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The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Evolution of Catalytic Nucleic Acids

G.F. Joyce, G.C. Johns, B.M. Paegel, G. Springsteen, S.B. Voytek

Biochemists traditionally have treated biological enzymes as found objects and have sought to describe the structural and functional properties of the enzymes in much the same way that a naturalist would describe different species of organisms. With the emergence of molecular genetics and comparative structural biology, enzymes increasingly are regarded as descendants of a common evolutionary heritage. We seek to understand the processes by which enzymes evolve. We have developed technologies that allow us to evolve nucleic acid enzymes in the test tube. Nucleic acids are especially amenable to in vitro evolution because they have both genetic and catalytic properties. In addition, they are naturally occurring biomolecules and thus can readily be brought to bear on biological systems. Our work has resulted in novel RNA and DNA enzymes that have interesting properties and potential applications in biomedicine.

Continuous In Vitro Evolution

In our most powerful method for in vitro evolution, we use RNA enzymes that catalyze the joining of RNA molecules and can evolve in a continuous manner. With this method, all the processes of evolution occur within a common reaction mixture. The only intervention required is refreshing the supply of reaction materials periodically, usually by transferring a small part of a completed reaction mixture to a fresh reaction vessel. In this way, we can “culture” evolving enzymes, just as one would culture cells, but in an entirely acellular context.

Recently, we made a significant advance in continuous in vitro evolution by doing away with intervention by the experimenter. Such intervention is laborious and introduces an element of artificiality and imprecision in the evolution process. We have used microfluidic technology to carry out the evolution of RNA enzymes in precisely metered volumes of a few hundred nanoliters that are confined to fluidic circuits within a fabricated glass wafer. This mode of “evolution on a chip” allows continuous monitoring of the evolving population via a confocal laser microscope and relies on computer-controlled microvalves to supply the reagents as needed. It has enabled us to witness darwinian evolution in real time. For example, when we reduced the concentration of the substrate for the RNA-joining reaction, the evolving population adapted to the more stringent conditions within several hours.

We are carrying out 3 other projects related to continuous in vitro evolution. The first involves the development of on-chip methods for random mutagenesis of nucleic acids. In this project. we use deoxyribonucleotide analogs that increase the error rate of the polymerase enzymes used to carry out RNA amplification. The second project concerns the development of a second “species” of RNA enzyme that can undergo continuous in vitro evolution. With a second species, 2 distinct species could coevolve under a common set of environmental conditions, providing an experimental means for testing hypotheses about evolutionary competition and cooperation. A third line of research concerns expanding the function of the continuously evolving RNA enzymes so that they catalyze multiple RNA-joining reactions involving short oligonucleotide substrates. This expansion will facilitate the development of RNA enzymes that can act as both ligases and polymerases.

Novel RNA and DNA Enzymes

We are using conventional in vitro evolution methods to develop other RNA and DNA enzymes, especially enzymes that can catalyze the template-directed joining of RNA or DNA substrates. Such enzymes have potential applications in clinical diagnostics pertaining to the target-specific amplification of nucleic acids. For example, the R3C RNA enzyme, developed previously in our laboratory, catalyzes the joining of 2 RNA substrates, 1 with a 3′ hydroxyl and 1 with a 5′ triphosphate (Fig. 1A). Recently, we converted this enzyme to a corresponding DNA enzyme through in vitro evolution. The resulting DNA enzyme has activity similar to that of its RNA ancestor but with a distinct sequence and slightly modified structure.

We also are studying the DSL RNA enzyme (Fig. 1B), first developed by T. Inoue and colleagues at Kyoto University, Kyoto, Japan. It too catalyzes the joining of 2 RNA substrates but with a relatively slow catalytic rate and a restricted range of reaction conditions. We added 35 random-sequence nucleotides to this enzyme and through in vitro evolution improved its catalytic rate by more than 150-fold. This increase was achieved by applying stringent selection pressure, with a rapid-quench method to select molecules that could react in less than 0.1 second. We are continuing the evolution process to explore the performance limits of this enzyme.

Fig. 1. Two RNA enzymes with RNA-joining activity. A, The R3C enzyme contains 72 nucleotides. B, The DSL enzyme contains 109 nucleotides plus an insertion of 35 random-sequence nucleotides (N35). The curved arrows indicate the site of the reaction.


Oberhuber, M., Joyce, G.F. A DNA-templated aldol reaction as a model for the formation of pentose sugars in the RNA world. Angew. Chem. Int. Ed. 44:7580, 2005.

Paul, N., Springsteen, G., Joyce, G.F. Conversion of a ribozyme to a deoxyribozyme through in vitro evolution. Chem. Biol. 13:329, 2006.

Paegel, B.M., Grover, W.H., Skelley, A.M., Mathies, R.A., Joyce, G.F. Microfluidic serial dilution circuit. Anal. Chem. 78:7522, 2006.


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

Joyce Web Site