DNA Molecular Engineering-based Living Cell Membrane Proteins Analysis

Membrane proteins are the main carriers of cell membrane activities,playing an important role in many important biological activities,such as cell signal transduction,material exchange,and cell recognition.The abnormal behavior of membrane protein usually accompanies the development of diseases.The analysis of living cell membrane proteins can help to elucidate the basic laws of cell life activities,clarify the dialectical relationship between the abnormalities of membrane proteins and the development of diseases,and thus provide theoretical basis for the development of disease drugs and disease diagnosis and treatment.However,there are still a series of difficulties and challenges in the analysis of living cell membrane proteins.First,the distribution and movement behavior of membrane proteins on the surface of living cells are highly dynamic and heterogeneous.It is hard to obtain in-situ dynamic information of membrane proteins highly expressed on the surface of living cells at the singlemolecular level using traditional research strategies.Second,cell-cell interaction interface often involves the coordination of multiple ligand-receptors and other functional proteins to cooperate with cells to carry out specific life activities.However,existing strategies are difficult to achieve the research on the orderly regulation and coordination of multiple membrane proteins under natural conditions.DNA molecule has excellent programmability and good biocompatibility.With the development of DNA nanotechnology,researchers have not only constructed a large number of dynamic DNA molecular systems and static DNA assembly structures using DNA molecules,but also developed a series of DNA molecules with special functions,namely functional nucleic acids.In addition,thanks to mature DNA synthesis and modification technologies,DNA molecules are easily coupled with many functional units,further enriching the toolbox for functional design of DNA molecules.These characteristics of DNA provide a flexible and powerful programmable platform for molecular engineering design,which greatly promote the development of chemical biology,molecular medicine and other fields.In this dissertation,aiming at many important scientific problems in the field of analysis of living cell membrane proteins,a series of DNA molecular tools were constructed based on DNA molecular engineering strategies,which were used to 1)realize the in-situ monitoring and analysis of membrane proteins on the surface of living cells at the single-molecule level,2)realize the research on the relationship between the interaction of multiple membrane proteins at the cell-cell interface and cell recognition response under physiological conditions.The specific research includes the following two parts:1)In situ monitoring and analysis of membrane proteins on living cell surface at the single-molecule level.In Chapter 2,based on the principles of single-molecule localization in fluorescent imaging and random sampling in mathematics,this dissertation proposed a single molecule imaging and analysis strategy,termed ligand dilution analysis.Given sufficient dilutions,the distance between two fluorescent bindings can be large enough to be resolved by single-molecule localization.Theoretical derivation and model verification have confirmed that the ligand dilution technique has the random sampling property,which is not affected by the density of the receptor and the non-specific adsorption of molecules with high binding affinity.Subsequently,the feasibility of the ligand dilution analysis was verified by using buffer solution and glass supported phospholipid bilayer system,respectively.Finally,the ligand dilution analysis was applied to the single-molecule imaging of aptamer sgc8 c and its target molecule PTK7,preliminarily clarifying its potential application in the analysis of membrane receptors highly expressed on the living cell surface.In Chapter3,ligand dilution analysis was further applied to the imaging analysis of the membrane receptor densely expressed on living cell surface at the single molecular level.Ligand dilution-based static analysis of membrane receptors included: nonlinear regressionbased receptor density analysis,cooperative ligand dilution strategy-based local superresolution colocalization,and computer docking-based aptamer binding mode simulation.The dynamic studies of membrane receptors included: time-dependent pattern of receptor dynamic distribution on live cell membrane,and two-and threedimensional motion analysis of highly expressed receptors on the live cell membrane.2)Studying the relationship between the interaction of multiple membrane proteins at the cell-cell interface and cell recognition response under physiological conditions.In Chapter 4,a series of DNA nanojunctions with distinct sizes and excellent membrane anchoring performance for precise control over the intermembrane spacing at the cell-cell interface were constructed by using membrane-anchored DNA tetrahedrons.Through VA-TIRFM,CLSM,and TEM imaging,the ability of DNA nanojunctions to precisely regulate the intermembrane distance of the cell-cell interface was proved.In Chapter 5,the prepared DNA nanojunctions were used to regulate the intermembrane spacing at the DC-T cell interface under physiological conditions and further studied the molecular mechanism behind T cell signal transduction.Unlike conventional genetic engineering strategies,no protein modification was required in this system.Through DNA hybridization,DNJs with high surface density could facilitate the DC-T cell adhesion and stabilize the contact zone,which led to enhanced amplitude of T cell signaling.At a critical low surface density,DNJ-37 with a size beyond the dimension of the TCR-p MHC complex could extend the intermembrane distance that resulted in inhibited T cell triggering,possibly owing to the impairment of CD45 segregation.Whereas DNJ-7 with a small size could compress the DC-T cell interface and achieved an improvement of T cell activation,presumably from the combined effect of strict CD45 exclusion and TCR conformation exchange resulted from additional mechanical forces.Taken together,these results suggested that the axial dimension of the close contact zone plays an important role in T cell activation,and T cell receptor signaling might be related to the distribution of CD45 and the conformation of TCR.In summary,in order to solve the problem of obtaining in-situ dynamic information of membrane proteins highly expressed on the surface of living cells at the molecular level,this dissertation proposed a single molecule imaging and analysis strategy termed ligand dilution analysis,and studied the different effects of sampling process of traditional sparse labeling strategy and ligand dilution technique on the analysis results.Based on ligand dilution analysis,static and dynamic analysis of receptor molecules highly expressed on the surface of living cells at the single molecular level was realized.In order to solve the problem of difficult to achieve orderly regulation of multiple membrane proteins under natural conditions,this dissertation constructed DNA nanojunctions of different sizes to accurately manipulate the intermembrane spacing at the DC-T cell interface,achieving effective control of multiple membrane proteins.The relationship between T cell receptor signaling and CD45 distribution and TCR conformation had been studied at the molecular level.A series of studies carried out in this dissertation provide efficient molecular tools for insitu analysis of membrane proteins on the surface of living cells,which is expected to clarify the relationship between the abnormalities of membrane proteins and the development of diseases at the molecular level,and thus provide a strong theoretical guidance for accurate diagnosis and treatment of diseases.