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The Skaggs Institute
for Chemical Biology


Scientific Report 2008




Continuous In Vitro Evolution of RNA Enzymes

G.F. Joyce, B.J. Lam, T.A. Lincoln, B.M. Paegel, K.L. Petrie

We have devised methods for evolving nucleic acid enzymes in the test tube. These methods have enabled us to develop a variety of RNA and DNA enzymes, some of which have had applications in biomedicine. We continue both to advance the technology of directed molecular evolution and to seek novel applications for our evolved enzymes.

Advanced Directed Evolution Technology

Most in vitro evolution studies involve a powerful but laborious process in which a population of molecules is first challenged to perform a biochemical task, segregated on the basis of whether or not the molecules performed the task, and then amplified to produce "progeny" molecules that resemble but are not identical to their parents. This entire set of procedures is repeated until the population adapts to the task at hand, a process typically requiring weeks to months to complete. We have developed methods that allow us to carry out evolution in a continuous manner, within a single reaction mixture. The only required manipulation is to refresh periodically the supply of reagents, a task that is accomplished by either a serial transfer or serial dilution procedure. The continuous evolution method allows adaptation to occur within a period of only a few days.

Recently, we developed a marked enhancement of continuous in vitro evolution in which the process is carried out within a computer-controlled microfluidic chip. The circuits on the chip contain a population of billions of RNA enzymes with RNA-joining activity, and these molecules can be challenged to adapt to various imposed selection constraints. The growth of the population is monitored continuously by using a laser confocal microscope. Whenever the population size reaches a predetermined threshold, chip-based operations are executed to isolate a fraction of the population and mix it with fresh reagents. In a recently published article, we described the first example of "evolution on a chip," in which a population of RNA enzymes underwent 500 iterations of 10-fold exponential growth followed by 10-fold dilution, carried out during a period of 70 hours. During that time, the molecules evolved to use progressively lower concentrations of a required substrate; each step of that adaptation was observed in real time.

We recently devised 2 further enhancements of the continuous evolution method. The first involves a controlled mutagenesis technique that can be applied throughout selective amplification. This technique allows us to maintain a diverse population of individuals, even in the face of stringent selection pressure, thus enabling a more comprehensive exploration of potentially advantageous variants. The second enhancement involves a method for isolating and then propagating individual RNA enzymes within water-in-oil compartments within a microfluidic chip. A novel multiport injector design allows us to produce millions of individual fluidic compartments of precisely controlled size (Fig. 1), ranging from 20 to 100 μm in diameter (containing 4–500 pL). These microcompartments allow each enzyme to express a "cellular" phenotype based on the enzyme's catalytic function.
Fig. 1. High-throughput production of water droplets in oil, carried out within a microfluidic chip. The liquid (containing a blue dye) enters the chip and fans out radially to meet the oil, where it forms microdroplets (here 100 μm in diameter), which are then collected. Each droplet contains, on average, 1 starting RNA enzyme and the materials necessary for that enzyme to undergo continuous evolution.

Ligand-Dependent Exponential Amplification Of RNA

We previously developed an RNA enzyme that catalyzes its own replication by joining 2 RNA substrates to form additional copies of itself. This enzyme was converted to a cross-catalytic format whereby 2 RNA enzymes catalyze each other's synthesis from a total of 4 RNA substrates. We then used in vitro evolution to improve substantially the activity of the cross-replicating RNA enzymes. The enzymes now can undergo efficient exponential amplification, generating about a billion copies in 30 hours at a constant temperature of 42°C.

Recently, we inserted a ligand-binding domain adjacent to the catalytic domain of the cross-replicating enzymes so that the enzymes undergo exponential amplification in the presence, but not the absence, of the corresponding ligand. The catalytic domain assumes its active conformation only when the ligand is present, resulting in a large signal that can be used to detect and quantify compounds of biomedical interest, such as proteins, drugs, and metabolites. For example, the cross-replicating enzymes were made dependent on theophylline, a drug commonly used to treat respiratory diseases, for which the dose must be carefully adjusted on the basis of its level in the serum. Strong exponential amplification occurred in the presence of theophylline, but amplification in the presence of caffeine was undetectable (Fig. 2), even though the 2 compounds differ by only a single methyl group. Furthermore, the exponential growth rate of the enzymes depended on the concentration of theophylline, a characteristic that allowed us to construct standardized curves that could be used to determine the concentration of theophylline in an unknown sample. The method is analogous to quantitative polymerase chain reaction for the detection of nucleic acids but can be generalized to a wide variety of targets relevant to medical diagnostics and environmental monitoring.
Fig. 2. Exponential amplification of cross-replicating RNA enzymes dependent on the compound theophylline. Amplification occurs in the presence of 1 mM theophylline (filled circles) but not in the presence of 1 mM caffeine (open circles).

Publications

Paegel, B.M., Joyce, G.F. Darwinian evolution on a chip. PLoS Biol. 6:e85, 2008.

 

Gerald F. Joyce, M.D., Ph.D.
Professor
Dean, Faculty

Joyce Web Site