Evolution of RNA enzymes occurs within a volume of 50500 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 min1.
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 min1. 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 min1, 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.
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.