205 Ultrathin nanopores for structural analysis of small nucleic acids

Abstract

Pinpointing the mechanisms behind function in biological macromolecules is essential for understanding the emerging and evolving nature of life. Biological macromolecules have evolved over billions of years to function efficiently in the highly heterogeneous, crowded environment of a cell. In particular, non-coding ribonucleic acids (RNAs) and deoxyribonucleic acids (DNAs) are omnipresent in cells, preforming both regulatory and catalytic functions by virtue of their structure. One class of such nucleic acids, ironically termed “junk DNA,”1 is significantly involved in orchestrating gene regulation. The RNA subunits of the ribosome2 are an integral part of the translational machinery for protein synthesis. Many ribozymes and other viral RNAs such as the hammerhead ribozyme and the canonical internal ribosome entry site3 exhibit enzymatic activity. Although the exact mechanisms remain unclear, the uniting theme in these non-coding nucleic acids is that their tertiary (spatial) structure yields specific chemical activities and functions. While existing techniques (e.g. nuclear magnetic resonance, X-ray crystallography) provide detailed structural information, inherent drawbacks such as ensemble averaging errors, crystallization artifacts, low time resolutions, and the need for ample amounts of material limit the availability and relevancy of the obtained structural information. Bioinformatics-based tools aim to bridge the gap in knowledge by proposing a homology-based approach to structural prediction, although viable experimental techniques are required in order to unequivocally support and improve such predictions. In this talk, I will present our group's efforts to fabricate nanoscale pore devices for extracting useful structural information about nucleic acids that display in vivo function. Electron-beam irradiation of various thin freestanding membranes affords nanopores with controlled dimensions and interfacial properties, to a quality level that allows highly sensitive analysis of individual nucleic acids in solution at high-throughput. I will describe the properties of our nanopores, as well as some of our recent explorations that have permitted the analysis of DNA and RNA structures.