve high-efficiency enzyme-free signal transduction and is expected to become a widely used biosensing tool.3.Zippered G-quadruplex/hemin DNAzyme: Exceptional catalyst for diverse bioanalytical applicationsG-quadruplex/hemin DNAzymes with horseradish peroxidase(HRP)-mimic activity are becoming promising candidates for applications in biosensing,nano-materials,and biomolecular devices.Nevertheless,enhancing their catalytic performance remains a goal for researchers.Here,we explore a novel G4/hemin proximity assembly with exceptional HRP mimic catalytic properties,namely zippered G4/hemin DNAzymes(Z-G4/H).The detailed investigation of the catalytic efficiency and mechanisms of Z-G4/H verified that the hybridization of the hemin-grafted oligonucleotide with complementary strand containing G4 sequence and adenine nucleotide residues conducted to mimic the tight configuration of protease cofactors and apoenzyme backbone,resulting in higher activity,more rapid rate and lower environmental dependency than classical G4/hemin DNAzymes.Furthermore,the sensitive detection of the BCR-ABL fusion gene in vitro and situ imaging of human epidermal growth factor receptor 2(HER2)dimers on cell surface demonstrated the universal potential of Z-G4/H for the applications in clinical diagnostics and researches of biomacromolecule interactions.The presented zippered G4/hemin assembly may offer significant opportunities and inspiration for the engineering of the mimic enzyme to be comparable with natural protease.