Hongje Jang

A Graphene‐Based Platform for the Assay of Duplex‐DNA Unwinding by Helicase

Time to unwind: Graphene oxide (GO) enables the quantitative measurement of helicase‐dependent double‐stranded DNA (dsDNA) unwinding activity in real time. GO selectively binds to unwound fluorescent‐dye‐labeled single‐stranded DNA and quenches its fluorescence (see picture). The helicase activity is monitored by following the change in fluorescence.WILEY-VCH

The effective nuclear delivery of doxorubicin from dextran-coated gold nanoparticles larger than nuclear pores

To date, gold nanoparticles (AuNPs) have been investigated for diverse bioapplications. Generally, AuNPs are engineered to possess surface coating with organic/inorganic shells to increase colloidal stability in biological solutions and to facilitate chemical conjugation. In the present study, we developed a strategy to prepare dextran-coated AuNPs with control over its size by simply boiling an aqueous solution of Au salt and dextran, in which dextran serves as both reducing agent for AuNP (Au(0)) formation from Au(III) and AuNP surface coating material. The prepared dextran-coated AuNPs (dAuNPs) maintained its colloidal stability under high temperature, high salt concentration, and extreme pH. Importantly, the dAuNP remarkably improved efficacy of an anti-cancer agent, doxorubicin (Dox), when harnessed as a Dox delivery carrier. The half-maximal inhibitory concentration (EC50) of Dox-conjugated dAuNP with diameter of 170 nm was ∼9 pM in HeLa cells, which was 1.1 × 10(5) times lower than that of free Dox and lower than any previously reported values of Dox-nanoparticle complex. Interestingly, smaller AuNPs with 30 and 70 nm showed about 10 times higher EC50 than 170 nm AuNPs when treated to HeLa cells after conjugation with Dox. To achieve high cytotoxicity as cancer therapeutics, Dox should be delivered into nucleus to intercalate with DNA double helix. We show here that Dox-AuNPs was far more efficient as an anti-cancer drug than free Dox by releasing from AuNPs through spontaneous degradation of dextran, allowing free diffusion and nuclear uptake of Dox. We also revealed that larger AuNPs with lower degree of dextran crosslinking promoted faster degradation of dextran shells.

Discovery of Hepatitis C Virus NS3 Helicase Inhibitors by a Multiplexed, High‐Throughput Helicase Activity Assay Based on Graphene Oxide

A GO‐to solution: A simple graphene oxide (GO)‐based assay to screen for selective inhibitors of a hepatitisu2005C virus (HCV) helicase along with inhibitors of a severe acute respiratory syndrome coronavirus (SARS CoV) helicase was tested (see scheme). A single screen found five inhibitors highly selective for the HCV helicase orthologous to the SARS CoV helicase. Some of these hits were validated using the same GO‐based assay.WILEY-VCH

Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown

DNAzyme is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific manner and thus, silencing target gene. A particularly important challenge in achieving the successful down-regulation of gene expression is to efficiently deliver DNAzymes to disease sites and cells. Here, we report the nanoparticle-assisted functional delivery of therapeutic DNAzyme for the treatment of hepatitis C by inducing knockdown of hepatitis C virus (HCV) gene, NS3. HCV NS3 gene encodes helicase and protease which are essential for the virus replication. The nanocomplex showed efficient NS3 knockdown while not evoking undesired immune responses or notable cytotoxicity. We also demonstrated the DNAzyme conjugated nanoparticle system could be applicable in vivo by showing the accumulation of the nanoparticles in liver, and more specifically, in hepatocytes. We believe that the present work is a successful demonstration of effective, functional, non-immunostimulatory DNAzyme delivery system based on inorganic nanoparticles with high potential for further therapeutic application of DNAzyme in the treatment of hepatitis C.

Cytoprotective effects of graphene oxide for mammalian cells against internalization of exogenous materials

To date, graphene oxide (GO), an oxidized version of graphene, has been utilized in many research areas including bioapplications such as drug delivery and bioanalysis. Unlike other spherical or polygonal nanomaterials, GO exhibits a sheet-like structure, which in itself suggests interesting applications based on its shape. Here we show that GO can protect cells from internalization of toxic hydrophobic molecules, nanoparticles, and nucleic acids such as siRNA and plasmid DNA by interacting with cell surface lipid bilayers without noticeably reducing cell viability. Furthermore, the cytoprotective effect of GO against the internalization of extracellular materials enabled spatial control over gene transfection through region-selective gene delivery only into GO-untreated cells, and not into the GO-treated cells.

Graphene oxide for fluorescence-mediated enzymatic activity assays

Graphene oxide (GO), an oxidized 2-dimensional carbon material, has been studied actively in both academia and industry due to its unique physicochemical properties including high surface area, excellent aqueous dispersibility, quenching capability of contiguous fluorophores and adsorption affinity with diverse biomolecules. Particularly, GO has been widely used in biological applications involving small molecule biosensors, enzymatic reaction monitoring, drug delivery and therapeutic applications. Among these rapidly developing fields, we focused on the current advancements of fluorescence mediated activity assay platforms for enzymes including nucleases, methyltransferases, protein kinases, caspases and helicases by using affinity and fluorescence quenching capability of GO with fluorophore labelled biomolecule substrates in the aqueous solution phase, and its important applications with future perspectives in this article.

A new helicase assay based on graphene oxide for anti-viral drug development

Recently, graphene oxide (GO), one of the carbon nanomaterials, has received much attention due to its unique physical and chemical properties and high potential in many research areas, including applications as a biosensor and drug delivery vehicle. Various GO-based biosensors have been developed, largely based on its surface adsorption properties for detecting biomolecules, such as nucleotides and peptides, and real-time monitoring of enzymatic reactions. In this review, we discuss recent advances in GO-based biosensors focusing on a novel assay platform for helicase activity, which was also employed in high-throughput screening to discover selective helicase inhibitors.