DNA Nanoprobes-based And Toehold-mediated Strand Displacement Reaction For Study On Tumor Exosomes Detection

Tumor-derived exosomes play a vital part in tumor development,tumor metastasis,immune response inhibition,angiogenesis,and other physiological processes.Therefore,the analysis and detection of tumor exosomes will greatly contribute to the early diagnosis,curative effect evaluation,and prognosis analysis of tumors.However,currently developed methods still have many key scientific problems that need to be solved urgently,such as low sensitivity,poor specificity,poor accuracy,and high cost.In this thesis,aiming at the exploration of novel tumor exosomes detection techniques with high sensitivity,high specificity and high accuracy,a series of nanoprobes have been constructed by taking the ability of base-pairing principles and DNA double helix properties to assemble specific functions and geometric structures.By combing toehold-mediated strand displacement reaction with the characteristics of fast response and sensitive signal conversion and aptamer as molecular recognition tools,using breast cancer,human acute leukemia and human hepatocellular carcinoma cells as the research object,studies on the detection of exosomal mi RNAs and proteins combined with have been systematically investigated.The detailed research content is listed as follows:1.In situ multiplex detection of breast cancer exosomal mi RNAs based on Ytype DNA nanoprobes and DNA strand replacementIn view of the insufficient accuracy of single target detection and the need for RNA extraction,a multicolor DNA nanoprobe for in-situ detection of three mi RNAs derived from tumor exosomes was developed.In this design,three DNA single strands labeled with quenchers,as recognition probes,were autonomously assembled into Ytype DNA scaffolds by the complementary base pairing rule,and three reporter probes were bound to the Y-type DNA scaffolds to construct a self-quenched multicolor DNA.When the nanoprobe entered into exosomes and bound to the target mi RNA,DNA strand replacement reaction was triggered and the reporter sequences was then released,thus producing fluorescence signals for in-situ detection of multiple exosomal mi RNAs.Taking mi R-21,mi R-27 a and mi R-375,highly expressed in MCF-7 exosomes,as model targets,the experimental results showed that the fluorescence intensities generated from MCF-7 exosomes was positively correlated with their concentrations,when compared with MCF-10 A exosomes.By measuring the expression levels of three mi RNAs(mi R-21,mi R-27 a,and mi R-375)in MCF-7 exosomes,the detection limits of exosomes were 0.116 μg/m L,0.125 μg/m L,and 0.287 μg/m L,respectively.Meanwhile,its high selectivity to target exosomal mi RNAs was performed in analyzing mixed exosomes samples.In addition,by analyzing the expression levels of three mi RNAs in clinical plasma exosomes,the nanoprobe could be used to effectively differentiate breast cancer patients from healthy donors,showing high accuracy and high specificity,which was expected to provide more accurate information for early tumor diagnosis.2.Aptamer recognition-based 3D multipedal DNA walker and catalyzed hairpin assembly for ultrasensitive Detection of CCRF-CEM exosomesAiming at the problem that the exosomal surface protein markers may not be detected due to their low abundance in the early stage of the tumor,three-dimensional(3D)multipedal DNA walker with strong signal amplification ability has been successfully developed by combing aptamer with highly specific recognition performance and micron-sized agarose bead(MB)-based 3D track.Once recognizing the target protein on the surface of exosomes,the aptamer probe could self-assemble into a multipedal DNA walker with exosome as a core.Propelled by catalyzed hairpin assembly(CHA),the prepared DNA walker could move progressively along 3D track,giving rise to the continuous generation of fluorescent output molecules for amplified detection of tumor target exosomes.As proof of concept,PTK7 aptamer against CCRFCEM exosomes was used as model target.Results from flow cytometry analysis displayed that this method held a good sensitivity,leading to a 23–time increase in signal to back ratio(SBR).When compared to that of the non-catalytic reaction,the limit of detection(LOD)(1 particle/μL)of the DNA walker was increased by about 66-fold,generating nearly two orders of magnitude magnification.Furthermore,this method could be able to quantitatively detect target in mixed exosomes samples and the LOD of this method in 5% exosomes-depleted(UC)FBS was nearly 3 particles/μL.Besides,Good recoveries(79.31 to 98.71%)were received by spiking different amounts of CCRF-CEM EXs in 60% UC serum sample.The versatility of the method was certified by using another aptamer targeting MUC1 mucin on the surface of breast cancer(MCF-7)exosomes.By testing the expression levels of PTK7 protein in clinical serum samples,this probe could be able to differentiate breast cancer patients from healthy donors,further confirming the potential of PTK7 protein markers in clinical diagnosis,which was expected to provide new ideas for the development of new methods for early clinical diagnosis of tumors.3.Aptamer recognition and catalyzed hairpin assembly-based 3D DNA walker with a self-serviced interfacial track for ultrasensitive detection of SMMC-7721exosomesIn order to overcome the complicated operation,low walking efficiency and insufficient sensitivity of traditional DNA walkers,an aptamer recognition and catalytic hairpin assembly(CHA)-based 3D DNA walker with self-serviced interfacial tracks was designed by using exosomes as 3D track and the split aptamer with high specific recognition ability,in which the surface phospholipid bilayer of exosomes were used to anchor the cholesterol-modified catalytic hairpin strands.When target exosomes were present,the split aptamer probes could recognize and be self-assembled on the surface of target protein,leading to the two split trigger sequences closed to each other to form a complete trigger sequence.Then,the DNA walker could be powered by CHA reaction,which was triggered by the complete trigger sequence,and walked freely and continuously,thereby achieving the fluorescence resonance energy transfer(FRET)signal for washing-free and amplified detection of tumor exosomes.As proof of concept,N-acetylgalactosamine(Gal NAc),highly expressed on the SMMC-7721 exosomes,was used for studies.Compared to the control probe without cholesterol modification,the walking efficiency of the DNA walker was significantly improved with a fast response(within 60 min),which may be benefited from the increased local reaction concentration and collision probability of the probes due to the exosomal membrane fluidity.Using exosomes to anchor the hairpin,the ability of the probe to resist the nuclease digestion could be significantly improved.Meanwhile,using FRET strategy,the probe effectively overcame false positive signals caused by DNA enzymes and chemicals in the serum samples.Benefiting from a low background by split trigger and CHA signal amplification,the signal-to-noise ratio of the probe was increased to 5 times than that of the control,and the detection limit was down to1 particle/μL.Also,its good selectivity and high sensitivity to target exosomes was demonstrated in analyzing mixed exosomes samples.In addition,a detection limit of127 particle/μL in 5% UC serum and a good recovery(93%-100%)in 10% UC serum samples were obtained.Moreover,this method could also be used for direct detection of micro-supernatants(μL)from cells and clinical plasma.On this basis,the DNA walker could be further applied in the clinical diagnosis with an accuracy of 100%,confirming that the N-glycoprotein markers have great potential in clinical diagnosis,thus it was expected to provide technical methods for early tumor diagnosis and important information.