MnO₂ Nanosheets Mediated "DD-A" Fret Nucleic Acid Probes For Intracellular RNA Detection

RNA exists in a wide variety of organisms not only having the storage and transfer of genetic information function but also as a ribozyme play a direct role in the metabolism of cells.It can be through the splicing,editing and recoding to achieve the development and differentiation of biological regulation.The expression level of certain RNAs is closely related to tumor formation,development and metastasis.Therefore,the detection of these RNAs can provide important information for the progress of tumor research and has far-reaching significance for early diagnosis and effective treatment of tumor.Fluorescence Resonance Energy Transfer(FRET)is an earlier developed technology,and many FRET-based nucleic acid probes have been used for RNA detection.whereas double donor-single acceptor(DD-A)probes have been shown to be capable of improve FRET efficiency.Hybrid chain reaction(HCR)is a signal amplification technology in constant temperature without enzyme-assisted which can effectively improve the detection sensitivity.In this thesis,two kinds of nucleic acid nanoprobes were designed based on double donor-single acceptor FRET probe and HCR signal amplification technique.The main contents are as follows:(1)A novel double donor-single acceptor FRET nucleic acid nanoprobe with MnO2 nanosheets carrier was constructed to detect intracellular mRNA.The double donor-single acceptor binary probes were adsorbed on the surface of the MnO2 nanosheets and can be efficiently delivered into the cells.The MnO2 nanosheets were reduced by intracellular glutathione(GSH)and the probes were released.when encountered the target mRNA,the released binary probes were hybridized with it which made the fluorescent dyes labeled on the probe closed to each other to produce a "DD-A" FRET signal.The design of probe sequences is simple and the use of FRET signal mode can effectively avoid the false positive signal on the impact of the experiment.Meanwhile the probe has a higher FRET efficiency than the traditional single donor-single acceptor(D-A)binary probe which the detection limit is about one order of magnitude higher than "D-A" model.Besides,it has a higher cell imaging contrast and can be used as a simple and effective tool for basic research and clinical diagnosis.(2)A "DD-A" HCR nucleic acid nanoprobe based on MnO2 nanosheets,double donor-single acceptor FRET model and hybridization chain reaction(HCR)was designed for intracellular miRNA detection.Hairpin probes were adsorbed onto the MnO2 nanosheets and efficiently delivered into the cells.The MnO2 nanosheets were reduced by intracellular glutathione(GSH),causing hairpin probes to be released.When the target miRNA exists,it can open the hairpin probe to trigger the HCR reaction to form a long nicked double-stranded DNA.The two donor fluorophore and acceptor fluorophore labeled on the probe will be closed to each other to generate energy transfer,resulting in "DD-A" FRET signal.A plurality of FRET signal units distributed over the long duplex cause the detection signal to be enhanced,thereby achieving amplification detection of the target.While the "DD-A" probe can improve the efficiency of FRET,the introduction of signal amplification strategy is more conducive to realize a sensitive detection of low concentration target.