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Scientific Report 2008

Molecular Biology

Directed Evolution of Nucleic Acid Enzymes

G.F. Joyce, S.E. Hamilton, D.P. Horning, T.A. Lincoln, B.J. Lam, B.M. Paegel, K.L. Petrie, S.B. Voytek

The scientific community will soon celebrate the 200th anniversary of the birth of Charles Darwin and the 150th anniversary of the publication of his seminal work On the Origin of Species by Means of Natural Selection. The principles of darwinian evolution are fundamental to understanding biological organization at the level of populations of organisms and for explaining the development of biological genomes and macromolecular function. In our laboratory, darwinian evolution has become a chemical tool for discovering and optimizing functional macromolecules in the test tube. We have developed powerful methods for the in vitro evolution of nucleic acids and are applying those methods to the discovery of molecules of biochemical and biomedical importance. In addition, we are studying the processes of darwinian evolution itself, carried out at the level of molecules rather than at the level of cells or organisms.

Continuous In Vitro Evolution

We have devised a system for the continuous in vitro evolution of RNA enzymes that have RNA-joining activity. The system operates at a constant temperature within a common reaction vessel. RNA enzymes in a population of trillions are challenged to attach themselves to an RNA substrate, and as a consequence, the reacted enzymes become amplified by polymerase proteins (also present in the reaction mixture) to generate progeny. The progeny enzymes in turn have the opportunity to perform the reaction, causing the population to expand exponentially. Whenever the supply of substrates becomes exhausted, a fresh supply of reactants can be provided, allowing exponential amplification to continue indefinitely.

Until recently, all continuous in vitro evolution experiments were done with a single species of RNA enzyme derived from the CL1 ligase. We recently established a second continuously evolving enzyme based on descendants of the DSL ligase. This new enzyme was propagated for hundreds of successive generations to optimize its catalytic activity.

Recently, we challenged the 2 distinct species of continuously evolving enzymes to operate within the same environment (Fig. 1). Initially, variants of the CL1 ligase dominated the mixture, prompting us to add an inhibitory molecule to modulate their growth. Subsequently, variants of the DSL ligase became dominant, and these too were kept in check by adding a species-specific inhibitor. Eventually, we succeeded in maintaining the 2 species without the use of inhibitors by supplying 5 different RNA substrates. Each of the 2 enzymes evolved to use different substrates as its preferred resource, exploiting distinct niches within the common environment. This situation is a demonstration of niche formation at the molecular level, analogous to processes of biological evolution essential for maintaining species diversity within natural ecosystems.
Fig. 1. Continuous coevolution of 2 distinct species of RNA enzymes with RNA-joining activity. Zigzag lines indicate the concentration of the CL1 (blue) and DSL (red) enzymes before and after each cycle of exponential growth and dilution (based on the concentration of the corresponding cDNA).

Self-Sustained Replication of RNA

The continuous in vitro evolution system depends on 2 protein enzymes, a retroviral reverse transcriptase and a bacteriophage RNA polymerase, to bring about the amplification of reacted RNA enzymes. Recently, we developed a system in which the RNA enzymes catalyze their own replication. We began with the R3C ligase developed previously in our laboratory. This molecule has a simple architecture amenable to various rearrangements. Previously, the R3C ligase was configured so that it would join 2 pieces of RNA to produce additional copies of itself, thus achieving RNA-catalyzed self-replication. Next, the enzyme was converted to a cross-catalytic format whereby 2 RNA enzymes brought about each other's synthesis from a total of 4 component RNA substrates. During the past year, we used in vitro evolution to enhance the activity of the cross-replicating RNA enzymes, improving their catalytic rate by more than 20-fold and increasing their extent of reaction from 15% to 90%. The resulting enzymes are able to undergo self-sustained exponential amplification at a constant temperature, achieving nearly billion-fold amplification in 30 hours.

The cross-replicating RNA enzymes can amplify themselves indefinitely in the absence of proteins or any other biological materials. As in the continuous evolution system, we allow the molecules to expand exponentially until the supply of substrates is exhausted and then provide fresh reactants to allow exponential amplification to continue indefinitely. We have prepared several versions of the cross-replicating enzymes that differ with respect to their genotype and corresponding phenotype. The genotype specifies the identity of the cross-replicating partners, and the phenotype is reflected in the catalytic properties of the molecules. In this way, we are striving to construct an artificial genetic system that can undergo self-sustained darwinian evolution. Thus far, we have shown selection among various cross-replicators that undergo exponential amplification within a common reaction mixture. Occasionally, a novel recombinant arises that amplifies more efficiently than either of its parents. With this system, it may be possible to explore alternative solutions to different environmental constraints, as occur in the natural evolution of biological organisms.


Joyce, G.F. Forty years of in vitro evolution. Angew. Chem. Int. Ed. 46:6420, 2007.

Paegel, B.M., Joyce, G.F. Darwinian evolution on a chip. PLoS Biol. 6:e85, 2008.

Voytek, S.B., Joyce, G.F . Emergence of a fast-reacting ribozyme that is capable of undergoing continuous evolution. Proc. Natl. Acad. Sci. U. S. A. 104:15288 2007.


Gerald F. Joyce, M.D., Ph.D.
Dean, Faculty

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