Using graphene to protect DNA from cleavage during cellular deliveryw

Genetic engineering holds great promise and opportunities for medicine and biomedical research. For example, RNA interference (RNAi) and antisense (AS) therapies are potentially powerful candidates for clinical treatments of various ailments, including cancer and AIDS. However, these therapeutic oligonucleotides are easily degraded by cellular enzymes or digested by cellular nucleases. Some measures have been taken to protect the oligonucleotides from cleavage and deliver oligonucleotides into cells, such as adding inhibitors in DNA solutions and complexing the nucleic acid with cationic polymers or lipids. Recently, inorganic nanomaterials have been applied as useful molecular transporters due to their unique properties such as large surface area, embedded effect and size effect. Some inorganic nanomaterials can protect oligonucleotides from cleavage due to their steric hindrance effect. Up to now, only a few types of nanomaterials have been used in oligonucleotides delivery, such as silica nanoparticles, gold nanoparticles and single-walled carbon nanotubes (SWCNTs). However, the search for new carriers which can protect oligonucleotides from cleavage and which possess low toxicity, is still highly active. Graphene, a single layer of carbon atoms in a closely packed honeycomb two-dimensional structure, is a new kind of carbon nanostructure material which was first produced in 2004. It has attracted great attention because of its remarkable electronic, mechanical and thermal properties. Graphene has been exploited in many applications, such as composites, Li-ion batteries, and electrochemical biosensors. However, little has been done to explore the use of graphene in the biomedical field. Recently, Dai and co-workers uncovered a unique ability of functionalized graphene in the attachment and delivery of aromatic, water-insoluble drugs. Most recently, we demonstrated the ability of water-soluble graphene oxide as a platform for highly sensitive and selective detection of DNA. However, the application of graphene as a transporter to deliver oligonucleotides for gene detection and therapy have not been reported yet. Herein, we first report that functionalized nanoscale graphene oxide (NGO) sheets can protect oligonucleotides from cleavage and deliver oligonucleotides into cells (Fig. 1). The model oligonucleotide used in this paper is molecular beacon (MB). MB is a hairpin-shaped DNA with a self-complementary stem that brings a terminal-labeled fluorophore and a quencher into close proximity, causing the fluorescence of the fluorophore to be quenched by energy transfer. When a MB hybridizes with its complementary target, the beacon undergoes a spontaneous conformational reorganization with the opening of the stem, leading to a fluorescence restoration. MBs have widespread use in visualization of mRNA expression in living cells owing to their unique structural property. The MB sequence used in this paper is 50-Dabcyl-CGA CGG AGA AAG GGC TGC CAC GTC G-Cy5-30, the loop of which was designed to incorporate a complementary region for the survivin transcript, a target that has received significant attention due to its potential use in cancer therapeutics and diagnostics. The functionalized nanoscale graphene oxide (NGO) was synthesized according to the report of Dai and co-workers. The sizes of NGO sheets were mostly lower than 100 nm according to atomic force microscopy (AFM) characterization (ESIw). This will increase the transfer efficiency of NGO. We then investigated the binding of MB to NGO. In a previous paper, we have proved that single-stranded DNA can be adsorbed onto graphene oxide sheets and graphene oxide can efficiently quench the fluorescence of the labeled fluorophore in single-strand DNA. In this work, we found NGO can also adsorb MB and decrease the background fluorescence of MB (Fig. 2(a) and (b)). However, in the presence of target DNA, the fluorescence intensity was significantly enhanced (Fig. 2(c)). This result indicated that the target DNA can