Evolution in the Test Tube

Sol Spiegelman and his group carried out the first successful evolution experiments in vitro in the 1960s (for a review see, for example, Spiegelman, 1971). These experiments were based on replication of single-strand RNA through catalysis by a bacteriophage-specific enzyme, mostly Qp replicase. A sample of RNA molecules carrying the recognition site for the replicase is introduced into a buffer solution that contains the four nucleotide triphosphates (ATP, CTP, GTP, and UTP) as well as the replicase. Consumed materials are replenished through transfer of small samples into fresh stock solution. After a sufficient number of repeated transfers RNA molecules are isolated that are adapted to the stock solution, in the sense that they replicate most efficiently. Although the molecular details of the replication process and the nature of the optimization process were largely unknown in the early days, the interpretation of the serial transfer experiments as Darwinian evolution in the test was essentially correct. The Qp RNA evolution experiments were repeated and put on a firm molecular basis later on, and finally, they were applied to the design of RNA molecules with predefined properties (for a collection of reviews see Watts and Schwarz, 1997).

The mechanism of RNA replication by virus-specific enzymes like Qp replicase consists of two multistep RNA polymerization cycles, each synthesizing the complementary strand of an RNA template molecule, plus-I or minus-I, respectively (Biebricher and Eigen, 1988):

plus-I + activated monomers p minus-I + plus-I minus-I + activated monomers p plus-I + minus-I

After an initial period the plus-minus ensemble grows like a single entity with a rate parameter k = V(k+ X k_), where k+ and k_ are the parameters for the two reactions shown above. Depending on relative amounts of template and enzyme three different regimes of RNA replication are distinguished: (1) exponential growth of RNA concentration at excess concentration of the replicase, (2) linear growth of RNA concentration at moderate excess of template, and (3) saturation or approach to constant RNA concentration at large excess of template leading to product inhibition of the reaction. Darwinian selection occurs in phase (1) and in phase (2) but the selection criteria are different. Only the kinetic parameters, k+ and k_, are relevant in the exponential growth regime, whereas both the kinetic parameters and the binding equilibrium of template and replicase determine the reproductive success of molecules in the linear phase.

Two results of these studies turned out to be central for understanding the mechanisms of evolutionary optimization: (1) the Darwinian mechanism of optimization through variation and selection is not bound to the existence of cellular life, and (2) populations of RNA molecules under conditions that sustain plus-minus replication are optimized in test tube experiments provided RNA replication operates either in the exponential or the linear growth regime. Another important feature of RNA evolution in the test tube is that it is more system specific and cannot be readily extended to more complex evolving assays: the dichotomy of genotype and phenotype (variation occurs on genotypes being nucleic acid sequences and selection operates on phenotypes determining fitness) is confined to a single RNA molecule with the sequence as genotype and the structure as phenotype.

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