Research Of DNA And RNA By Nanopore Single-molecule Analytical System

Nanopore single-molecule techology is a highly innovative technology,which is widely used in DNA sequencing,disease diagnosis,drug screening and environmental monitoring.Biological nanopores receive the extensive attentions because of its advantage of real-time,sensitive,selective and stable.The principle of single-molecule nanopore detection is that the analyte is driven into the nanopore under the influence of a voltage and emerging a measurable ion current block.According to the dwell time,frequency,and even amplitude of characteristic currents,we can reveal the characteristics of analyte.Scientists have not stopped to research the genetic materials（DNA and RNA）.The researchers hope that understanding more about the genetic materials may lift the life sciences to a new level.Here,we use PEGs as crowders to simulate crowding conditions and protein nanopore as sensor to analysing dynamics of ssDNA in solution.In the second part,we study the interaction between pre-microRNA-21 and peptide at the single-molecule level.The main research contents are included as follows:1 Research ReviewWe give a summary of the development,detection principle and application of nanopore single-molecule technology.2 Crowding-induced DNA translocation through a protein nanoporeCrowded cellular environment is highly associated with many significant biological processes.However,the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored.Here,we use nanopore single-molecule analytical technique to quantify the thermodynamic and kinetics of DNA transport under heterogeneous co-solute PEGs.The results demonstrate that the frequency of translocation event exhibits a non-monotonic dependence on the crowding agent size,while both the event frequency and translocation time increase monotonically with increasing crowder concentration.In the presence of PEGs,the rate of DNA capture into the nanopore elevates 118.27-fold in contrast to the decrease of translocation velocity in the pore（from 20μs/base to 120μs/base）.Interestingly,the impact of PEG 4k on the DNA-nanopore interaction is the most notable,with up to△△G16.27 kJ mol^-1 changes in free energy and 764.5-fold increase in the binding constant at concentration of 40%（w/v）.The study presents a powerful tool for exploring single-molecule kinetics of molecular crowding,and furthermore,the nanopore molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.3 Study on the reaction between pre-microRNA-21 and peptide based onα-HL nanoporeReactions of pre-microRNA and small molecule have insiginicant importance in regulating the level of microRNA.Here,we chooseα-HL protein as nanochannel.By analyzing the events of peptide-pre-microRNA-21 mixture,we discover the mean residence time(τ_off)of level 1 of type C is 828.8±61.61 ms,which is 2-fold longer than that of events in the absence of peptide（419.50±33.29 ms）.The above results testify that peptide does interact with the pre-microRNA-21 and enhance the stability of pre-microRNA-21 hairpin structure,thus leading to a significant increase in unzipping time.Adding different molar ratio of peptide to pre-microRNA-21 in the nanopore,the greater molar ratio,the more events with long residence time and the longer the average residence time.Reach a certain mole ratio of reacants that no longer increase average dwell time of peptide-pre-microRNA-21 mixcure.This experiment provides a new platform for drug screening.