The Skaggs Institute
for Chemical Biology

Catalytic Nucleic Acids for Treating the Molecular Basis of Disease

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

Genetic information and the functional macromolecules that it encodes are the products of darwinian evolution based on natural selection. We have developed methods for evolving functional macromolecules under a controlled set of conditions. These methods allow us to produce molecules that conform to a chosen set of selection constraints. Our aim is both to understand the evolutionary processes by which functional macromolecules arise and to produce novel compounds that might have applications in biomedicine. Most of our work has concerned the in vitro evolution of RNA or DNA enzymes, taking advantage of the fact that these molecules have both genetic and catalytic properties and can readily applied to biological systems.

Continuous In Vitro Evolution

Previously, we developed a powerful method for the in vitro evolution of RNA enzymes that catalyze the joining of RNA molecules. With this method, all the processes of evolution occur continuously within a common reaction mixture. Enzymes that perform the reaction are amplified to produce “progeny” molecules, which in turn can perform another RNA-joining reaction. After about 15 minutes, as the supply of substrates becomes exhausted, a small portion of the population of evolving molecules is transferred to a fresh reaction vessel, allowing evolution to continue indefinitely. In this way, we have been able to “culture” evolving enzymes, analogous to the way one cultures bacteria.

The chief drawback of the continuous in vitro evolution system is the need for human intervention to prepare reagents and transfer material from one reaction vessel to the next. These steps are labor intensive, consume a substantial amount of reagents, and are difficult to implement with high precision. During the past year, we developed a microfluidic-based continuous evolution system that allows us to carry out “evolution on a chip” (Fig. 1).

Fig. 1. Microfluidic-based continuous evolution. A, Diagram (left) and operation (right) of the dilution circuit. The circular channel (1 cm in diameter) is pumped by mixing valves 1, 2, and 3. Fluidic channels connect the input and output reservoirs (Rin and Rout) to the circular channel via bus valves (I and O, respectively). Operation of the circuit was visualized by using fluorescein dye (i–vi). B, Continuous evolution of an RNA enzyme, involving 70 successive 10-fold dilutions. Each dilution was triggered when the enzyme concentration reached approximately 50 nM.

Evolution of RNA enzymes occurs within a volume of 50–500 nL that is confined to a microfluidic circuit within a fabricated glass wafer. The fluid is manipulated by pneumatically controlled membrane valves that are operated by a computer. The concentration of RNA enzymes is monitored continuously via a confocal laser microscope. Whenever the concentration reaches a predetermined threshold, a small aliquot is retained and diluted with a fresh supply of reagents. We are using this method to carry out in vitro evolution in an automated and highly precise manner.

Novel RNA and DNA Enzymes

In addition to the RNA enzyme with RNA-joining activity that is used in the continuous in vitro evolution system, we have developed other RNA and DNA enzymes that can join RNA or DNA substrates. These enzymes have potential applications in clinical diagnostics for target-specific amplification of nucleic acids. One of the RNA enzymes being studied is the R3C RNA ligase. We obtained the ancestor of this enzyme by in vitro evolution, starting from a population of random-sequence RNAs that contained only 3 different nucleotide building blocks: adenosine, guanosine, and uridine. The ancestral enzyme then was evolved to the R3C ligase, which contains all 4 building blocks: adenosine, guanosine, uridine, and cytosine. The ligase consists of 59 nucleotides and catalyzes the joining of 2 RNA substrates with a rate of 0.32 min^–1.

The R3C RNA enzyme prepared as a DNA molecule of the same sequence (by replacing uridine with thymidine) had no activity. Through a process of in vitro evolution, however, we were able to develop a DNA version of the enzyme. The DNA version contains 10 mutations relative to the starting sequence and operates with a catalytic rate of 0.05 min^–1. The DNA enzyme, which was evolved to join 2 RNA substrates, cannot join 2 DNA substrates. We now are using in vitro evolution to complete the transition from an RNA enzyme with RNA-joining activity to a DNA enzyme with DNA-joining activity.

We also are studying the DSL RNA enzyme, first developed by Tan Inoue and colleagues at Kyoto University in Japan. This enzyme joins 2 RNA substrates with a catalytic rate of 0.12 min^–1, about 3-fold slower than the rate of the R3C RNA enzyme. However, unlike the R3C enzyme, the DSL enzyme binds the 2 substrates through an uninterrupted region of Watson-Crick pairing. We are evolving the DSL enzyme, both to improve its catalytic rate and to reduce its dependence on specific binding interactions between the enzyme and substrates. Our aim is to evolve an optimized form of the enzyme that can undergo continuous in vitro evolution. A variant of the DSL enzyme with this behavior would allow us to pit 2 different “species” of RNA enzymes against each other in a molecular battle of the fittest.

Publications

Johns, G.C., Joyce, G.F. The promise and peril of continuous in vitro evolution. J. Mol. Evol. 61:253, 2005.

Joyce, G.F., Orgel, L.E. Progress toward understanding the origin of the RNA world. In: The RNA World, 3rd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2006, p. 23.