Scientific Report 2005

Molecular Biology

Directed Evolution of Nucleic Acid Enzymes

G.F. Joyce, T.A. Jackson, G.C. Johns, H.R. Kalhor, C.-Y. Lai, M. Oberhuber, B.M. Paegel, G.G. Springsteen, S.B. Voytek

All life known to exist on Earth today is based on DNA genomes and protein enzymes, but strong evidence indicates that it was preceded by a simpler form of life based on RNA. This earlier era is referred to as the “RNA world.” During that time, genetic information resided in the sequence of RNA molecules, and phenotype was derived from the catalytic behavior of RNA. By studying the properties of RNA in the laboratory, especially with regard to the evolution of catalytic function, we can gain insight into the RNA world. In addition, we can develop novel nucleic acid enzymes that have applications in biology and medicine.

SYnthesis and Derivatization of Ribose

Ribose, the sugar component of RNA, is a minor component among the many products of the condensation of formaldehyde. In addition, ribose is more reactive than most other sugars and degrades more rapidly than they do. Thus, it is difficult to understand why ribose is included in the genetic material.

We exploited the greater reactivity of ribose by allowing it to react preferentially with cyanamide to form a stable product. This product crystallized spontaneously in aqueous solution under a broad range of conditions; the corresponding cyanamides derived from other sugars did not. Furthermore, the ribose-cyanamide crystals reacted with cyanoacetylene to form cytosine α-nucleoside in nearly quantitative yield.

The RNA-catalyzed synthesis of ribose from simple starting materials would have been an essential reaction in the RNA world. We approached this problem by examining the ability of a nucleic acid template to direct the synthesis of ribose from 2 aldehyde-bearing oligonucleotides, one with glyceraldehyde at its 3´ end and the other with glycoaldehyde at its 5´ end. The 2 oligonucleotides were allowed to bind at adjacent positions along a complementary template, resulting in an aldol reaction that gave rise to pentose sugars (Fig. 1).

Fig. 1. RNA-directed synthesis of pentose sugars via aldol condensation. Two oligonucleotides, one with glyceraldehyde at its 3´ end (S1) and the other with glycoaldehyde at its 5´ end (S2), are joined in the presence of a complementary template to form a pentose-linked product.

No reaction was detected in the absence of the template. Adding lysine to the mixture increased the reaction rate substantially. This reaction will be used as the basis for in vitro evolution experiments to obtain RNAs that catalyze the formation of ribose.

Cross-Replicating RNA Enzymes

The central process of the RNA world was the RNA-catalyzed replication of RNA. We previously developed an RNA enzyme, termed the R3C ligase, that catalyzes the template-directed joining of 2 RNA molecules. This enzyme was converted to a format that allows it to produce additional copies of itself through the joining of 2 component subunits. The copies in turn give rise to additional copies, resulting in an exponential increase in the number of enzyme molecules over time. We further modified the reaction system so that it would operate cross-catalytically, whereby 2 RNA enzymes catalyze each other’s synthesis from a total of 4 substrates (Fig. 2).

Fig. 2. Cross-catalytic replication of RNA enzymes. The enzyme E binds the substrates S1´ and S2´ and catalyzes their joining to form the enzyme E´. Similarly, the enzyme E´ binds and joins the substrates S1 and S2 to form the enzyme E.

The newly formed copies of each enzyme give rise to additional copies of the cross-catalytic products, and the rate of formation of both enzymes increases during the course of the reaction. Currently, the cross-replicating system operates with a highly restricted set of RNA sequences, but it provides an opportunity for developing more efficient and more complex networks of replicating RNAs.

Continuous Evolution Of RNA Enzymes

Previously, we developed a powerful method for the in vitro evolution of RNA enzymes that catalyze the joining of RNA molecules. Rather than manipulating the RNAs through successive steps of reaction, selection, and amplification, we devised a way to have these steps occur continuously within a common reaction vessel. Evolution can be carried out indefinitely by a serial transfer procedure, whereby a small part of a completed reaction mixture is transferred to a new reaction vessel that contains a fresh supply of substrates and the other components necessary for selective amplification.

During the past year, we began 3 new lines of investigation involving continuous in vitro evolution. First, we modified the system so that an increased frequency of random mutations would occur during amplification. This modification allows us to generate and exploit genetic diversity within the system, providing a more realistic model of biological evolution. Second, using either 2 distinct variants of 1 enzyme or 2 different enzymes, we sought to evolve 2 different RNA enzymes within a common environment. These evolved enzymes will be used to study competition and cooperation in the context of RNA-based evolution.

Third, we implemented a novel microfluidic system for continuous in vitro evolution. In this system, the population of enzymes is confined to a microfluidic circuit within a fabricated glass wafer that contains a middle layer of an elastomeric material that functions as control valves. The concentration of RNA is monitored by using a confocal fluorescence microscope, and serial transfer is triggered automatically whenever the population size reaches a predetermined threshold. The microfluidic system makes it possible to conduct thousands of generations of in vitro evolution in a highly precise manner with little intervention by the experimenter.

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, in press.

Kim, D.-E., Joyce, G.F. Cross-catalytic replication of an RNA ligase ribozyme. Chem. Biol. 11:1505, 2004.

Paul, N., Joyce, G.F. Minimal self-replicating systems. Curr. Opin. Chem. Biol. 8:634, 2004.

Springsteen, G., Joyce, G.F. Selective derivatization and sequestration of ribose from a prebiotic mix. J. Am. Chem. Soc. 126:9578, 2004.