| Telomeres are complexes of proteins and DNA that located at the ends of chromosomes in eukaryotic cells.Telomere DNA maintains a circular structure in the cell to protect the end of the chromosome.They are essential for maintaining the integrity of the genome,the life of the cell,and cancer diagnosis and treatment.In most organisms,telomere DNA is a highly repeating short-chain sequence and many telomere DNA consists of two to five adjacent guanines repeating on the same strand and the corresponding cytosine on its complementary strand.The repeat is 5'-TTAGGG and the length is between 7Kb and 15 Kb.The structure is not a simple complementary double helix structure on these G-rich telomere DNA sequences.This telomere G-rich DNA strand can form a kind of three-dimensional structure: G-quadruplex.The structure of G-quadruplex(G4)in different salts is also very different.And the structure of G4 formed by telomere DNA is closely related to its function,thus the research on G-quadruplex has been gradually deepened in recent years.There are a wealth of traditional methods reported for the investigation of G4 conformation along with the folding dynamics at both single-molecular and singleatomic level,include ultraviolet spectrum(UV),circular dichroism spectroscopy(CD),nuclear magnetic resonance spectroscopy(NMR),X-ray diffraction,gel electrophoresis,thermal denaturation and surface plasmon resonance(SPR).These detection methods can only study the structure of G-quadruplex after its formation,and cannot monitor it in real time at single-molecule level.On the basis of the above technologies,singlemolecule fluorescence and optical tweezers emerged.They have disadvantages as tedious operation,time-consuming and high cost.Nanopore single-molecule detection technology provided a new method for the study on G-quadruplex structure.Inspired by the Coulter principle,the idea of material detection through devices containing nanopores was proposed.Nanopore detection technology has been rapidly developed in the past two decades,now this technology has been widely used to detect various substances such as DNA,proteins,sugar,antigen,antibody and even ions.According to the difference of nanopore carrier materials,nanopores are divided into biological nanopores and solid-state nanopores.Biological nanopores are channels that are channels formed by self-assembled protein polymers under certain conditions.The channels have stable structures and properties and have a defined nanometer size.The application of biological nanopores is extensive,for example,?-hemolysin(nanopore formed by the amino acid heptamer inserted in the phospholipid bilayer)can be used to detect single base in phospholipid bilayer.Biological nanopores are also used to detect proteins,small biological molecules,and metal ions.The advantages of biological pores include good reproducibility with natural assembly pore structure,good biocompatibility and low system noise.However,the lipid bilayer that supported the nanopores is unstable and is affected by the buffer system and temperature.The sensitivity of the lipid layer,the size of the biological pores,and the diversity of the charge distribution on the wall of pores greatly limit the conditions and scope of biological pore detection,and also limit its scale-up generation.In order to build nanochannel systems with different functions,solid nanopores with more stable structural properties were later developed.The rapid development of micro and nanomanufacturing technology has provided conditions for the development of solidstate pores.Solid-state nanopores are fabricated by physically or chemically processing gaps or holes with nanopore sizes on an insulating two-dimensional material film.Compared with biological nanopores,the pore diameter of solid nanopores can be designed according to the size of the analytes,and it has obvious advantages in chemical and thermodynamic tolerance.The solid-state nanopores are prepared through physical or chemical methods such as Transmission Electron Microscope(TEM),plasma etching,focused ion beam(Focus Ion Bean,FIB),and dielectric breakdown methods.The preparation of nanopore by FIB and TEM is expensive and complicated to operate.Chemical etching has low resolution and is difficult to control.Therefore,the preparation of nanopores by dielectric breakdown with simple operation and low cost has been rapidly developed and widely used.This article introduces a CSDB(Current-Stimulus Dielectric Breakdown)model and a method for preparing nanopores via LabVIEW computer program control dielectric breakdown.In view of the limitations of existing methods in the rapid and convenient detection of G-quadruplex single molecule structure,this paper proposes a method for monitoring the structure and formation process of G-quadruplex using solid-state nanopore single molecule detection technology.Compared with existing methods,this method has the advantages of real-time,fast,low cost,simple operation and does not require large instruments.The research work of this paper includes the following items:Preparation of solid nanopores by dielectric breakdown method.Nanopores with different pore diameters were prepared on a SiNx film with a thickness of 10 to 20 nm by using Current-Stimulus Dielectric Breakdown(CSDB).Optimization of the preparation process of nanopores by adjusting the voltage of breakdown and the pore expansion.Immobilization of telomere sequence on solid-state nanopore and monitoring the G4 folding process.Via covalent modification of telomere sequences on the inner surface of the nanopore,the telomere sequences DNA were induced to fold into different G4 structures in distinct metal ion salt solutions.The blockage duration in nanopore deciphered the tolerance and stability of the stereostructure of G4 under electrophoretic force,realizing dynamic monitoring of the G-quadruplexes at singlemolecule level.Label-Free single-molecule identification and exploration of folding process on telomere G4 with a solid-state nanopore sensor.This work introduced a single-molecule solid-state nanopore method for the determination of intermolecular and intramolecular G4 with distinct structure in varied cation buffers with a tailored aperture and meanwhile realized capturing single-molecule G4 folding process.Furthermore,realtime test demonstrated a decline in lower current blockage ratio(?I/I0=0-0.2)that represents unstructured DNA molecule translocation and an increase in higher current blockage ratio(?I/I0=0.7-1)that represents G4 translocation.This indicates that the G4 structure is constantly forming with the passage of time.The ratio of its number to the total amount of DNA continues to increase. |
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