It is possible to evolve RNA enzymes in a continuous manner employing a simple serial transfer procedure. This method previously had been applied only to descendants of one unusually fast-reacting RNA enzyme with RNA ligase activity. This work establishes a second continuously evolving RNA enzyme, also with RNA ligase activity, but with a completely independent evolutionary origin. Once established, continuous evolution was carried out for 80 successive transfers, maintaining the population against an overall dilution of 10 207-fold. The resulting RNA enzymes exhibited ∼105-fold improvement in catalytic efficiency compared to the starting molecules, and had become dependent on a structural feature of the substrate that previously conferred no selective advantage. This adaptation was eliminated by deleting the substrate feature and then carrying out 20 additional transfers of continuous evolution, which resulted in molecules with even greater catalytic activity.;With two distinct "species" of continuously evolving enzymes established, it was possible to conduct molecular ecology experiments in which the two were made to compete for limited amounts of substrate within a common environment, with utilization of the substrate being necessary for amplification of the RNA. Evolution in the presence of a single substrate led to the extinction of one or the other enzyme, whereas evolution in the presence of five alternative substrates led to the accumulation of mutations that allowed each enzyme to exploit a different preferred resource. The evolved enzymes were capable of sustained coevolution within a common environment, exemplifying the emergence of stable ecological niche behavior in a model system. Biochemical characterization of the two evolved enzymes revealed marked differences in their kinetic properties and adaptive strategies. One enzyme reacted with its preferred substrate about 100-fold faster than the other, but the slower-reacting species produced two- to three-fold more progeny per reacted parent molecule. The in vitro coevolution of two or more species of RNA enzymes will make possible further studies in molecular ecology, including the exploration of more complex behaviors, such as predation or cooperation, under controlled laboratory conditions.
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