A Single-molecule Study Of The Effect Of Macromolecular Crowding On The DNA Hybridization Process

About 20%-40% of the cell volume is full of DNA,protein and other biological macromolecules,and DNA plays an important role in the regulation of genetic information.Macromolecular crowding effect has significant effects on DNA molecular interaction and gene expression,and the mechanism is still unclear.With the rise of single molecules biology,the research of life process on single molecule level has been developed.So,based on single molecule magnetic tweezers technology,this paper explored the influence of macromolecular crowding effect on DNA hybridization at the single molecule level.Based on DNA hairpin structure and magnetic tweezers technology,the effects of different concentrations of macromolecules(PEG8000)on DNA hybridization were investigated by using oligonucleotide chains complementary to DNA hairpin base.With the increase of PEG8000,the hybridization probability of DNA oligonucleotide chain increased significantly.On this basis,the effects of the number and position of base defects on the hybridization process of oligonucleotide chain were systematically studied.When there is a base defect at one end of the oligonucleotide chain,the DNA hybridization probability first increases and then decreases with the increase of the concentration of PEG8000.When the defective base is located in the middle of the oligonucleotide chain,the hybridization process of the oligonucleotide chain is the most affected,almost impossible to hybridize.The mechanism of DNA hairpin stability degradation induced by fluoride was determined by Isothermal Titration Calorimetry(ITC),particle size potentiometry and molecular dynamics simulation.The dissociation constants between oligonucleotide chains,fluorine ions and oligonucleotide chains were measured by Isothermal Titration Calorimetry(ITC)There was no hydrogen bond between DNA oligonucleotides,but increased fluorine ion concentration leads to a gradual decrease in Zeta potential,causing decreased the stability of DNA hairpin.On this basis,molecular dynamics simulation was used to determine the reason for the decrease of zeta potential of oligonucleotide chain due to the increase of fluoride concentration.Radial distribution functions(RDFS)indicated that the distribution of fluoride ions was far away from oligonucleotides than that of chloride ions,which directly led to the decrease of zeta potential with the increase of fluoride concentration.