Studies Of Novel Biosensors For The Detection Of DNA-modifying Enzyme

Enzymes are proteins that act as bio logical catalysts. There are different types ofenzymes in living organis m, and their activities are highly related to many biolo gicalprocesses, inc luding the growth, development and metabolis m o f all living organism.Cell is the functiona l basic unit of life, and its whole life process is closely linked withenzyme activities. Continuous growth and reproduction of cell could be obtained onlyby ma intaining the enzyme activity. The life process of cell will come to an end whenthe enzyme is deactivated. In addition, the enzyme activity is also closely related tomany huma n diseases. Therefore, enzyme activity assay is very import ant forproviding essential information. Here,we developed several nove l b iosensors for thedetection of important DNA-modifying enzyme in living organis m and the relatedinhib itor screening by means of electroche mistry, chemiluminescence and fluorescencespectroscopy. The proposed biosensors have several advantages, such ascost-effectiveness, easy operation, rapid response, high sensitivity and specificity. Thedetailed methods are as fo llows:In chapte r2, a sens itive electrochemical biosensor for detec tion o f DNAmethyltrans ferase activity by comb ining DNA methylation-sensitive cleavage andtermina l trans ferase-mediated-extension was develped. In this assay, all the steps wereperformed on the surface of an e lectrode. When the biosensor was used to screeninhib itors, the DNA MTase and methylation sensitive restrictive endonuc lease could beadded, respective ly, whic h would overcome the drawback in ma ny previous methods.The TdTase-mediated base extens ion techniq ue was used to incorporate biotinylateddUTP （dUTP-biotin） into the3’-OH terminal of the new primer produced by thecleavage of methylation sensitive restrictive endonuclease. Depending on the highexactitude of TdTase, the dUTP-biotin was only incorporated into the3’-OH terminal,thus it would reduce the unspecific reaction and yie ld a lower background. Theextended biotin could conjugate with the streptavid in-alkaline phosphatase （SA-ALP）.ALP catalyzed the conversion of electrochemicallyinactive1-naphthyl phosphate（1-NP） into an e lectroche mically active pheno l for the ge neration of a namp lifiedelectrochemica l signa l.The detection limit was0.04U/mL.In chapte r3, a novel DNAzyme-based colorimetric assay for the detection ofhOGG1activity with la mbda exonuc lease cleavage was developed. DNA duplex containing an8-oxoG was used as probe for hOGG1. The hOGG1selective ly cleavedthe duple x at the8-oxoG site, yie ld ing a new DNA duple x with a recessed5’-phosphate terminus. Digestion of the new DNA duplex by λ exo leaded to therelease of G-rich ssDNA. The ssDNA comb ined with hemin to formG-quadruple x–hemin complex and acting as a peroxidase-mimicking DNAzyme, whichcan catalyze H₂O₂-med iated oxidation of ABTS2-to generate the colored productABTS-（λmax=418nm）. The activity of hOGG1is directly related to UV/Visabsorption intens ity. The hOGG1concentratio n is quantied by monitoring the UV/Visspectra changes. The detection limitation of was0.01U/mL.In chapte r4, a sens itive fluorescence strategy for the detection of hOGG1activitybased on nick ing enzyme isotherma l amp lification and DNAzyme cascadeamplificatio n was developed. A duplex DNA probe with an8-oxoG was used as probefor hOGG1. After recognition with hOGG1, a hairp in DNA was formed for nickingenzyme and DNA polymerase to continuous ly generate a great deal of nicked products,which was as part of Mg²⁺-dependent DNAzyme. Next, the release nicked productshybridized with the quenched catalytic and molecular beacon （CAMB）. In the presenceof Mg²⁺，DNAzyme catalyzed the cleavage of the molecular beacon （MB） substrate,caus ing the quenched MB fluorophore/quencher pair to be separated, therebyproducing a dramatic increase of fluorescent signal. After enzymatic multip le turnoverof MB substrate, sensitive detection of hOGG1activity could be realized. Thedetection limit of this method was0.17U/mL.