Study On DNA Detection And Activity Of G-quadruplex/hemin DNAzymes Strategries Based On DNA Assembly

With the accomplishment of Human Genome Project and the progress of functional gene,gene diagnosis has become an important research field of molecular biology and biomedicine.The quantitative detection of sequence-specific DNA fragment shows its great significance in the areas of gene analysis,disease diagnosis,food safety,judicial identification and environmental monitoring.Based on the principle of nucleic acid base pairing,DNA sensors have been developed very fast in recent years,which can acchieve rapid,sensitive,and selective detection of specific sequences of gene fragments.Developing highly sensitive and selective DNA detection methodologies are crucial due to the low abundance of the sequence-specific DNA fragment in real sample as well as the complexity of the real sample composition.In addition,DNA has been found to possess the properties of enzymes under certain conditions,such as G-quadruplex structure combined with hemin has peroxidase activity and has a wide range of applications in life analysis.However,the relatively low catalytic efficiency of G-quadruplexe/hemin DNAzymes restrict their further applications and efficiently improve G-quadruplexes DNAzyme activity still remains a significant challenge.This thesis focuses on exploring novel DNA detection methodologies,which utilizes the DNA assembly technology to achieve signal amplification or develop new signal transduction pathway and on improving the activity of DNA enzyme,developing new applications.The thesis includes the following sections:1.Target-driven DNA Association to Initiate Cyclic Assembly of Hairpins for Biosensing and Logic Gate OperationA target-driven DNA association was designed to initiate cyclic assembly of hairpins,which led to an enzyme-free amplification strategy for detection of nucleic acid or aptamer substrate and flexible construction of logic gates.The cyclic system contained two ssDNA(S1 and S2)and two hairpins(H1 and H2).These ssDNA could co-recognize the target to produce a S1-target-S2 structure,which brought their toehold and branch-migration domains into close proximity to initiate the cyclic assembly of hairpins.The assembly product further induced the dissociation of a double-stranded probe DNA(Q:F)via toehold-mediated strand displacement to switch the fluorescence signal.This method could detect DNA and ATP as model analytes down to 21.6 pM and 38 nM,respectively.By designing different DNA input strands,the "AND","INHIBIT" and "NAND" logic gates could be activated to achieve the output signal.The proposed biosensing and logic gate operation platform showed potential applications in disease diagnosis.2.A Plasmonic Colorimetric Strategy for Biosensing through Enzyme Guided Growth of Silver Nanoparticles on Gold NanostarsA plasmonic colorimetric strategy was designed for sensitive detection of biomolecules through enzyme guided silver nanoparticles(AgNPs)growth on gold nanostars(AuNS).The growth of AgNPs on AuNS led to a substantial blue shift of the localized surface plasmon resonance(LSPR)peak and the color change of AuNS from blue to dark blue,purple and ultimately orange.Both the LSPR blueshift wavelength and the color of detection solution containing AuNS,Ag+ and ascorbic acid 2-phosphate(AAP)depend on the amount of enzyme that catalyzed the dephosphorylation of AAP to reduce Ag+ on AuNS surface.Thus this strategy could be used for LSPR and naked-eye detections of both the enzyme such as alkaline phosphatase(ALP)and other biomolecules involved in biorecognition events using ALP as a tag.The LSPR detection method for ALP showed a linear range from 1.0 pM to 25 nM with a detection limit of 0.5 pM.Using DNA as a mode target molecule,this technique showed a detection range from 10 fM to 50 pM DNA with a detection limit of 2.6 fM through the convenient combination with hybridization chain reaction amplification.The proposed plasmonic colorimetric strategy could be extended as a general analytical platform for design of immunosensors and aptasensors with ALP as a label.3.A pH-responsive Colorimetric Strategy for DNA Detection by Acetylcholinesterase Catalyzed Hydrolysis and Cascade AmplificationA pH-responsive colorimetric strategy was designed for sensitive and convenient biosensing by introducing acetylcholinesterase(AChE)catalyzed hydrolysis of acetylcholine to change solution pH and phenol red as an indicator.Using DNA as a target model,this technique was successfully employed for sensitive DNA analysis by labelling AChE to DNA.The sensitivity could be greatly improved by coupling a newly designed magnetic probe with target DNA-triggered nonenzymatic cascade amplification.In the presence of a help DNA(H)and the functional probe,the cascade assembly via toehold-mediated strand displacement released the AChE-conjugated sequence from magnetic beads,which could be simply separated from the reaction mixture to catalyze the hydrolysis of ACh in detection solution.The color change of detection solution from pink to orange-red,orange-yellow and ultimately yellow could be used for target DNA detection by naked eye and colorimetry with the absorbance ratio of detection solution at 558 nm to 432 nm as the signal.The nonenzymatically sensitized colorimetric strategy showed a linear range from 50 pM to 50 nM with a detection limit of 38 pM,indicating a promising application in DNA analysis.4.Thermophilic Tetramolecular G-quadruplex/hemin DNAzymeQuadruplex-based DNAzyme system is one of the most useful artificial enzymes or catalysts due to their unique properties that make them reliable alternatives to proteins for performing catalytic transformation.Herein,we report on the first prototype of a thermally stable DNAzyme system,referred to as a thermophilic DNAzyme,capable of oxidizing substrates in harsh experimental conditions,i.e.,high temperature(up to 95 ?)and long reaction times(up to 18 h at 75 ?).The step-by-step design of this unique heat-activated G-quadruplex/hemin catalyst,including the modification of adenines at both ends of G-tracts,the choice of cation and its concentration for DNAzyme stabilization,is described.This work provides the first insights into the mechanistic origins of its outstanding properties as well as an example of an industrially relevant application.