Scientific Report 2008
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
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.
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.
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.