Transient Hybridization Directed Nanoflare For Single Molecule MiRNA Imaging

MiRNA is a non-coding RNA that acts as a gene regulator in higher eukaryotes.The miRNA gene accounts for approximately 2%of the human genome,exceeding the combination of all protein-coding genes.miRNAs are involved in virtually all cellular pathways,including cancer,associated with human health and disease.Expression levels of multiple miRNAs in tumor cells Blood abnormalities in cancer patients suggest that miRNAs are potential biomarkers for cancer diagnosis.Accurate quantifications of cellular miRNAs are important not only for accelerating them becoming reliable diagnostics biomarkers,and also for deeply understanding their influence on central signaling pathways.Although single-molecule miRNA imaging permits quantifying biomolecules at single-molecule level,it is limited by the sensitivity and specificity of hybridization-based probes.Based on the above background:We develop a miRNA single-molecule imaging method by using conjugated polymer nanoparticle(CPN)labeled short DNA probe termed as nanoflare.The transient hybridization of the nanoflares and target miRNAs yields featured single-molecule kinetics signal rendering high single-molecule sensitivity and specificity.MiRNA can be detected with a remarkable detection limit of 1 fM without any amplification steps.Taking advantage of the featured single-molcule signal of the nanoflare,we are able to directly count single miR-21 molecules in single cells by using highly inclined and laminated optical sheet(HILO)microscopy.The statistics of the counting reveals miR-21's cell-to-cell fluctuation and differential expression of tumor cells and normal cells jobs:(1)Design of a synthetic conjugated polymer PFBT[(9,9-dioctylfluorene)-alternate-co-(1,4-benzo-(2,1 ',3)-thiadiazole)].(2)The conjugated polymer PFBT and the PEG having a carboxyl group form nanoparticles by a nano-coprecipitation method.The nanoparticles are coupled to an oligonucleotide chain to form a fluorescent probe.The characterization of the detection,optical characteristic detection,stability experiments,etc.proved that the nanoparticles have good stability and high brightness,indicating that they can be used as fluorescent signal probes for experiments.(3)At the single molecule level,explore the number of different complementary bases and different concentrations of buffer solution for single molecule experiments,select the best signal probe for miRNA cell imaging method research.(4)At the cell imaging level,the optimal signal probe is selected to affect the distribution of miRNAs in the cells by affecting acquisition,image reconstruction,kinetic filtration,single-cell miRNA counting,and statistical analysis.