Studies On New Methods For The Detection Of Nucleic Acid Modifying Enzymes Activity Based On The Signal Amplification Technology Of Exonuclease And Nanomaterials

Biosensor technology with the advantages of has short analysis time, cost-efficiency, good selectivity and high sensitivity, has greatly promoted the application of Analytical Chemistry in the field of life sciences. Due to the advantages of without isolation, simple operation, can be performed in-situ measurement, low cost and so on, fluorescence sensing technology has been widely applied for detection of nucleic acids, proteins, bioactive small molecules, viruses, etc. The introduced Nanomaterials with special optical and electrical characteristics provides a new perspective to construct the optical biosensor for Analytical Chemistry. In recent years, nucleic acid-mediated signal amplification method provides for the highly sensitive detection of biological important technical support, it has been widely used in various types of biomolecules detection. Gene modification process is closely related with the development of cancer, and genetic modification subject to various nucleic acid modifying enzymes coordinately regulated. Therefore, to establish new methods of nucleic acid modification techniques activity detection is important in the early diagnosis and treatment of cancer and other serious diseases, as well as the pathogenesis of cancer research.In this hesis, nanomaterials and nucleic acid signal amplification technology were employed for fluorescence sensing system. This thesis has developed threenew methods for detecting Dam methyltransferase. base excision repair enzyme activity analysis and Inhibitor screening. The main contents of this work can be concluded as follows:Part one:In this work, a simple fluorescence strategy based on the graphene oxide (GO) platform and T7 exonuclease (T7 Exo)-assisted cyclic signal amplification is developed for the fast and sensitive detection of DNA methyltransferase (MTase) activity and inhibition. In the sensing design, Dam MTase was used as amodel analyte. In the presence of Dam MTase. a hairpin probe (HP) was methylated, and then speciallyrecognized and cleaved by Dpn I endonuclease, releasing a ssDNA fragment. The released ssDNA subsequently hybridized witha FAM-labeled signal probe (DP) to form a duplex with a blunt 5’-terminal of DPand a 4-mer overhang at the 5’-end of the released ssDNA. This would trigger the T7 Exo-assisted cyclicsignal amplification by repeating the hybridization and digestion of DP, liberating the fluorophore. The liberated fluorophore could not be adsorbed on the GO surface due to low affinity and the fluorescence signal was retained. In contrast, no enzymatic degradation of the DP occurred in the absence of Dam MTase. Thus the intact DP was then adsorbed on the GO surface, resulting influorescence quenching. By combining the efficient digestion ability of T7 Exo and the super fluorescence quenching efficiency of GO, the present strategy exhibits a high signal-to-background ratio, providing a satisfying sensitivity for the Dam MTase activity assay. In addition, this method does not require a specific recognition sequencefor enzymatic cyclic amplification and dual labels with fluorophore/quencher pairs, making the designeasy and low cost. Furthermore, the proposed method was also applied to assay the inhibition of Dam MTase activity.Part two:Herein, we introduced a tungsten disulfide (WS2) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity.Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as thesubstrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3-terminal of DP. This design facilitated the Exo Ill-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS2 nanosheet. Thus, the final system exhibited a small FP signal.However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS2 nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, thismethod was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics.Part three:We introduced a simple, fast and label free fluorescent assay for uracil DNA glycosylase activity based on the signal amplification of exonuclease I. In our design, a hairpin-structured probe (HP) with a blunt 3-end in the duplex stem was used as the UDG. Four uracil deoxyribonucleotides were modified in the stem region. In the absence of UDG, the HP probe with a hairpin-structure was the predominant form. SG I dye would bind with the stem region by the both intercalation andminor groove binding, producing a strong fluorescence signal. After adding the UDG, uracil bases were removed from the deoxyribose phosphate backbone of HP. AP sites were generated simultaneously, which lowered the melting temperature of HP. Thereby, the HP probe was opened to form a single strandstructure. The Exo I would then catalyze the digestion of new generated ssDNA into mononucleotides. Taking the advantage of the weaker affinity between SG I and mononucleotides, the fluorescence signal of final system was further remarkably reduced. This method exhibits a high sensitivity for UDG activity assaywith a low detection limit of 0.0070 U mL. The developed method exhibits an approving performance in complex biological samples, and can be usedfor evaluating inhibition effect of 5-fluorouracil on UDG activity.