In vitro evolution of a ribozyme that contains 5-bromouridine

The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reactio n compared to the behavior of the unmodified ribozyme. A population of 10(1 3) variant ribozymes was constructed, each containing 5-bromouridine in pla ce of uridine. Five successive 'generations' of in vitro evolution were car ried out, selecting for improved phosphoester transferase activity. The evo lved molecules exhibited a 27-fold increase in catalytic efficiency compare d to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutation s were found to be responsible for this altered behavior. These mutations e nhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out the ir catalytic function.