Nucleic Acid Isothermal Amplification And Its Application In Biochemical Analysis

Isothermal amplification of nucleic acids is a simple process that can rapidly and efficiently synthesize multiple copies of nucleic acid molecules at constant temperature.The process does not require the thermal cycler,which has the advantages of high sensitivity,high specificity,time-saving and simple operation.In recent years,various isothermal amplification techniques have been developed,mainly including DNA replication with enzymes and nucleic acid assembly without enzymes.These isothermal amplification methods have been used for various targets analysis such as DNA,RNA,cells,proteins,small molecules,and ions.Most of these methods have a good development and application for the detection of nucleic acids,and some have achieved commercial success.Meanwhile,isothermal amplification also provides abundant nucleic acids as materials for constructing higher-ordered nucleic acid nanostructures,nucleic acid templated metal nanostructures,and nucleic acid hydrogels,which hold great promise in biosensing,bioimaging,and nanomedicine.Therefore,more opportunities have been opened for biochemical analysis.On the other hand,due to the limited amount of some analytical targets and the limitations of poor environmental conditions,it is still necessary to develop new DNA isothermal amplification technology with high efficiency,programmable and mild operating conditions to realize nondestructive and highly sensitive analysis,and even real-time detection of biological molecules or cells.In this thesis,we have developed some new isothermal signal amplification technologies on the basis of the existing researches through nucleic acid sequence programming,as well as the optimization of buffer and other conditions which have greatly improved the sensitivity,specificity and gentleness of the experimental environment of DNA signal amplification,so as to achieve efficient,rapid and non-destructive analysis of the targets.1.pH-responsive novel netlike rolling circle amplification for the visual detection of miRNA based on test paper.Based on the extension of primers in the process of netlike rolling circle amplification(NRCA)and the release of H+,we have designed a new test strips-based POCT method to detect biomarker miRNA.This method adopts efficient isothermal amplification,thus avoiding the use of expensive thermal cycler.Due to the generation of H+,the pH value in the system decreased significantly from alkaline to acidic.Therefore,we can use pH-sensitive indicator or pH test paper to detect miRNA,which is also applicable to home health care and those harsh circumstances.Moreover,tumor-associated miRNA-21 in both spiked serum samples and cells is successfully achieved.For the determination of miR-21 positive or negative sample,a pH test paper may help;and if accurate quantitation is required,colorimetric assay of the pH-sensitive indicator is preferred,which is also applicable to professional laboratory for quick quantification.This work not only enables the detection of miRNA with favorable efficiency,specificity and sensitivity,but also provides a new idea for the detection of other tumorassociated nucleic acids or viruses.2.Logic-gate responsive nucleic acid rolling circle amplification for high accuracy imaging analysis of cancer cells.Based on aptamers,we have developed a novel logic-gate responsive molecular machine for bispecific recognition and computation of cell membrane surfaces.The DNA molecular machine simply assembles and consists of three functional DNA single-strand hybridization to form a DNA ternary complexes including a circular DNA template,an aptamer probe and a preprimer.Only when the target cell exists,the DNA molecular machine performs "AND" logic operation and amplified "ON" fluorescence signal generates through rolling circle amplification,which improves the accuracy of cell recognition.However,when the target cell does not exist,the molecular machine will not generate fluorescence signal when it is in the"OFF" state.In addition,we tried to apply the logic-gate responsive DNA molecular machine to analyze target cells in complex samples,and the results showed that the molecular machine could accurately distinguish target cells from other cells,which demonstrated our method owns good specificity and could solve the false positive in the detection process.Therefore,the logicgate responsive DNA molecular machine performs logical operation on the cell membrane surface and amplifies the signal,which has realized efficient and accurate imaging analysis of cancer cells.Therefore,this method has great potential for disease diagnosis,and provides new tools and ideas for biomedical research.3.Intramolecular proximity strand displacement(IPSD)-mediated isothermal nucleic acid amplification for nondestructive analysis of cells and cell membrane proteins.Based on our previous independent development of a process called intramolecular proximity strand displacement(IPSD)mediated isothermal nucleic acid amplification and its related technology,we further designed and proposed immuno-IPSD that was operated in the cell culture containing a variety of complex growth factor in this work.Thus,ultrasensitive and nondestructive detection of circulating tumor cell membrane protein has been realized.Meanwhile,through the application of immuno-IPSD technology for the analysis of different tumor cell membrane proteins,we found that IPSD has favorable specificity and signal amplification ability,which can be utilized for the analysis of very few or even single cells,thus providing a new technical support for the detection of a variety of low-abundance proteins on the cell membrane.In addition,we also made a comprehensive assessment of the cell state and activity after the immuno-IPSD analysis.After the immuno-IPSD analysis,we found that there was almost no difference in cell morphology,activity and proliferation ability,and surprisingly,the drug sensitivity of cells was almost the same before and after the immuno-IPSD analysis.Therefore,our proposed immuno-IPSD can not only achieve high sensitive detection for highly heterogeneous cells,but also can maintain the basic morphological and physiological activity of cells,which even can be continued culturing and can be used for drug sensitive test and the analysis of critical protein.Furthermore,this method lays a foundation for the in-depth analysis of circulating tumor cells molecular phenotype and its future application in drug development.4.Exponential hybridization chain reaction and imaging analysis of membrane protein interactions.Based on the hybridization chain reaction and catalytic self-assembly of hairpins,we have proposed the exponential chain replacement amplification method based on the double hairpin structure,and developed a new exponential hybridization chain reaction(E-HCR)strategy by introducing the double hairpin structure into the strand replacement amplification system.In detail,in the presence of the trigger strand,the trigger strand could open the double hairpin structure through the toehold initial strand replacement reaction,and then open a new double hairpin structure with the help of the auxiliary hairpin.Due to a new round of amplification generated from each trigger strand sequence,the amount of trigger strand sequences is doubled,and the signal is also increased which can realize exponential signal amplification.Therefore,combining this new signal amplification technique with the proximity strategy,we have proposed a new method for the determination of protein interaction analysis.Taking the two cell membrane protein named epidermal growth factor receptor(EGFR and HER2)as an example,the trigger strands respectively recognizing with EGFR and HER2,could form a new combinational trigger strand and open the double hairpin,then inducing the subsequent E-HCR self-assembly on the cell membrane due to the interaction between EGFR and HER2.This method could realize the amplification of the membrane protein interaction analysis,and effectively monitor the effects of protein activators and inhibitors on the protein interactions in real time.Meanwhile,E-HCR cascade self-assembly is a DNA isothermal signal amplification technology without the involvement of nucleic acid tool enzymes and various biochemical reactions are not involved during the process.Therefore,through the evaluation of cell state from cell activity,cell apoptosis,cell proliferation,critical protein,the results showed that EHCR can minimize the possible damage caused by the analysis system to cells,maintain the integrity of the physiological activity of cells,and facilitate the in-depth analysis of cells,such as in vitro cell culture and drug screening.Therefore,our proposed exponential HCR for analysis of membrane protein interaction provides a new research idea for the dynamic analysis and non-destructive analysis of non-genetic engineering living cell membrane protein interaction,which will promote the application of DNA isothermal signal amplification technology for more complex cell analysis.