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Review
, 74, 17-23

Evolution in an RNA World

Affiliations
Review

Evolution in an RNA World

G F Joyce. Cold Spring Harb Symp Quant Biol.

Abstract

A long-standing research goal has been to develop a self-sustained chemical system that is capable of undergoing Darwinian evolution. The notion of primitive RNA-based life suggests that this goal might be achieved by constructing an RNA enzyme that catalyzes the replication of RNA molecules, including the RNA enzyme itself. This reaction was demonstrated recently in a cross-catalytic system involving two RNA enzymes that catalyze each other's synthesis from a total of four component substrates. The cross-replicating RNA enzymes undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of approximately 1 hour and can be continued indefinitely. Small populations of cross-replicating RNA enzymes can be made to compete for limited resources within a common environment. The molecules reproduce with high fidelity but occasionally give rise to recombinants that also can replicate. Over the course of many "generations" of selective amplification, novel variants arise and grow to dominate the population based on their relative fitness under the chosen reaction conditions. This is the first example, outside of biology, of evolutionary adaptation in a molecular genetic system.

Figures

Figure 1
Figure 1
Scheme for selective amplification of RNA molecules that catalyze the RNA-templated joining of RNA. The putative catalytic domain consists of random-sequence nucleotides that are attached to a template region, which is complementary to the 3′ end of an oligonucleotide substrate and to the 5′ end of the population of RNAs. Any RNA molecule that catalyzes ligation of the substrate to itself (curved arrow) will contain two primer binding sites (boxed regions) that are necessary for reverse transcription and PCR amplification.
Figure 2
Figure 2
Time course of continuous evolution of the class I RNA ligase enzyme in a serial transfer experiment involving 100 successive rounds of ~1,000-fold growth and 1,000-fold dilution. The concentration of RNA enzymes was measured before and after each transfer (zigzag line). The time between transfers was decreased as tolerated, initially 1 h and eventually 15 min (figure based on Wright and Joyce 1997).
Figure 3
Figure 3
Sequence and secondary structure of various forms of the R3C ligase enzyme. A, The enzyme (E) adopts a three-way junction structure upon binding two oligonucleotide substrates (A and B), which become ligated (curved arrow) to form the product. Conserved nucleotides that are essential for catalytic function are shown. B, The self-replicating or cross-replicating enzyme ligates two substrates to yield a new copy of the enzyme or its cross-catalytic partner, respectively. Open boxes indicate regions of Watson-Crick pairing between enzyme and substrates that can have any complementary sequence. C, The central stem-loop of the enzyme can be replaced by an aptamer domain, configured such that binding of the corresponding ligand is required to stabilize the active structure of the enzyme. The aptamer domains for theophylline (theo) and flavin mononucleotide (FMN) are shown.
Figure 4
Figure 4
Self-sustained cross-replication of the R3C RNA ligase enzyme in a serial transfer experiment. The concentrations of E (black) and E′ (gray) were measured before and after each transfer. A, A single cross-replicator was propagated for six successive rounds of ~25-fold growth and 25-fold dilution. B, A starting population of 12 different cross-replicators were propagated for 20 successive rounds of ~20-fold growth and 20-fold dilution, with the opportunity for recombination throughout (figure based on Jackson and Joyce 2009).
Figure 5
Figure 5
Ligand-dependent exponential amplification of cross-replicating RNA enzymes that contain the theophylline aptamer (see Figure 3C). A, Amplification of E (black) and E′ (gray) occurs in the presence of 5 mM theophylline, but not 5 mM caffeine (figure based on Lam and Joyce 2009). B, The exponential growth rate depends on the concentration of theophylline, which was either 50, 100, 200, or 500 μM. The chemical structure of theophylline is shown.

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