Nanopore Single Molecule Study Of Crowding Effect-induced DNA Hairpin Folding Kinetics And Single-stranded DNA Condensation

In living cells,normal life activities are inseparable from the crowded environment of macromolecules within the cell.How a cell carries out various life processes in a crowded environment has long attracted researchers’ attention.At the same time,DNA molecule is an important genetic material in cells,and understanding its real structural changes in cells will help people to have a deeper understanding of life activities.Therefore,studying the effects of molecular crowding on hairpin DNA folding and single-stranded DNA condensation is important for understanding the functional and structural dynamics of nucleic acids.Currently,many research methods have been used to analyze the behavior of biomolecules in complex crowded environments.But for complex systems,measuring individual molecules may be a more efficient approach.Single-molecule techniques are powerful tools for studying complex systems,which can directly measure population distribution and kinetic parameters,and can reveal static and dynamic changes in biomolecules.In recent years,nanopore technology has been widely used as a label-free,rapid and sensitive single-molecule analysis method in the fields of single-molecule analysis,single-molecule DNA sequencing and single-molecule chemical reaction.The folding-unfolding kinetics of hairpin DNA in the environment and molecular crowding-induced single-stranded DNA condensation was studied at the single-molecule level.The specific research contents are as follows:1.Nanopore single-molecule studies of hairpin DNA folding-unfolding dynamics in crowded environmentsHairpin DNA is widely involved in the process of DNA replication,transcription and translation,and can interact with proteins and also affect the normal life activities in cells.Therefore,it is necessary to study the folding and unfolding kinetics of hairpin DNA.Using the α-HL protein nanopore as the analytical element,we demonstrated the two-state folding dynamics of hairpin DNA by studying the relationship between the folding process of hairpin DNA and the size,mass and volume fraction of the crowding agent.The study found that with the increase of the molecular weight and mass volume fraction of the crowding agent,the number of DNA molecules converted from the single-stranded state to the hairpin state increased,which was mainly due to the faster loop closing speed of the hairpin DNA.Thermodynamic studies of hairpin DNA formation show that the crowding effect can significantly reduce the Gibbs free energy of hairpin DNA formation,which is thermodynamically powerful;at the same time,kinetic studies show that with the increase of the crowding agent mass and volume fraction,the rate of folding The constant increased by1.25 times,and the unfolding rate constant decreased by nearly 3 times,that is,the unfolding process dominated the formation of hairpin DNA.This study has important implications for further understanding the structure and function of biomolecules in the crowded environment of cells.2.Single-molecule analysis of crowding effect-induced single-stranded DNA condensationDNA condensation and decondensation processes play an important role in biological systems.For example,histones condense DNA molecules into chromatin and depolymerize during gene expression,cell division,and other processes.Therefore,studying DNA condensation is of great significance for understanding normal life activities.We constructed an electrochemical biosensing platform using biological nanopores to investigate the condensation process of single-stranded DNA(ss DNA)in a crowded environment at the single-molecule level.The study found that with the increase of the size of the crowding agent,the degree of DNA condensation increased significantly,and four DNA forms were found in the condensation process.By studying the effect of crowding agent mass and volume fraction on DNA coagulation process,it was found that 20% PEG 8000 had the best coagulation effect on DNA.The aggregation process of ss DNA was monitored in real time in 20% PEG 8000.In addition,we monitored the condensation process of ss DNA in 20% PEG 8000 in real time and found that the condensation of ssDNA is a simple to complex process.