Stabilization of ssRNA on Graphene Oxide Surface: An Effective Way to Design Highly Robust RNA Probes Liang Cui, Zirong Chen, Zhi Zhu, Xiaoyan Lin, Xi Chen, and Chaoyong James Yang* State Key Laboratory of Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Science, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China * S Supporting Information ABSTRACT: RNA probes constitute an important class of functional nucleic acids (FNAs). However, because of their notorious vulnerability to enzymatic degradation, extremely careful and special protocols must be followed when dealing with RNA probes. To fully use the large number of RNA FNAs available for bioanalysis and biomedicine, it is important to explore effective methods to protect RNA probes from enzymatic digestion. In this work, we systematically demon- strate that graphene oxide (GO) can effectively protect RNA probes from enzymatic digestion. Based on this finding, we propose an effective way to design robust RNA biosensors by simply mixing RNA probes with GO for analysis of nucleic acids, proteins, and small molecules. The entire assay is sensitive, selective, rapid, and more importantly, does not require any special protocols. The ability to protect ssRNA from enzymatic digestion by GO offers an exciting new way to stabilize ssRNA, which will not only provide new opportunities to utilize the large number of currently available, yet rarely explored, RNA FNAs for bioanalysis but also offer a new solution to protect important ssRNA molecules, such as microRNA and antisense ssRNA, for a great variety of biomedical applications. O ver the last two decades, numerous functional nucleic acids (FNAs), including aptamers, riboswitches, ribo- zymes, and DNAzymes have been discovered. 1-3 These FNAs have found wide applications in bioanalysis and biomedicine, including biomolecule sensing, biomarker discovery, drug screening, targeted delivery, gene regulation, and disease diagnosis. 4-7 Specifically, a wide variety of FNA probes have been proposed for sensitive and selective detection of cells, nucleic acids, proteins, small molecules, and metal ions. 5,6 However, most of the reported probes are based on DNA, while RNA probes are rarely explored. Taking aptamers as an example, although there have been a great number of RNA aptamer sequences discovered, only a few RNA aptamers, such as VEGF, thrombin, and theophylline, have been utilized for biosensing. 8-10 The primary reason for this lack of popularity is the vulnerability of RNA probes to enzymatic degradation. Because extremely careful and special protocols must be followed when RNA probes are used, more stable DNA probes are preferred in bioassay development. 11-13 To fully use the large number of RNA FNAs available for bioanalysis and biomedicine, it is essential that effective methods to protect RNA from enzymatic digestion be explored. In recent years, a novel inorganic nanomaterial, graphene, has become extremely popular in nanoelectronic and biological applications. 14-16 It has been reported that graphene oxide (GO) can bind and quench dye-labeled single-stranded DNA (ssDNA) probes, while it has less affinity toward double- stranded DNA (dsDNA) or secondary and tertiary structured ssDNA. 17,18 On the basis of this finding, GO has been used for the detection of a variety of analytes. 14,19,20 It has also been found that DNA absorbed on GO surfaces can be effectively protected from nuclease digestion. For example, Lin’s group reported that ssDNA absorbed on GO surfaces can be effectively protected from enzymatic cleavage by DNase I. 21 Zhang’s group and Li’s group found that in the presence of sufficient GO, dsDNA can also be adsorbed and exhibit enhanced resistance to several types of nucleases. 22,23 The excellent protective property of GO against DNA nuclease digestion has, thus, further extended the applications of DNA probes both in vivo and in vitro. 24-27 Furthermore, dsRNA has been immobilized on positively charged functionalized GO via electrostatic interaction with the aid of 1-pyrenemethylamine hydrochloride or polyethylenimine for siRNA delivery. 28,29 The ssRNA has also been immobilized on GO via directed chemical conjugation 30 for toxin sensing. It has been found that ssRNA chemically immobilized on GO can be effectively protected against nuclease digestion. Surprisingly, until now, the non- covalent binding ability of ssRNA to GO and the nuclease resistance property of the resulting ssRNA/GO complex has not been explored. In an attempt to develop an effective method to construct highly stable RNA probes for biosensing and bioapplications, in this work, the binding ability and stability of ssRNA on GO was Received: October 31, 2012 Accepted: January 16, 2013 Article pubs.acs.org/ac © XXXX American Chemical Society A dx.doi.org/10.1021/ac303179z | Anal. Chem. XXXX, XXX, XXX-XXX