RESUMO
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.
Assuntos
Biomarcadores Tumorais , Neoplasias , DNA , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Nanogéis , Neoplasias/tratamento farmacológicoRESUMO
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.
Assuntos
Neoplasias da Mama/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Grafite/química , Neoplasias Pancreáticas/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Difusão Dinâmica da Luz , Feminino , Grafite/farmacologia , Humanos , Células MCF-7 , Nanocompostos/química , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Pontos Quânticos/químicaRESUMO
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.
Assuntos
Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais/métodos , DNA/química , Endonucleases Flap/metabolismo , Nanoestruturas/química , Proteínas de Neoplasias/metabolismo , Neoplasias , Linhagem Celular Tumoral , Humanos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
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.
Assuntos
Endonucleases Flap/metabolismo , Endonucleases Flap/antagonistas & inibidores , Humanos , Especificidade por SubstratoRESUMO
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 (0.1g/mL oil/acetone, 24h, -80°C, precooled Büchner filtration) and interesterification (Lipozyme TL IM, N2 flow, 2.5h, 40°C) successfully doubled the omega-3 fatty acid content to 43.20 mol%. (1)H 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.
Assuntos
Óleo de Fígado de Bacalhau/química , Ácidos Graxos Ômega-3/química , Esterificação , SolventesRESUMO
Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures.
Assuntos
DNA Helicases/metabolismo , DNA Cruciforme/química , Quadruplex G , Dano ao DNA , Replicação do DNA , DNA de Forma B/química , DNA de Forma B/metabolismo , DNA Cruciforme/metabolismo , Microscopia de Força Atômica , Conformação de Ácido NucleicoRESUMO
It is demonstrated that positive supercoiling affiliated with nucleosome formation can act as the driving force to repair the G-quadruplex, cruciform as well as a stable non-B DNA structure caused by peptide nucleic acid.
Assuntos
DNA de Forma B/química , Nucleossomos/metabolismo , Reparo do DNA , DNA Cruciforme/química , Quadruplex G , Conformação de Ácido Nucleico , Nucleossomos/química , Ácidos Nucleicos Peptídicos/químicaRESUMO
It is demonstrated that the prokaryote-exclusively-owned DNA gyrase is capable of facilitating the generation of a G-quadruplex from a long perfectly matched duplex DNA at physiological concentrations of cations.
Assuntos
DNA Girase/metabolismo , DNA Circular/metabolismo , Quadruplex G , Plasmídeos/metabolismo , DNA Circular/química , Plasmídeos/químicaRESUMO
It was demonstrated in our studies that norfloxacin, a representative member of quinolone antibiotics, can indeed stabilize the gyrase-DNA complex formed during enzymatic cycle. In addition, the formation of the drug-induced complex has been firstly visualized through our atomic force microscopic examination.
Assuntos
DNA Girase/metabolismo , Microscopia de Força Atômica , Quinolinas/farmacologia , Antibacterianos/farmacologia , Eletroforese em Gel de Ágar , Estabilidade Enzimática/efeitos dos fármacos , Estrutura Molecular , Norfloxacino/farmacologiaRESUMO
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.