Hierarchical Accessibility of Double-Helical RNA and DNA Processing Signals in Highly Dense Self-Assemblies

Abstract

The ability to accurately detect biomolecules in single-cell amounts is an important goal of basic and translational research. The inherent capability of nucleic acids to self-assemble allows the spontaneous formation of highly dense, self-assembled monolayers (SAMs). These assemblages can provide the basis for development of nano-based devices with programmable, single-molecule detection capability. Double-stranded(ds) RNA is an important biomarker of viral infection and certain cancers. While dsRNA behavior in solution has been extensively characterized by diverse physicochemical approaches, the properties of highly dense assemblies of dsRNA are largely unknown, and may be qualitatively different from those in solution. High-density, 39 bp, chimeric dsRNA:dsDNA (ds-chimera) SAMs have been prepared on modified gold surfaces. using atomic force microscopy (AFM)-based nanomanipulation and analysis, and fluorescence microscopy, we demonstrate the preferential binding of ethidium to the ds-chimera SAM compared to the single-stranded form. As revealed by AFM detection of height change, the ds-chimera SAM is reactive towards the dsRNA-specific RNase III of Aquifex aeolicus (Aa-RNase III) and restriction endonuclease BamHI, each having a recognition site in the dsRNA and dsDNA segments, respectively. We also show that the reactivity of the BamHI cleavage site, which is proximal to the gold surface, can be controlled by SAM density, and that access to the BamHI site is dependent upon the prior action of RNase III at its site in the center of the ds-chimera. These results reveal novel properties of protein-nucleic acid interactions within a high-density array environment that are relevant to nanoscale detection methodologies.