The Skaggs Institute
for Chemical Biology
Evolution of Catalytic Nucleic Acids
G.F. Joyce, G.C. Johns, B.M. Paegel, G. Springsteen, S.B. Voytek
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
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.