Study On The Self-assembling Of Supermolecular Aggregation Using DNA With The Mediation Of Protein

The self-assembly of biomacromolecule is an effective way to create new material with nano-sized and micron-grade.In recent years,more and more scientists are interested in researching on the self-assembly of various materials.Self-assembly not only can build various materials to be used for many areas,but also can provide basic research for many research fields.In this thesis,the main work of this thesis is divided into three sections,as follows: Firstly,the content of this research considers the use of apoferritin-assisted DNA to guide gold nanoparticles to assemble into flower-like structures.This chapter selects protein apoferritin,which can respond to pH value.When the apoferritin solution pH value is adjusted to 2,apoferritin will dissociate and 24 subunits are obtained.The prepared gold nanoparticles(particle size of 5 nm)are then mixed with the above subunit solution.The next process involves the pH value of the mixed solution being slowly adjusted to neutral,the subunits become recombinant and the protein will recover the original globular structure.In the process of reorganisation of apoferritin,the gold nanoparticles are caged in the cage of apoferritin.After the excess gold nanoparticles in the solution are removed and concentrated,the solution is divided into two parts,with each part added to a thioled DNA solution.Moreover,the base sequence of the added thioled DNA is complementary to each other.Thioled DNA combines with gold nanoparticles caged in the apoferritin through the hydrophilic channel of the protein.The excess thioled DNA of the two solutions is removed and concentrated respectively,following which the two solutions are mixed.The annealing of the mixture is performed following a programmed cooling using a single cycle of Polymerase Chain Reaction(PCR)procedure.This approach can lead to further self-assembly of gold nanoparticles.The assembly flowers are then characterised by transmission electron microscopy,scanning electron microscopy,polyacrylamide gel electrophoresis and atomic force microscopy.The components of the assembled flowers are confirmed by various means of testing,initially confirming that the protein,DNA and gold nanoparticles self-assemble into flower-like structures.It is preliminarily confirmed that protein,DNA and gold nanoparticles assemble into the obtained flower-structure through screw dislocations.Secondly,the content of this chapter describes how a protein(apoferritin)is connected with thioled DNA using an amphoteric crosslinking agent Sulfo-SMCC and the protein apoferritin is further assembled into micron fibre through further DNA hybridisation.Firstly,crosslinker Sulfo-SMCC reacts with the amino-group of apoferritin to produce a maleimide-activated protein,Apoferritin-SMCC.After removing the excess of crosslinker and by-products,the maleimide-activated Apoferritin-SMCC solution is divided into two parts,and thioled DNA solution is added to the above solutions respectively.Moreover,the base sequence of the added thioled DNA is complementary base paring.The maleimide-activated protein(Apoferritin-SMCC)reacts with thiolated DNAs in the appropriate molar ratio and produces Apoferritin-DNAs through addition reaction.The H-S bond of thiolated DNA is added to the double bond in the SMCC of ApoferritinSMCC.Subsequently,the above two Apoferritin-DNA complex solutions are concentrated and mixed after removing the excess of thioled DNA.Apoferritin molecules are connected to each other through strict complementary base pairing of DNA function.Finally,the polymer Apoferritin-DNAs self-assemble into fibres with half helical structure via further complementary base paring of the DNAs.The fibres are presented by Scanning Electron Microscopy(SEM),agarose gel electrophoresis,Optical Microscopy(OM),confocal fluorescence microscopy and atomic force microscope(AFM)respectively.The microstructure of the assembled fibre is observed by using atomic force microscopy.The results suggest that DNA and apoferritin first self-assemble lamellar structure and then these lamellar structures assemble into fibres in a parallel way.Finally,this chapter focuses on using an amphoteric crosslinking agent,Sulfo-SMCC,to connect Bovine Serum Albumin(BSA)and DNA.Bovine Serum Albumin is then connected through the complementary base pairing of DNA.After that,BSA is further assembled into micron fibre through further DNA hybridisation.Firstly,crosslinker SulfoSMCC reacts with the amino-group of protein BSA to produce a maleimide-activated protein BSA-SMCC.After removing the excess of crosslinker and by-products,the maleimide-activated BSA-SMCC solution is divided into two parts,and thioled DNA solution is added to the above solutions respectively.Moreover,the base sequence of the added thioled DNA is complementary base paring.The maleimide-activated protein(BSA-SMCC)reacts with thiolated DNAs in the appropriate molar ratio and produces BSA-SMCC-DNAs through an addition reaction.The H-S bond of thiolated DNA is added to the double bond in the SMCC of BSA-SMCC.Subsequently,the above two BSADNA complex solutions are concentrated and mixed after removing the excess of thioled DNA.BSA molecules are connected with each other through the strict complementary base pairing of DNA function.Finally,the polymer BSA-SMCC-DNAs are selfassembled into fibres with half helical structure via further complementary base paring of the DNAs.In addition,fluorescently labelled protein(FITC-BSA)and DNA(Cy5-DNA)are used to repeat the experiment under the same conditions.The results further confirm that the assembly is composed of protein BSA and DNA.The microstructure of the fibres is observed using atomic force microscopy(AFM).The assembled mechanism of the fibre is that BSA and DNA firstly assemble into lamellar structure and these lamellae are then assembled into fibres in parallel.The self-assembly materials constructed by macromolecules have potential applications in biomedical,materials and chemical fields because of their biocompatibility and biodegradability.