Construction Of New Methodologies For Nucleic Acid Detection Based On G-quadruplex And Junction Probe

G-quadruplex(also known as G4)is secondary structure formed in nucleic acids by sequences that are rich in guanine(G).Four guanines in a G-quadruplex can associate through Hoogsteen hydrogen bonding to form guanine tetrad,and two or more such guanine tetrads can stack on each other to form a G-quadruplex.In the solution with monovalent cation,G-quadruplex can form into complex with hemin monomer and results in peroxidase-mimicking properties(peroxidase-mimicking DNAzyme).The use of peroxidase-mimicking DNAzyme as a signal detection element has a strong portability for nucleic acid detection.However,the nucleic acid homogeneous detection method based on peroxidase-mimicking DNAzyme has a large detection background,which hinders its wide application in nucleic acid homogeneous detection.The concept of junction probe(JP)is derived from binary probe(BP).A detectable signal can only be generated when both probes simultaneously hybridize to the target sequence.This shows strong hybridization specificity,and the potential to be applied to homogeneous detection of nucleic acids.Conventional junction probes have been subjected to complex sequence design and intricate optimization,which increase the cost of detection.Based on the previous studies,we hereby construct junction probes based on strand displacement and G-quadruplex for portable detection of nucleic acid,and evaluates its detection efficiency.This thesis is divided into the following two chapters:In the first chapter,we present a three-way junction based system where two split G-quadruplex forming sequences are coupled into two probes.Only upon the introduction of target gene transcript that offering a specific recognizable splicing site do the two probes assembled into three way junction conformation in a devised process,thus providing a functional G-quadruplex conformation that greatly enhances hemin peroxidation.A notable resolution for gene splicing site detection is achieved.The detection limitation by colorimetric assay is 0.063 ?M,and this system has been proved to discriminate even in a single base false level around splicing site(about 3 times of single mismatched analyte to gain an equal signal by perfect analyte),showing a capacity to further exploit a simple equipped device for gene transcript detection.In the second chapter,we describe a method for DNA target recognition and signal amplification that is based on the target-induced formation of three way junction.The subsequent assembly of two DNA probes releases the inhibitory strand and triggers a downstream strand displacement amplification.This causes the formation of a G-rich single sequence that combines with hemin monomer with its peroxidase-mimicking properties.The resulting peroxidase(POx)activity is quantified by using H2O2 and TMB as the substrate.In the presence of an inhibitor,in contrast,the POx-like activity is strongly reduced.This forms the basis for a highly sensitive DNA assay.It has a 0.8 pM detection limit when operated at a wavelength of 450 nm.And it was applied to the isothermal determination of target DNA with high selectivity.