Single-molecule Study Of Double-stranded DNA And A? Protein In Macromolecule Crowded Environment

The existence of various organelles and cytoskeletons will lead to the formation of crowded environment in the cell.crowded environment will further affect the structure and function of biological macromolecules.Many studies have shown that crowded environments can lead to changes in the structure and stability of biomolecules.However,how the crowded environment affects the structure and interaction of biomolecules is still a worth considering problem.Nanopore single-molecule technology means when the analyte to pass through the nanopore to formate the ionic current blocking signal,achieves the characterization of the restructure,size,and charge of the biological sample.Due to this technology has the advantages of simplicity,speed,and label-free,it is widely used in research fields such as DNA,RNA,protein interaction,and detection of various metal ions.In addition,investigating to DNA,proteins,enzymes interactions has important value at the single-molecule level.It has taken a big step in promoting the development of diagnosis and treatment methods for human-related diseases.In addition,at the single-molecule level,investigating to DNA,proteins,enzymes interactions has important value,and has taken a big step in promoting the development of diagnosis and treatment methods for human-related diseases.it researchs different molecular weight,different concentrations of PEG on the behavior of perforations analyte,construct a new platform for DNA analysis and protein interactions.The main contents are as follows:1.Research on double-stranded DNA in crowded environment based on?-HL nanopore As one of the main genetic materials of life,DNA plays an important role in the process of gene expression,transcription and translation.By adding crowding agents of different concentrations and different molecular weights to the electrolyte solution,the interaction of double-stranded DNA with nanopores under the action of an electric field was explored.By analyzing the residence time and frequency of double-stranded DNA perforation,it is found that as the concentration and molecular weight of the crowding agent increase,the probability of double-stranded DNA perforation gradually increases,and its residence time increases.Further research has been made on the fact that the crowded environment contains a mismatch.The effect of bases on the perforation behavior of double-stranded DNA.It was found that the presence of crowding agents increased the stability of double-stranded DNA containing mismatched base pairs,and significantly increased the residence time of perforation.At the same time,we also found that the perforation behavior of fully complementary double-stranded DNA was delayed to varying degrees under asymmetric crowded environmental conditions2.Research on A?Aggregation Mediated by Metal Ions in Crowded Environment Based on?-HL Nanopores.How to fold the nascent peptide chain into a protein with a specific structure and function,how to solve the problem of misfolding and aggregation that can easily occur during the folding process.This has always been the research focus of researchers.In the experiment,15%(w/v)PEG 4000 was used as a crowding reagent to simulate the cell crowding environment,by analyzing the current blocking amplitude(I/I₀)and residence time(?_off)when A?protein passing through the nanopore,the folding and aggregation of A?peptide mediated by metal ions are studied.Studies have shown that the crowding of macromolecules promotes the conformation of A?_1-40peptide from a folded structure to a random coiled structure,resulting in a conformation that is easy to aggregate.At the same time,A?aggregation mediated by metal ion Cu²⁺was observed at the single-molecule level by analyzing the residence time and perforation frequency.The analysis of the ion current relative blocking ratio confirmed that the addition of Cu²⁺induced the conformation of A?to change.Further explored the conformational changes of the complexes formed by different metal ions and A?peptides in crowded environments,as well as the difference in binding force with nanopores.This research provides an experimental basis for understanding the conformational changes of proteins in cells,and provides an effective method for exploring related pathogenesis such as Alzheimer's disease and Parkinson's disease.