2.8 angstrom crystal structure of the malachite green aptamer

Previous in vitro selection experiments identified an RNA aptamer that reco gnizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactiva tion, we have determined the 2.8 Angstrom crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-b inding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize th e folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base q uadruple to effectively encapsulate the ligand. Even in the absence of spec ific stabilizing hydrogen bonds, discrimination between related fluorophore s and chromophores is possible due to tight packing in the RNA binding pock et, which severely limits the size and shape of recognized ligands. The sit e of laser-induced cleavage lies relatively far from the bound TMR (similar to 15 Angstrom). The unusual backbone conformation of the cleavage site nu cleotide and its high level of solvent accessibility may, combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanis ms for cleavage, such as conformational changes in the aptamer or long-rang e electron transfer between the bound ligand and the cleavage site nucleoti de. (C) 2000 Academic Press.