Studies On The Construction Of Functionalized Nucleic Acid Nanoprobes And Their Applications In MicroRNA Detection

Cancer is a major disease that threatens human life.Early cancer diagnosis is very important in controlling the global cancer patient population.It helps doctors to identify the stage of cancer in time,and to select the appropriate treatment and prognosis evaluation.micro RNA(mi RNAs,mi R)is one of the potential tumor markers for early cancer diagnosis.However,due to its low abundance,short sequence and high sequence similarity,it is a challenge to develop accurate and highly sensitive biosensors in mi RNAs detection and imaging.Peptide nucleic acid(PNA)holds many merits,including excellent hybridization characteristics,flexible molecular design,"electrical neutrality" and stability.These unique properties make it an ideal sensing material for complex biological environments,even in whole cells.Although many kinds of PNAbased detection probes had been reported so far,most of them show low sensitivity,complicated modification and synthesis,and difficulty in cell imaging.Therefore,it’s very important to develop PNA nanoprobes with efficient signal amplification and cell delivery properties.This thesis presented studies on the application of PNA nanoprobe for mi RNAs detection using enzyme-free strand replacement technique and DNA nanostructure combing with electrochemical or portable device.The research topics of this paper are as follows:In chapter 1,we firstly reviewed the research progress in the correlation between mi RNAs and cancer,and the problems in mi RNA detection.Secondly,the research on PNA including physicochemical properties,modification,delivery and potential application were introduced.Then,we focused on the construction principle and applications of dynamic DNA nanotechnology with enzyme-free strand replacement technique and DNA nanostructures.Finally,based on the unaddressed issues in mi RNAs detection and PNA detection mode,the significance of the construction of PNA nanoprobe for more sensitive mi RNA detection using PNA and DNA nanotechnology is emphasized.In chapter 2,catalytic hairpin assembly(CHA)-mediated PNA nanoprobes with high yield,good stability and versatility were successfully prepared by carefully optimizing the stem-ring sequences of mi RNA-mediated CHA reaction and PNA concentrations.Specifically,the hybridization stability of hairpin DNA was improved,in which the signal leakage caused by spontaneous hybridization between hairpin DNA and the direct combination of PNA and stem ring domain was effectively avoided by optimizing stem-ring domain.The probe is inherently modular and scalable,providing a template for low abundance and highly sensitive detection of mi RNAs.In chapter 3,an electrochemical sensor was constructed based on the combination of CHA-mediated PNA nanoprobe and a gold electrode.In the presence of the target mi RNAs,the CHA was triggered selectively between two hairpins with one ferrocene(Fc)or methylene blue(MB)labelled.It was confirmed that the simultaneous reaction by dual CHA amplification was feasible.The resulting redox-active group modified CHA products were then specifically captured by the PNA probes attached on the surface of a gold electrode,which bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals for sensitive and simultaneous detecting of low amount mi R-21 and mi R-155 in cancer cells.This result was consistent with the results of real-time quantitative polymerase chain reaction(q RT-PCR).This assay was also highly selective for discriminating mi RNAs with similar sequences and has detection limits of 2.49 f M and 11.63 f M for mi R-21 and mi R-155,respectively.Due to the simplify,sensitivity and accuracy,this method thus has great potential to be applied for detecting a variety of mi RNA biomarkers simultaneously for clinic applications.In chapter 4,a personal glucose meter(PGM)sensor is constructed with CHAmediated PNA nanoprobe on 96-well plate.Firstly,mi R-155 in serum triggers the CHA reaction,and then the CHA product is specifically captured by the PNA probes attached on the surface of a 96-well plate,which in turn triggers the hybridization chain reaction(HCR),resulting in the local enrichment of invertase.Nextly,introducing a sucrose substrate for the invertase results in the generation of glucose,which can be detected by PGM.In this sensor,the synergy isothermal amplification reaction between CHA and HCR makes the sensor capable to achieve a broad dynamic range from 1 f M to 10 n M with a detection limit down to 0.36 f M(3 orders of magnitude lower than that without HCR),and capable of distinguishing single-base mismatched sequences.The portable,robust,low-cost,rapid readout and easy-to-operate PGM sensor makes it possible for on-site nucleic acids detection,suggesting its great application potentials as a promising POCT device in cancer diagnostics.In chapter 5,because of the charge-neutrality and overall lipophilic character,unmodified PNA are basically not taken up by cells.To overcome this weakness,PNA oligomers were conjugated with the DNR to facilitate membrane association/endocytosis.The DNR-mediated PNA nanoprobes are periodic,regular and functional,and they are very attractive for low cost,unique stability,controllable structure and easy modification,which is ideal for the efficient targeted cell delivery of PNA.These advantageous PNA features may enable a number of applications on the imaging and gene regulation of intracellular mi RNAs.