Pyrite footprinting of RNA.

ABSTRACT

In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ((·)OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS(2)) can produce sufficient (·)OH to footprint DNA. The 49-nt Diels-Alder RNA enzyme catalyzes the C-C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme's active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels-Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to (·)OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop's flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated (·)OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.