Study Of Biomolecules And Their Interaction Based On Atomic Force Microscopy

Atomic force microscopy(AFM)has been widely used in biological research due to its advantages of high resolution,high sensitivity and imaging under nearphysiological conditions.Atomic force microscopy can be used not only to characterize the surface morphology of samples,but also to quantify the interactions between biomolecules,identify and locate of specific molecules on the cell surface,and characterize the nanomechanical properties of living cells.In this dissertation,the following researches have been carried out based on atomic force microscopy.1.The interaction between the Methyl-CpG-Binding Domain(MBD)of MethylCpG Binding Protein 2(MeCP2)and methylated DNA was investigated using Atomic Force Microscopy based Single-Molecule Force Spectroscopy(AFM-SMFS).The binding forces between MeCP2 MBD domain and methylated DNA at different loading rates were obtained at the single-molecule level.The dynamic parameters of MeCP2 MBD domain/methylated DNA complex during dissociation process were also obtained.In addition,the interactions between MeCP2 MBD domain and methylated DNA,hemi-methylated DNA and unmethylated DNA molecules were compared,and it was obtained that MeCP2 MBD domain had a strong interaction with methylated DNA molecules,indicating that MeCP2 MBD domain had a specificity recognition with symmetrical methyl-CpG sites on methylated DNA molecules.This work provided new insights into the molecular mechanism of MeCP2-mediated methylation signaling.2.The interactions between programmed cell death 1 ligand 1(PD-L1)and PDL1 antibody or programmed cell death protein 1(PD-1)were studied using AFM-SMFS.The expression of PD-L1 on the cell surface were investigated using Force-Volume imaging.The unbinding forces and dynamic parameters of PD-L1/PD-L1 antibody and PD-L1/PD-1 complexes were obtained by AFM-SMFS,it was found that the binding of PD-L1 and PD-1 was stronger than PD-L1 and PD-L1 antibody.The expression of PD-L1 on the surface of T24 cells before and after interferon-γ(IFN-γ)treatment were obtained at the single cell level by Force-Volume imaging.The results indicated that IFN-γ could up-regulate the expression of PD-L1 on the surface of T24 cells,but did not affect the interaction forces between PD-L1 and PD-1 or PD-L1 antibody.This work further revealed the molecular mechanism of the interaction between PD-L1 and PD-1 or PD-L1 antibody.In addition,the expression of PD-L1 on the surface of tumor cells was quantified,which is of great significance for the prediction and diagnosis of tumor immunotherapy.3.The effects of aptamer conjugated polydopamine-coated gold nanoparticles(Au@PDA-Apt)on amyloid β-protein(Aβ1-40)and cell morphology as well as mechanical properties induced by Aβ1-40 protein aggregation were investigated by AFM imaging and quantitative nanomechanical measurement.It was found that Au@PDAApt nanoparticles can not only inhibit the fibrillation of Aβ1-40 protein,but also disaggregate Aβ1-40 fibrils.Cell experiments further showed that Au@PDA-Apt nanoparticles had good cytocompatibility and could effectively scavenge the intracellular reactive oxygen species,and had a protective effect on PC 12 cells.In addition,AFM quantitative nanomechanical imaging showed that Au@PDA-Apt nanoparticles could effectively prevent the damage of Aβ1-40 protein aggregation on cell morphology and Young’s modulus.This work provided a potential nano-drug candidate for Aβ-targeted Alzheimer’s disease therapy,and broadened the application of AFM in evaluation the efficacy of nanoparticles.