Background

Catalytic RNAs, possessing both a genotype and a phenotype, are ideal molecules for in vitro evolution experiments. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification, and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population.

Results

Beginning with a pool of 10¹³ variants of the Tetrahymena ribozyme, we evolved ribozymes with the ability to cleave an RNA substrate in the presence of Ca²⁺, an activity that does not exist for the wild-type molecule. Over the course of 12 "generations", a seven-error variant emerged that has substantial Ca²⁺-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics: logarithmic, linear, and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme.

Conclusion

In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonably short time scale.