Application Of Functionalized Probes/Vectors In Gene Detection And Therapy

With the urgent demand for precision medicine and in-depth research on functional genomics,gene detection and therapy have been placed many expectations.Scientists look forward to bring lasting improvements and treatments to complex diseases in many fields of human beings from a genetic perspective.Discovery of miRNA and siRNA in non-coding small RNAs adds potential targets and therapeutic tools for gene detection and therapy.However,their clinical application is hindered by their inner instability and disturbance of the physiological environment.Nowadays,developing feasible probes and vectors to facilitate the application of non-coding small RNAs in gene therapy and detection is a very promising solution.Thus,this thesis focuses on the development and application of functional probes and vectors centering on miRNA and siRNA.The work mainly includes the preparation of three tension-promoted highly specific recognition probes and the study of miRNA detection,as well as the preparation of stimulus-responsive covalently cross-linked nanocarriers and the study of siRNA transportation.(1)Sealed circular probe for miRNA detection and imaging in live cellsA sealed circular probe with large tension has been prepared with efficient copper-free click ligation.The comparative analysis of the circular probe and the linear probe shows that the large tension force triggered by the ring structure promotes the high specific recognition ability.Impressively,single base mismatch could be discriminated without any enzyme assistance and room temperature.More importantly,the circular probe was transferred into living cells of different cell lines,and the signal intensity was consistent with the miRNA expression level in the cells,indicating that the circular probe can be applied to the detection and imaging of endogenous miRNA in living cells.The work provides a new strategy for miRNA detection and intracellular imaging by the tension promoted specific recognition.(2)Highly sensitive and quantitative miRNA detection based on closed circular probe and dual signal amplificationA highly selective and sensitive miRNA detection platform was developed by integration of closed ring probe and dual signal amplification system including autocatalytic DNAzyme and light harvesting cationic conjugated polymer(PFP).Precisely because of the high signal amplification efficiency triggered by target miRNA activated autocatalytic cyclic cleavage of DNAzyme together with the unique fluorescence resonance energy transfer(FRET)property between conjugated polymer and small molecule dye,the sensitivity of this probe has been effectively improved,and enabling highly sensitive detection of let-7a down to 1.5 f M.Besides,on the basis of circular tension and toehold-initiated strand displacement,this assay is also highly selective and can easily discriminate single base mismatch difference among miRNA family members.More importantly,this probe could quantitatively define the content of let-7a in three cell lines and the results are consistent well with that of q RT-PCR.Thus dual signal amplification probe could be very useful for sensitive and selective miRNA profiling and be potential candidate for early diagnosis of miRNA related diseases.(3)Logic gate and dual signal amplification-based probe for sensitive multiplex detection of miRNAsSensitive multiplex miRNA detection probe was prepared,which continues the high specificity and sensitivity of the dual amplified circular probe.With the introduction of Y shape DNA and streptavidin magnetic beads,multiple signals can be read out simultaneously by single excitation through efficient multiple fluorescence resonance energy transfer(FRET)between conjugated polymer and different dye-labeled substrate chains.Furthermore,different types of logic gate can also be operated by observing the emission intensities of the labeling dyes with corresponding miRNAs as inputs,thus proposed a new way for the specific detection of certain miRNAs according to the logic signals.More importantly,we successfully applied the strategy for multiple miRNAs detection in cell lysates and the results agree well with that of q RT-PCR.Anyway,we believe that this platform holds great potential for miRNA detection in biological samples.(4)pH-responsive siRNA covalently cross-linked nanoparticles for the treatment of acute liver injury in miceNew siRNA delivery strategy was proposed(PNSDS),which was positive charge free nanocarriers,composed of multi-arm polyethylene glycol(PEG)backbone,hydrophobic chain with acid-sensitive groups and azide groups and cyclooctyne-modified siRNA and mannose by covalent cross-linking.The unique siRNA cross-linked construction of PNSDS allows it to have minimal cytotoxicity,high siRNA loading efficiency,and a stimulus-responsive property that enables the selective intracellular release of siRNA in response to pH conditions.The results demonstrated that PNSDS can deliver tumor necrosis factor alpha(TNF-?)siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media.Moreover,PNSDS with mannose targeting ligands can selectively accumulate in mice liver,induce specific inhibition of TNF-? expression in vivo,and consequently protect mice from inflammation induced liver damages.Therefore,this work provides a new way for modularization and functionalization of siRNA vector systems to effectively improve the therapeutic potential of RNAi-based therapies.