Studies of DNA-interacting properties of certain topological enzymes

Topoisomerases are topological enzymes that are involved in the regulation of DNA supercoiling. In this thesis, two of the type IIA topoisomerases, Topoisomerase II and DNA Gyrase, will be discussed and their DNA-interacting properties will be studied. Firstly, the change in topology of Kinetoplast DNA (kDNA) catalysed by Topoisomerase II was examined. With the aid of Atomic Force Microscopy (AFM), the different forms of kDNA during the decatenation process were observed. It was found out that the optimum conditions for gel electrophoresis were set at 0.5 unit of human Topoisomerase II alpha at the reaction time of 15 minutes. Furthermore, AFM images of two minicircles and three minicircles kDNA molecules were observed. We have concluded that the decatenation process of kDNA by Topo II is a ‘step-wise’ reaction that involved one or more reaction intermediates. In the next part of the studies, the development of stable duplex oligonucleotides and its inhibitory effect on DNA gyrase were examined. Due to the increase in bacterial resistance to antibacterial agents, it is important to explore new types of antibacterial agents. One of the “dumbbell-shaped” oligonucleotides that we have designed has been shown to have inhibitory effect on DNA gyrase and has an IC50 value of 31.62 nM. In addition, it is demonstrated that the positions of the nick sites on the oligonucleotides will affect the inhibitory action on E.coli gyrase significantly. The DNA-interacting properties of the two enzymes, Topoisomerase II and DNA gyrase were explored. These studies and findings have increased our knowledge between the topological properties of DNA and these enzymes. In addition, the information provided may contribute and benefit to the future studies of these topological enzymes.Master of Scienc

Enrichment of omega-3 fatty acids in cod liver oil via alternate solvent winterization and enzymatic interesterification

Enrichment of omega-3 fatty acids in cod liver oil via alternate operation of solvent winterization and enzymatic interesterification was attempted. Variables including separation method, solvent, oil concentration, time and temperature were optimized for the winterization. Meanwhile, Novozyme 435, Lipozyme RM IM and Lipozyme TL IM were screened for interesterification efficiency under different system air condition, time and temperature. In optimized method, alternate winterization (0.1 g/mL oil/acetone, 24 h, −80 °C, precooled Büchner filtration) and interesterification (Lipozyme TL IM, N2 flow, 2.5 h, 40 °C) successfully doubled the omega-3 fatty acid content to 43.20 mol%. 1H NMR was used to determine omega-3 fatty acid content, and GC–MS to characterize oil product, which mainly contained DHA (15.81 mol%) and EPA (20.23 mol%). The proposed method offers considerable efficiency and reduce production cost drastically. Oil produced thereof is with high quality and of particular importance for the development of omega-3 based active pharmaceutical ingredients.MOE (Min. of Education, S’pore)Accepted versio

Cancer biomarker-triggered disintegrable DNA nanogels for intelligent drug delivery

Even though various techniques have been developed thus far for targeted delivery of therapeutics, design and fabrication of cancer biomarker-triggered disintegrable nanogels, which are exclusively composed of nucleic acid macromolecules, are still challenging nowadays. Here, we describe for the first time our creation of intelligent DNA nanogels whose backbones are sorely disintegrable by flap endonuclease 1 (FEN1), an enzymatic biomarker that is highly overexpressed in most cancer cells but not in their normal counterparts. It is the catalytic actions of intracellular FEN1 on bifurcated DNA structures that lead to the cancer-specific disintegration of our DNA nanogels and controlled release of drugs in target cancer cells. Consequently, the brand-new strategies introduced in the current report could break new ground in designing drug carriers for eliminating unwanted side effects of chemotherapeutic agents and live-cell probes for cancer risk assessment, diagnosis, and prognosis

Graphene quantum dot-based nanocomposites for diagnosing cancer biomarker APE1 in living cells

As an essential DNA repair enzyme, apurinic/apyrimidinic endonuclease 1 (APE1) is overexpressed in most human cancers and is identified as a cancer diagnostic and predictive biomarker for cancer risk assessment, diagnosis, prognosis, and prediction of treatment efficacy. Despite its importance in cancer, however, it is still a significant challenge nowadays to sense abundance variation and monitor enzymatic activity of this biomarker in living cells. Here, we report our construction of biocompatible functional nanocomposites, which are a combination of meticulously designed unimolecular DNA and fine-sized graphene quantum dots. Upon utilization of these nanocomposites as diagnostic probes, massive accumulation of fluorescence signal in living cells can be triggered by merely a small amount of cellular APE1 through repeated cycles of enzymatic catalysis. Most critically, our delicate structural designs assure that these graphene quantum dot-based nanocomposites are capable of sensing cancer biomarker APE1 in identical type of cells under different cell conditions and can be applied to multiple cancerous cells in a highly sensitive and specific manners. This work not only brings about new methods for cytology-based cancer screening but also lays down a general principle for fabricating diagnostic probes that target other endogenous biomarkers in living cells.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)This work was supported by the Agency for Science, Technology and Research, Singapore (SERC A1883c0007 to T.L.); Zhejiang University (the start-up grant to F.S.); and Singapore Ministry of Education (RG122/18(S) and RG117/ 17(S) to T.L.)

Investigation of human flap structure-specific endonuclease 1 (FEN1) activity on primer-template models and exploration of a substrate-based FEN1 inhibitor

Flap structure-specific endonuclease 1 (FEN1) is one of the enzymes that involve in Eukaryotic DNA replication and repair. Recent studies have proved that FEN1 is highly over-expressed in various types of cancer cells and is a drug target. However, a limited number of FEN1 inhibitors has been identified and approved. Herein, we investigate the catalytic activity of FEN1, and propose a substrate-based inhibitor. As a consequence, one of the phosphorothioate-modified substrates is proved to exhibit the most efficient inhibitory effect in our in vitro examinations. A novelly-designed substrate-based FEN1 inhibitor was accordingly constructed and determined a remarkable IC50 value.MOE (Min. of Education, S’pore)Accepted versio

Versatile types of DNA-based nanobiosensors for specific detection of cancer biomarker FEN1 in living cells and cell-free systems

Flap structure-specific endonuclease 1 (FEN1) is overexpressed in various types of human cancer cells and has been recognized as a promising biomarker for cancer diagnosis in the recent years. In order to specifically detect the abundance and activity of this cancer-overexpressed enzyme, different types of DNA-based nanodevices were created during our investigations. It is shown in our studies that these newly designed biosensors are highly sensitive and specific for FEN1 in living cells as well as in cell-free systems. It is expected that these nanoprobes could be useful for monitoring FEN1 activity in human cancer cells, and also for cell-based screening of FEN1 inhibitors as new anticancer drugs.MOE (Min. of Education, S’pore)Accepted versio

An Alternative Method for Evaluating Stabilities of DNA Hairpin Structures

We have developed an alternative methodology for determining the stabilities of DNA hairpin structures based on a specially designed oligonucleotide template and the T4 DNA ligase. This method was systematically tested by evaluation of DNA hairpin stabilities when varying the loop size and the nature of the loop sequence.MOE (Min. of Education, S’pore

Interference of intrinsic curvature of DNA by DNA-intercalating agents

It has been demonstrated in our studies that the intrinsic curvature of DNA can be easily interrupted by low concentrations of chloroquine and ethidium bromide. In addition, the changes of DNA curvature caused by varying the concentration of these two DNA intercalators can be readily verified through using an atomic force microscope