Development Of New Assays For Enzyme Activity Based On Nucleic Acid Identification

As a biological catalytic function of biological macromolecules, enzymes exist inall the living animals and plants, dominating the organism’s metabolism process andmaintaining the body’s normal functions. They not only relevant for biological growth,genetic and nerve conduction, and other life activities, also, aging and cell damage inthe pathogenesis and various kinds of diseases such as cancer. In recent decades,people get more and more attention about the study of the enzymes activity. people notonly explore the mechanism between the enzymes and DNA, but also study a series ofdetermination method for enzymes activity, which brought a new dawn for the furtherresearches of the related performance and is of great significance in biology. Theresearch of biosensor is one of the most important topics in analytical chemistry inrecent years which has been applied in many fields widely. The develop DNAbiosensors using nucleic acids as molecular recognition elements based on thespecificity bases complementary hybridization in DNA molecules have attracted manyresearches attention. Since it has simple and rapid, high sensitivity, strong stability, aswell as good biocompatibility and has been widely applied in clinical medicine,environmental monitoring, etc. This paper mainly established several DNA biosensorswhich are simple and rapid, low cost sensitive and selective detection of RNAendonuclease activity of Argonaute2(Ago2) protein and uracil DNA glycosylase(UDG) activity.There are four chapters in this paper:1. The first chapter systematically introduces the basic components, detectionprinciple, classification, application and tendency of biosensors. And then introducesthe working principle, types and application of DNA biosensors, and most attentionis focued on the electrochemical DNA biosensors and fluorescent DNA biosensors. Subsequently, it introduces the graphene oxide nanomaterials (GO), a special DNAsecondary structure G-quadruplex and nicking endonucleasesc. Finally, summarizethe related content of Ago2protein and UDG briefly.2、A novel graphene oxide (GO)-based amplified fluorescence assay for RNAendonuclease activity of Mammalian Argonaute2(Ago2) was developed. Ago2protein and guide miRNA can form Ago2-miRNA complex which exhibits RNAendonuclesae activity. Owing to the extraordinarily high quenching efficiency of GOand the different affinity with single stranded DNA (ssDNA) containing differentamount of bases, the probe labeled with a fluorophore caboxyfluorescein (FAM)exhibits minimal background fluorescence, while strong fluorescence intensity isobserved with the addition of Ago2-miRNA complex due to the scission of the targetmRNA sequences and the release of FAM-linked oligonucleotide fragments.Furthermore, Ago2-miRNA complex may be released to initiate another cycle ofcleavage. The newly designed simple and cost-effective method exhibits highsensitivity with a detection limit of1.7nM (3σ) for Ago2endonuclease activity.3、A novel and label-free electrochemical sensing platform for sensitive detectionof uracil DNA glycosylase (UDG) activity has been developed, which is based onUDG–catalyzed removal of uracil bases inducing strand release with methylene blue(MB) as an electrochemical hybridization indicator. The capture DNA containing athiol group and its complementary single-strand DNA containing four uracil baseswere first self-assembly on the gold electrode whose surface was pre-modified withdendritic gold nanoparticles by direct electrodeposition. Subsequently, MB wasabundantly intercalated into the formed DNA duplex structure, resulting in ameasurable electrochemical signal. In the presence of UDG, the uracil bases werespecifically hydrolyzed, which could induce the unwinding of the DNA duplex,accompanied by the release of the intercalated MB from the electrode surface,leading to the corresponding proportional reduction of the redox signal.Experimental results revealed that the biosensor showed sensitive response to thelogarithm of UDG concentration in the range from0.025U mL–1to2U mL–1with adetection limit of0.012U mL–1. In addition, the proposed strategy shows advantagesof desirable sensitivity, high selectivity and low cost. It is the first example thatemploys the electrochemical assay for the detection of UDG activity.4、Specific and sensitive detection of base is essential to many fundamentalbiochemical process researches. Here, we propose a novel label-free homogeneous strategy for visualized excision repair enzyme activity activity assay based onnicking enzyme assisted signal amplification. In this method, uracil DNAglycosylase (UDG) as a model, two hairpin probes were employed for thecolorimetric detection, namely hairpin probe1(HP1) carrying two uracil residues inthe stem, and hairpin probe2(HP2) containing a G-riched DNAzyme segment, andthe recognition sequence as well as the cleavage site for the nicking enzyme. In thepresence of UDG, the uracil bases in the stem of HP1can be specifically recognizedand hydrolyzed by UDG, which leads to the followed series of reactions, and thencombine the function of nicking enzyme to induce the cycle process. Finally,generate enormous amount of G-riched DNAzyme segments which could bindhemin to form a catalytically active G-quadruplex-hemin DNAzyme which cancatalyze the H2O2-mediated oxidation of (ABTS2–) to the colored ABTS.–, providinga visible signal for UDG activity detection. This assay exhibits several advantagessuch as simplicity, low-cost, high selectivity and desirable sensitivity, which showsgreat potential of providing a promising platform for visualized analysis of otherbiomolecules.