Study on the Applications of Functional G-quadruplex in Biosensing and Logic Gates

Author:Wang Shuang

Supervisor:yang xiu rong

Database:Doctor

Degree Year:2019

Download:35

Pages:167

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DNA,also called deoxyribonucleic acid,is one of most important life fundamental molecule.It existed in all plant,animal cell,and in the microbe and organisms.Different arrangement order of DNA bases composed of different genetic instructions to guide biological heredity,biological development and life functioning.As the development of science and techonology,DNA’s roles and functions are far beyond what was first thought as genetic material.Due to multiple merits including specific recognized ability,predictability,programmability,biocompatibility,and structural polymorphism,DNAs were regarded as one of the most potential materials,thus were widely employed in recognizing special target,sophisticated nanostructure,constructing DNA molecule robot,data storage,logic operation and so on.In this thesis,DNA worked as important material were used in constructing biosensing interfcaes,changing the microenvironment of fluorescent dyes,and exploring the signal transduction mechanism between fluorescent dye and other fluorophores like dye and siver nanoclusters via modulation of DNA structure.Based on these new design strategy and findings,a series of biosensors and logic operations systems were achieved.The detailed contents were shown below:1.In this work,we utilized the direct conformational signal changes in specific interaction process between Pb2+ and guanine-rich(G-rich)oligonucleotides to establish a sensing system.The binding of Pb2+ and G-rich oligonucleotides results in conformational transition from single strand to G-quadruplex,which was recorded real-time by dual polarization interferometry(DPI).The sensing platform was established via layer-by-layer assembly strategy that employing polyethyleneimine and ploythymine as media layers to link the G-rich DNA on the DPI chip,which is simple,facile and without need of synthesizing sophisticated nanomaterial.On the basis of above sensing platform and quantifiable conformational changes,a simple,sensitive,specific and label-free Pb2+ biosensor was established.This research provides us a simple and sensitive method for detection of ions,also encourages us to combine surface technique to biosensing application and explore the relationship between structures and functions.2.A DNA tetrahedron platform with high reproducibility and regenerative ability was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA(G-rich ssDNA)and Pb2+.The oriented immobilization of probes greatly decreased the spatial hindrance effect and improved the accessibility of the probes to the Pb2+ions.Through real-time monitoring of the whole formation process of the G-quadruplex,we speculated that the probes on the tetrahedron platform initially stood on the sensing surface with a random coil conformation,then the G-rich ssDNA preliminarily formed unstable G-quartets by H-bonding and cation binding,subsequently forming a completely folded and stable quadruplex structure through relatively slow strand rearrangements.On the basis of these studies,we also developed a novel sensing platform for the specific and sensitive determination of Pb2+and its chelating agent ethylenediaminetetraacetic acid.This study not only provides a proof-of-concept for conformational dynamics of G-quadruplex-related drugs and pathogenes,but also enriches the biosensor tools by combining nanomaterial with interfaces technique.3.For the first time,we established a label-free logic gate by regarding the structure-related signal as output.DPI was employed to reveal the detailed conformational transitions occurring in the multiple-factor biomolecule interactions and then was utilized as a detection tool of logic gate.As a vital merit of this system,the dependence of the density output signal on the interaction with multiple-factor input can mimic the function of signal communication in OR,INHIBIT,and IDENTITY logic gates and the INHIBIT-OR cascade circuit.Additionally,the DPI signal with logic stringency can unambiguously distinguish conformational polymorphisms and compare structural stability.This study provides a new way for the construction of a label-free logic gate,supplements information deficiency of reaction details,and extends the application of DPI in logic operation.4.A simple but versatile platform was developed by integrating split G-quadruplex and fluorophore into a molecular beacon,where the photoinduced electron transfer could occur when the fluorophore approached the preformed G-quadruplex/hemin complexes.Such design subtly combined the G4 disruption-induced fluorescent tum-on strategy and the photoinduced electron transfer property into one platform for constructing the logic circuits.On the basis of such a universal platform,a series of binary logic gates(OR,INHIBIT,AND,and XOR),a combinatorial gate(INHIBIT-OR),and even a complex logic operation for discrimination of multiples of three from natural numbers less than ten have been successfully achieved only by employing such platform as work unit and single-strand DNAs as inputs.The set-reset function of this platform could be realized by alternatively introducing blocking and releasing strands.In addition,this platform could operate in a biological matrix stably and precisely.Therefore,such a universal platform lays the foundation for complicating the logic systems,realizing the biocomputing and also points out a new direction for target detection.5.We firstly integrate the catalyzed hairpin assembly(CHA)and rolling circle amplification(RCA)into an electrochemical biosensor for sensitive and specific detection of miR-21.Meanwhile,an electric potential was employed to modulate the efficiency of CHA occurred on the electrode,which offer a simple but effective method to surmount the accessibility problem of probes.The biosensor achieved an ultrasensitive determination of miR-21 with a low limit of detection of 13.5 fM and a linear range from 15 fM to 250 pM.This research encourages us to challenge the hyphenated multiple amplification strategies and provides a stable and effective method for the detection of diseases-related miRNAs in peripheral biofluids,as well as paves a road for the future clinical diagnostics and treatment of disease.6.For the first time,we found and verified that a duplex connection at one end of G4 could dramatically enhance the fluorescence of G4/CV.The fluorescence enhancement mechanism was made clear with the aid of CD,fluorescence spectra and DFT calculations,which was in line with our conception that the“G4 nano-accommodation with a duplex roof",had a higher efficiency to confine the twisted intramolecular charge transfer(TICT)of CV.Additionally we systematically explored the effect of sequence length and base type on the fluorescence enhancement,and the results demonstrated that lengths over 16 bases and purine bases like A and G had more obvious promotion on the emission properties of CV in G4.The new phenomenon also was utilized in target nucleic acid detection,and showed excellent performance in sensitivity,specificity,and biocompatibility.Our finding offered a new approach to effectively enhance the fluorescence of dyes by assembling a modular DNA nanostructure and also supplied a new signal transduction strategy for bioanalysis and bioimaging.7.DNA radar was first built by assigning the silver nanocluster(AgNC)as the radar transmitter,the middle single strand DNA-bridge connected on the AgNCs as the electromagnetic wave,and the G4/crystal violet complex as the radar antenna.The radar antenna could receive the signal of the target DNA that met the electromagnetic wave and give a location via light-up fluorescence.Here,G4 is chosen as the suitable template to connect potential nanomaterial AgNCs with the G4 binder(crystal violet,CV)since the rich guanine in G4 could not only enhance the fluorescence of AgNCs but also form quartets offering powerful binding sites for the G4 binder.Meanwhile,the hybridization behavior of the middle single strand-bridge produced contrary effects decreasing the fluorescence of AgNCs and increasing the fluorescence of G4/CV,which vests a ratiometric feature in such DNA radar.Additionally,this DNA radar model could realize a cascade of logic circuits,the construction of a 1-to-2 decoder,and the ratiometric detection of target DNA.This system could also be employed for DNA detection in a biological matrix,which could be potentially usable as a unique means for monitoring the pathological process of disease,and lays the foundation for the future treatment of diseases.