Study Of Novel Methods For In Situ Detection Of Intracellular MicroRNAs

MicroRNAs(miRNAs)are a group of non-coding,endogenous and small-sized RNAs.They play significant regulatory roles in a wide variety of biological processes including cell development,differentiation,metabolism,and apoptosis.Specifically,the aberrant expression and misregulation of miRNAs can lead to various diseases.Therefore,miRNAs have been recognized as a clinically important class of diagnostic and prognostic biomarkers and reliable indicators for cellular status,and their detection has formed a rapidly emerging field for further understanding the biochemical function of miRNAs and exploring useful diagnostic and prognostic markers of diseases.This thesis focuses on exploring new methods to analyze and detect miRNA.1.In situ quantitation of intracellular microRNA in the whole cell cycle with a functionalized carbon nanosphere probeA cell-specific method for in situ quantitative detection of intracellular microRNA(miRNA)in whole cell cycle was proposed using an energetic carbon nanosphere(f-CNS)probe.The f-CNS probe was prepared by assembling Cy5 labeled single-strand DNA(Cy5-ssDNA)and folic acid on CNS surface.The interaction between ssDNA and CNS led to the fluorescence quenching of Cy5.Upon the recognition of the ssDNA to complementary miRNA,the hybridization product detached from CNS surface,which led to the fluorescence recovery of Cy5 labeled to ssDNA and provided a specific method for detection of miRNA.The presence of folic acid endowed the probe with good specificity to folate acceptor overexpressed cells Thus this probe could be used for cell-specific intracellular miRNA sensing with a confocal microscope.Coupled with cell synchronization to gain the whole cycle cells,the proposed method allowed the intracellular miRNA detection in whole cell cycle.Using miRNA-18a as a target model,its average quantity in single S-,G2/M-and G1-phase HepG2 cell could be obtained to be 0.779,1.03 and 1.23 pg,respectively.This method provided the first protocol for in situ quantitation of intracellular miRNA in whole cell cycle and possessed promising application in the study of miRNA related cell cycle bioprocesses and biomedicine2.Simultaneous sensing of intracellular microRNAs with a multi-functionalized carbon nitride nanosheet probeThis work successfully assembled two dye-ssDNAs and FA on CNNS to design a mf-CNNS probe for target-cell-specific simultaneous monitoring of multiple intracellular miRNAs and intracellular imaging.The CNNS showed a strong FL quenching ability on the dye labels,which could be recovered upon the specific recognition of the dye-ssDNAs to miRNAs due to the release of the formed DNA-miRNA duplex helix from CNNS surface.The presence of folic acid endowed the probe with good specificity to folate acceptor overexpressed cells,which leads to the specific receptor-mediated endocytosis.The subcellular localization analysis indicates the efficient escape of mf-CNNS probe from endocytic vesicles in cytoplasm,where the ssDNAs assembled on the probe surface can be recognized by the miRNAs target to recover the dye fluorescence.The good protection properties of CNNS for avoiding nuclease digestion and SSB interaction of ssDNAs and low cytotoxicity led to a specific method for in situ monitoring of multiple intracellular miRNAs.The HepG2 cells were incubated with different miRNA inhibitors to induce different levels of miRNAs,dynamic changes in miRNA expression levels was monitored by the proposed strategy in living cells.The designed sensing strategy realized the simultaneous detection of multiple miRNAs in living cells.In virtue of these advantages,the proposed strategy implied the potential for biomedical research and clinical diagnostics.3.Peptide nucleic acid-functionalized carbon nitride nanosheet as a probe for in situ monitoring of intracellular microRNAA novel probe for recognition of both folate acceptor overexpressed cancer cells and intracellular microRNA(miRNA)is designed by functionalizing carbon nitride nanosheet(f-CNNS)with Cy5-labeled peptide nucleic acid(Cy5-PNA)and folate.The interaction between Cy5-PNA and CNNS quenches the fluorescence of Cy5,and the presence of folate endows the probe with good specificity to folate acceptor overexpressed cells.The probe can be specifically uptaken by cancer cells with an incubation step.Upon the recognition of the PNA to complementary miRNA,the hybridization product is released from CNNS surface,which leads to the fluorescence recovery and provided a specific method for sensing of miRNA.Thus this probe can be used for cell-specific intracellular miRNA sensing with a confocal microscope.Compared to DNA,the PNA endows the f-CNNS probe with higher specificity,higher protection properties and lower background signal.The Using miRNA-18a as a target model,the dynamic changes of its expression level inside living cells can be monitored with the proposed method.This method possesses promising application in the study of miRNA related bioprocesses and biomedicine.