RESUMO
A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.
Assuntos
DNA/química , Inibidores Enzimáticos/química , Exonucleases/antagonistas & inibidores , Fluoresceína/química , Grafite/química , Sequência de Bases , Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Impressão Tridimensional , Espectrometria de Fluorescência/métodosRESUMO
Artificial, synthetic chaperones have attracted much attention in biomedical research due to their ability to control the folding of proteins and peptides. Here, we report bio-inspired multifunctional porous nanoparticles to modulate proper folding and intracellular delivery of therapeutic α-helical peptide. The Synthetic Nano-Chaperone for Peptide (SNCP) based on porous nanoparticles provides an internal hydrophobic environment which contributes in stabilizing secondary structure of encapsulated α-helical peptides due to the hydrophobic internal environments. In addition, SNCP with optimized inner surface modification not only improves thermal stability for α-helical peptide but also supports the peptide stapling methods in situ, serving as a nanoreactor. Then, SNCP subsequently delivers the stabilized therapeutic α-helical peptides into cancer cells, resulting in high therapeutic efficacy. SNCP improves cellular uptake and bioavailability of the anti-cancer peptide, so the cancer growth is effectively inhibited in vivo. These data indicate that the bio-inspired SNCP system combining nanoreactor and delivery carrier could provide a strategy to expedite the development of peptide therapeutics by overcoming existing drawbacks of α-helical peptides as drug candidates.
Assuntos
Chaperonas Moleculares , Peptídeos , Sequência de Aminoácidos , Chaperonas Moleculares/metabolismo , Peptídeos/química , Conformação Proteica em alfa-Hélice , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.
Assuntos
Edição de Genes , Nanopartículas , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Ribonucleoproteínas/genéticaRESUMO
Graphene oxide (GO), an oxidized derivative of graphene, has received much attention for developing novel fluorescent bioanalytic platforms due to its remarkable optical properties and biocompatibility. The reliable performance and robustness of GO-based biosensors have enabled various applications in the biomedical field including diagnosis and drug discovery. Here, recent advances in the development of GO-based fluorescent biosensors are overviewed, particularly nucleic acid detection and enzyme activity assay. In addition, practical applications in biomarker detection and high-throughput screening are also examined. Lastly, basic design principles and remaining challenges of these types of biosensors are discussed for further progress.
Assuntos
Técnicas Biossensoriais , Descoberta de Drogas , Corantes Fluorescentes/química , Grafite/química , Biomarcadores/análise , Corantes Fluorescentes/síntese química , Grafite/síntese química , Humanos , Estrutura MolecularRESUMO
RNA demethylase has recently been known to be associated with cancer development but its selective inhibitors as anti-cancer agents have rarely been investigated to date. Herein, we have developed a fluorescent nanobiosensor which enables efficient quantitative analysis of RNA demethylase ALKBH5 activity and shows a high potential for robust inhibitor screening.
Assuntos
Homólogo AlkB 5 da RNA Desmetilase/química , Técnicas Biossensoriais , Proteínas de Ligação a DNA/química , Endorribonucleases/química , Proteínas de Escherichia coli/química , Grafite/química , Nanopartículas/química , Desmetilação , Espectrometria de FluorescênciaRESUMO
MicroRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes. Therefore, control over the disease-related miRNA expression is important both for basic research and for a new class of therapeutic modality to treat serious diseases such as cancer. Here, we present a high-throughput screening strategy to identify small molecules that modulate miRNA expression in living cells. The screen enables simultaneous monitoring of the phenotypic cellular changes associated with the miRNA expression by measuring quantitative fluorescent signals corresponding to target miRNA level in living cells based on a novel biosensor composed of peptide nucleic acid and nano-sized graphene oxide. In this study, the biosensor based cellular screening of 967 compounds (including FDA-approved drugs, enzyme inhibitors, agonists, and antagonists) in cells identified four different classes of small molecules consisting of (i) 70 compounds that suppress both miRNA-21 (miR-21) expression and cell proliferation, (ii) 65 compounds that enhance miR-21 expression and reduce cell proliferation, (iii) 2 compounds that suppress miR-21 expression and increase cell proliferation, and (iv) 21 compounds that enhance both miR-21 expression and cell proliferation. We further investigated the hit compounds to correlate cell morphology changes and cell migration ability with decreased expression of miR-21.
Assuntos
Técnicas Biossensoriais/métodos , Grafite/química , Ensaios de Triagem em Larga Escala/métodos , MicroRNAs/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análiseRESUMO
Herein, hybrid nanocomposite of praseodymium doped TiO2 nanocrystals and graphene oxide nanosheets are prepared by facile hydrothermal treatment. As-synthesized Pr-TiO2 /NGO hybrid nanocomposite exhibits enhanced photocatalytic activity under visible light irradiation by the intact graphene oxide and doped lanthanide mediated band gap narrowing compared to TiO2 . Moreover, high payload and controlled release of doxorubicin by charge reversal of hybrid nanocomposite at endosomal pH and near-infrared irradiation mediated efficient photothermal conversion provide highly favorable features in therapeutic applications. Through the combination of these three distinctive therapeutic modalities, highly efficient trimodal cancer cell ablation is demonstrated.