Study On Isothermal Amplification Method Based On Functional DNA Nanostructure

In recent years,with the rapid development of nanotechnology,DNA as a nanometer-scale flexible material is widely used to construct biomolecular recognition probes due to its superior programmability,easy modification and functionality.DNA nanostructure-based biomolecular recognition probes play an important role in the field of analytical chemistry and clinical diagnosis.At the same time,nucleic acid isothermal amplification technology is a new type of in vitro amplification technology that developed after polymerase chain reaction technology,which can achieve rapid and highly sensitive amplification of target molecules under constant temperature conditions.This technology is easy to operate,and does not need to rely on accurate instruments and equipment.Recently,it has been widely used in the detection of various small molecule target substances in vitro and in vivo.In this work,by integrating the latest researches of analytical chemistry,nanoscience and clinical laboratory diagnostics,based on employing the controllable and functional DNA nanostructure,as well as combining with the isothermal amplification techniques,three novel and rapid methods have been developed for sensitive analysis of proteins and nucleic acids.These new methods provide potential analytical platforms for early diagnosis and prognosis monitoring of diseases.This study mainly includes the following three parts:1.Collapse of DNA tetrahedron nanostructure for “off-on” fluorescence detection of DNA methyltransferase activityAs a potential detection technique,highly rigid and versatile functionality of DNA tetrahedron nanostructures is often used in biosensing systems.In this work,a novel multifunctional nanostructure has been developed as an “off-on” fluorescent probe for detection of target methyltransferase by integrating the elements of DNA tetrahedron,target recognition,and dual-labeled reporter.This sensing system is initially in an “OFF” state owing to the close proximity of fluorophores and quenchers.After the substrate is recognized by target methyltransferase,the DNA tetrahedron can be methylated to produce methylated DNA sites.These sites can be recognized and cut by the restriction endonuclease DpnI to bring about the collapse of the DNA tetrahedron,which leads to the separation of the dual-labeled reporters from the quenchers,and thus the recovery of fluorescence signal to produce an “ON” state.The proposed DNA tetrahedron-based sensing method can detect Dam methyltransferase in the range of 0.1 U/mL to 90 U/mL with a detection limit of 0.045 U/mL,and shows good specificity and reproducibility for detection of Dam methyltransferase in real sample.Thus,this work possesses a promising prospect for detection of DNA methyltransfrase in the field of clinical diagnostics.2.A simple method for ultrasensitive detection of BCR-ABL1 fusion gene based on three-way junction DNA structure induced dual-isothermal amplificationA one-step,rapid fluorescent biosensing method has been developed for ultrasensitive detection of BCR-ABL1 fusion gene based on the polymerase/nicking endonuclease assisted dual-isothermal amplification.In the method,BCR-ABL1 fusion gene can be specifically identified by dual probe to form three-way junction DNA structure(3-WJ).Then the 3-WJ DNA structure is driven by polymerase and nicking endonuclease to generate a large number of triggers,initiating downstream rolling circle amplification reaction,achieving exponential growth of signal.Benefiting from the cascade amplification,the developed method generates a wide linear response from 10 fM to 1 nM with a low detection limit of 5.52 fM.In addition,the one-step operation allows the assay to be completed within 60 min and acceptable recovery is obtained in complex samples.These merits endow the biosensing strategy a certain potential for the clinical diagnosis and scientific research of BCR-ABL1 fusion gene.3.An ultrasensitive fluorescence detecting method for detection and in situ imaging of chronic myeloid leukemia-related BCR-ABL1 mRNAA fluorescent detecting method has been developed for rapid and ultrasensitive detection of BCR-ABL1 mRNA in chronic myeloid leukemia(CML)based on DNAzyme cleavage-induced rolling circle amplification.In the presence of BCR-ABL1 mRNA,DNAzyme is activated to split the target sequence into two fragments,producing the 3’ terminus on the forward cleavage fragments.After T4 polynucleotide kinase modification reaction,the forward cleavage fragments are extended by rolling circle amplification.Plenty of long single DNA strands are produced and partially hybridized with the fluorescence-quenching decorator probes,thus inducing the separation of fluorophore and quencher decorator probes and recovery of fluorescence.Highly sensitive detection of BCR-ABL1 is achieved with a limit of detection at 9.4 fM.In addition,this method is adopted to successfully in situ image the BCR-ABL1 mRNA in the cytoplasm of human leukemia bone marrow cells.Moreover,results of the BCR-ABL1 mRNA expression in clinical samples achieved by this method are well consistent with that of reverse transcription PCR and fluorescence in situ hybridization analysis.Therefore,this developed method might provide a potential alternative tool for precise diagnosis of CML.