Studies Of Enzyme Kinetics And Cellular Application Based On Liposome And DNA

Phospholipid bilayers are one of the most common and important self-assembling structure in nature.They not only provide protective containers for cells and subcellular compartments,but also carry many machines for cell communication and transmembrane transport.Artificially prepared liposomes and solid-supported lipid bilayer membranes(SLB)serve as the simplest cell model or cell membrane system,providing an excellent platform for studying cellular biochemical reactions,transmembrane transport,and membrane surface chemistry.DNA is another material with excellent self-assembly ability in nature.In recent years,DNA nanostructures and functional modification technologies have developed rapidly and are widely used in biosensing,drug delivery,and logic computing.The rational combination of lipid bilayers with DNA nanotechnology and the use of current single-molecule technology make it possible to study enzymatic reactions,drug delivery,etc.in the intracellular environment or on the cell membrane surface.In the present thesis,we explored the single molecule kinetics and intracellular delivery of enzymes by using lipid bilayers and DNA nanotechnology.The main contents are as follows:First,for the lack of single-molecule enzyme kinetics in the cellular environment,we built a platform for the visualization of single-molecule enzymes in a simulated cellular environment.By encapsulating individual horseradish peroxidase(HRP)in a crowding agent containing liposome,and attaching it to SLB,the change of fluorescence intensity in single-molecule enzymatic reactions can be monitored by total internal reflection microscopy(TIRFM)in real-time.The analysis results showed that the correlation between the activity state and the inhibition state of HRP was weakened in the crowding and confined environment,and the degree of product inhibition was reduced.Furthermore,we used small angle X-ray scattering(SAXS)experiments and model reconstruction to establish the relationship between enzyme dynamics and conformation changes in crowding environment,which has important guiding significance for understanding biological processes and studying intracellular signal transduction.Second,it is difficult to observe the dynamic process of rapidly diffusing proteins in solution environment at single molecule level.To solve this problem,we constructed a platform for visualizing the single-molecule enzyme cascade reaction on SLB.Upstream(catalase)and downstream(glucose oxidase)were anchored on SLB in different ways,and the movement behaviors of upstream and downstream enzymes in the 2D space were monitored via TIRFM in real time.By analyzing the trajectories of individual enzymes,the diffusion coefficient of catalase showed substrate dependence,but the direction of motion was random.Experiment and theoretical derivation indicated that the 'trending movement' of enzyme was the result of a balance between translational and rotational competition.This work provides new ideas for studying the dynamic interaction of proteins,screening of inhibitors,etc.Third,direct delivery of proteins to living cells remains a challenge in basic research and therapeutic applications.Thus,we developed a new strategy to achieve membrane fusion of liposomes and cells and intracellular delivery of proteins via DNA anti-parallel hybridization.HRP molecules were encapsulated into liposomes and were delivered to the cytoplasm through this strategy.The visualization of fluorescence products generated from catalytic reaction verified the occurrence of membrane fusion,and the fusion process was independent of endocytosis.In addition,the chaindisplacement reaction and the hybridization chain reaction studied the spatiotemporal controllability of membrane fusion,and the DNA-mediated specific fusion was achieved in the multi-system.Finally,cytochrome C was delivered to the cytosol to induce apoptosis.This membrane fusion strategy has good application potential in protein drug delivery and gene editing.