Living Cell MicroRNA Imaging Based On Enhanced Catalytic Hairpin Assembly

MicroRNAs are a dominating family of small noncodingRNAs that act as posttranscriptional regulators in crucial cellular processes,including proliferation,differentiation,migration and apoptosis.The dysregulation of microRNAs is predictive of many cancerous diseases,making microRNAs-based diagnostics more appealing for earlier cancers diagnosis and prognosis.In view of the cell-to-cell variation in microRNA expression,in situ imaging of microRNA in living cells would greatly facilitate the monitoring of detailed spatial expression of microRNA and clinic research on microRNA-related cellular processes and diseases.However,it remains a challenge to visualize intracellular microRNA due to their short nature and extremely low abundance.To advance in situ microRNA imaging in living cells,some enzyme-free isothermal amplification approaches based on nucleic acid have become increasingly attractive,such as catalytic hairpin assembly(CHA),hybridization chain reaction(HCR)and DNAzyme biocatalyst.Especially,CHA can continuously reset target microRNA for microRNA imaging in living cells,thus rarely affecting cellular life activities.In this thesis,two kinds of isothermal enzyme-free biosensing platforms based on CHA reaction were constructed for monitoring and imaging of lowly expressed microRNAs in living cells.The main contents are as follows:1.Cross-catalytic CHA-DNAzyme circuit for living cell microRNA imaging.A novel enzyme-free cross-catalytic circuit is engineered based on CHA circuit and DNAzyme biocatalyst under homogeneous isothermal conditions.The CHA reaction and DNAzyme execution participate in the reaction alternatively,thus realizing the cross-triggered cascading amplifier circuit.As a versatile and modular sensing platform,the present cross-catalytic system guarantees the highly sensitive and selective identification of the DNA and microRNA analyte,originating from the inherent reaction accelerations and the multiple-guaranteed stringent recognitions.Significantly,this method was further applied for in situ intracellular imaging of mi R-21 from different living cells,and achieving a robust monitoring of the varied microRNA expressions as well as different distributions.2.Hairpin-dimmer-based localized CHA reaction for microRNA detection.In this work,three kinds of dimer hairpins tethered by palindromic sequences are designed for constructing localized CHA reaction.Target microRNA initiates the self-assembly process among the hairpins and results in the formation of reticulate DNA structures that restrict the hairpins in a confined space.Benefiting from the spatial-confinement effect,the relative concentrations of reacting hairpins increase thereby speeding up the reaction kinetics.Compared to the conventional CHA reaction,the present localized CHA reaction enhances the reaction speed and efficiency.Our design enables the highly sensitive and selective identification of mi R-21,and may provide a new platform for visualization of microRNA in living cells.