Functional DNA Based Single-molecular Level Protein Dynamic And Live Cell Protein Delivery Study

The function of oligodeoxyribonucleic acid(DNA)is to carry the genetic information.However,due to the properties of DNA,including the specificity and editablity of DNA hybridization,adjustability of length and sequence and conductivity,it has been widely used in cross-disciplinary research fields,such as sensing,nanomaterials,logic calculations and drug delivery,becoming a kind of shining star material.The diversity of DNA functional modifications is another important reason why DNA can be widely used.Functional modifications can greatly enrich the function of DNA and make DNA more likely to bind to other materials.At the same time,benifiting from the properties of DNA,various materials integrated with DNA are more powerful and more versatile.The main content described in this dissertation is using functionalized DNA and DNA nanostructures to develop methods capable of observing the dynamic interaction of proteins at single-molecule level and methods for specific live cell protein delivery that does not depend on destructing endocytotic vesicles.The main contents are as follows:First,we combined functionalized DNA and DNA nanostructures with fluidic phospholipid bilayer for developing a method which allows real-time imaging of the enzymatic cascade(glucose oxidase-catalase)dynamic at single-molecule level.Since cholesterol can spontaneously insert into phospholipid bilayer,we utilized dynamic phospholipid bilayer composed of unsaturated phospholipids and cholesterol-modified DNA to construct a mobile DNA monolayer.DNA and fluorescent molecules modified catalase can be tethered onto the fluidic phospholipid bilayer via DNA hybridization and freely move.In order to use total internal reflection fluorescence microscopy to observe the kinematic behavior and distribution behavior of catalase around glucose oxidase in the cascade reaction,we introduce triangle DNA origami raft to anchor glucose oxidase on the phospholipid bilayer and localize glucose oxidase with fluorescently labeled DNA on the origami raft.Through single-molecule fluorescence tracking and imaging,we found that the diffusion coefficient of catalase around glucose oxidase increases when the cascade reaction occurs,but its distribution is random,and the reason may be its own rapid rotation leads to that the power from catalytic reaction does not have a fixed direction.Second,based on the first work,we established a method for observing the dynamic interaction of cell signaling proteins at single-molecular level.We selected two kinases AKT2(upstream)and GSK3?(downstream)in the PI3K/AKT pathway that are directly related to cell life activities in this study and expressed biotin-labeled AKT2 and GSK3? in mammalian cells.We use the specific binding of biotinstreptavidin and biotin functionalized DNA for DNA modification of both kinases.And we use fluorescent antibodies for labeling the two kinases.Total internal reflection fluorescence microscopy imaging demonstrated the dynamic tethering of AKT2 on the phospholipid bilayer and the assembly of GSK3? on the DNA origami.Using this method,we have successfully imaged the interaction of AKT2 and GSK3? at the singlemolecule level.This method can be easily applied to the study of other protein interactions.Compared with the traditional qualitative methods for protein interactions,this method allows real-time observing transient protein interactions and acquisiting kinetic information.Third,we combined cholesterol-functionalized DNA with liposomes to construct a specific live cell protein delivery method that mimics natural membrane fusion process.We encapsulated horseradish peroxidase(HRP)as exogenous protein cargos in about 100 nm of electrically neutral liposome,and then modified the surface of the liposome with 3'-end cholesterol modified DNA.The surface of the target cell membrane was modified with 5'-end cholesterol modified complementary DNA.The two DNAs could bring the liposomes and cell membrane into close contact by a "zipper" like hybridization,which promotes membrane fusion and in turn releases cargo proteins into the cytosol.The HRP-catalyzed fluorescence reaction demonstrates the feasibility of DNA hybridization mediated membrane fusion.We also explored the temporal and spatial controllability of membrane fusion using strand displacement and hybridization strands.This method does not rely on destructing endocytic vesicles and has high biocompatibility.The above content shows new ideas arising from the combination of functionalized DNA or DNA nanostructures with other materials.In the end,we summarize the above work and predict the development of functionalized DNA or DNA nanostructures.