Construction Of Cell Membrane Localized Nucleic Acid Probe

Cell membrane,containing a phospholipid bilayer with a thickness of 5-10 nm,maintains a stable intracellular environment by controlling the selective entry and exit of substances in the cell.It plays an essential role in many life activities such as cell metabolism,reproduction,differentiation,apoptosis,intercellular communication and so on.The distribution of some specific substances in the inner and outer microenvironment of the cell membrane reflects the physiological state of the cell to a certain extent.In addition,various important information is provided by them for the studies of cellular and molecular biological processes.For example,the concentration distribution of magnesium ions in the inner and outer sides of the cell membrane is strictly regulated by ion channels.Their dysregulation will disrupt intracellular homeostasis,which may lead to the various diseases such as cancer,autoimmune diseases,neurodegenerative diseases and so on.Therefore,real-time monitoring of the dynamic distribution of the specific substances in the inner and outer microenvironment of the cell membrane,makes it important for the study of the mechanism of cell physiological activity and the occurrence and development of diseases.Conventional detection techniques,such as mass spectrometry,molecular imprinting,etc.,are not suitable for dynamic monitoring of cell microenvironment due to their inherent analysis characteristics such as discontinuity,static state,and cumbersome operation.Biosensing probes localized on the outside of the cell membrane have been successfully constructed,which show unique advantages in realtime monitoring of the physiological activities of secreted targets.Functional nucleic acids refer to oligonucleotides with catalytic activity or molecular recognition ability,including deoxyribozyme(DNAzyme)and aptamer.Characterized by easy access,easy modification,high programmability,high chemical stability,and low immunogenicity,the emergence of functional nucleic acids has received extensive attentions in the field of biosensing probe research.In recent years,functional nucleic acid probes located on the outside of the cell membrane have been well developed,however,how to precisely modify the functional nucleic acid probes on the inside of the cell membrane is still a challenging scientific issue.In this thesis,inspired by the interaction between exosomes and cells,a membrane fusion strategy based on nanolipid vesicles was developed.Utilizing amphiphilic functional nucleic acid as the basic molecular tool,we constructed a functional nucleic acid probe located on the inner side of cell membrane.The core idea is that the functional nucleic acids can be anchored on the inner surface of cell membrane efficiently,through the directional control of functional nucleic acids on the inner side of nanosized lipid vesicles.In addition,the efficient anchoring of amphiphilic functional nucleic acid probes on the outside of the cell membrane was achieved by combining with hydrophilic and hydrophobic interactions.The successful construction of functional nucleic acid probes located on the inside and outside the cell membrane will provide a new tool for the simultaneous dynamic monitoring of the environment inside and outside the cell membrane.The main contents are as follows:In Chapter 2: We made a nanolipid vesicle with membrane fusion ability.By modifying amphiphilic functional nucleic acid molecules on the inner side of the vesicle,we realized the effective labeling of functional nucleic acid on the inner side of cell membrane.Then,we investigated the directivity of functional nucleic acids in nanolipid vesicles by confocal imaging.Meanwhile,we labeled lipid membrane dye on nano lipid vesicles and encapsulating propidium iodide(PI)dye,indicating that the localization of functional nucleic acids to the inner side of the membrane was realized through the membrane fusion process between nanolipid vesicles and cell membrane.Because of the advantages in easy operation,fast fusion rate and high efficiency,this strategy will showcase great promise for the internal surface modification of cell membrane.In Chapter 3: We characterized the functional nucleic acid probes which localized on the inside and outside of the cell membrane,respectively.For the probes on the inner side,we investigated the stability,biocompatibility and universality.For the probes on the outer ones,the insertion efficiency and stability of the probe by confocal imaging were investigated.The results showed that both the probes on inside and outside of the cell membrane have highly distinct membrane orientation,good stability,biocompatibility and universality,thus providing a new tool for dynamic monitoring of the inner and outer microenvironment of the cell membrane.In Chapter 4: Using the DNAzyme,which could specifically respond to magnesium ions,as the model probe,we verified the feasibility of constructing the probe on the inner and outer sides of the cell membrane.Firstly,we designed and synthesized the DNAzyme fluorescent probe,and checked the sensitivity and specificity of the probe for magnesium ion detection in a buffer system.Next,combining the membrane fusion strategy and hydrophobic interaction,we successfully developed DNAzyme probes labeling with different fluorophores on the inner and outer sides of the plasma membrane,respectively,which can offer technical support for the dynamic monitoring of magnesium ions in the inner and outer microenvironment of cell membrane.