Non-unity molecular heritability demonstrated by continuous evolution in vitro

Introduction: When catalytic RNA is evolved in vitro, the molecule's chemic al reactivity is usually the desired selection target. Sometimes the phenot ype of a particular RNA molecule cannot be unambiguously determined from it s genotype, however. This can occur if a nucleotide sequence can adopt mult iple folded states, an example of non-unity heritability (i.e. one genotype gives rise to more than one phenotype). In these cases, more rounds of sel ection are required to achieve a phenotypic shift. We tested the influence of non-unity heritability at the molecular level by selecting for variants of a ligase ribozyme via continuous evolution. Results: During 20 bursts of continuous evolution of a 152-nucleotide ligas e ribozyme in which the Mg2+ concentration was periodically lowered, a nine -error variant of the starting 'wild-type' molecule became dominant in the last eight bursts. This variant appears to be more active than the wild typ e. Kinetic analyses of the mutant suggest that it may not possess a higher first-order catalytic rate constant, however. Examination of the multiple R NA conformations present under the continuous evolution conditions suggests that the mutant is superior to the wild type because it is less likely to misfold into inactive conformers. Conclusions: The evolution of genotypes that are more likely to exhibit a p articular phenotype is an epiphenomenon usually ascribed only to complex li ving systems. We show that this can occur at the molecular level, demonstra ting that in vitro systems may have more life-like characteristics than pre viously thought, and providing additional support for an RNA world.