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Directed Evolution of RNA and DNA Enzymes

G.F. Joyce, T.L. Sheppard, J.K. Rogers, J. Nowakowski, P.T. Ordoukhanian, X. Dai, R.K. Bruick, S.W. Santoro, K.E. McGinness, R. Kumar, M.W. Anderson, T.A. Staples

The principles of darwinian evolution can be applied to a large, heterogeneous population of RNA or DNA molecules to obtain particular molecules that have desired biochemical properties, including the ability to catalyze a particular chemical reaction. A population of variant molecules is subjected to repeated rounds of selective amplification in the test tube. Only those molecules that perform a chosen catalytic task are amplified, so that through successive rounds, the population adapts to the task at hand. We can select from among trillions of variant molecules in less than 1 hour. This ability enables us to evolve nucleic acids rapidly, compared with the rate at which whole organisms evolve.

Several years ago, beginning with a large pool of random-sequence DNA molecules and using in vitro selection, we produced the first example of a DNA enzyme. More recently, we used a similar procedure to obtain a general-purpose RNA-cleaving DNA enzyme that can operate under cellular conditions. The latter enzyme can be directed to cleave almost any RNA substrate in a sequence-specific manner. It has the highest catalytic efficiency of any known nucleic acid enzyme, even exceeding that of the protein enzyme ribonuclease A. The catalytic efficiency of the DNA enzyme is determined by the rate of association of the enzyme and substrate, which is limited only by the rate of duplex formation between complementary DNA and RNA molecules.

Another of our projects is the in vitro evolution of RNA enzymes that catalyze the joining of template-bound RNA molecules. This joining is a stepping stone to the development of RNA enzymes that catalyze the replication of RNA. Recently, we developed the ability to carry out continuous in vitro evolution of RNA enzymes with RNA-joining activity. During continuous evolution, RNA molecules that catalyze this reaction are immediately eligible for amplification, and newly produced RNAs are immediately eligible to catalyze another reaction. This situation enables us to maintain laboratory "cultures" of evolving RNA enzymes, analogous to the way cultures of bacteria are maintained. Thus, darwinian evolution can be continued indefinitely, by using a population of about 10¹³ molecules that are amplified with a doubling time of about 1.5 minutes.

We also used continuous in vitro evolution to provide a real-time model of the evolution of viral resistance. The RNA-cleaving DNA enzyme described earlier was directed to cleave RNA enzymes that were undergoing continuous evolution. The cleavage site within the RNA enzyme was at a position critical for substrate recognition. At first, the effect of the cleavage was devastating. No detectable survival of functional RNA enzymes occurred in the presence of 100 nM DNA enzyme. However, after 33 hours of continuous evolution, the RNA enzymes could tolerate 10 µM DNA enzyme. They had acquired specific mutations that conferred resistance to the DNA enzyme but did not disrupt their own catalytic function. On the basis of these mutations, a new DNA enzyme was constructed that regained the ability to cleave the RNA enzyme, renewing the game of molecular cat and mouse.

In another study, we used continuous in vitro evolution to study how a genetic macromolecule can alter its chemical composition while undergoing darwinian evolution. RNA molecules are composed of 4 building blocks: A, G, C, and U. The RNA enzyme that is the subject of continuous evolution cannot tolerate replacement of these building blocks by certain chemical analogs. For example, replacement of C by "sulfur-C," the phosphorothioate derivative of C, abolishes activity. When C is replaced by sulfur-C in the continuous evolution mixture, catalysis and replication come to a halt. However, by steadily increasing the proportion of sulfur-C relative to C, we were able to wean the evolving population over to sulfur-C (Fig. 1). Similarly, by increasing the proportion of sulfur-U relative to U, we were able to wean RNAs that already contained sulfur-C to a diet of A, G, sulfur-C, and sulfur-U.

These studies of RNA-based evolving systems are relevant to understanding the early history of life on Earth. It is thought that an RNA-based genetic system, often referred to as the "RNA world," preceded the DNA and protein-based genetic system that has existed on this planet for the past 3.6 billion years. One aim of our research program is to recapitulate the biochemistry of the RNA world in the laboratory. We are using in vitro evolution to explore the catalytic potential of RNA and, in particular, to search for RNA enzymes that have the ability to catalyze their own replication. In vitro evolution, most notably continuous in vitro evolution, provides a laboratory tool for re-creating functional aspects of the RNA world.

PUBLICATIONS

Joyce, G.F. Nucleic acid enzymes: Playing with a fuller deck. Proc. Natl. Acad. Sci. U.S.A. 95:5845, 1998.

Joyce, G.F., Orgel, L.E. The origins of life: A status report. Am. Biol. Teacher 60:10, 1998.

Joyce, G.F., Orgel, L.E. Prospects for understanding the origin of the RNA world. In: The RNA World, 2nd ed. Gesteland, R.F., Atkins, J.F. (Eds.). Cold Spring Harbor Press, Cold Spring Harbor, NY, in press.

Santoro, S.W., Joyce, G.F. Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry, in press.