Complex RNA molecules are at the heart of a number of fundamental biological processes, including translation, tRNA maturation, and RNA splicing. [1] The unique threedimensional structures adopted by these molecules determine their activity and in some cases, such as the assembly of the spliceosome, a dynamic series of rearrangements of the RNA structure is required to assemble an active catalytic complex. Considerable progress has been made, using phylogenetic, X-ray, NMR, and chemical probing techniques, towards understanding the two-and three-dimensional structures adopted by large RNAs. [2±8] The mechanisms and pathways by which large RNAs fold into these structures are obviously of interest, and there is a need for novel approaches to study these processes.
One of the most powerful techniques for probing nucleic acid structure is chemical footprinting. Diffusable hydroxyl radicals produced from Fe-ethylenediaminetetraacetate (EDTA) or Cu-phenanthroline complexes effect cleavage of the phosphodiester backbone of both DNA and RNA; cleavage of radio-labeled molecules can be easily analyzed on high-resolution polyacrylamide gels. [9, 10] Protection against such cleavage through binding of a protein or formation of a higher order structure is detected as an area of reduced cleavage or footprint. These commonly used footprinting techniques may be regarded as thermodynamic since the radicals are typically generated over a 10 ± 60 minute time period and thus are useful for examining a system at equilibrium. Elegant kinetic footprinting studies were performed by Chance, Brenowitz, Woodson, and co-workers to elucidate the folding pathway of the Group I ribozymeÐ radicals were produced on a 50 ± 100 ms timescale by synchrotron X-ray irradiation of folding reactions and allowed the visualization of discrete intermediates on the folding pathway. [11] Our interest in the dynamics of RNA structure has prompted us to explore the application of other readily available chemical footprinting reagents to kinetic studies of RNA folding. Here we describe the first use of such a reagent, peroxynitrous acid, as a tool for examining the folding pathway of a complex RNA: the Tetrahymena ribozyme.
Peroxynitrous acid (HOONO) is unstable in aqueous solution, undergoing homolytic cleavage to generate hydroxyl radical and nitrogen dioxide, [12, 13] and has been employed in green color persisted for more than 20 min. Ethylene oxide was condensed to a liquid (0.21 mL) and added to the reaction mixture. The flask was sealed and heated at 45 8C for 8 h. After cooling to room temperature, the THF was removed by rotatory evaporation. Filtration through a plug of silica gel removed DMSO and naphthalene by-products (7: 0.61 g, 98 % yield). 1