Study On The Structural Characteristics Of Protein-nucleic Acid Complexes Based On Solid-state Nanopore

DNA replication and amplification reactions based on DNA polymerase are one of the most important technical methods in molecular biology research.Studying the interaction process between DNA polymerase and nucleic acid in a solution environment is of great significance for a deeper understanding of DNA polymerization.Compared with traditional protein characterization methods,nanopore single-molecule sensors can characterize the conformational changes and dynamic processes of proteins in a solution environment in real time without labels,and have significant technical advantages and important application potential.This topic applies a silicon nitride solid-state nanopore sensor with a pore size of about 35 nm to detect DNA polymerase I Klenow fragment(KF),KF-DNA binary complex and Klenow fragments mixed with different DNA substrates,aiming to analyze the conformational changes and transport kinetics characteristics of the Klenow fragment before and after binding to the substrate,and explore the factors that affect the efficiency of polymerase binding to DNA substrates,including the base mismatches in the substrate and the concentration ratio of polymerase and substrate during sample preparation.The main research contents of this subject are as follows:1.Building a single-molecule detection platform based on solid-state nanoporesTo build a single-molecule detection platform,the silicon nitride solid-state nanopores with a diameter of 30 nm and less than 20 nm were processed respectively by using focused ion beam and transmitted electron beam.Based on the existing analysis methods and models,a 13-nm solid-state nanopore was used to explore the structural characteristics of single-stranded DNA and hairpin DNA,for verifying the ability of solid-state nanopores to recognize DNA conformation,and establishing a complete detection and analysis process for subsequent experiments.The results show that the hairpin DNA takes a shorter time to pass through the hole,causes a smaller current change,and generates more simple signals,corresponding to its more stable conformation and simpler translocation behavior.2.Studying the structural characteristics of the "protein-nucleic acid" binary complexA solid-state nanopore sensor with a pore size of 35 nm was used to characterize the conformation and transport dynamics of Klenow fragment and KF-DNA binary complex.The results of conformational characteristics show that the hydrodynamic radius of Klenow fragment is reduced by about 0.36 nm after binding to the substrate,and the conformational flexibility is reduced.The characteristics of transport kinetics point out that the binary complex with smaller volume and more compact structure is more difficult to be captured by nanopores and more difficult to pass through.By comparing the proportions of different types of translocation signals,it can be seen that due to the interference of the exposed template chain and the strong interaction between the binary complex and the hole wall,the binary complex has a more complex translocation behavior.3.Exploring the factors that affect the efficiency of polymerase binding to DNA substrateA solid-state nanopore sensor with a pore size of 35 nm was used to explore the factors that affect the efficiency of polymerase binding to DNA substrates,including the base mismatches in the substrate and the concentration ratio of polymerase and substrate during sample preparation to the polymerase binding substrate,by designing DNA substrates with different sticky ends.The results show that when the 3' end of the substrate primer chain contains mismatched bases,the binding efficiency of the Klenow fragment to the substrate is reduced;as the polymerase concentration ratio increases during sample preparation,the binding efficiency of the Klenow fragment to the substrate is improved.