Application Of Functionalized Nanoprobes In MicroRNA Analysis

The accomplishlment of the Human Genome Project and the progress in research of the functional genomics make gene diagnoses a hot pot in the areas of molecular biology and biomedicine.MicroRNAs(miRNAs)are short endogenous noncoding small RNAs that exist in genes of humans,plants,and animals.miRNAs play critical roles in multiple cell biological processes including cell proliferation,differentiation,apoptosis,metabolism and tumorigenesis.miRNAs expression detection and analysis is a basic and preliminary procedure in most miRNA studies such as understanding its biological functions,diagnosis of diseases,as well as discovering new drug targets.Combining with the nanomaterials of diverse structure and property,this project is dedicated to functionalized nanoprobes in biosensing and celluar analysis.1.A multiple and sensitive miRNA detection method has been designed that uses conventional gel electrophoresis and rational designed catalytic hairpin assembly(CHA)system without any complex nanomaterials or enzymatic amplification.The CHA gel assay can detect miRNA at fM levels and shews good capability of discriminating miRNA family members and base-mis1matched miRNAs.It has the good feasibility in the analysis of miRNAs extracted from cell lysates,and the results are in agreement with the conventional polymerase chain reaction(PCR)amplification-based results.Notably,depending on the length of the designed hairpin probes,the hybridized strucutures appear differently in the gel electrophoresis results.The CHA gel assay consist of different hairpin probes for various target miRNAs can effectively discriminated and simultaneously detected multiple miRNAs in homogenous solution and miRNAs extracted from cell lysates.The work paves a new avenue for practical use of a conventional gel electrophoresis for sensitive interesting nucleic acids sequence detection without conventional PCR and comlex nanomaterials-based amplification.2.Two programmable oligonucleotide hairpin probes functionalized gold nanorods(THP-AuNRs)were designed to develop a near-infrared(NIR)laser triggered target strand displacement amplification(SDA)approach for sensitive miRNA imaging quantitative analysis in single living cells and muticellular mmor spheroids(MCTSs).Such a NIR-trig-ered SDA strategy achieves facile and sensitive monitoring of a model oncogenic miRNA-373 in various cancer lines and MCTS simulated tumor tissue.Notably,using a linear regression equation derived from miRNA milics,a quantitative method of miRNA in single living cells was realized due to the high sensitivity.This provides a new way for sensitive real-time monitoring of intracellular miRNA,and may be promising for miRNAbased biomedical applications.3.Two types of molecular beacon(MB)with good mature and pre-miRNAs discrimination capability were rationally designed.A class of bioconpatible and water-soluble monolayer Mo2C QDs was synthesized with high-yield via a facile liquid exfoliation route for MB gene delivery.The resulting Mo2C QDs demonstrated large surface availability,good fluorescence quench capability,low cytotoxicity and gene protection property against DNase I degradation during transfection.It enabled efficient loading of the MB for intracellular mature and pre-miRNAs distinguishably simultaneous detection.Moreover,the Mo2C QDs showed a strong near-infrared(NIR)absorbance,which showed good feasibility for photothermal(PT)and photoacoustic(PA)imaging guided photothennal therapy(PTT)in vivo.The Mo2C QDs nanoprobe may hold great potential for biogenesis and biomedical application.4.A fluorescent labeling Dicer substrate and effectively deliver it into cell by exosome derived from the target parent cells was designed for intracellular Dicer expression level monitor and gene therapy.Using pre-miRNA let-7a as a model,it was demonstrated that the Dicer substrate was effectively cleaved by Dicer to produce mature miRNA let-7a with intrinsic gene silence ability.After packaging substrates into exosomes with little immunogenicity and good innate biocompatibility by electrcoporation and dclivered to target cells,the Dicer mediated substrate cleavage was effectively monitored by the · flyorcscence recovery.providing a powerful tool for Dicer analysis.Importantly,the cleaved miRNA let-7a product exhibited signficant suppression toward tumor cell growth and regulated cancer cells cycle.This work might open a new avenuet for Dicer analysis and Dicer-related clinical application.