| DNA is not only a biological macromolecule which was used to store and transmit genetic information in the life system,but also a highly programmable biological material.DNA material is very stable,simple to design,easy to synthesize and modify,and possesses outstanding programmability and predictability.Based on these facts,researchers have developed emerging nanotechnology based on the self-assembly of DNA material.This technology can realize precise and controllable self-assembly of DNA nanostructure at the molecular level,as well as addressable modification and dynamic control of guest molecules.Thus,it shows great potential in the design and construction of molecular devices.In recent years,using DNA nanostructures to precisely control the functions of biomolecules has become an important hot topic,among which semi-synthesis DNA-protein complexes have attracted extensive attention.Semi-synthesis DNA-protein complex is a biological macromolecule constructed by semi-artificial synthesis of DNA and protein,which either contains the programmability,predictability,and controllability of DNA or the functionality of proteins.Based on the high controllability of DNA,semi-synthesis DNA-protein complexes have been used to analyze the structure of proteins,study the interaction of proteins and build protein dynamic networks.Moreover,taking advantage of the biochemical functions of proteins,they also hold great promise in biosensing,disease treatment and molecular catalysis.Although semi-synthesis DNA-protein complexes have great application prospects,most research is basically focused on the construction of models in test tubes,while the applications in biological systems are relatively rare.To expand the application of semi-synthesis DNA-protein complexes in biological systems,this thesis integrates the methods of DNA nanotechnology into the functional diversity of semi-synthesis DNA-protein complexes,design and constucting a series of novel semi-synthesis DNA-protein complexes for the applications in biosensing,cell functionalization and cancer therapy.The details are as follows:（1）The point of care analysis is of great importance in the early diagnosis of the disease,the monitoring of the course of the disease,and the timely feedback on treatment effect.However,traditional optical probes have some application limitations in point of care analysis,such as the requirement of large detection instruments,as well as background interference from complex samples and light scattering interference.To solve these problems,in Chapter2,we constructed a ratiometric Nano Luc-DNA probe based on bioluminescence resonance energy transfer.Using bioluminescence to report signal,this probe does not require large detection instruments,and can avoid the high background signal and light scattering interference.In addition,the self-correcting nature of the ratiometric probe is capable of precisely quantitative detection of targets.Through the design of the DNA sequence,we have constructed probes for the detection of ATP and Zn2+.These probes show good selectivity and sensitivity to the targets.Finally,these probes are used for quantitative detection of ATP and Zn2+in blood samples with satisfactory results.（2）To explore more functions of cells,bioengineers use genetic encoding to introduce functional proteins into the membranes of cell for the construction of engineered cells,such as CAR-T cells.However,the application of genetic engineering technology in cell engineering has some shortcomings,such as time-consuming process and inability to controllable assembly of the target protein.Thus,in Chapter 3 we propose a new strategy for the controllable assembly of exogenous proteins on the cell membrane.Based on the predictability and dynamic regulation of DNA nanotechnology,we have achieved rapid,efficient,and controllable assembly of exogenous proteins on the cell membrane surface through the complementary hybridization reaction and strand displacement reactions between the DNA-protein conjugates and the DNA scaffold.（3）In Chapter 4,based on the formerly mentioned exogenous proteins assembly strategy,we have constructed a series of functionalized cells with different functions.Based on the rapid and controllable advantages of this strategy,β-glucosidase was assembled on the surface of mammalian cell membranes to realize its functionalization in intolerable cellobiose hydrolysis.Subsequently,the DNA strand displacement reaction was used to realize the cell functional replacement from cellobiose hydrolysis to lactose hydrolysis.In addition,cells functionalized with a three-enzyme cascade system ofβ-galactosidase,glucose oxidase and horseradish peroxidase have also been successfully constructed.（4）The applicability of traditional enzyme activated prodrug strategy,particularly HRP-IAA therapy,is limited by the insufficient endogenous substrate H2O2.To solve this problem,in Chapter 5,DNA nanostructures were used to assemble the glucose oxidase-horseradish peroxidase（GOx-HRP）cascade system for synergistic cancer therapy.Due to the spatial confinement on DNA nanostrcuture,the efficiency of intermediate metabolites transportation between the enzyme cascades was improved,which improve the conversion rate of prodrug by the cascade.And GOx can trigger efficient glucose depletion for tumor starvation therapy.The DNA nanostructure-based confined cascade enzyme system has a good cancer treatment effect in vitro and in vivo.And this strategy is expected to be used in a broader field of synthetic biology and biomedicine. |
