Cell Membrane Interface Regulation Based On DNA Nanotechnology

This dissertation uses DNA nanotechnology for the cell membrane interface regulation.Cell membrane plays an important role in cell proliferation,metabolism,endocytosis,exocytosis,signal transduction,migration and apoptosis.How to characterize and control the physiological process of cell membrane is an important theme in cell biology,but traditional materials are intertwined on the cell membrane,making it difficult to achieve precise regulation of the cell membrane.This dissertation is based on the cell membrane interface regulation,using the precise base-pairing principle and programmability of DNA molecules to synthesize various DNA nanostructures,acquiring quantitative regulation of cell membrane interface,and providing a new perspective and method for the study of cell membrane.The main contents of this thesis include modification of cell membrane with cholesterol-DNA,DNA tetrahedral framework nucleic acids(FNA)monitoring vesicle membrane fusion dynamics,DNA nanomachines controlling cell membrane cholesterol oligomerization and deoligomerization,DNA Origami-mediated cell conjugation and cell cluster communication,and monitoring artificially constructed soma-soma synaptic neurotransmitter exocytosis.The main results are as follows:(1)After DNA is modified on the cell membrane,the principle of base pairing and programmability of DNA are used to characterize and control the cell membrane,then we can study the function of the cell.The prerequisite for these studies is modification of cell membrane with DNA.We use cholesterol-DNA to directly modify the cells.This method can quickly modify not only suspend cells,but also adherent cells.(2)The vesicle fusion is an important process in the information transmission of neuronal cell.We constructed a DNA tetrahedron-based FNA which rapidly responds to pH and anchors to the PC12 cell membrane by hybridization with a cholesterol-DNA strand.After entering into the cell vesicle by endocytosis,FNA is stably anchored to the vesicle inner membrane;and the vesicle contains multiple FNAs.When vesicle exocytosis,these properties of FNA ensure them to be used to monitor vesicle fusion dynamics in real time,with clearly distinguishing vesicle complete fusion and partial fusion model,and indicating vesicle fusion speed during complete fusion.(3)Oligomerization and deoligomerization of cell membrane molecules are common mechanisms for their physiological functions.We presented a novel DNA nanomachine which can realize cholesterol oligomerization(including dimerization and trimerization)on living cell membranes through toehold mediated DNA strand displacement reactions,and can realize its reverse process--deoligomerization.In addition,we found that cholesterol oligomerization is mainly distributed in the cell membrane raft region,which means the method has the potential for biological application.This DNA nanotechnology provides a new method for the study and regulation of cell membrane molecules.(4)Cell clusters are critical for intercellular communication,tissue and organ formation,immune response,and cancer metastasis.However,precise control of cell cluster formation and cluster interactions research remains a great challenge.We developed a nano-structured cell cluster construction technology based on DNA Origami.Using the programmability of DNA Origami,we have constructed a variety of cell cluster patterns.Based on simple cell cluster models,we investigated their contact-type intercellular communication: gap junction based molecular diffusion,tunneling nanotube based organelle transport and cellular immunity.(5)There are a variety of synapses in the biology organism,but analyzing the transmitters exocytosis between individual isolated-synapses remains a great challenge.We constructed a single soma-soma synapse in vitro by DNA Origami and monitored neurotransmitter exocytosis between synapses in real time by carbon fiber nanoelectrodes.This method of coupling cell conjugation and nanoelectrode provides a new perspective for the research of inter-synapse transmitter monitoring.