Single-molecule Analysis And Sensing Based On Biological Nanopore

Single-molecule analytical techniques that analyze the structure,confomatinal change,dynamic and interaction of the analytes at single-molecule level can provide more information of molecular structure and their function or mechanism.As a potential technique for the third generation gene sequencing,nanopore has provided a rapid and real-time single-molecular analytical platform for molecular biology and nanoscience.Based on the nanostructure of a biological nanopore?-Hemolysin(?-HL)and its great ability to sense and distinguish single-molecular structures,this thesis has proposed strategies for highly specific molecule recognition and developed single-molecular analytical methods and sensors for the cancer-related biomarker detection,small molecule drug mechanism and thermodynamic analysis,and nanocluster structure identification.The details are as follows:A highly selective nanopore proximity bioassay for protein biomarker platelet-derived growth factor B-chain(PDGF-BB)detection was presented based on a proximity binding-induced DNA strand displacement strategy.An individual target protein was recognized by two specific oligonucleotide aptamers which were linked with complementary sequences as probes.The protein recognition induced the proximity of two probes and an output DNA was released and translocated through?-HL nanopore with an output signal,allowing the quantification of the target protein.This rapid and label-free bioassay had great selectivity without the need of chemical modification or signal amplification,and could be applied in serum samples.Human telomere sequence interactions with a small molecule drug pyridostatin(PDS)were analyzed in single-molecule level.Different telomeric DNA G-quadruplex structures were captured and discriminated by the vestibule of?-HL nanopore.The potent stabilization effect of drug on G-quadruplex structure was demonstrated by analysis of the unraveling time of G-quadruplexes.Signature two-level electronic blocks were discovered.The translocation studies and the free-energy analysis demonstrated a coordinated effect of K~+and drug on G-quadruplex stabilization.Additionally,the nanopore platform permits the efficient and accurate determination of drug affinity constants without the requirement for labeling,amplification,or ligand/receptor titration,possessing great potential for the design and screen of anticancer drugs.Multiple nanoclusters including kinds of polyoxometalates and Au nanocluster were analysed in single-molecule level via?-HL nanopore,by providing structure-dependent 2D dwell time-current blockage spectrums and normalized capture rates for the cluster translocation events.The?-hemolysin ion channel permits the discrimination of nanocluster structures with atomic precision,and shows 1000-fold higher sensitivity in analyte concentration than other classical methods.Molecular dynamics(MD)simulations furtherly revealed the nanocluster translocation dynamics and related the experimental result with the nanocluster structure.The nanopore platform provides a novel powerful tool for nanocluster characterization.