Construction Of DNA Functional Materials And Their Application In Cell-free Protein Synthesis System

Cell is the basic structural unit and functional unit of life.The complex and delicate intracellular organization is the structural foundation of cell for self-regulation and efficient life activities.Investigation of cellular structure-function relationship presents significant implications for understanding the operational mechanism of cell system.In this thesis,we fabricated a series of DNA assembly nanostructures,and employed these assemblies in cell-free protein synthesis(CFPS)system to systematically explore the effects of genetic DNA proximity and gene circuit compartment on gene expression;in addition,we investigated the regulation of magnesium ion(Mg²⁺)on the lyophilized CFPS system.This thesis includes five chapters:In Chapter 1,we summarized the characteristics of cell system and the organization of genomic DNA in living cells,summerized the development,composition,advantages and applications of CFPS system,and reviewed the advantages,building blocks and assembly methods of DNA materials.And then we put forward our research in this thesis.In Chapter 2,we designed and synthesized a branched gene architecture in which three transcription units were integrated into one framework through assembly based on the polymerase chain reaction(PCR),together with the exploitation of these constructs in CFPS system,probing the impact of this spitical physical factor on gene transcription and translation.The results showed that the branched gene system enhanced gene expression yields,in particular at low concentrations of DNA and RNA polymerase;furthermore,in a crowded microenvironment that mimicked the intracellular microenvironment,gene expression from branched genes maintained a relatively high level.In Chapter 3,we fabricated a gene circuit compartment by coanchoring two function-related genes(regulatory gene and regulated reporter gene)on gold nanoparticles,and investigated the compartment effect on cascade gene expression in CFPS system.In order to steadily anchor the genes on the surface of gold nanoparticles,both the regulatory gene and regulated reporter gene were attached on a Y-shaped DNA nanostructure whose other two branches were mercapto-modified via PCR assembly.In CFPS system,the gene circuit compartment on nanoparticle made the regulatory gene and regulated reporter gene spatially proximal at nanoscale,thus effectively improving the transfer efficiency of the synthesized regulatory proteins from regulatory genes to the regulated reporter genes in the compartments,and consequently,the biochemical reaction efficiency could be significantly increased.Experimental results demonstrated that both the yield and initial expression rate of the fluorescent reporter protein in the gene circuit compartment system were enhanced compared with those in free gene circuit system.In addition,we found that the gene circuit compartment effect was gene concentration-dependent that the gene circuit compartment on the nanointerface could remarkably enhance cascade gene expression with a relatively low concentration of genes via favorable spatial proximity.In Chapter 4,we reported the protection role of Mg²⁺on lyophilized CFPS system,and proposed two-metal-ion regulation:Mg²⁺(I)acted as an allosteric role for enzymes to prevent the conformational damage of enzymes from direct binding with substrates during lyophilization which was proven could lock up inactive enzyme-substrate complex;Mg²⁺(II)consequently bound to enzymes to activate the CFPS system.In Chapter 5,we made a conclusion and prospect for our investigation.We have fabricated several functional DNA nanostructures and revealed the biological significance of genetic DNA orgnization in CFPS system,which presents significant implications for the cellular gene structure-function relationship.In addition,we have reaveled the regulation of Mg²⁺on lyophilized CFPS system,which provides implications for understanding the functions of Mg²⁺in biological systems.