Structural and biochemical analysis of in vitro evolved RNA

The general objectives of this study were to analyze the essential relationship between form and function in structured RNA molecules. In particular, the aims were to (1) identify the role of divalent metals in the catalysis of an in vitro evolved self-alkylating ribozyme; and (2) use x-ray crystallography to solve the structure of an RNA aptamer specific for cyanocobalamin and investigate the mechanism of molecular recognition, any observed structural motifs, and the role of water and metal ions in defining tertiary structure.; Biochemical studies involving the self-alkylating ribozyme indicate a purely structural role for divalent metal ions in catalytic enhancement. It is shown that this role can be obviated by the addition of non-metallic structural stabilizers including spermidine and cobalt hexamine. Further, the degree of catalytic enhancement of different metal ions can be roughly correlated to their atomic radii and ability to stabilize RNA structure as measured indirectly by determining their relative effects on melting point.; Structural analysis of the vitamin B12 binder (1) provides examples of novel structural motifs, (2) demonstrates how RNA can use a water core to stabilize non-canonical base pairs and base triples, and (3) suggests a mechanism of binding unique among aptamers studied thus far. Through analysis of crystal contacts we can predict a novel loop/triplex docking motif that could be used to mediate interactions between domains of large biological RNA molecules. Results from a modified in vitro selection experiment show a context dependence for base triple formation.; These results expand our knowledge of RNA structure and function, further demonstrate the molecular adaptability of RNA, and confirm prior conclusions based on the role divalent metals play in naturally occurring ribozymes. In addition, these results set up ground rules for base triple formation, which (in the future) can be used to predict triplex formation and possibly overall tertiary structure.