| Cell membrane is an important organelle composed of lipid,protein and carbohydrate.It not only structurally maintains the integrity of the cell and constitutes the boundary between the cell and the cell-surface microenvironment,but is also functionally related to vital activities such as cell proliferation,migration,and apoptosis.Cell membrane is the crucial mediator for intercellular substance exchange and signal communication between cell and the environment,cell-cell communication.Cellular microenvironment,the local environment surrounding the cell,is the place where these interactions happen.p H of the cellular microenvironment is of great significance in many physiological and pathological processes,and the decrease of p H will inhibit various cell membrane-related physiological reactions.Neurotransmitters,a class of extracellular secreted chemical small molecules,are essential chemical mediators for neuronal communication in various neuromodulations.Abnormal homeostasis of neurotransmitters is implicated in the pathogenesis of a wide range of neurological or psychiatric disorders,such as Parkinson's disease,schizophrenia,and depression.The analyses and visual measurements of important factors in cellular microenvironment are highly desirable for understanding the cell membrane-associated physiological and pathological processes.Recently,related researches have received broad interest.However,in-situ detection of factors in cellular microenvironment remains challenging because of the complexity and variability of molecules with high dynamics.DNA molecule is the genetic material of life with rigorous base-pairing and programmable property.DNA nanotechnology has been developed rapidly in the past thirty years,and various DNA nanostructures with different dimensions,sizes,and shapes have been fabricated based on bottom-up modular self-assembly and DNA origami.DNA nanostructures are easy to functional modificate with prominent biocompatibility,and present stronger nuclease resistance over ss DNA and ds DNA.Thus,they are excellent materials for fabricating new-type biosensing platforms.Based on the background mentioned above,this thesis demonstrates the construction of two distinct DNA nanostructure that can be stably anchored on the cell membrane,achieving live-cell imaging of the dynamics of p H or signaling molecules in the cellular microenvironment.The research projects have been carried out as follows:(1)Construction of a p H-responsive dynamic reversible DNA tweezer.With a functional nucleotide acid(i-motif)as the motor,we constructed a p H-responsive dynamic reversible DNA tweezer.The DNA tweezer contains three key elements:(a)a framework of DNA tweezer composed of three short DNA strands,(b)a p H-sensitive i-motif sequence to dynamically control the switch between the“open”and“closed”states of the DNA tweezer in response to different p H,(c)and a pair of FRET fluorophores(rhodamine green and rhodamine red)to produce the ratiometric fluorescent signal.The structure of the DNA tweezer was ascertained with native polyacrylamide-gel electrophoresis.Fluorescence spectra verified that the FRET signal of the DNA tweezer can excellently reflect different p H in buffer ranging from 5.0?7.5,covering the p H of internal and external of normal and pathological cells.The DNA tweezer can quickly and reversibly switch between the state“open”and“closed”in less than 1 min when the p H in buffer is cyclically switched between 5.0 and 7.5,and the signal maintained 95%even after six cycles,showing its potential in p H dynamic detection.(2)Cell surface and apoplastic p H imaging based on the DNA Tweezer.Taking advantage of the easy functional modification of DNA molecules,we labeled the DNA tweezer with cholesterol grous.Laser confocal fluorescence imaging and flow cytometry confirmed that the DNA tweezer can not only anchor on cell membrane with high efficiency,but also anchor on different kinds of cancer cells(Hela,Hep G2)and normal cells(LO2),and it occurs on almost all cells.In addition,MTT assays proved that DNA tweezer anchored on cell membrane with good biocompatibility,laying the foundation for further application in biological cells.It was more meaningful that the live-cell imaging and semi-quantitative analysis of the cell surface p H was performed by the DNA tweezer,and further the dynamic change of p H on tumor cell surface was dynamically observed and monitored in real-time.Furthermore,the alkalization process of plant roots caused by the rapid-alkalinization factor(RALF1)has been directly determined using the DNA tweezer,which provides a novel tool to explore the molecular mechanisms in plant growth.(3)Construction and cell membrane imaging of DNA triangular prism.Based on the geometric spatial symmetry of nanostructures and DNA palindrome sequence,a cell membrane-anchored DNA triangular prism through modular was constructed through self-assembly.The prepared DNA triangular prism extends cholesterol-tags on the bottom three vertices and three sticky ends(poly-A,21 nt)on the top three vertices for further assembly of functional nucleotide acid or sensory module.The DNA triangular prism was labled with fluorophore through the assembly of sticky ends with FAM-labeled stand R.Cell membrane colocalization imaging showed that the DNA triangular prism can quickly(10 min)and accurately anchor on cell membrane with significantly high colocalization rate of 94.33%.The fluorescence intensity of the experimental group increased by 150 times to control group,as exhibited in flow cytometry assay.Compared with one cholesterol modified linear DNA structure(one dimension,1D-Cho),the proposed three cholesteryl-tagged DNA triangular prism(three dimensions,3D-Cho)presented improved stability on cell membrane without intercellular tranfertion,providing a new tool for target molecule measurements in cellular microenvironment.(4)Cell-surface-anchored DNA nanoprism probe for neurotransmitter imaging.Based on the above studies,taking dopamine(DA)as a detection model,we further designed a DNA nanoprism probe through the modular self-assembly of the prism with the sensory module(specific target-binding aptamer).The probe can detect DA in solution with excellent sensitivity and selectivity,with a linear relationship in the concentration range of 0-100 n M and a detection limit of 0.45 n M,and realized the cell surface imaging of exogenous DA.It was more significant that we have successfully visualized the transient DA release on the cell surface with high spatiotemporal precision,and discovered the dynamic correlation between DA release and calcium influx,triggered by high K~+.Because of its modular design and construction,this probe can be applied for cell surface single-target or multi-target detection and analysis,just by changing the specific aptamer sequence in the sensory module.Thus,this probe is expected to be developed as a universal platform for in situ sensoring and imaging of target molecules in cell surface microenvironment. |
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