Development of graphene oxide based enzyme activity assays and its application for drug delivery

Abstract

Graphene oxide is one of the carbon nanomaterials which has been received much attention due to its unique physical and chemical properties and high potential in many research areas including application s as biosensors and drug delivery vehicles, recently. Various GO-based biosensors largely based on its surface adsorption properties, have been developed not only for the detection of biomolecules including oligonucleotides, peptides and essential proteins, but also for the monitoring of enzymatic reactions. Especieally we focused on the essential motro protein, helicase which unwind the double stranded nucleic acids into single stranded ones using energy from nucleoside triphosphate hydrolysis. They have been implicated in core cellular processes that require single stranded nucleic acids, including DNA replication, repair and transcription. Their position in vital process and highly conserved structures give advantages to use in disease treating target for drug discovery. Previously reported numerous helicase activity assay by direct observation and through the radio- and fluorescence- labeled approaches were contributed to unveiling helicase unwinding reaction and inhibitor screening, but they exhibited only poor activities in cell-based analysis. To overcome the current limitation of helicase activity assay platforms, we adopted the graphene oxide as trapping and sensing materials with fluorescenctly labeled substrates. Our newly reported graphene oxide based platform for the assay of duplex-DNA unwinding activity against SARS CoV helicase repre-sented several advantages over the conventional methods, including real-time monitoring, cost-effectiveness, easiness in preparation and assay, and high-throughput screening feasibility.

Next, we improved the system to multiplexed helicase assay for high-throughput screening of inhibitors of incurable disease target protein HCV NS3 helicase and SARS CoV helicase. The measuring of HCV and SARS CoV helicase activity was easily accomplished in a single mixed solution using two distinct DNA substrates tethered to different fluorophores. One round of library screening to discover highly selective small-molecules consisted antiviral direct acting drug candidates. In conclusion, a multiplexed screen by using graphene oxide based helicase activity assay platform is highly advfantageous not only because multiple rounds of screening are combinded into one screen to reduce the overall cost and labor, but also because just one round of multiplexed screening provides information on the relative selectivity of the hit compounds towards each target disease helicase. This multiplexed platform can be used to evaluate helicase activities in a highly parallel manner and of helicase-inhibitor-based antiviral drugs for various diseases.