Construction Of DNA Biosensor Based On G-quadruplex

In recent years,simple,facile,accurate and efficient DNA biosensor with specific recognition and sequence polytropism has attracted considerable interest.G-rich DNA sequence structures known as G-quadruplexes,emerging as a three-dimensional structure of special interest are widely dispered in eukaryotic genomes.Using S1 nuclease and adnosine triphosphate as models,based on the specific binding of G-rich sequences with thioflavin T,the digestion of exo III to catalyze the hydrolyzation of the blunt and recessed 3'-hydroxyl terminus of duplex DNA,the high activity of peroxidase-mimicking DNAzyme and single-stranded nucleic acid endonuclease of S1 nuclease that can catalyze ssDNA and ssRNA cleavage to yield small oligonucleotide fragments or mono-oligonucleotide.The high sensitive and specific DNA biosensors have been proposed.1.A label-free fluorescent adenosine triphosphate biosensor via overhanging aptamer-triggered enzyme protection and target recycling amplification is proposed based on the digestion of exo ? to catalyze the hydrolyzation of the blunt and recessed 3'-hydroxyl terminus of duplex DNA.Herein,a label-free fluorescent adenosine triphosphate(ATP)aptasensor is fabricated with a DNA hairpin and an overhanging aptamer.In the presence of ATP,the overhanging sequences of the aptamer may form preferred substrates of exo ?,and thus trigger the enzyme-assisted amplification,which results in the release of G-rich sequences.Free G-rich sequences subsequently generate an enhanced flourescent signal by binding with thioflavin T.However,if ATP is absent,the overhanging sequence can induce steric hindrance and protect the DNA hairpin against the digestion of exo ?,significantly reducing the noise of this biosensor.Accordingly,the signal-to-noise ratio of the sensing system is greatly improved,which ensures the desirable analytical performance of the proposed aptasensor both in pure samples and real samples.2.The constraction of metal-ion-free G-quadruplex-Hemin DNAzyme is proposed based on the high activity of peroxidase-mimicking DNAzyme and single-stranded nucleic acid endonuclease of S1 nuclease that can catalyze ssDNA and ssRNA cleavage to yield small oligonucleotide fragments or mono-oligonucleotide.In this work,a new kind of peroxidase-mimicking DNAzyme(G-quadruplex-hemin DNAzyme,G4-hemin)is constructed by using hemin-modified G-rich DNA(hemin-G-DNA).Experimental results demonstrate that the G-rich DNA can form a G-quadruplex structure by the inducement of terminally modified hemin,rendering the assembly of hemin and G-quadruplex structure spontaneously and efficiently.As a result,G-hemin revealed higher peroxidase activity than traditional G-quadruplex/hemin DNAzyme(G4/hemin).Besides,different from G4/hemin,G4-hemin is constructed in one step without the participation of metal ions and adscititious hemin.Accordingly,the construction procedure is significantly simplified and the background signal from dissociative hemin is remarkably reduced.In a proof-of-concept trial,according to the colorimetric signals of G4-hemin,a novel biosensor for the detection of S1 nuclease activity is established,which provides a novel perspective for designing peroxidase-mimicking DNAzyme-based biosensors.3.An enzymatic assay for Co(?)and Ni(?)with G-quadruplex/hemin DNAzyme is proposed based on the high activity of peroxidase-mimicking DNAzyme and inhibition of Co(?)and Ni(?)to G-quadruplex/hemin DNAzyme.In this work,a new kind of peroxidase-mimicking DNAzyme(G-quadruplex-hemin DNAzyme,G4-hemin)is constructed by using hemin-modified G-rich DNA(hemin-G-DNA).Different from traditional G4/hemin,G4-hemin is constructed in one step without the participation of metal ions and adscititious hemin.Accordingly,the construction procedure is significantly simplified and the background signal from dissociative hemin is remarkably reduced.Co(?)and Ni(?)could increase the thermodynamic stability of duplexes containing a G-G mismatched base pair,presumably through the formation of a G-Co?-G and G-Ni?-G motifs,strongly inhibiting the enzymatic activity of G-quadruplex/hemin DNAzyme by Co(?)and Ni(?),which can be used to assay Co(?)and Ni(?).The detection of Co(?)and Ni(?)in solution can be achieved with naked eyes.This assay can be used to construct a "NOR" colorimetric logic gate.